Structural and biophysical characterisation of agrin laminin G3 domain constructs.

PubMed

Tidow, Henning; Mattle, Daniel; Nissen, Poul

2011-01-01

Agrin mediates accumulation of acetylcholine receptors (AChRs) at the developing neuromuscular junction, but has also been implicated as a regulator of central nervous system (CNS) synapses. A C-terminal region of agrin (Ag-C20) binds to the α3 subunit of the sodium-potassium ATPase (NKA) in CNS neurons suggesting that α3NKA is a neuronal agrin receptor, whereas a shorter agrin fragment (Ag-C15) was shown to act as a competitive antagonist. Here, we show that the agrin C22 construct, which represents the naturally occurring neurotrypsin cleavage product, constitutes a well-folded, stable domain, while the deletion of 48 residues that correspond to strands β1-β4 of the agrin laminin G3 domain imposed by the agrin C15 construct leads to a misfolded protein.

Laminin-111 protein therapy reduces muscle pathology and improves viability of a mouse model of merosin-deficient congenital muscular dystrophy.

PubMed

Rooney, Jachinta E; Knapp, Jolie R; Hodges, Bradley L; Wuebbles, Ryan D; Burkin, Dean J

2012-04-01

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a lethal muscle-wasting disease that is caused by mutations in the LAMA2 gene, resulting in the loss of laminin-α2 protein. MDC1A patients exhibit severe muscle weakness from birth, are confined to a wheelchair, require ventilator assistance, and have reduced life expectancy. There are currently no effective treatments or cures for MDC1A. Laminin-α2 is required for the formation of heterotrimeric laminin-211 (ie, α2, β1, and γ1) and laminin-221 (ie, α2, β2, and γ1), which are major constituents of skeletal muscle basal lamina. Laminin-111 (ie, α1, β1, and γ1) is the predominant laminin isoform in embryonic skeletal muscle and supports normal skeletal muscle development in laminin-α2-deficient muscle but is absent from adult skeletal muscle. In this study, we determined whether treatment with Engelbreth-Holm-Swarm-derived mouse laminin-111 protein could rescue MDC1A in the dy(W-/-) mouse model. We demonstrate that laminin-111 protein systemically delivered to the muscles of laminin-α2-deficient mice prevents muscle pathology, improves muscle strength, and dramatically increases life expectancy. Laminin-111 also prevented apoptosis in laminin-α2-deficient mouse muscle and primary human MDC1A myogenic cells, which indicates a conserved mechanism of action and cross-reactivity between species. Our results demonstrate that laminin-111 can serve as an effective protein substitution therapy for the treatment of muscular dystrophy in the dy(W-/-) mouse model and establish the potential for its use in the treatment of MDC1A. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Jab1 regulates Schwann cell proliferation and axonal sorting through p27

PubMed Central

Porrello, Emanuela; Rivellini, Cristina; Dina, Giorgia; Triolo, Daniela; Del Carro, Ubaldo; Ungaro, Daniela; Panattoni, Martina; Feltri, Maria Laura; Wrabetz, Lawrence; Pardi, Ruggero; Quattrini, Angelo

2014-01-01

Axonal sorting is a crucial event in nerve formation and requires proper Schwann cell proliferation, differentiation, and contact with axons. Any defect in axonal sorting results in dysmyelinating peripheral neuropathies. Evidence from mouse models shows that axonal sorting is regulated by laminin211– and, possibly, neuregulin 1 (Nrg1)–derived signals. However, how these signals are integrated in Schwann cells is largely unknown. We now report that the nuclear Jun activation domain–binding protein 1 (Jab1) may transduce laminin211 signals to regulate Schwann cell number and differentiation during axonal sorting. Mice with inactivation of Jab1 in Schwann cells develop a dysmyelinating neuropathy with axonal sorting defects. Loss of Jab1 increases p27 levels in Schwann cells, which causes defective cell cycle progression and aberrant differentiation. Genetic down-regulation of p27 levels in Jab1-null mice restores Schwann cell number, differentiation, and axonal sorting and rescues the dysmyelinating neuropathy. Thus, Jab1 constitutes a regulatory molecule that integrates laminin211 signals in Schwann cells to govern cell cycle, cell number, and differentiation. Finally, Jab1 may constitute a key molecule in the pathogenesis of dysmyelinating neuropathies. PMID:24344238

Probing the acidic residue within the integrin binding site of laminin-511 that interacts with the metal ion-dependent adhesion site of α6β1 integrin.

PubMed

Taniguchi, Yukimasa; Li, Shaoliang; Takizawa, Mamoru; Oonishi, Eriko; Toga, Junko; Yagi, Emiko; Sekiguchi, Kiyotoshi

2017-06-03

Laminins are major cell-adhesive proteins of basement membranes that interact with integrins in a divalent cation-dependent manner. Laminin-511 consists of α5, β1, and γ1 chains, of which three laminin globular domains of the α5 chain (α5/LG1-3) and a Glu residue in the C-terminal tail of chain γ1 (γ1-Glu1607) are required for binding to integrins. However, it remains unsettled whether the Glu residue in the γ1 tail is involved in integrin binding by coordinating the metal ion in the metal ion-dependent adhesion site of β1 integrin (β1-MIDAS), or by stabilizing the conformation of α5/LG1-3. To address this issue, we examined whether α5/LG1-3 contain an acidic residue required for integrin binding that is as critical as the Glu residue in the γ1 tail; to achieve this, we undertook exhaustive alanine substitutions of the 54 acidic residues present in α5/LG1-3 of the E8 fragment of laminin-511 (LM511E8). Most of the alanine mutants possessed α6β1 integrin binding activities comparable with wild-type LM511E8. Alanine substitution for α5-Asp3198 and Asp3219 caused mild reduction in integrin binding activity, and that for α5-Asp3218 caused severe reduction, possibly resulting from conformational perturbation of α5/LG1-3. When α5-Asp3218 was substituted with asparagine, the resulting mutant possessed significant binding activity to α6β1 integrin, indicating that α5-Asp3218 is not directly involved in integrin binding through coordination with the metal ion in β1-MIDAS. Given that substitution of γ1-Glu1607 with glutamine nullified the binding activity to α6β1 integrin, these results, taken together, support the possibility that the critical acidic residue coordinating the metal ion in β1-MIDAS is Glu1607 in the γ1 tail, but no such residue is present in α5/LG1-3. Copyright © 2017 Elsevier Inc. All rights reserved.

Recognition of fibronectin by Penicillium marneffei conidia via a sialic acid-dependent process and its relationship to the interaction between conidia and laminin.

PubMed

Hamilton, A J; Jeavons, L; Youngchim, S; Vanittanakom, N

1999-10-01

Adhesion of Penicillium marneffei conidia to the extracellular matrix protein laminin via a sialic acid-dependent process has previously been demonstrated. This study describes the interaction of P. marneffei conidia with fibronectin and examines the relationship of this process to the recognition of laminin via conidia. Immunofluorescence microscopy demonstrated that fibronectin bound to the surface of conidia and to phialides, but not to hyphae, in a pattern similar to that reported for laminin. Conidia were able to bind to fibronectin immobilized on microtiter plates in a concentration-dependent manner. However, binding to fibronectin (at any given concentration of protein and conidia) was less than that to laminin under equivalent conditions. Soluble fibronectin and antifibronectin antibody inhibited adherence of conidia to fibronectin in the plate adherence assay; soluble laminin also caused pronounced inhibition. Various monosaccharides and several peptides had no effect on adherence to fibronectin. However, N-acetylneuraminic acid abolished adherence to fibronectin, indicating that the interaction was mediated through a sialic acid-dependent process; the latter parallels observations of laminin binding by conidia. Fibronectin binding (and binding of laminin) was considerably reduced by prolonged preincubation of conidia with chymotrypsin, suggesting the protein nature of the binding site. Conidia from older cultures were more adherent to both immobilized fibronectin and laminin than conidia from younger cultures. Ligand affinity binding demonstrated the presence of a 20-kDa protein with the ability to bind both fibronectin and laminin. There would therefore appear to be a common receptor for the binding of fibronectin and laminin on the surface of P. marneffei, and the interaction described here maybe important in mediating attachment of the fungus to host tissue.

Laminin γ3 plays an important role in retinal lamination, photoreceptor organisation and ganglion cell differentiation.

PubMed

Dorgau, Birthe; Felemban, Majed; Sharpe, Alexander; Bauer, Roman; Hallam, Dean; Steel, David H; Lindsay, Susan; Mellough, Carla; Lako, Majlinda

2018-05-23

Laminins are heterotrimeric glycoproteins of the extracellular matrix. Eleven different laminin chains have been identified in vertebrates. They are ubiquitously expressed in the human body, with a distinct tissue distribution. Laminin expression in neural retina and their functional role during human retinogenesis is still unknown. This study investigated the laminin expression in human developing and adult retina, showing laminin α1, α5, β1, β2 and γ1 to be predominantly expressed in Bruch's membrane and the inner limiting membrane. Laminin-332 and laminin γ3 expression were mainly observed in the neural retina during retinal histogenesis. These expression patterns were largely conserved in pluripotent stem cell-derived retinal organoids. Blocking of laminin γ3 function in retinal organoids resulted in the disruption of laminar organisation and synapse formation, the loss of photoreceptor organisation and retinal ganglion cells. Our data demonstrate a unique temporal and spatial expression for laminins and reveal a novel role for laminin γ3 during human retinogenesis.

Efficient delivery of connective tissue growth factor shRNA using PAMAM nanoparticles.

PubMed

Huang, Z J; Yi, B; Yuan, H; Yang, G P

2014-08-28

The aim of this study was to detect the anti-fibrosis activity of connective tissue growth factor (CTGF) small hairpin RNA (shRNA) mediated by polyamidoamine dendrimer nanoparticles in rat myocardial cell lines and myocardium. CTGF shRNAs were constructed from inverted oligonucleotides and a polyamidoamine nanoparticle vector was used to transfer shRNA into H9c2 myocardial cells and spontaneously hypertensive rats. The expression of CTGF, transforming growth factor-b1, and laminin were measured by semi-quantitative reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry. pCTGF-shRNA significantly reduced CTGF upregulation induced by angiotensin II in H9c2 myocardial cells. The mRNA and protein expression of CTGF and laminin in pCTGF-shRNA-transferred spontaneously hypertensive rats decreased significantly compared to the control group and pHK-shRNA group (P < 0.05). The expression of transforming growth factor-b1 showed no significant difference among the 3 groups (P > 0.05). pCTGF-shRNA mediated by polyamidoamine can be used to successfully reduce myocardial CTGF and laminin expression, suggesting that this system can be used to improve myocardial fibrosis therapy.

Integrin β1 regulates leiomyoma cytoskeletal integrity and growth

PubMed Central

Malik, Minnie; Segars, James; Catherino, William H.

2014-01-01

Uterine leiomyomas are characterized by an excessive extracellular matrix, increased mechanical stress, and increased active RhoA. Previously, we observed that mechanical signaling was attenuated in leiomyoma, but the mechanisms responsible remain unclear. Integrins, especially integrin β1, are transmembrane adhesion receptors that couple extracellular matrix stresses to the intracellular cytoskeleton to influence cell proliferation and differentiation. Here we characterized integrin and laminin to signaling in leiomyoma cells. We observed a 2.25 ± 0.32 fold increased expression of integrin β1 in leiomyoma cells, compared to myometrial cells. Antibody-mediated inhibition of integrin β1 led to significant growth inhibition in leiomyoma cells and a loss of cytoskeletal integrity. Specifically, polymerization of actin filaments and formation of focal adhesions were reduced by inhibition of integrin p1. Inhibition of integrin β1 in leiomyoma cells led to 0.81 ± 0.02 fold decrease in active RhoA, and resembled levels found in serum-starved cells. Likewise, inhibition of integrin β1 was accompanied by a decrease in phospho-ERK. Compared to myometrial cells, leiomyoma cells demonstrated increased expression of integrin α6 subunit to laminin receptor (1.91 ± 0.11 fold), and increased expression of laminin 5α (1.52±0.02), laminin 5β (3.06±0.92), and laminin 5γ (1.66 ± 0.06). Of note, leiomyoma cells grown on laminin matrix appear to realign themselves. Taken together, the findings reveal that the attenuated mechanical signaling in leiomyoma cells is accompanied by an increased expression and a dependence on integrin β1 signaling in leiomyoma cells, compared to myometrial cells. PMID:23023061

Function of the alpha 6 Integrins in Breast Carcinoma

DTIC Science & Technology

2001-10-01

motility on laminin-1. Time-lapse was resistant to solubilization with Triton X-100. Cy- videomicroscopy of clone A cells on laminin-1 revealed...represent SEM. The Integrin a6/34 Participates in the Dynamic Formation of Actin-based Motility Structures: Filopodia lapse videomicroscopy in serum-free...threefold greater time-lapse videomicroscopy to understand how the cu6P34 than on an equivalent concentration of collagen type I, integrin contributes to

Analysis of Nidogen-1/Laminin γ1 Interaction by Cross-Linking, Mass Spectrometry, and Computational Modeling Reveals Multiple Binding Modes

PubMed Central

Lössl, Philip; Kölbel, Knut; Tänzler, Dirk; Nannemann, David; Ihling, Christian H.; Keller, Manuel V.; Schneider, Marian; Zaucke, Frank; Meiler, Jens; Sinz, Andrea

2014-01-01

We describe the detailed structural investigation of nidogen-1/laminin γ1 complexes using full-length nidogen-1 and a number of laminin γ1 variants. The interactions of nidogen-1 with laminin variants γ1 LEb2–4, γ1 LEb2–4 N836D, γ1 short arm, and γ1 short arm N836D were investigated by applying a combination of (photo-)chemical cross-linking, high-resolution mass spectrometry, and computational modeling. In addition, surface plasmon resonance and ELISA studies were used to determine kinetic constants of the nidogen-1/laminin γ1 interaction. Two complementary cross-linking strategies were pursued to analyze solution structures of laminin γ1 variants and nidogen-1. The majority of distance information was obtained with the homobifunctional amine-reactive cross-linker bis(sulfosuccinimidyl)glutarate. In a second approach, UV-induced cross-linking was performed after incorporation of the diazirine-containing unnatural amino acids photo-leucine and photo-methionine into laminin γ1 LEb2–4, laminin γ1 short arm, and nidogen-1. Our results indicate that Asn-836 within laminin γ1 LEb3 domain is not essential for complex formation. Cross-links between laminin γ1 short arm and nidogen-1 were found in all protein regions, evidencing several additional contact regions apart from the known interaction site. Computational modeling based on the cross-linking constraints indicates the existence of a conformational ensemble of both the individual proteins and the nidogen-1/laminin γ1 complex. This finding implies different modes of interaction resulting in several distinct protein-protein interfaces. PMID:25387007

The role of alpha3beta1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes.

PubMed

deHart, Gregory W; Healy, Kevin E; Jones, Jonathan C R

2003-02-01

Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the incorporation of laminin-5 into its proper higher-order structure within the extracellular matrix of keratinocytes and (2) that the organizational state of laminin-5 has an influence on laminin-5 matrix function. Copyright 2003 Elsevier Science (USA)

A fractal nature for polymerized laminin.

PubMed

Hochman-Mendez, Camila; Cantini, Marco; Moratal, David; Salmeron-Sanchez, Manuel; Coelho-Sampaio, Tatiana

2014-01-01

Polylaminin (polyLM) is a non-covalent acid-induced nano- and micro-structured polymer of the protein laminin displaying distinguished biological properties. Polylaminin stimulates neuritogenesis beyond the levels achieved by ordinary laminin and has been shown to promote axonal regeneration in animal models of spinal cord injury. Here we used confocal fluorescence microscopy (CFM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize its three-dimensional structure. Renderization of confocal optical slices of immunostained polyLM revealed the aspect of a loose flocculated meshwork, which was homogeneously stained by the antibody. On the other hand, an ordinary matrix obtained upon adsorption of laminin in neutral pH (LM) was constituted of bulky protein aggregates whose interior was not accessible to the same anti-laminin antibody. SEM and AFM analyses revealed that the seed unit of polyLM was a flat polygon formed in solution whereas the seed structure of LM was highly heterogeneous, intercalating rod-like, spherical and thin spread lamellar deposits. As polyLM was visualized at progressively increasing magnifications, we observed that the morphology of the polymer was alike independently of the magnification used for the observation. A search for the Hausdorff dimension in images of the two matrices showed that polyLM, but not LM, presented fractal dimensions of 1.55, 1.62 and 1.70 after 1, 8 and 12 hours of adsorption, respectively. Data in the present work suggest that the intrinsic fractal nature of polymerized laminin can be the structural basis for the fractal-like organization of basement membranes in the neurogenic niches of the central nervous system.

Presynaptic congenital myasthenic syndrome with a homozygous sequence variant in LAMA5 combines myopia, facial tics, and failure of neuromuscular transmission.

PubMed

Maselli, Ricardo A; Arredondo, Juan; Vázquez, Jessica; Chong, Jessica X; Bamshad, Michael J; Nickerson, Deborah A; Lara, Marian; Ng, Fiona; Lo, Victoria L; Pytel, Peter; McDonald, Craig M

2017-08-01

Defects in genes encoding the isoforms of the laminin alpha subunit have been linked to various phenotypic manifestations, including brain malformations, muscular dystrophy, ocular defects, cardiomyopathy, and skin abnormalities. We report here a severe defect of neuromuscular transmission in a consanguineous patient with a homozygous variant in the laminin alpha-5 subunit gene (LAMA5). The variant c.8046C>T (p.Arg2659Trp) is rare and has a predicted deleterious effect. The affected individual, who also carries a rare homozygous sequence variant in LAMA1, had muscle weakness, myopia, and facial tics. Magnetic resonance imaging of brain showed mild volume loss and periventricular T2 prolongation. Repetitive nerve stimulation revealed 50% decrement of compound muscle action potential amplitudes and 250% facilitation immediately after exercise, Endplate studies identified a profound reduction of the endplate potential quantal content and endplates with normal postsynaptic folding that were denuded or partially occupied by small nerve terminals. Expression studies revealed that p.Arg2659Trp caused decreased binding of laminin alpha-5 to SV2A and impaired laminin-521 cell-adhesion and cell projection support in primary neuronal cultures. In summary, this report describing severe neuromuscular transmission failure in a patient with a LAMA5 mutation expands the list of phenotypes associated with defects in genes encoding alpha-laminins. © 2017 Wiley Periodicals, Inc.

Hedgehog signaling and laminin play unique and synergistic roles in muscle development.

PubMed

Peterson, Matthew T; Henry, Clarissa A

2010-03-01

Hedgehog (Hh) signaling and laminin-111, a basement membrane protein, are required for early muscle development. Hh signaling specifies different populations of muscle fibers and laminin-111 is critical for early muscle morphogenesis. However, additional requirements for Hh signaling and laminin during later phases of muscle development are not known. Furthermore, interactions between Hh signaling and laminin in this context are unknown. We used laminin gamma1 mutant zebrafish and cyclopamine to block Hh signal transduction separately and in combination to investigate their functions and interactions. We found that both Hh signaling and laminin are required for normal myosin chain expression. In addition, Hh signaling and laminin act synergistically during fast-twitch fiber elongation: fast muscle cells do not elongate in embryos deficient for both Hh signaling and laminin. Finally, we present evidence that suggests that Hh signaling is indirectly required via slow fiber specification for recovery of fast fiber elongation in laminin gamma1 mutant embryos. Copyright (c) 2010 Wiley-Liss, Inc.

Partial rescue of glomerular laminin alpha5 mutations by wild-type endothelia produce hybrid glomeruli.

PubMed

Abrahamson, Dale R; St John, Patricia L; Isom, Kathryn; Robert, Barry; Miner, Jeffrey H

2007-08-01

Both endothelial cells and podocytes are sources for laminin alpha1 at the inception of glomerulogenesis and then for laminin alpha5 during glomerular maturation. Why glomerular basement membranes (GBM) undergo laminin transitions is unknown, but this may dictate glomerular morphogenesis. In mice that genetically lack laminin alpha5, laminin alpha5beta2gamma1 is not assembled, vascularized glomeruli fail to form, and animals die at midgestation with neural tube closure and placental deficits. It was previously shown that renal cortices of newborn mice contain endothelial progenitors (angioblasts) and that when embryonic day 12 kidneys are transplanted into newborn kidney, hybrid glomeruli (host-derived endothelium and donor-derived podocytes) result. Reasoning that host endothelium may correct the glomerular phenotype that is seen in laminin alpha5 mutants, alpha5 null embryonic day 12 metanephroi were grafted into wild-type newborn kidney. Hybrid glomeruli were identified in grafts by expression of a host-specific LacZ lineage marker. Labeling of glomerular hybrid GBM with chain-specific antibodies showed a markedly stratified distribution of laminins: alpha5 was found only on the inner endothelial half of GBM, whereas alpha1 located to outer layers beneath mutant podocytes. For measurement of the contribution of host endothelium to hybrid GBM, immunofluorescent signals for laminin alpha5 were quantified: Hybrid GBM contained approximately 50% the normal alpha5 complement as wild-type GBM. Electron microscopy of glomerular hybrids showed vascularization, but podocyte foot processes were absent. It was concluded that (1) endothelial and podocyte-derived laminins remain tethered to their cellular origin, (2) developing endothelial cells contribute large amounts of GBM laminins, and (3) podocyte foot process differentiation may require direct exposure to laminin alpha5.

Localization of laminin B1 mRNA in retinal ganglion cells by in situ hybridization

PubMed Central

1990-01-01

In the nervous system, neuronal migration and axonal growth are dependent on specific interactions with extracellular matrix proteins. During development of the vertebrate retina, ganglion cell axons extend along the internal limiting (basement) membrane and form the optic nerve. Laminin, a major component of basement membranes, is known to be present in the internal limiting membrane, and might be involved in the growth of ganglion cell axons. The identity of the cells that produce retinal laminin, however, has not been established. In the present study, we have used in situ hybridization to localize the sites of laminin B1 mRNA synthesis in the developing mouse retina. Our results show that there are at least two principal sites of laminin B1 mRNA synthesis: (a) the hyaloid vessels and the lens during the period of major axonal outgrowth, and (b) the retinal ganglion cells at later development stages. Muller (glial) cells, the major class of nonneuronal cells in the retina, do not appear to express laminin B1 mRNA either during development or in the adult retina. In Northern blots, we found a single transcript of approximately 6-kb size that encodes the laminin B1 chain in the retina. Moreover, laminin B1 mRNA level was four- to fivefold higher in the postnatal retina compared to that in the adult. Our results show that in addition to nonneuronal cells, retinal ganglion cells also synthesize laminin. The function of laminin in postnatal retinas, however, remains to be elucidated. Nevertheless, our findings raise the possibility that neurons in other parts of the nervous system might also synthesize extracellular matrix proteins. PMID:2351694

Human umbilical cord mesenchymal stromal cells exhibit immature nucleus pulposus cell phenotype in a laminin-rich pseudo-three-dimensional culture system.

PubMed

Chon, Brian H; Lee, Esther J; Jing, Liufang; Setton, Lori A; Chen, Jun

2013-10-02

Cell supplementation to the herniated or degenerated intervertebral disc (IVD) is a potential strategy to promote tissue regeneration and slow disc pathology. Human umbilical cord mesenchymal stromal cells (HUCMSCs) - originating from the Wharton's jelly - remain an attractive candidate for such endeavors with their ability to differentiate into multiple lineages. Previously, mesenchymal stem cells (MSCs) have been studied as a potential source for disc tissue regeneration. However, no studies have demonstrated that MSCs can regenerate matrix with unique characteristics matching that of immature nucleus pulposus (NP) tissues of the IVD. In our prior work, immature NP cells were found to express specific laminin isoforms and laminin-binding receptors that may serve as phenotypic markers for evaluating MSC differentiation to NP-like cells. The goal of this study is to evaluate these markers and matrix synthesis for HUCMSCs cultured in a laminin-rich pseudo-three-dimensional culture system. HUCMSCs were seeded on top of Transwell inserts pre-coated with Matrigel™, which contained mainly laminin-111. Cells were cultured under hypoxia environment with three differentiation conditions: NP differentiation media (containing 2.5% Matrigel™ solution to provide for a pseudo-three-dimensional laminin culture system) with no serum, or the same media supplemented with either insulin-like growth factor-1 (IGF-1) or transforming growth factor-β1 (TGF-β1). Cell clustering behavior, matrix production and the expression of NP-specific laminin and laminin-receptors were evaluated at days 1, 7, 13 and 21 of culture. Data show that a pseudo-three-dimensional culture condition (laminin-1 rich) promoted HUCMSC differentiation under no serum conditions. Starting at day 1, HUCMSCs demonstrated a cell clustering morphology similar to that of immature NP cells in situ and that observed for primary immature NP cells within the similar laminin-rich culture system (prior study). Differentiated HUCMSCs under all conditions were found to contain glycosaminoglycan, expressed extracellular matrix proteins of collagen II and laminin α5, and laminin receptors (integrin α3 and β4 subunits). However, neither growth factor treatment generated distinct differences in NP-like phenotype for HUCMSC as compared with no-serum conditions. HUCMSCs have the potential to differentiate into cells sharing features with immature NP cells in a laminin-rich culture environment and may be useful for IVD cellular therapy.

Cellular Interaction of Integrin α3β1 with Laminin 5 Promotes Gap Junctional Communication

PubMed Central

Lampe, Paul D.; Nguyen, Beth P.; Gil, Susana; Usui, Marcia; Olerud, John; Takada, Yoshikazu; Carter, William G.

1998-01-01

Wounding of skin activates epidermal cell migration over exposed dermal collagen and fibronectin and over laminin 5 secreted into the provisional basement membrane. Gap junctional intercellular communication (GJIC) has been proposed to integrate the individual motile cells into a synchronized colony. We found that outgrowths of human keratinocytes in wounds or epibole cultures display parallel changes in the expression of laminin 5, integrin α3β1, E-cadherin, and the gap junctional protein connexin 43. Adhesion of keratinocytes on laminin 5, collagen, and fibronectin was found to differentially regulate GJIC. When keratinocytes were adhered on laminin 5, both structural (assembly of connexin 43 in gap junctions) and functional (dye transfer) assays showed a two- to threefold increase compared with collagen and five- to eightfold over fibronectin. Based on studies with immobilized integrin antibody and integrin-transfected Chinese hamster ovary cells, the interaction of integrin α3β1 with laminin 5 was sufficient to promote GJIC. Mapping of intermediate steps in the pathway linking α3β1–laminin 5 interactions to GJIC indicated that protein trafficking and Rho signaling were both required. We suggest that adhesion of epithelial cells to laminin 5 in the basement membrane via α3β1 promotes GJIC that integrates individual cells into synchronized epiboles. PMID:9852164

Nano-structure of the laminin γ-1 short arm reveals an extended and curved multidomain assembly.

PubMed

Patel, Trushar R; Morris, Gordon A; Zwolanek, Daniela; Keene, Douglas R; Li, Jianhua; Harding, Stephen E; Koch, Manuel; Stetefeld, Jörg

2010-09-01

Laminins are multidomain glycoproteins that play important roles in development and maintenance of the extracellular matrix via their numerous interactions with other proteins. Several receptors for the laminin short arms revealed their importance in network formation and intercellular signaling. However, both the detailed structure of the laminin γ-1 short arm and its organization within the complexes is poorly understood due to the complexity of the molecule and the lack of a high-resolution structure. The presented data provide the first subatomic resolution structure for the laminin γ-1 short arm in solution. This was achieved using an integrated approach that combined a number of complementary biophysical techniques such as small angle X-ray scattering (SAXS), analytical ultracentrifugation, dynamic light scattering and electron microscopy. As a result of this study, we have obtained a significantly improved model for the laminin γ-1 short arm that represents a major step forward in molecular understanding of laminin-mediated complex formations. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Presence of Laminin Receptors in Staphylococcus aureus

NASA Astrophysics Data System (ADS)

Lopes, J. D.; Dos Reis, M.; Brentani, R. R.

1985-07-01

A characteristic feature of infection by Staphylococcus aureus is bloodstream invasion and widespread metastatic abscess formation. The ability to extravasate, which entails crossing the vascular basement membrane, appears to be critical for the organism's pathogenicity. Extravasation by normal and neoplastic mammalian cells has been correlated with the presence of specific cell surface receptors for the basement membrane glycoprotein laminin. Similar laminin receptors were found in Staphylococcus aureus but not in Staphylococcus epidermidis, a noninvasive pathogen. There were about 100 binding sites per cell, with an apparent binding affinity of 2.9 nanomolar. The molecular weight of the receptor was 50,000 and pI was 4.2. Eukaryotic laminin receptors were visualized by means of the binding of S. aureus in the presence of laminin. Prokaryotic and eukaryotic invasive cells might utilize similar, if not identical, mechanisms for invasion.

Tetraspanin CD151 regulates alpha6beta1 integrin adhesion strengthening

NASA Technical Reports Server (NTRS)

Lammerding, Jan; Kazarov, Alexander R.; Huang, Hayden; Lee, Richard T.; Hemler, Martin E.

2003-01-01

The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including alpha6beta1. To probe strength of alpha6beta1-dependent adhesion to laminin-1, defined forces (0-1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-alpha6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening alpha6beta1 integrin-mediated adhesion to laminin-1.

Melanoma Spheroid Formation Involves Laminin-Associated Vasculogenic Mimicry

PubMed Central

Larson, Allison R.; Lee, Chung-Wei; Lezcano, Cecilia; Zhan, Qian; Huang, John; Fischer, Andrew H.; Murphy, George F.

2015-01-01

Melanoma is a tumor where virulence is conferred on transition from flat (radial) to three-dimensional (tumorigenic) growth. Virulence of tumorigenic growth is governed by numerous attributes, including presence of self-renewing stem-like cells and related formation of patterned networks associated with the melanoma mitogen, laminin, a phenomenon known as vasculogenic mimicry. Vasculogenic mimicry is posited to contribute to melanoma perfusion and nutrition in vivo; we hypothesized that it may also play a role in stem cell–driven spheroid formation in vitro. Using a model of melanoma in vitro tumorigenesis, laminin-associated networks developed in association with three-dimensional melanoma spheroids. Real-time PCR analysis of laminin subunits showed that spheroids formed from anchorage-independent melanoma cells expressed increased α4 and β1 laminin chains and α4 laminin expression was confirmed by in situ hybridization. Association of laminin networks with melanoma stem cell–associated nestin and vascular endothelial growth factor receptor-1 also was documented. Moreover, knockdown of nestin gene expression impaired laminin expression and network formation within spheroids. Laminin networks were remarkably similar to those observed in melanoma xenografts in mice and to those seen in patient melanomas. These data indicate that vasculogenic mimicry–like laminin networks, in addition to their genesis in vivo, are integral to the extracellular architecture of melanoma spheroids in vitro, where they may serve as stimulatory scaffolds to support three-dimensional growth. PMID:24332013

Increased Expression of Laminin Subunit Alpha 1 Chain by dCas9-VP160.

PubMed

Perrin, Arnaud; Rousseau, Joël; Tremblay, Jacques P

2017-03-17

Laminin-111 protein complex links the extracellular matrix to integrin α7β1 in sarcolemma, thus replacing in dystrophic muscles links normally insured by the dystrophin complex. Laminin-111 injection in mdx mouse stabilized sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damages. These results suggested that increased laminin-111 is a potential therapy for DMD. Laminin subunit beta 1 and laminin subunit gamma 1 are expressed in adult human muscle, but laminin subunit alpha 1 (LAMA1) gene is expressed only during embryogenesis. We thus developed an alternative method to laminin-111 protein repeated administration by inducing expression of the endogenous mouse Lama1 gene. This was done with the CRSPR/Cas9 system, i.e., by targeting the Lama1 promoter with one or several gRNAs and a dCas9 coupled with the VP160 transcription activation domain. Lama1 mRNA (qRT-PCR) and proteins (immunohistochemistry and western blot) were not detected in the control C2C12 myoblasts and in control muscles. However, significant expression was observed in cells transfected and in mouse muscles electroporated with plasmids coding for dCas9-VP160 and a gRNA. Larger synergic increases were observed by using two or three gRNAs. The increased Lama1 expression did not modify the expression of the α7 and β1 integrins. Increased expression of Lama1 by the CRISPR/Cas9 system will have to be further investigated by systemic delivery of the CRISPR/Cas9 components to verify whether this could be a treatment for several myopathies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Hepatocyte attachment to laminin is mediated through multiple receptors

PubMed Central

1990-01-01

The interaction of hepatocytes with the basement membrane glycoprotein laminin was studied using synthetic peptides derived from laminin sequences. Rat hepatocytes bind to laminin and three different sites within the A and B1 chains of laminin were identified. Active laminin peptides include the PA22-2 peptide (close to the carboxyl end of the long arm in the A chain), the RGD-containing peptide, PA21 (in the short arm of the A chain) and the pentapeptide YIGSR (in the short arm of the B1 chain). PA22-2 was the most potent peptide, whereas the other two peptides had somewhat lower activity. Furthermore, hepatocyte attachment to laminin was inhibited by the three peptides, with PA22-2 being the most active. Various proteins from isolated membranes of cell- surface iodinated hepatocytes bound to a laminin affinity column including three immunologically related binding proteins : Mr = 67,000, 45,000, and 32,000. Several proteins--Mr = 80,000, 55,000, and 38,000- 36,000--with a lower affinity for laminin were also identified. Affinity chromatography on peptide columns revealed that the PA22-2 peptide specifically bound the Mr = 80,000, 67,000, 45,000, and 32,000 proteins, the PA21 peptide bound the Mr = 45,000 and 38,000-36,000 proteins and the YIGSR peptide column bound the 38,000-36,000 protein. Antisera to a bacterial fusion protein of the 32-kD laminin-binding protein (LBP-32) reacted strongly with the three laminin-binding proteins, Mr = 67,000, 45,000, and 32,000, showing that they are immunologically related. Immunoperoxidase microscopy studies confirmed that these proteins are present within the plasma membrane of the hepatocyte. The antisera inhibited the adhesion of hepatocytes to hepatocytes to laminin by 30%, supporting the finding that these receptors and others mediate the attachment of hepatocytes to several regions of laminin. PMID:2136861

Immunohistochemical Study of Laminin-332 γ2 Chain and MMP-9 in High Risk of Malignant Transformation Oral Lesions and OSCC.

PubMed

Silva, Eline Manhães Reid; Freitas, Vanessa Morais; Bautz, Willian Grassi; de Barros, Liliana Aparecida Pimenta; da Gama de Souza, Letícia Nogueira

2018-01-01

Oral squamous cell carcinoma is associated with alterations in basement membrane. Laminin-332 is present in basal lamina and performs multiple biologic effects by γ2 chain. Matrix metalloproteinase acts disrupting extracellular components and was related to poor prognosis in cancer. Here, molecular profile of laminin-332 γ2 chain and matrix metalloproteinase-9 was assessed in oral lesions. The expression of laminin-332 γ2 chain and matrix metalloproteinase-9 (MMP-9) was examined by immunohistochemistry in 10 patients with high risk of malignant transformation oral lesions and 26 cases of oral squamous cell carcinoma (OSCC). Associations between microscopic and clinicopathologic features were established. Immunostaining of laminin-332 γ2 chain in high risk oral lesions was most detected in basement membrane which is continuous, while the majority of OSCC cases showed a discontinuous membrane (P = 0.001). It was observed a positive reaction for γ2 chain in invasive fronts and a higher expression in epithelial compartment of smoking patients with OSCC (P < 0.0001). In epithelium, MMP-9 expression was presented in all layers with no difference between lesions. However, an elevated immunostaining in stromal cells was associated with male patients (P = 0.0054), older than 60 years (P = 0.0101) and with OSCC. Present study results support the hypothesis of changes in molecules expression in high risk oral lesions and oral squamous cell carcinoma. A relation between clinical and molecule profile was observed. Those molecules may represent a useful tool to predict oral cancer behaviour.

Loss of laminin alpha 1 results in multiple structural defects and divergent effects on adhesion during vertebrate optic cup morphogenesis

PubMed Central

Bryan, Chase D.; Chien, Chi-Bin; Kwan, Kristen M.

2016-01-01

The vertebrate eye forms via a complex set of morphogenetic events. The optic vesicle evaginates and undergoes transformative shape changes to form the optic cup, in which neural retina and retinal pigmented epithelium enwrap the lens. It has long been known that a complex, glycoprotein-rich extracellular matrix layer surrounds the developing optic cup throughout the process, yet the functions of the matrix and its specific molecular components have remained unclear. Previous work established a role for laminin extracellular matrix in particular steps of eye development, including optic vesicle evagination, lens differentiation, and retinal ganglion cell polarization, yet it is unknown what role laminin might play in the early process of optic cup formation subsequent to the initial step of optic vesicle evagination. Here, we use the zebrafish lama1 mutant (lama1UW1) to determine the function of laminin during optic cup morphogenesis. Using live imaging, we find, surprisingly, that loss of laminin leads to divergent effects on focal adhesion assembly in a spatiotemporally-specific manner, and that laminin is required for multiple steps of optic cup morphogenesis, including optic stalk constriction, invagination, and formation of a spherical lens. Laminin is not required for single cell behaviors and changes in cell shape. Rather, in lama1UW1 mutants, loss of epithelial polarity and altered adhesion lead to defective tissue architecture and formation of a disorganized retina. These results demonstrate that the laminin extracellular matrix plays multiple critical roles regulating adhesion and polarity to establish and maintain tissue structure during optic cup morphogenesis. PMID:27339294

Review: Lutheran/B-CAM: a laminin receptor on red blood cells and in various tissues.

PubMed

Kikkawa, Yamato; Miner, Jeffrey H

2005-01-01

The Lutheran blood group glycoprotein (Lu), also known as basal cell adhesion molecule (B-CAM), is a transmembrane receptor with five immunoglobulin-like domains in its extracellular region; it is therefore classified as a member of the immunoglobulin (Ig) gene family. Lu/B-CAM is observed not only on red blood cells, but also on a subset of muscle and epithelial cells in various tissues. Recently, several groups have reported that Lu/B-CAM is a novel receptor for laminin a5. The laminin a5 chain is a component of the laminin-511 (alpha 5 beta 1 gamma 1), -521 (alpha 5 beta 2 gamma 1), and -523 (alpha 5 beta 2 gamma 3) heterotrimers and is expressed throughout the mammalian body. We also have shown that Lu/B-CAM is co-localized with laminin alpha 5 in various tissues. Although the biological role of Lu/B-CAM remains unclear, the specific binding of Lu/B-CAM to laminin alpha 5 suggests that it plays an important role in developmental and physiological processes. It also is necessary to investigate further the interaction between Lu/B-CAM and laminin a5 in pathological processes, including sickle cell disease and cancer.

Chimeric protein identification of dystrophic, Pierson and other laminin polymerization residues

PubMed Central

McKee, Karen K.; Aleksandrova, Maya; Yurchenco, Peter D.

2018-01-01

Laminin polymerization is a key step of basement membrane self-assembly that depends on the binding of the three different N-terminal globular LN domains. Several mutations in the LN domains cause LAMA2-deficient muscular dystrophy and LAMB2-deficient Pierson syndrome. These mutations may affect polymerization. A novel approach to identify the amino acid residues required for polymerization has been applied to an analysis of these and other laminin LN mutations. The approach utilizes laminin-nidogen chimeric fusion proteins that bind to recombinant non-polymerizing laminins to provide a missing functional LN domain. Single amino acid substitutions introduced into these chimeras were tested to determine if polymerization activity and the ability to assemble on cell surfaces were lost. Several laminin-deficient muscular dystrophy mutations, renal Pierson syndrome mutations, and Drosophila mutations causing defects of heart development were identified as ones causing loss of laminin polymerization. In addition, two novel residues required for polymerization were identified in the laminin γ1 LN domain. PMID:29408412

Preventing tissue fibrosis by local biomaterials interfacing of specific cryptic extracellular matrix information

PubMed Central

Horejs, Christine-Maria; St-Pierre, Jean-Philippe; Ojala, Juha R. M.; Steele, Joseph A. M.; da Silva, Patricia Barros; Rynne-Vidal, Angela; Maynard, Stephanie A.; Hansel, Catherine S.; Rodríguez-Fernández, Clara; Mazo, Manuel M.; You, Amanda Y. F.; Wang, Alex J.; von Erlach, Thomas; Tryggvason, Karl; López-Cabrera, Manuel; Stevens, Molly M.

2017-01-01

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin β1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the β1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor β1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets. PMID:28593951

Preventing tissue fibrosis by local biomaterials interfacing of specific cryptic extracellular matrix information

NASA Astrophysics Data System (ADS)

Horejs, Christine-Maria; St-Pierre, Jean-Philippe; Ojala, Juha R. M.; Steele, Joseph A. M.; da Silva, Patricia Barros; Rynne-Vidal, Angela; Maynard, Stephanie A.; Hansel, Catherine S.; Rodríguez-Fernández, Clara; Mazo, Manuel M.; You, Amanda Y. F.; Wang, Alex J.; von Erlach, Thomas; Tryggvason, Karl; López-Cabrera, Manuel; Stevens, Molly M.

2017-06-01

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin β1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the β1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor β1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets.

Interdependence of laminin-mediated clustering of lipid rafts and the dystrophin complex in astrocytes.

PubMed

Noël, Geoffroy; Tham, Daniel Kai Long; Moukhles, Hakima

2009-07-17

Astrocyte endfeet surrounding blood vessels are active domains involved in water and potassium ion transport crucial to the maintenance of water and potassium ion homeostasis in brain. A growing body of evidence points to a role for dystroglycan and its interaction with perivascular laminin in the targeting of the dystrophin complex and the water-permeable channel, aquaporin 4 (AQP4), at astrocyte endfeet. However, the mechanisms underlying such compartmentalization remain poorly understood. In the present study we found that AQP4 resided in Triton X-100-insoluble fraction, whereas dystroglycan was recovered in the soluble fraction in astrocytes. Cholesterol depletion resulted in the translocation of a pool of AQP4 to the soluble fraction indicating that its distribution is indeed associated with cholesterol-rich membrane domains. Upon laminin treatment AQP4 and the dystrophin complex, including dystroglycan, reorganized into laminin-associated clusters enriched for the lipid raft markers GM1 and flotillin-1 but not caveolin-1. Reduced diffusion rates of GM1 in the laminin-induced clusters were indicative of the reorganization of raft components in these domains. In addition, both cholesterol depletion and dystroglycan silencing reduced the number and area of laminin-induced clusters of GM1, AQP4, and dystroglycan. These findings demonstrate the interdependence between laminin binding to dystroglycan and GM1-containing lipid raft reorganization and provide novel insight into the dystrophin complex regulation of AQP4 polarization in astrocytes.

Laminin promotes neuritic regeneration from cultured peripheral and central neurons

PubMed Central

1983-01-01

The ability of axons to grow through tissue in vivo during development or regeneration may be regulated by the availability of specific neurite-promoting macromolecules located within the extracellular matrix. We have used tissue culture methods to examine the relative ability of various extracellular matrix components to elicit neurite outgrowth from dissociated chick embryo parasympathetic (ciliary ganglion) neurons in serum-free monolayer culture. Purified laminin from both mouse and rat sources, as well as a partially purified polyornithine-binding neurite promoting factor (PNPF-1) from rat Schwannoma cells all stimulate neurite production from these neurons. Laminin and PNPF-1 are also potent stimulators of neurite growth from cultured neurons obtained from other peripheral as well as central neural tissues, specifically avian sympathetic and sensory ganglia and spinal cord, optic tectum, neural retina, and telencephalon, as well as from sensory ganglia of the neonatal mouse and hippocampal, septal, and striatal tissues of the fetal rat. A quantitative in vitro bioassay method using ciliary neurons was used to (a) measure and compare the specific neurite-promoting activities of these agents, (b) confirm that during the purification of laminin, the neurite-promoting activity co- purifies with the laminin protein, and (c) compare the influences of antilaminin antibodies on the neurite-promoting activity of laminin and PNPF-1. We conclude that laminin and PNPF-1 are distinct macromolecules capable of expressing their neurite-promoting activities even when presented in nanogram amounts. This neurite-promoting bioassay currently represents the most sensitive test for the biological activity of laminin. PMID:6643580

Forced expression of laminin β1 in podocytes prevents nephrotic syndrome in mice lacking laminin β2, a model for Pierson syndrome

PubMed Central

Suh, Jung Hee; Jarad, George; VanDeVoorde, Rene G.; Miner, Jeffrey H.

2011-01-01

Pierson syndrome is a congenital nephrotic syndrome with ocular and neurological defects caused by mutations in LAMB2, the gene encoding the basement membrane protein laminin β2 (Lamβ2). It is the kidney glomerular basement membrane (GBM) that is defective in Pierson syndrome, as Lamβ2 is a component of laminin-521 (LM-521; α5β2γ1), the major laminin in the mature GBM. In both Pierson syndrome and the Lamb2−/− mouse model for this disease, laminin β1 (Lamβ1), a structurally similar homolog of Lamβ2, is marginally increased in the GBM, but it fails to fully compensate for the loss of Lamβ2, leading to the filtration barrier defects and nephrotic syndrome. Here we generated several lines of Lamβ1 transgenic mice and used them to show that podocyte-specific Lamβ1 expression in Lamb2−/− mice abrogates the development of nephrotic syndrome, correlating with a greatly extended lifespan. In addition, the more Lamβ1 was expressed, the less urinary albumin was excreted. Transgenic Lamβ1 expression increased the level of Lamα5 in the GBM of rescued mice, consistent with the desired increased deposition of laminin-511 (α5β1γ1) trimers. Ultrastructural analysis revealed occasional knob-like subepithelial GBM thickening but intact podocyte foot processes in aged rescued mice. These results suggest the possibility that up-regulation of LAMB1 in podocytes, should it become achievable, would likely lessen the severity of nephrotic syndrome in patients carrying LAMB2 mutations. PMID:21876163

Forced expression of laminin beta1 in podocytes prevents nephrotic syndrome in mice lacking laminin beta2, a model for Pierson syndrome.

PubMed

Suh, Jung Hee; Jarad, George; VanDeVoorde, Rene G; Miner, Jeffrey H

2011-09-13

Pierson syndrome is a congenital nephrotic syndrome with ocular and neurological defects caused by mutations in LAMB2, the gene encoding the basement membrane protein laminin β2 (Lamβ2). It is the kidney glomerular basement membrane (GBM) that is defective in Pierson syndrome, as Lamβ2 is a component of laminin-521 (LM-521; α5β2γ1), the major laminin in the mature GBM. In both Pierson syndrome and the Lamb2(-/-) mouse model for this disease, laminin β1 (Lamβ1), a structurally similar homolog of Lamβ2, is marginally increased in the GBM, but it fails to fully compensate for the loss of Lamβ2, leading to the filtration barrier defects and nephrotic syndrome. Here we generated several lines of Lamβ1 transgenic mice and used them to show that podocyte-specific Lamβ1 expression in Lamb2(-/-) mice abrogates the development of nephrotic syndrome, correlating with a greatly extended lifespan. In addition, the more Lamβ1 was expressed, the less urinary albumin was excreted. Transgenic Lamβ1 expression increased the level of Lamα5 in the GBM of rescued mice, consistent with the desired increased deposition of laminin-511 (α5β1γ1) trimers. Ultrastructural analysis revealed occasional knob-like subepithelial GBM thickening but intact podocyte foot processes in aged rescued mice. These results suggest the possibility that up-regulation of LAMB1 in podocytes, should it become achievable, would likely lessen the severity of nephrotic syndrome in patients carrying LAMB2 mutations.

Exclusion of candidate genes from the chromosome 1q juvenile glaucoma region and mapping of the peripheral cannabis receptor gene (CNR2) to chromosome 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sunden, S.L.F.; Nichols, B.E.; Alward, W.L.M.

Juvenile onset primary open angle glaucoma has been mapped by linkage to 1q21-q31. Several candidate genes were evaluated in the same family used to identify the primary linkage. Atrionatriuretic peptide receptor A (NPR1) and laminin C1 (LAMC1) have been previously mapped to this region and could putatively play a role in the pathogenesis of glaucoma. A third gene, the peripheral cannabis receptor (CNR2) was not initially mapped in humans but was a candidate because of the relief that cannabis affords some patients with primary open angle glaucoma. Microsatellites associated with NPR1 and LAMC1 revealed multiple recombinations in affected members ofmore » this pedigree. CNR2 was shown to be on chromosome 1 by PCR amplification of a 150 bp fragment of the 3{prime} untranslated region in monochromosomal somatic cell hybrids (NIGMS panel No. 2). These primers also revealed a two allele single strand conformation polymorphism which showed multiple recombinants with juvenile onset primary open angle glaucoma in large pedigrees, segregating this disorder. The marker was then mapped to 1p34-p36 by linkage, with the most likely location between liver alkaline phosphatase (ALPL) and alpha-L-1 fucosidase (FUCA1).« less

Inhibition of hair follicle growth by a laminin-1 G-domain peptide, RKRLQVQLSIRT, in an organ culture of isolated vibrissa rudiment.

PubMed

Hayashi, Kazuhiro; Mochizuki, Mayumi; Nomizu, Motoyoshi; Uchinuma, Eijyu; Yamashina, Shohei; Kadoya, Yuichi

2002-04-01

We established a serum-free organ culture system of isolated single vibrissa rudiments taken from embryonic day 13 mice. This system allowed us to test more than 30 laminin-derived cell adhesive peptides to determine their roles on the growth and differentiation of vibrissa hair follicles. We found that the RKRLQVQLSIRT sequence (designated AG-73), which mapped to the LG-4 module of the laminin-alpha1 chain carboxyl-terminal G domain, perturbed the growth of hair follicles in vitro. AG-73 is one of the cell-binding peptides identified from more than 600 systematically synthesized 12 amino acid peptides covering the whole amino acid sequence of the laminin-alpha1, -beta1, and -gamma1 chains, by cell adhesion assay. Other cell-adhesive laminin peptides and a control scrambled peptide, LQQRRSVLRTKI, however, failed to show any significant effects on the growth of hair follicles. The AG-73 peptide binds to syndecan-1, a transmembrane heparan-sulfate proteoglycan. Syndecan-1 was expressed in both the mesenchymal condensation and the epithelial hair peg of developing vibrissa, suggesting that AG-73 binding to the cell surface syndecan-1 perturbed the epithelial-mesenchymal interactions of developing vibrissa. The formation of hair bulbs was aberrant in the explants treated with AG-73. In addition, impaired basement membrane formation, an abnormal cytoplasmic bleb formation, and an unusual basal formation of actin bundles were noted in the AG-73-treated-hair matrix epithelium, indicating that AG-73 binding perturbs various steps of epithelial morphogenesis, including the basement membrane remodeling. We also found a region-specific loss of the laminin-alpha1 chain in the basement membrane at the distal region of the invading hair follicle epithelium, indicating that laminins play a part in hair morphogenesis.

Secondary reduction of alpha7B integrin in laminin alpha2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle.

PubMed

Cohn, R D; Mayer, U; Saher, G; Herrmann, R; van der Flier, A; Sonnenberg, A; Sorokin, L; Voit, T

1999-03-01

The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The alpha7beta1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the alpha7 integrin are laminin-1 (alpha1-beta1-gamma1), laminin-2 (alpha2-beta1-gamma1) and laminin-4 (alpha2-beta2-gamma1). Targeted deletion of the gene for the alpha7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the alpha7B and beta1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the alpha7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the beta1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of alpha7B integrin was significantly reduced at the sarcolemma in six patients with laminin alpha2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin alpha2 chain expressed. In contrast, the expression of the laminin alpha2 chain was not altered in the skeletal muscle of the alpha7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the alpha7 integrin subunit and that of the laminin alpha2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of alpha7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even in young boys (age <2 years). The expression of the beta1D integrin subunit was not altered in any of our patients with different types of muscular dystrophy. In contrast, sarcolemmal expression of beta1D integrin was significantly reduced in the alpha7 integrin knock-out mice, whereas the expression of the components of the DGC was not altered. The secondary loss of alpha7B in laminin alpha2 chain deficiency defines a biochemical change in the composition of the plasma membrane resulting from a primary protein deficiency in the basal lamina. These findings, in addition to the occurrence of a muscular dystrophy in alpha7 deficient mice, implies that the alpha7B integrin is an important laminin receptor within the plasma membrane which plays a significant role in skeletal muscle function and stability.

Inhibition on Apoptosis Induced by Elevated Hydrostatic Pressure in Retinal Ganglion Cell-5 via Laminin Upregulating β1-integrin/Focal Adhesion Kinase/Protein Kinase B Signaling Pathway.

PubMed

Li, Yi; Chen, Yan-Ming; Sun, Ming-Ming; Guo, Xiao-Dan; Wang, Ya-Chen; Zhang, Zhong-Zhi

2016-04-20

Glaucoma is a progressive optic neuropathy characterized by degeneration of neurons due to loss of retinal ganglion cells (RGCs). High intraocular pressure (HIOP), the main risk factor, causes the optic nerve damage. However, the precise mechanism of HIOP-induced RGC death is not yet completely understood. This study was conducted to determine apoptosis of RGC-5 cells induced by elevated hydrostatic pressures, explore whether laminin is associated with apoptosis under pressure, whether laminin can protect RGCs from apoptosis and affirm the mechanism that regulates the process of RGCs survival. RGC-5 cells were exposed to 0, 20, 40, and 60 mmHg in a pressurized incubator for 6, 12, and 24 h, respectively. The effect of elevated hydrostatic pressure on RGC-5 cells was measured by Annexin V-fluorescein isothiocyanate/propidium iodide staining, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Western blotting of cleaved caspase-3 protein. Location and expression of laminin were detected by immunofluorescence. The expression of β1-integrin, phosphorylation of focal adhesion kinase (FAK) and protein kinase B (PKB, or AKT) were investigated with real-time polymerase chain reaction and Western blotting analysis. Elevated hydrostatic pressure induced apoptosis in cultured RGC-5 cells. Pressure with 40 mmHg for 24 h induced a maximum apoptosis. Laminin was declined in RGC-5 cells after exposing to 40 mmHg for 24 h. After pretreating with laminin, RGC-5 cells survived from elevated pressure. Furthermore, β1-integrin and phosphorylation of FAK and AKT were increased compared to 40 mmHg group. The data show apoptosis tendency of RGC-5 cells with elevated hydrostatic pressure. Laminin can protect RGC-5 cells against high pressure via β1-integrin/FAK/AKT signaling pathway. These results suggest that the decreased laminin of RGC-5 cells might be responsible for apoptosis induced by elevated hydrostatic pressure, and laminin or activating β1-integrin/FAK/AKT pathway might be potential treatments to prevent RGC loss in glaucomatous optic neuropathy.

Inhibition on Apoptosis Induced by Elevated Hydrostatic Pressure in Retinal Ganglion Cell-5 via Laminin Upregulating β1-integrin/Focal Adhesion Kinase/Protein Kinase B Signaling Pathway

PubMed Central

Li, Yi; Chen, Yan-Ming; Sun, Ming-Ming; Guo, Xiao-Dan; Wang, Ya-Chen; Zhang, Zhong-Zhi

2016-01-01

Background: Glaucoma is a progressive optic neuropathy characterized by degeneration of neurons due to loss of retinal ganglion cells (RGCs). High intraocular pressure (HIOP), the main risk factor, causes the optic nerve damage. However, the precise mechanism of HIOP-induced RGC death is not yet completely understood. This study was conducted to determine apoptosis of RGC-5 cells induced by elevated hydrostatic pressures, explore whether laminin is associated with apoptosis under pressure, whether laminin can protect RGCs from apoptosis and affirm the mechanism that regulates the process of RGCs survival. Methods: RGC-5 cells were exposed to 0, 20, 40, and 60 mmHg in a pressurized incubator for 6, 12, and 24 h, respectively. The effect of elevated hydrostatic pressure on RGC-5 cells was measured by Annexin V-fluorescein isothiocyanate/propidium iodide staining, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Western blotting of cleaved caspase-3 protein. Location and expression of laminin were detected by immunofluorescence. The expression of β1-integrin, phosphorylation of focal adhesion kinase (FAK) and protein kinase B (PKB, or AKT) were investigated with real-time polymerase chain reaction and Western blotting analysis. Results: Elevated hydrostatic pressure induced apoptosis in cultured RGC-5 cells. Pressure with 40 mmHg for 24 h induced a maximum apoptosis. Laminin was declined in RGC-5 cells after exposing to 40 mmHg for 24 h. After pretreating with laminin, RGC-5 cells survived from elevated pressure. Furthermore, β1-integrin and phosphorylation of FAK and AKT were increased compared to 40 mmHg group. Conclusions: The data show apoptosis tendency of RGC-5 cells with elevated hydrostatic pressure. Laminin can protect RGC-5 cells against high pressure via β1-integrin/FAK/AKT signaling pathway. These results suggest that the decreased laminin of RGC-5 cells might be responsible for apoptosis induced by elevated hydrostatic pressure, and laminin or activating β1-integrin/FAK/AKT pathway might be potential treatments to prevent RGC loss in glaucomatous optic neuropathy. PMID:27064044

Type XVII collagen (BP180) can function as a cell-matrix adhesion molecule via binding to laminin 332

PubMed Central

Van den Bergh, F.; Eliason, S.L.; Giudice, G.J.

2010-01-01

Collagen XVII (COL17) is a transmembrane glycoprotein that is expressed on the basal surface of basal epidermal keratinocytes. Previous observations have led to the hypothesis that an interaction between COL17 and laminin 332, an extracellular matrix protein, contributes to the attachment of the basal keratinocyte to the basement membrane. In order to isolate and manipulate COL17 interactions with ECM components, we induced COL17 expression in two cells lines, SK-MEL1 and K562, that exhibit little or no capacity to attach to our test substrates, including laminin 332, types I and IV collagen, and fibronectin. Cells expressing high levels of COL17 preferentially adhered to a laminin 332 matrix, and, to a lesser extent, type IV collagen, while showing little or no binding to type I collagen or fibronectin. A quantitative analysis of cell adhesive forces revealed that, compared with COL17-negative cells, COL17-positive cells required over 7-fold greater force to achieve 50% detachment from a laminin 332 substrate. When a cell preparation (either K562 or SK-MEL1) with heterogeneous COL17 expression levels was allowed to attach to a laminin 332 matrix, the COL17-positive and COL17-negative cells differentially sorted to the bound and unbound cell fractions, respectively. COL17-dependent attachment to laminin 332 could be reduced or abolished by siRNA-mediated knockdown of COL17 expression or by adding to the assay wells specific antibodies against COL17 or laminin 332. These findings provide strong support for the hypothesis that cell surface COL17 can interact with laminin 332 and, together, participate in the adherence of a cell to the extracellular matrix. PMID:21034821

β1D chain increases α7β1 integrin and laminin and protects against sarcolemmal damage in mdx mice

PubMed Central

Liu, Jianming; Milner, Derek J.; Boppart, Marni D.; Ross, Robert S.; Kaufman, Stephen J.

2012-01-01

The dystrophin–glycoprotein complex connects myofibers with extracellular matrix laminin. In Duchenne muscular dystrophy, this linkage system is absent and the integrity of muscle fibers is compromised. One potential therapy for addressing muscular dystrophy is to augment the amount of α7β1 integrin, the major laminin-binding integrin in skeletal muscle. Whereas transgenic over-expression of α7 chain may alleviate development of muscular dystrophy and extend the lifespan of severely dystrophic mdx/utrn−/− mice, further enhancing levels of α7 chain provided little additional membrane integrin and negligible additional improvement in mdx mice. We demonstrate here that normal levels of β1 chain limit formation of integrin heterodimer and that increasing β1D chain in mdx mice results in more functional integrin at the sarcolemma, more matrix laminin and decreased damage of muscle fibers. Moreover, increasing the amount of β1D chain in vitro enhances transcription of α7 integrin and α2 laminin genes and the amounts of these proteins. Thus manipulation of β1D integrin expression offers a novel approach to enhance integrin-mediated therapy for muscular dystrophy. PMID:22180459

Correlation Between Extravasation and Alterations of Cerebrovascular Laminin and β-Dystroglycan Immunoreactivity Following Cryogenic Lesions in Rats.

PubMed

Kálmán, Mihály; Tóth, László; Szöllosi, Dávid; Oszwald, Erzsébet; Mahalek, Judit; Sadeghian, Sam

2017-11-01

The blood-brain barrier becomes "leaky" following lesions. Former studies revealed that following lesions the immunoreactivity of cerebrovascular laminin becomes detectable whereas that of β-dystroglycan disappears. These alterations may be indicators of glio-vascular decoupling that may result in the impairment of the blood-brain-barrier. This study investigates correlation between the post-lesion extravasation and the above-mentioned immunohistochemical alterations. Following cryogenic lesions, the survival periods lasted 5, 10, 30 minutes, 1 or 12 hours, or 1 day. Some brains were fixed immediately post-lesion. Immunofluorescent reactions were performed in floating sections. The extravasation was detected with immunostaining for plasma fibronectin and rat immunoglobulins. When the survival period was 30 minutes or longer, the area of extravasation corresponded to the area of altered laminin and β-dystroglycan immunoreactivities. Following immediate fixation some laminin immunoreactivity was already detected. The extravasation seemed to precede this early appearance of laminin immunoreactivity. The β-dystroglycan immunoreactivity disappeared later. When the extravasation spread into the corpus callosum, vascular laminin immunoreactivity appeared but the β-dystroglycan immunoreactivity persisted. It seems that extravasation separates the glial and vascular basal laminae, which results in the appearance of laminin immunoreactivity. The disappearance of β-dystroglycan immunoreactivity is neither a condition nor an inevitable consequence of the 2 other phenomena. © 2017 American Association of Neuropathologists, Inc. All rights reserved.

Axonal sprouting and laminin appearance after destruction of glial sheaths.

PubMed Central

Masuda-Nakagawa, L M; Muller, K J; Nicholls, J G

1993-01-01

Laminin, a large extracellular matrix molecule, is associated with axonal outgrowth during development and regeneration of the nervous system in a variety of animals. In the leech central nervous system, laminin immunoreactivity appears after axon injury in advance of the regenerating axons. Although studies of vertebrate nervous system in culture have implicated glial and Schwann cells as possible sources, the cells that deposit laminin at sites crucial for regeneration in the living animal are not known. We have made a direct test to determine whether, in the central nervous system of the leech, cells other than ensheathing glial cells can produce laminin. Ensheathing glial cells of adult leeches were ablated selectively by intracellular injection of a protease. As a result, leech laminin accumulated within 10 days in regions of the central nervous system where it is not normally found, and undamaged, intact axons began to sprout extensively. In normal leeches laminin immunoreactivity is situated only in the basement membrane that surrounds the central nervous system, whereas after ablation of ensheathing glia it appeared in spaces through which neurons grew. Within days of ablation of the glial cell, small mobile phagocytes, or microglia, accumulated in the spaces formerly occupied by the glial cell. Microglia were concentrated at precisely the sites of new laminin appearance and axon sprouting. These results suggest that in the animal, as in culture, leech laminin promotes sprouting and that microglia may be responsible for its appearance. Images Fig. 1 Fig. 2 Fig. 3 PMID:8506343

Laminin promotes vascular network formation in 3D in vitro collagen scaffolds by regulating VEGF uptake.

PubMed

Stamati, Katerina; Priestley, John V; Mudera, Vivek; Cheema, Umber

2014-09-10

Angiogenesis is an essential neovascularisation process, which if recapitulated in 3D in vitro, will provide better understanding of endothelial cell (EC) behaviour. Various cell types and growth factors are involved, with vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 key components. We were able to control the aggregation pattern of ECs in 3D collagen hydrogels, by varying the matrix composition and/or having a source of cells signalling angiogenic proteins. These aggregation patterns reflect the different developmental pathways that ECs take to form different sized tubular structures. Cultures with added laminin and thus increased expression of α6 integrin showed a significant increase (p<0.05) in VEGFR2 positive ECs and increased VEGF uptake. This resulted in the end-to-end network aggregation of ECs. In cultures without laminin and therefore low α6 integrin expression, VEGFR2 levels and VEGF uptake were significantly lower (p<0.05). These ECs formed contiguous sheets, analogous to the 'wrapping' pathway in development. We have identified a key linkage between integrin expression on ECs and their uptake of VEGF, regulated by VEGFR2, resulting in different aggregation patterns in 3D. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake*

PubMed Central

Chung, Heesung; Jung, Hyejung; Lee, Jung-hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

2014-01-01

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. PMID:24951591

Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

PubMed

Chung, Heesung; Jung, Hyejung; Lee, Jung-Hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

2014-08-01

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Evidences that beta1 integrin and Rac1 are involved in the overriding effect of laminin on myelin-associated glycoprotein inhibitory activity on neuronal cells.

PubMed

Laforest, Sullivan; Milanini, Julie; Parat, Fabrice; Thimonier, Jean; Lehmann, Maxime

2005-11-01

During neurite elongation, migrating growth cones encounter both permissive and inhibitory substrates, such as laminin and MAG (myelin-associated glycoprotein), respectively. Here, we demonstrated on two neuronal cell lines (PC12 and N1E-115), that laminin and collagen hampered, in a dose-dependent manner, MAG inhibitory activity on several integrin functions, i.e., neurite growth, cell adhesion and cell spreading. Using a function blocking antibody, in PC12 cells, we showed that alpha1beta1 integrin is required in these phenomena. In parallel, we observed that MAG perturbs actin dynamics and lamellipodia formation during early steps of cell spreading. This seemed to be independent of RhoA activation, but dependent of Rac-1 inhibition by MAG. Laminin overrode MAG activity on actin and prevented MAG inhibition NGF-induced Rac1 activation. In conclusion, we evidenced antagonistic signaling between MAG receptors and beta1 integrins, in which Rac-1 may have a central function.

E-cadherin-defective gastric cancer cells depend on Laminin to survive and invade.

PubMed

Caldeira, Joana; Figueiredo, Joana; Brás-Pereira, Catarina; Carneiro, Patrícia; Moreira, Ana M; Pinto, Marta T; Relvas, João B; Carneiro, Fátima; Barbosa, Mário; Casares, Fernando; Janody, Florence; Seruca, Raquel

2015-10-15

Epithelial-cadherin (Ecad) deregulation affects cell-cell adhesion and results in increased invasiveness of distinct human carcinomas. In gastric cancer, loss of Ecad expression is a common event and is associated with disease aggressiveness and poor prognosis. However, the molecular mechanisms underlying the invasive process associated to Ecad dysfunction are far from understood. We hypothesized that deregulation of cell-matrix interactions could play an important role during this process. Thus, we focussed on LM-332, which is a major matrix component, and in Ecad/LM-332 crosstalk in the process of Ecad-dependent invasion. To verify whether matrix deregulation was triggered by Ecad loss, we used the Drosophila model. To dissect the key molecules involved and unveil their functional significance, we used gastric cancer cell lines. The relevance of this relationship was then confirmed in human primary tumours. In vivo, Ecad knockdown induced apoptosis; nonetheless, at the invasive front, cells ectopically expressed Laminin A and βPS integrin. In vitro, we demonstrated that, in two different gastric cancer cell models, Ecad-defective cells overexpressed Laminin γ2 (LM-γ2), β1 and β4 integrin, when compared with Ecad-competent ones. We showed that LM-γ2 silencing impaired invasion and enhanced cell death, most likely via pSrc and pAkt reduction, and JNK activation. In human gastric carcinomas, we found a concomitant decrease in Ecad and increase in LM-γ2. This is the first evidence that ectopic Laminin expression depends on Ecad loss and allows Ecad-dysfunctional cells to survive and invade. This opens new avenues for using LM-γ2 signalling regulators as molecular targets to impair gastric cancer progression. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

E-cadherin-defective gastric cancer cells depend on Laminin to survive and invade

PubMed Central

Caldeira, Joana; Figueiredo, Joana; Brás-Pereira, Catarina; Carneiro, Patrícia; Moreira, Ana M.; Pinto, Marta T.; Relvas, João B.; Carneiro, Fátima; Barbosa, Mário; Casares, Fernando; Janody, Florence; Seruca, Raquel

2015-01-01

Epithelial-cadherin (Ecad) deregulation affects cell–cell adhesion and results in increased invasiveness of distinct human carcinomas. In gastric cancer, loss of Ecad expression is a common event and is associated with disease aggressiveness and poor prognosis. However, the molecular mechanisms underlying the invasive process associated to Ecad dysfunction are far from understood. We hypothesized that deregulation of cell–matrix interactions could play an important role during this process. Thus, we focussed on LM-332, which is a major matrix component, and in Ecad/LM-332 crosstalk in the process of Ecad-dependent invasion. To verify whether matrix deregulation was triggered by Ecad loss, we used the Drosophila model. To dissect the key molecules involved and unveil their functional significance, we used gastric cancer cell lines. The relevance of this relationship was then confirmed in human primary tumours. In vivo, Ecad knockdown induced apoptosis; nonetheless, at the invasive front, cells ectopically expressed Laminin A and βPS integrin. In vitro, we demonstrated that, in two different gastric cancer cell models, Ecad-defective cells overexpressed Laminin γ2 (LM-γ2), β1 and β4 integrin, when compared with Ecad-competent ones. We showed that LM-γ2 silencing impaired invasion and enhanced cell death, most likely via pSrc and pAkt reduction, and JNK activation. In human gastric carcinomas, we found a concomitant decrease in Ecad and increase in LM-γ2. This is the first evidence that ectopic Laminin expression depends on Ecad loss and allows Ecad-dysfunctional cells to survive and invade. This opens new avenues for using LM-γ2 signalling regulators as molecular targets to impair gastric cancer progression. PMID:26246502

Dystroglycan loss disrupts polarity and beta-casein induction inmammary epithelial cells by perturbing laminin anchoring

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weir, M. Lynn; Oppizzi, Maria Luisa; Henry, Michael D.

2006-02-17

Precise contact between epithelial cells and their underlying basement membrane is critical to the maintenance of tissue architecture and function. To understand the role that the laminin receptor dystroglycan (DG) plays in these processes, we assayed cell responses to laminin-111 following conditional ablation of DG expression in cultured mammary epithelial cells (MECs). Strikingly, DG loss disrupted laminin-111-induced polarity and {beta}-casein production, and abolished laminin assembly at the step of laminin binding to the cell surface. DG re-expression restored these deficiencies. Investigations of mechanism revealed that DG cytoplasmic sequences were not necessary for laminin assembly and signaling, and only when themore » entire mucin domain of extracellular DG was deleted did laminin assembly not occur. These results demonstrate that DG is essential as a laminin-111 co-receptor in MECs that functions by mediating laminin anchoring to the cell surface, a process that allows laminin polymerization, tissue polarity, and {beta}-casein induction. The observed loss of laminin-111 assembly and signaling in DG-/-MECs provides insights into the signaling changes occurring in breast carcinomas and other cancers, where DG's laminin-binding function is frequently defective.« less

Stromal laminin chain distribution in normal, hyperplastic and malignant oral mucosa: relation to myofibroblast occurrence and vessel formation.

PubMed

Franz, Marcus; Wolheim, Anke; Richter, Petra; Umbreit, Claudia; Dahse, Regine; Driemel, Oliver; Hyckel, Peter; Virtanen, Ismo; Kosmehl, Hartwig; Berndt, Alexander

2010-04-01

The contribution of stromal laminin chain expression to malignant potential, tumour stroma reorganization and vessel formation in oral squamous cell carcinoma (OSCC) is not fully understood. Therefore, the expression of the laminin chains alpha2, alpha3, alpha4, alpha5 and gamma2 in the stromal compartment/vascular structures in OSCC was analysed. Frozen tissue of OSCC (9x G1, 24x G2, 8x G3) and normal (2x)/hyperplastic (11x) oral mucosa was subjected to laminin chain and alpha-smooth muscle actin (ASMA) immunohistochemistry. Results were correlated to tumour grade. The relation of laminin chain positive vessels to total vessel number was assessed by immunofluorescence double labelling with CD31. Stromal laminin alpha2 chain significantly decreases and alpha3, alpha4, alpha5 and gamma2 chains and also ASMA significantly increase with rising grade. The amount of stromal alpha3, alpha4 and gamma2 chains significantly increased with rising ASMA positivity. There is a significant decrease in alpha3 chain positive vessels with neoplastic transformation. Mediated by myofibroblasts, OSCC development is associated with a stromal up-regulation of laminin isoforms possibly contributing to a migration promoting microenvironment. A vascular basement membrane reorganization concerning alpha3 and gamma2 chain laminins during tumour angioneogenesis is suggested.

α1β1 Integrin/Rac1-Dependent Mesangial Invasion of Glomerular Capillaries in Alport Syndrome

PubMed Central

Zallocchi, Marisa; Johnson, Brianna M.; Meehan, Daniel T.; Delimont, Duane; Cosgrove, Dominic

2014-01-01

Alport syndrome, hereditary glomerulonephritis with hearing loss, results from mutations in type IV collagen COL4A3, COL4A4, or COL4A5 genes. The mechanism for delayed glomerular disease onset is unknown. Comparative analysis of Alport mice and CD151 knockout mice revealed progressive accumulation of laminin 211 in the glomerular basement membrane. We show mesangial processes invading the capillary loops of both models as well as in human Alport glomeruli, as the likely source of this laminin. l-NAME salt–induced hypertension accelerated mesangial cell process invasion. Cultured mesangial cells showed reduced migratory potential when treated with either integrin-linked kinase inhibitor or Rac1 inhibitor, or by deletion of integrin α1. Treatment of Alport mice with Rac1 inhibitor or deletion of integrin α1 reduced mesangial cell process invasion of the glomerular capillary tuft. Laminin α2–deficient Alport mice show reduced mesangial process invasion, and cultured laminin α2–null cells showed reduced migratory potential, indicating a functional role for mesangial laminins in progression of Alport glomerular pathogenesis. Collectively, these findings predict a role for biomechanical insult in the induction of integrin α1β1–dependent Rac1-mediated mesangial cell process invasion of the glomerular capillary tuft as an initiation mechanism of Alport glomerular pathology. PMID:23911822

Laminin and Fibronectin in Cell Adhesion: Enhanced Adhesion of Cells from Regenerating Liver to Laminin

NASA Astrophysics Data System (ADS)

Carlsson, Roland; Engvall, Eva; Freeman, Aaron; Ruoslahti, Erkki

1981-04-01

Laminin, a basement membrane glycoprotein isolated from cultures of mouse endodermal cells and rat yolk sac carcinoma cells, promoted the attachment of liver cells obtained from regenerating mouse liver. Cells from normal mouse liver attached readily to dishes coated with fibronectin but attached poorly to surfaces coated with laminin. Both proteins efficiently promoted the attachment of cells from livers undergoing regeneration. After regeneration, the attachment to laminin returned to the low levels found in animals not subjected to partial hepatectomy but attachment to fibronectin remained high. Immunofluorescent staining of sections of normal liver with antilaminin revealed the presence of laminin in or adjacent to the walls of the bile ducts and blood vessels. After induction of regeneration by partial hepatectomy, increased amounts of laminin appeared in the sinusoidal areas. After carbon tetrachloride poisoning, staining for laminin was especially pronounced in the necrotic and postnecrotic areas around the central veins. This additional expression of laminin was transient. It reached a maximum around 5-6 days after the injury and then gradually disappeared. These findings show that laminin is an adhesive protein. The increase of laminin in regenerating liver and the adhesiveness of cells from such livers to laminin suggest a role for laminin in the maintenance of a proper tissue organization during liver regeneration.

Laminin α1 is essential for mouse cerebellar development

PubMed Central

Ichikawa-Tomikawa, Naoki; Ogawa, Junko; Douet, Vanessa; Xu, Zhuo; Kamikubo, Yuji; Sakurai, Takashi; Kohsaka, Shinichi; Chiba, Hideki; Hattori, Nobutaka; Yamada, Yoshihiko; Arikawa-Hirasawa, Eri

2011-01-01

Laminin α1 (Lama1), which is a subunit of laminin-1 (laminin-111), a heterotrimeric ECM protein, is essential for embryonic development and promotes neurite outgrowth in culture. Because the deletion of Lama1 causes lethality at early embryonic stages in mice, the in vivo role of Lama1 in neural development and functions has not yet been possible to determine. In this study, we generated conditional Lama1 knockout (Lama1CKO) mice in the epiblast lineage using Sox2-Cre mice. These Lama1CKO mice survived, but displayed behavioral disorders and impaired formation of the cerebellum. Deficiency of Lama1 in the pial basement membrane of the meninges resulted in defects in the conformation of the meninges. During cerebellar development, Lama1 deficiency also caused a decrease in the proliferation and migration of granule cell precursors, disorganization of Bergmann glial fibers and endfeet, and a transient reduction in the activity of Akt. A marked reduction in numbers of dendritic processes in Purkinje cells was observed in Lama1CKO mice. Together, these results indicate that Lama1 is required for cerebellar development and functions. PMID:21983115

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) disrupts blood-brain barrier integrity through a mechanism involving P2X7 receptors.

PubMed

Rubio-Araiz, Ana; Perez-Hernandez, Mercedes; Urrutia, Andrés; Porcu, Francesca; Borcel, Erika; Gutierrez-Lopez, Maria Dolores; O'Shea, Esther; Colado, Maria Isabel

2014-08-01

The recreational drug 3,4-methylenedioxymethamphetamine (MDMA; 'ecstasy') produces a neuro-inflammatory response in rats characterized by an increase in microglial activation and IL-1β levels. The integrity of the blood-brain barrier (BBB) is important in preserving the homeostasis of the brain and has been shown to be affected by neuro-inflammatory processes. We aimed to study the effect of a single dose of MDMA on the activity of metalloproteinases (MMPs), expression of extracellular matrix proteins, BBB leakage and the role of the ionotropic purinergic receptor P2X7 (P2X7R) in the changes induced by the drug. Adult male Dark Agouti rats were treated with MDMA (10 mg/kg, i.p.) and killed at several time-points in order to evaluate MMP-9 and MMP-3 activity in the hippocampus and laminin and collagen-IV expression and IgG extravasation in the dentate gyrus. Microglial activation, P2X7R expression and localization were also determined in the dentate gyrus. Separate groups were treated with MDMA and the P2X7R antagonists Brilliant Blue G (BBG; 50 mg/kg, i.p.) or A-438079 (30 mg/kg, i.p.). MDMA increased MMP-3 and MMP-9 activity, reduced laminin and collagen-IV expression and increased IgG immunoreactivity. In addition, MDMA increased microglial activation and P2X7R immunoreactivity in these cells. BBG suppressed the increase in MMP-9 and MMP-3 activity, prevented basal lamina degradation and IgG extravasation into the brain parenchyma. A-438079 also prevented the MDMA-induced reduction in laminin and collagen-IV immunoreactivity. These results indicate that MDMA alters BBB permeability through an early P2X7R-mediated event, which in turn leads to enhancement of MMP-9 and MMP-3 activity and degradation of extracellular matrix.

CD90-positive cells, an additional cell population, produce laminin {alpha}2 upon transplantation to dy{sup 3k}/dy{sup 3k} mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukada, So-ichiro; Yamamoto, Yukiko; Segawa, Masashi

2008-01-01

Laminin {alpha}2 is a component of skeletal and cardiac muscle basal lamina. A defect of the laminin {alpha}2 chain leads to severe congenital muscular dystrophy (MDC1A) in humans and dy/dy mice. Myogenic cells including myoblasts, myotubes, and myofibers in skeletal muscle are a possible source of the laminin {alpha}2 chain, and myogenic cells are thus proposed as a cell source for congenital muscular dystrophy therapy. However, we observed production of laminin {alpha}2 in non-myogenic cells of normal mice, and we could enrich these laminin {alpha}2-producing cells in CD90{sup +} cell fractions. Intriguingly, the number of CD90{sup +} cells increased dramaticallymore » during skeletal muscle regeneration in mice. This fraction did not include myogenic cells but exhibited a fibroblast-like phenotype. Moreover, these cells were resident in skeletal muscle, not derived from bone marrow. Finally, the production of laminin {alpha}2 in CD90{sup +} cells was not dependent on fusion with myogenic cells. Thus, CD90{sup +} cells are a newly identified additional cell fraction that increased during skeletal muscle regeneration in vivo and could be another cell source for therapy for lama2-deficient muscular dystrophy.« less

Basal cell adhesion molecule/lutheran protein. The receptor critical for sickle cell adhesion to laminin.

PubMed Central

Udani, M; Zen, Q; Cottman, M; Leonard, N; Jefferson, S; Daymont, C; Truskey, G; Telen, M J

1998-01-01

Sickle red cells bind significant amounts of soluble laminin, whereas normal red cells do not. Solid phase assays demonstrate that B-CAM/LU binds laminin on intact sickle red cells and that red cell B-CAM/LU binds immobilized laminin, whereas another putative laminin binding protein, CD44, does not. Ligand blots also identify B-CAM/LU as the only erythrocyte membrane protein(s) that binds laminin. Finally, transfection of murine erythroleukemia cells with human B-CAM cDNA induces binding of both soluble and immobilized laminin. Thus, B-CAM/LU appears to be the major laminin-binding protein of sickle red cells. Previously reported overexpression of B-CAM/LU by epithelial cancer cells suggests that this protein may also serve as a laminin receptor in malignant tumors. PMID:9616226

α1β1 integrin/Rac1-dependent mesangial invasion of glomerular capillaries in Alport syndrome.

PubMed

Zallocchi, Marisa; Johnson, Brianna M; Meehan, Daniel T; Delimont, Duane; Cosgrove, Dominic

2013-10-01

Alport syndrome, hereditary glomerulonephritis with hearing loss, results from mutations in type IV collagen COL4A3, COL4A4, or COL4A5 genes. The mechanism for delayed glomerular disease onset is unknown. Comparative analysis of Alport mice and CD151 knockout mice revealed progressive accumulation of laminin 211 in the glomerular basement membrane. We show mesangial processes invading the capillary loops of both models as well as in human Alport glomeruli, as the likely source of this laminin. L-NAME salt-induced hypertension accelerated mesangial cell process invasion. Cultured mesangial cells showed reduced migratory potential when treated with either integrin-linked kinase inhibitor or Rac1 inhibitor, or by deletion of integrin α1. Treatment of Alport mice with Rac1 inhibitor or deletion of integrin α1 reduced mesangial cell process invasion of the glomerular capillary tuft. Laminin α2-deficient Alport mice show reduced mesangial process invasion, and cultured laminin α2-null cells showed reduced migratory potential, indicating a functional role for mesangial laminins in progression of Alport glomerular pathogenesis. Collectively, these findings predict a role for biomechanical insult in the induction of integrin α1β1-dependent Rac1-mediated mesangial cell process invasion of the glomerular capillary tuft as an initiation mechanism of Alport glomerular pathology. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Immortalized myogenic cells from congenital muscular dystrophy type1A patients recapitulate aberrant caspase activation in pathogenesis: a new tool for MDC1A research

PubMed Central

2013-01-01

Background Congenital muscular dystrophy Type 1A (MDC1A) is a severe, recessive disease of childhood onset that is caused by mutations in the LAMA2 gene encoding laminin-α2. Studies with both mouse models and primary cultures of human MDC1A myogenic cells suggest that aberrant activation of cell death is a significant contributor to pathogenesis in laminin-α2-deficiency. Methods To overcome the limited population doublings of primary cultures, we generated immortalized, clonal lines of human MDC1A myogenic cells via overexpression of both CDK4 and the telomerase catalytic component (human telomerase reverse transcriptase (hTERT)). Results The immortalized MDC1A myogenic cells proliferated indefinitely when cultured at low density in high serum growth medium, but retained the capacity to form multinucleate myotubes and express muscle-specific proteins when switched to low serum medium. When cultured in the absence of laminin, myotubes formed from immortalized MDC1A myoblasts, but not those formed from immortalized healthy or disease control human myoblasts, showed significantly increased activation of caspase-3. This pattern of aberrant caspase-3 activation in the immortalized cultures was similar to that found previously in primary MDC1A cultures and laminin-α2-deficient mice. Conclusions Immortalized MDC1A myogenic cells provide a new resource for studies of pathogenetic mechanisms and for screening possible therapeutic approaches in laminin-α2-deficiency. PMID:24314268

Immortalized myogenic cells from congenital muscular dystrophy type1A patients recapitulate aberrant caspase activation in pathogenesis: a new tool for MDC1A research.

PubMed

Yoon, Soonsang; Stadler, Guido; Beermann, Mary Lou; Schmidt, Eric V; Windelborn, James A; Schneiderat, Peter; Wright, Woodring E; Miller, Jeffrey Boone

2013-12-06

Congenital muscular dystrophy Type 1A (MDC1A) is a severe, recessive disease of childhood onset that is caused by mutations in the LAMA2 gene encoding laminin-α2. Studies with both mouse models and primary cultures of human MDC1A myogenic cells suggest that aberrant activation of cell death is a significant contributor to pathogenesis in laminin-α2-deficiency. To overcome the limited population doublings of primary cultures, we generated immortalized, clonal lines of human MDC1A myogenic cells via overexpression of both CDK4 and the telomerase catalytic component (human telomerase reverse transcriptase (hTERT)). The immortalized MDC1A myogenic cells proliferated indefinitely when cultured at low density in high serum growth medium, but retained the capacity to form multinucleate myotubes and express muscle-specific proteins when switched to low serum medium. When cultured in the absence of laminin, myotubes formed from immortalized MDC1A myoblasts, but not those formed from immortalized healthy or disease control human myoblasts, showed significantly increased activation of caspase-3. This pattern of aberrant caspase-3 activation in the immortalized cultures was similar to that found previously in primary MDC1A cultures and laminin-α2-deficient mice. Immortalized MDC1A myogenic cells provide a new resource for studies of pathogenetic mechanisms and for screening possible therapeutic approaches in laminin-α2-deficiency.

Neuronal migration on laminin in vitro.

PubMed

Liang, S; Crutcher, K A

1992-03-20

Chick sympathetic (E-9) or telencephalic (E-7) neurons were cultured at low density on poly-DL-ornithine (PORN), poly-L-lysine (POLS), laminin or laminin-covered PORN or POLS and monitored with time-lapse videomicroscopy. Neurons migrated on laminin, or laminin-covered PORN or POLS, but not on PORN or POLS alone. Neuronal migration did not involve interactions with other cells indicating that neurons are capable of independent migration when exposed to a laminin substrate.

Structural decoding of netrin-4 reveals a regulatory function towards mature basement membranes

PubMed Central

Reuten, Raphael; Patel, Trushar R.; McDougall, Matthew; Rama, Nicolas; Nikodemus, Denise; Gibert, Benjamin; Delcros, Jean-Guy; Prein, Carina; Meier, Markus; Metzger, Stéphanie; Zhou, Zhigang; Kaltenberg, Jennifer; McKee, Karen K.; Bald, Tobias; Tüting, Thomas; Zigrino, Paola; Djonov, Valentin; Bloch, Wilhelm; Clausen-Schaumann, Hauke; Poschl, Ernst; Yurchenco, Peter D.; Ehrbar, Martin; Mehlen, Patrick; Stetefeld, Jörg; Koch, Manuel

2016-01-01

Netrins, a family of laminin-related molecules, have been proposed to act as guidance cues either during nervous system development or the establishment of the vascular system. This was clearly demonstrated for netrin-1 via its interaction with the receptors DCC and UNC5s. However, mainly based on shared homologies with netrin-1, netrin-4 was also proposed to play a role in neuronal outgrowth and developmental/pathological angiogenesis via interactions with netrin-1 receptors. Here, we present the high-resolution structure of netrin-4, which shows unique features in comparison with netrin-1, and show that it does not bind directly to any of the known netrin-1 receptors. We show that netrin-4 disrupts laminin networks and basement membranes (BMs) through high-affinity binding to the laminin γ1 chain. We hypothesize that this laminin-related function is essential for the previously described effects on axon growth promotion and angiogenesis. Our study unveils netrin-4 as a non-enzymatic extracellular matrix protein actively disrupting pre-existing BMs. PMID:27901020

Laminin enhances the growth of human neural stem cells in defined culture media

PubMed Central

Hall, Peter E; Lathia, Justin D; Caldwell, Maeve A; ffrench-Constant, Charles

2008-01-01

Background Human neural stem cells (hNSC) have the potential to provide novel cell-based therapies for neurodegenerative conditions such as multiple sclerosis and Parkinson's disease. In order to realise this goal, protocols need to be developed that allow for large quantities of hNSC to be cultured efficiently. As such, it is important to identify factors which enhance the growth of hNSC. In vivo, stem cells reside in distinct microenvironments or niches that are responsible for the maintenance of stem cell populations. A common feature of niches is the presence of the extracellular matrix molecule, laminin. Therefore, this study investigated the effect of exogenous laminin on hNSC growth. Results To measure hNSC growth, we established culture conditions using B27-supplemented medium that enable neurospheres to grow from human neural cells plated at clonal densities. Limiting dilution assays confirmed that neurospheres were derived from single cells at these densities. Laminin was found to increase hNSC numbers as measured by this neurosphere formation. The effect of laminin was to augment the proliferation/survival of the hNSC, rather than promoting the undifferentiated state. In agreement, apoptosis was reduced in dissociated neurospheres by laminin in an integrin β1-dependent manner. Conclusion The addition of laminin to the culture medium enhances the growth of hNSC, and may therefore aid their large-scale production. PMID:18651950

Grip and slip of L1-CAM on adhesive substrates direct growth cone haptotaxis

PubMed Central

Abe, Kouki; Katsuno, Hiroko; Toriyama, Michinori; Baba, Kentarou; Mori, Tomoyuki; Hakoshima, Toshio; Kanemura, Yonehiro; Watanabe, Rikiya; Inagaki, Naoyuki

2018-01-01

Chemical cues presented on the adhesive substrate direct cell migration, a process termed haptotaxis. To migrate, cells must generate traction forces upon the substrate. However, how cells probe substrate-bound cues and generate directional forces for migration remains unclear. Here, we show that the cell adhesion molecule (CAM) L1-CAM is involved in laminin-induced haptotaxis of axonal growth cones. L1-CAM underwent grip and slip on the substrate. The ratio of the grip state was higher on laminin than on the control substrate polylysine; this was accompanied by an increase in the traction force upon laminin. Our data suggest that the directional force for laminin-induced growth cone haptotaxis is generated by the grip and slip of L1-CAM on the substrates, which occur asymmetrically under the growth cone. This mechanism is distinct from the conventional cell signaling models for directional cell migration. We further show that this mechanism is disrupted in a human patient with L1-CAM syndrome, suffering corpus callosum agenesis and corticospinal tract hypoplasia. PMID:29483251

Sarcospan integration into laminin-binding adhesion complexes that ameliorate muscular dystrophy requires utrophin and α7 integrin

PubMed Central

Marshall, Jamie L.; Oh, Jennifer; Chou, Eric; Lee, Joy A.; Holmberg, Johan; Burkin, Dean J.; Crosbie-Watson, Rachelle H.

2015-01-01

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in loss of the dystrophin–glycoprotein complex, a laminin receptor that connects the myofiber to its surrounding extracellular matrix. Utrophin, a dystrophin ortholog that is normally localized to the neuromuscular junction, is naturally upregulated in DMD muscle, which partially compensates for the loss of dystrophin. Transgenic overexpression of utrophin causes broad sarcolemma localization of utrophin, restoration of laminin binding and amelioration of disease in the mdx mouse model of DMD. We previously demonstrated that overexpression of sarcospan, a dystrophin- and utrophin-binding protein, ameliorates mdx muscular dystrophy. Sarcospan boosts levels of utrophin to therapeutic levels at the sarcolemma, where attachment to laminin is restored. However, understanding the compensatory mechanism is complicated by concomitant upregulation of α7β1 integrin, which also binds laminin. Similar to the effects of utrophin, transgenic overexpression of α7 integrin prevents DMD disease in mice and is accompanied by increased abundance of utrophin around the extra-synaptic sarcolemma. In order to investigate the mechanisms underlying sarcospan ‘rescue’ of muscular dystrophy, we created double-knockout mice to test the contributions of utrophin or α7 integrin. We show that sarcospan-mediated amelioration of muscular dystrophy in DMD mice is dependent on the presence of both utrophin and α7β1 integrin, even when they are individually expressed at therapeutic levels. Furthermore, we found that association of sarcospan into laminin-binding complexes is dependent on utrophin and α7β1 integrin. PMID:25504048

Bortezomib partially improves laminin α2 chain-deficient muscular dystrophy.

PubMed

Körner, Zandra; Fontes-Oliveira, Cibely C; Holmberg, Johan; Carmignac, Virginie; Durbeej, Madeleine

2014-05-01

Congenital muscular dystrophy, caused by mutations in LAMA2 (the gene encoding laminin α2 chain), is a severe and incapacitating disease for which no therapy is yet available. We have recently demonstrated that proteasome activity is increased in laminin α2 chain-deficient muscle and that treatment with the nonpharmaceutical proteasome inhibitor MG-132 reduces muscle pathology in laminin α2 chain-deficient dy(3K)/dy(3K) mice. Here, we explore the use of the selective and therapeutic proteasome inhibitor bortezomib (currently used for treatment of relapsed multiple myeloma and mantle cell lymphoma) in dy(3K)/dy(3K) mice and in congenital muscular dystrophy type 1A muscle cells. Outcome measures included quantitative muscle morphology, gene and miRNA expression analyses, proteasome activity, motor activity, and survival. Bortezomib improved several histological hallmarks of disease, partially normalized miRNA expression (miR-1 and miR-133a), and enhanced body weight, locomotion, and survival of dy(3K)/dy(3K) mice. In addition, bortezomib reduced proteasome activity in congenital muscular dystrophy type 1A myoblasts and myotubes. These findings provide evidence that the proteasome inhibitor bortezomib partially reduces laminin α2 chain-deficient muscular dystrophy. Investigation of the clinical efficacy of bortezomib administration in congenital muscular dystrophy type 1A clinical trials may be warranted. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

The biosynthesis, processing, and secretion of laminin by human choriocarcinoma cells.

PubMed

Peters, B P; Hartle, R J; Krzesicki, R F; Kroll, T G; Perini, F; Balun, J E; Goldstein, I J; Ruddon, R W

1985-11-25

Laminin, a glycoprotein component of basal laminae, is synthesized and secreted in culture by a human malignant cell line (JAR) derived from gestational choriocarcinoma. Biosynthetically labeled human laminin subunits A (Mr approximately 400,000) and B (Mr = 200,000 doublet) are glycoslyated with asparagine-linked high mannose oligosaccharides that are processed to complex oligosaccharides before the laminin molecule is externalized by the cell. The rate-limiting step in the processing of the asparagine-linked glycans of laminin is at the point of action of alpha-mannosidase I since the principal laminin forms that accumulate in JAR cells contain Man9GlcNAc2 and Man8GlcNAc2 oligosaccharide units. The combination of subunits to form the disulfide-linked laminin molecule (Mr approximately 950,000) occurs rapidly within the cell at a time when the subunits contain these high mannose oligosaccharides. The production of laminin is limited by the availability of the A subunit such that excess B subunit forms accumulate intracellularly as uncombined B and a disulfide-linked B dimer. Pulse-chase kinetic studies establish these B forms as intermediates in the assembly of the laminin molecule. The fully assembled laminin undergoes further oligosaccharide processing and translocation to the cell surface, but uncombined B and B dimer are neither processed nor secreted to any significant extent. Therefore, laminin subunit combination appears to be a prerequisite for intracellular translocation, processing, and secretion. The mature laminin that contains complex oligosaccharides does not accumulate intracellularly but is rapidly externalized upon completion, either secreted into the culture medium (25%) or associated with the cell surface (75%) as determined by susceptibility to degradation by trypsin. About one-third of the laminin molecules secreted or shed by JAR cells into the chase medium contain a smaller A subunit form that appears to have been modified by limited proteolytic cleavage. The putative proteolytic event is closely timed to the release of the laminin into the culture medium.

Laminin alterations after in vitro nonenzymatic glycosylation.

PubMed

Charonis, A S; Reger, L A; Dege, J E; Kouzi-Koliakos, K; Furcht, L T; Wohlhueter, R M; Tsilibary, E C

1990-07-01

Laminin, a basement membrane protein derived from the matrix of the Engelbreth-Holm-Swarm murine tumor, was nonenzymatically glycosylated in vitro in the presence of increasing glucose concentrations. The amount of glucose incorporated per laminin molecule was shown to be proportional to the molarity of glucose used. Nonenzymatic glycosylation resulted in formation of cross-links and alterations of the cruciform shape of laminin molecules; these alterations were dramatic when high concentrations of glucose were used. One of the functions of laminin, the process of self-assembly, was shown to be impaired after in vitro nonenzymatic glycosylation. Glucose incorporation resulted in a dramatic decrease of long-to-long laminin dimers, which normally form during the initial steps of assembly. Furthermore, nonenzymatic glycosylation of laminin reduced its ability to self-associate into complexes larger than dimers, as judged by turbidimetry. The observed decrease of maximal turbidity was proportional to the degree of nonenzymatic glycosylation. Aminoguanidine, which has been suggested to inhibit cross-link formation, was shown to restore to a large extent the shape of laminin, the percentage of long-to-long arm dimers, and the maximal turbidity when included in the mixtures of laminin and glucose. These data suggest that structural and functional alterations of laminin may be primarily due to formation of cross-links. Such modifications of laminin (along with our basement membrane components) may contribute to the morphological and physiological changes observed in basement membranes under diabetic conditions.

An antibody to the lutheran glycoprotein (Lu) recognizing the LU4 blood type variant inhibits cell adhesion to laminin α5.

PubMed

Kikkawa, Yamato; Miwa, Takahiro; Tohara, Yukiko; Hamakubo, Takayuki; Nomizu, Motoyoshi

2011-01-01

The Lutheran blood group glycoprotein (Lu), an Ig superfamily (IgSF) transmembrane receptor, is also known as basal cell adhesion molecule (B-CAM). Lu/B-CAM is a specific receptor for laminin α5, a major component of basement membranes in various tissues. Previous reports have shown that Lu/B-CAM binding to laminin α5 contributes to sickle cell vaso-occlusion. However, as there are no useful tools such as function-blocking antibodies or drugs, it is unclear how epithelial and sickled red blood cells adhere to laminin α5 via Lu/B-CAM. In this study, we discovered a function-blocking antibody that inhibits Lu binding to laminin α5 using a unique binding assay on tissue sections. To characterize the function-blocking antibody, we identified the site on Lu/B-CAM recognized by this antibody. The extracellular domain of Lu/B-CAM contains five IgSF domains, D1-D2-D3-D4-D5. The antibody epitope was localized to D2, but not to the D3 domain containing the major part of the laminin α5 binding site. Furthermore, mutagenesis studies showed that Arg(175), the LU4 blood group antigenic site, was crucial for forming the epitope and the antibody bound sufficiently close to sterically hinder the interaction with α5. Cell adhesion assay using the antibody also showed that Lu/B-CAM serves as a secondary receptor for the adhesion of carcinoma cells to laminin α5. This function-blocking antibody against Lu/B-CAM should be useful for not only investigating cell adhesion to laminin α5 but also for developing drugs to inhibit sickle cell vaso-occlusion.

The role of laminins in cartilaginous tissues: from development to regeneration.

PubMed

Sun, Y; Wang, T L; Toh, W S; Pei, M

2017-07-21

As a key molecule of the extracellular matrix, laminin provides a delicate microenvironment for cell functions. Recent findings suggest that laminins expressed by cartilage-forming cells (chondrocytes, progenitor cells and stem cells) could promote chondrogenesis. However, few papers outline the effect of laminins on providing a favorable matrix microenvironment for cartilage regeneration. In this review, we delineated the expression of laminins in hyaline cartilage, fibrocartilage and cartilage-like tissue (nucleus pulposus) throughout several developmental stages. We also examined the effect of laminins on the biological activities of chondrocytes, including adhesion, migration and survival. Furthermore, we scrutinized the potential influence of various laminin isoforms on cartilage-forming cells' proliferation and chondrogenic differentiation. With this information, we hope to facilitate the understanding of the spatial and temporal interactions between cartilage-forming cells and laminin microenvironment to eventually advance cell-based cartilage engineering and regeneration.

High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations.

PubMed

Schittny, J C; Timpl, R; Engel, J

1988-10-01

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.

Functional decline at the aging neuromuscular junction is associated with altered laminin-α4 expression.

PubMed

Lee, Kah Meng; Chand, Kirat K; Hammond, Luke A; Lavidis, Nickolas A; Noakes, Peter G

2017-03-14

Laminin-α4 is involved in the alignment of active zones to postjunctional folds at the neuromuscular junction (NMJ). Prior study has implicated laminin-α4 in NMJ maintenance, with altered NMJ morphology observed in adult laminin-α4 deficient mice ( lama 4 -/- ). The present study further investigated the role of laminin-α4 in NMJ maintenance by functional characterization of transmission properties, morphological investigation of synaptic proteins including synaptic laminin-α4, and neuromotor behavioral testing. Results showed maintained perturbed transmission properties at lama 4 -/- NMJs from adult (3 months) through to aged (18-22 months). Hind-limb grip force demonstrated similar trends as transmission properties, with maintained weaker grip force across age groups in lama 4 -/- . Interestingly, both transmission properties and hind-limb grip force in aged wild-types resembled those observed in adult lama 4 -/- . Most significantly, altered expression of laminin-α4 was noted at the wild-type NMJs prior to the observed decline in transmission properties, suggesting that altered laminin-α4 expression precedes the decline of neurotransmission in aging wild-types. These findings significantly support the role of laminin-α4 in maintenance of the NMJ during aging.

Basement membrane reconstruction in human skin equivalents is regulated by fibroblasts and/or exogenously activated keratinocytes.

PubMed

El Ghalbzouri, Abdoelwaheb; Jonkman, Marcel F; Dijkman, Remco; Ponec, Maria

2005-01-01

This study was undertaken to examine the role fibroblasts play in the formation of the basement membrane (BM) in human skin equivalents. For this purpose, keratinocytes were seeded on top of fibroblast-free or fibroblast-populated collagen matrix or de-epidermized dermis and cultured in the absence of serum and exogenous growth factors. The expression of various BM components was analyzed on the protein and mRNA level. Irrespective of the presence or absence of fibroblasts, keratin 14, hemidesmosomal proteins plectin, BP230 and BP180, and integrins alpha1beta1, alpha2beta1, alpha3beta1, and alpha6beta4 were expressed but laminin 1 was absent. Only in the presence of fibroblasts or of various growth factors, laminin 5 and laminin 10/11, nidogen, uncein, type IV and type VII collagen were decorating the dermal/epidermal junction. These findings indicate that the attachment of basal keratinocytes to the dermal matrix is most likely mediated by integrins alpha1beta1 and alpha2beta1, and not by laminins that bind to integrin alpha6beta4 and that the epithelial-mesenchymal cross-talk plays an important role in synthesis and deposition of various BM components.

Progesterone and gravidity differentially regulate expression of extracellular matrix components in the pregnant rat myometrium.

PubMed

Shynlova, Oksana; Mitchell, Jennifer A; Tsampalieros, Anne; Langille, B Lowell; Lye, Stephen J

2004-04-01

Myometrial growth and remodeling during pregnancy depends on increased synthesis of interstitial matrix proteins. We hypothesize that the presence of mechanical tension in a specific hormonal environment regulates the expression of extracellular matrix (ECM) components in the uterus. Myometrial tissue was collected from pregnant rats on Gestational Days 0, 12, 15, 17, 19, 21, 22, 23 (labor), and 1 day postpartum and ECM expression was analyzed by Northern blotting. Expression of fibronectin, laminin beta2, and collagen IV mRNA was low during early gestation but increased dramatically on Day 23 during labor. Expression of fibrillar collagens (type I and III) peaked Day 19 and decreased near term. In contrast, elastin mRNA remained elevated from midgestation onward. Injection of progesterone (P4) on Days 20-23 (to maintain elevated plasma P4 levels) delayed the onset of labor, caused dramatic reductions in the levels of fibronectin and laminin mRNA, and prevented the fall of collagen III mRNA levels on Day 23. Treatment of pregnant rats with the progesterone receptor antagonist RU486 on Day 19 induced preterm labor on Day 20 and a premature increase in mRNA levels of collagen IV, fibronectin, and laminin. Analysis of the uterine tissue from unilaterally pregnant rats revealed that most of the changes in ECM gene expression occurred specifically in the gravid horn. Our results show a decrease in expression of fibrillar collagens and a coordinated temporal increase in expression of components of the basement membrane near term associated with decreased P4 and increased mechanical tension. These ECM changes contribute to myometrial growth and remodeling during late pregnancy and the preparation for the synchronized contractions of labor.

Immunogold localisation of laminin in normal and exfoliative iris.

PubMed Central

Konstas, A. G.; Marshall, G. E.; Lee, W. R.

1990-01-01

Immunoelectron microscopic studies of exfoliative iris tissue (seven specimens) revealed the presence of laminin in the fibrillar component of exfoliation material. The immunogold label was uniformly distributed on the exfoliation fibres. Deposition of laminin labelled exfoliation material in the dilator muscle was a noteworthy feature, as was an apparent depletion of laminin in the basement membranes of ostensibly unaffected vessels. In control iris tissue (five enucleated eyes) laminin was identified in the basement membrane round vascular contractile cells, but not beneath the endothelium. Images PMID:2390517

Laminin coatings on implant surfaces promote osseointegration: Fact or fiction?

PubMed

Javed, Fawad; Al Amri, Mohammad D; Kellesarian, Sergio Varela; Al-Askar, Mansour; Al-Kheraif, Abdulaziz A; Romanos, Georgios E

2016-08-01

To our knowledge from indexed literature, the role of laminins in the expression of osteogenic biomarkers and osseointegration enhancement has not been systematically reviewed. The aim of the present systematic review was to assess the role of laminin coatings on implant surfaces in promoting osseointegration. To address the focused question, "Do laminin coatings on implant surfaces influence osseointegration?", indexed databases were searched from 1965 up to and including November 2015 using various combination of the following keywords: "Bone to implant contact"; "implant"; "laminins"; and "osseointegration". Letters to the Editor, case-reports/case-series, historic reviews, and commentaries were excluded. The pattern of the present systematic review was customized to primarily summarize the pertinent data. Nine studies were included. Six studies were prospective and were performed in animals and 5 studies were in vitro. Results from 8 studies showed that laminin coatings enhanced new bone formation around implants and/or bone-to-implant contact. One study showed that laminin coated implants surfaces did not improve osseointegration. On experimental grounds, laminin coatings seem to enhance osteogenic biomarkers expression and/or osseointegration; however, from a clinical perspective, further randomized control trials are needed to assess the role of laminin coatings in promoting osseointegration around dental implants. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Laminins affect T cell trafficking and allograft fate

PubMed Central

Warren, Kristi J.; Iwami, Daiki; Harris, Donald G.; Bromberg, Jonathan S.; Burrell, Bryna E.

2014-01-01

Lymph nodes (LNs) are integral sites for the generation of immune tolerance, migration of CD4+ T cells, and induction of Tregs. Despite the importance of LNs in regulation of inflammatory responses, the LN-specific factors that regulate T cell migration and the precise LN structural domains in which differentiation occurs remain undefined. Using intravital and fluorescent microscopy, we found that alloreactive T cells traffic distinctly into the tolerant LN and colocalize in exclusive regions with alloantigen-presenting cells, a process required for Treg induction. Extracellular matrix proteins, including those of the laminin family, formed regions within the LN that were permissive for colocalization of alloantigen-presenting cells, alloreactive T cells, and Tregs. We identified unique expression patterns of laminin proteins in high endothelial venule basement membranes and the cortical ridge that correlated with alloantigen-specific immunity or immune tolerance. The ratio of laminin α4 to laminin α5 was greater in domains within tolerant LNs, compared with immune LNs, and blocking laminin α4 function or inducing laminin α5 overexpression disrupted T cell and DC localization and transmigration through tolerant LNs. Furthermore, reducing α4 laminin circumvented tolerance induction and induced cardiac allograft inflammation and rejection in murine models. This work identifies laminins as potential targets for immune modulation. PMID:24691446

Structural determinants of the interaction between the Haemophilus influenzae Hap autotransporter and fibronectin.

PubMed

Spahich, Nicole A; Kenjale, Roma; McCann, Jessica; Meng, Guoyu; Ohashi, Tomoo; Erickson, Harold P; St Geme, Joseph W

2014-06-01

Haemophilus influenzae is a Gram-negative cocco-bacillus that initiates infection by colonizing the upper respiratory tract. Hap is an H. influenzae serine protease autotransporter protein that mediates adherence, invasion and microcolony formation in assays with human epithelial cells and is presumed to facilitate the process of colonization. Additionally, Hap mediates adherence to fibronectin, laminin and collagen IV, extracellular matrix (ECM) proteins that are present in the respiratory tract and are probably important targets for H. influenzae colonization. The region of Hap responsible for adherence to ECM proteins has been localized to the C-terminal 511 aa of the Hap passenger domain (HapS). In this study, we characterized the structural determinants of the interaction between HapS and fibronectin. Using defined fibronectin fragments, we established that Hap interacts with the fibronectin repeat fragment called FNIII(1-2). Using site-directed mutagenesis, we found a series of motifs in the C-terminal region of HapS that contribute to the interaction with fibronectin. Most of these motifs are located on the F1 and F3 faces of the HapS structure, suggesting that the F1 and F3 faces may be responsible for the HapS-fibronectin interaction. © 2014 The Authors.

Structural determinants of the interaction between the Haemophilus influenzae Hap autotransporter and fibronectin

PubMed Central

Spahich, Nicole A.; Kenjale, Roma; McCann, Jessica; Meng, Guoyu; Ohashi, Tomoo; Erickson, Harold P.

2014-01-01

Haemophilus influenzae is a Gram-negative cocco-bacillus that initiates infection by colonizing the upper respiratory tract. Hap is an H. influenzae serine protease autotransporter protein that mediates adherence, invasion and microcolony formation in assays with human epithelial cells and is presumed to facilitate the process of colonization. Additionally, Hap mediates adherence to fibronectin, laminin and collagen IV, extracellular matrix (ECM) proteins that are present in the respiratory tract and are probably important targets for H. influenzae colonization. The region of Hap responsible for adherence to ECM proteins has been localized to the C-terminal 511 aa of the Hap passenger domain (HapS). In this study, we characterized the structural determinants of the interaction between HapS and fibronectin. Using defined fibronectin fragments, we established that Hap interacts with the fibronectin repeat fragment called FNIII(1–2). Using site-directed mutagenesis, we found a series of motifs in the C-terminal region of HapS that contribute to the interaction with fibronectin. Most of these motifs are located on the F1 and F3 faces of the HapS structure, suggesting that the F1 and F3 faces may be responsible for the HapS–fibronectin interaction. PMID:24687948

A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins

PubMed Central

1995-01-01

To examine the function of the alpha 6 beta 4 integrin we have determined its ligand-binding ability and overexpressed two potentially dominant negative mutant beta 4 subunits, lacking either the cytoplasmic or extracellular domain, in bladder epithelial 804G cells. The results of cell adhesion and radioligand-binding assays showed that alpha 6 beta 4 is a receptor for several laminin isoforms, including laminin 1, 2, 4, and 5. Overexpression of the tail-less or head-less mutant beta 4 subunit did not suppress alpha 6 beta 4-mediated adhesion to laminins, as both types of transfectants adhered to these ligands in the presence of blocking anti-beta 1 antibodies as well as the controls. However, immunofluorescence experiments indicated that the endogenous alpha 6 beta 4 integrin and other hemidesmosomal markers were not concentrated in hemidesmosomes in cells overexpressing tail- less beta 4, while the distribution of these molecules was not altered in cells overexpressing the head-less subunit. Electron microscopic studies confirmed that cells overexpressing tail-less beta 4 had a drastically reduced number of hemidesmosomes, while cells expressing the head-less subunit had a normal number of these structures. Thus, expression of a tail-less, but not a head-less mutant beta 4 subunit leads to a dominant negative effect on hemidesmosome assembly without suppressing initial adhesion to laminins. We conclude that the alpha 6 beta 4 integrin binds to several laminins and plays an essential role in the assembly and/or stability of hemidesmosomes, that alpha 6 beta 4- mediated adhesion and hemidesmosome assembly have distinct requirements, and that it is possible to use a dominant negative approach to selectively interfere with a specific function of an integrin. PMID:7721947

Binding of extracellular matrix proteins to Aspergillus fumigatus conidia.

PubMed Central

Gil, M L; Peñalver, M C; Lopez-Ribot, J L; O'Connor, J E; Martinez, J P

1996-01-01

As detected by confocal immunofluorescence microscopy, binding of fibronectin and laminin appeared to be associated with the protrusions present on the outer cell wall layer of resting Aspergillus fumigatus conidia. Flow cytometry confirmed that binding of laminin to conidia was dose dependent and saturable. Laminin binding was virtually eliminated in trypsin-treated organisms, thus suggesting the protein nature of the binding site. Conidia were also able to specifically adhere to laminin immobilized on microtiter plates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) with laminin and antilaminin antibody of whole conidial homogenates allowed identification, among the complex array of protein and glycoprotein species, of one polypeptide with an apparent molecular mass of 37 kDa which specifically interacts with laminin. The fact that binding of conidia to soluble or immobilized laminin or fibronectin was inhibited by fibronectin or laminin, respectively, suggests the existence of common binding sites for both ligands on the surface of conidia. Intact conidia were also able to adhere to type I and IV collagen immobilized on microtiter plates; adhesion was found to be dose dependent and saturable. Adhesion to immobilized type I and IV collagen was markedly inhibited by laminin and weakly inhibited by fibronectin. Coincubation of conidia with Arg-Gly-Asp (RGD) peptides caused a dose-dependent decrease in binding of cells to immobilized or soluble fibronectin, yet interaction of cells with soluble or immobilized laminin and type I and IV collagen remained unaffected. Interactions described here could be important in mediating attachment of the fungus to host tissues, thus playing a role in the establishment of the disease. PMID:8945572

Laminin-411 Is a Vascular Ligand for MCAM and Facilitates TH17 Cell Entry into the CNS

PubMed Central

Flanagan, Ken; Fitzgerald, Kent; Baker, Jeanne; Regnstrom, Karin; Gardai, Shyra; Bard, Frederique; Mocci, Simonetta; Seto, Pui; You, Monica; Larochelle, Catherine; Prat, Alexandre; Chow, Samuel; Li, Lauri; Vandevert, Chris; Zago, Wagner; Lorenzana, Carlos; Nishioka, Christopher; Hoffman, Jennifer; Botelho, Raquel; Willits, Christopher; Tanaka, Kevin; Johnston, Jennifer; Yednock, Ted

2012-01-01

TH17 cells enter tissues to facilitate pathogenic autoimmune responses, including multiple sclerosis (MS). However, the adhesion molecules involved in the unique migratory capacity of TH17 cells, into both inflamed and uninflamed tissues remain unclear. Herein, we characterize MCAM (CD146) as an adhesion molecule that defines human TH17 cells in the circulation; following in vitro restimulation of human memory T cells, nearly all of the capacity to secrete IL-17 is contained within the population of cells expressing MCAM. Furthermore, we identify the MCAM ligand as laminin 411, an isoform of laminin expressed within the vascular endothelial basement membranes under inflammatory as well as homeotstatic conditions. Purified MCAM-Fc binds to laminin 411 with an affinity of 27 nM, and recognizes vascular basement membranes in mouse and human tissue. MCAM-Fc binding was undetectable in tissue from mice with targeted deletion of laminin 411, indicating that laminin 411 is a major tissue ligand for MCAM. An anti-MCAM monoclonal antibody, selected for inhibition of laminin binding, as well as soluble MCAM-Fc, inhibited T cell adhesion to laminin 411 in vitro. When administered in vivo, the antibody reduced TH17 cell infiltration into the CNS and ameliorated disease in an animal model of MS. Our data suggest that MCAM and laminin 411 interact to facilitate TH17 cell entry into tissues and promote inflammation. PMID:22792325

Imaging sulfur mustard lesions in human epidermal tissues and keratinocytes by confocal and multiphoton microscopy

NASA Astrophysics Data System (ADS)

Werrlein, Robert; Madren-Whalley, Janna S.

2002-06-01

Topical exposure to sulfur mustard (HD), a known theat agent, produces persistent and debilitating cutaneous blisters. The blisters occur at the dermal-epidermal junction following a dose-dependent latent period of 8-24 h, however, the primary lesions causing vesication remain uncertain. Immunofluorescent images reveal that a 5-min exposure to 400 (mu) M HD disrupts molecules that are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Using keratinocyte cultures and fluorochomes conjugated to two different keratin-14 (K14) antibodies (clones CKB1 and LL002), results have shown a statistically significant (p<0.1) 1-h decrease of 29.2% in expression of the CKB1 epitope, a nearly complete loss of CKB1 expression within 2 h, and progressive cytoskeletal (K14) collapse without loss in expression of the LL002 epitope. With human epidermal tissues, multi-photon images of (alpha) 6 integrin and laminin 5 showed disruptive changes in the cell-surface organization and integrity of these adhesion molecules. At 1 H postexposure, analyses showed a statistically significant (p<0.1) decrease of 27.3% in (alpha) 6 integrin emissions, and a 32% decrease in laminin 5 volume. Multi-photon imaging indicates that molecules essential for epidermal-dermal attachment are early targets in the alkylating events leading to HD-induced vesication.

Tissue distribution of very late activation antigens-1/6 and very late activation antigen ligands in the normal thymus and in thymoma.

PubMed Central

Ruco, L. P.; Paradiso, P.; Pittiglio, M.; Diodoro, M. G.; Gearing, A. J.; Mainiero, F.; Gismondi, A.; Santoni, A.; Baroni, C. D.

1993-01-01

The expression of very late activation antigens (VLAs)-1/6 was correlated with that of the VLA ligands fibronectin, laminin, collagen, and vascular cell adhesion molecule-1 in sections of normal thymus, in thymocyte suspensions, and in 10 cases of thymoma. Capsular epithelial cells are VLA-2+, VLA-3+, and VLA-6+ and face the thymic basement membrane, which is rich in fibronectin, laminin, and collagen type IV. Cortical epithelial cells are VLA-2+ and are embedded in a reticular meshwork of nonorganized extracellular matrix (ECM) that is rich in fibronectin. Cortical thymocytes, identified as CD3dim cells by using immunofluorescence in suspension, are highly positive for VLA-4, a fibronectin ligand. Most cortical macrophages are positive for vascular cell adhesion molecule-1, a molecule recognized by VLA-4. Medullary epithelial cells are VLA-2+/VLA-3+ and are codistributed with fibrous strands of organized ECM that are positive for fibronectin, collagen, and laminin. Medullary thymocytes, identified as CD3bright cells, are positive for VLA-4 and VLA-6, a ligand for laminin. Our findings suggest that intrathymic thymocyte maturation is associated with changes in expression of VLA molecules, which are apparently correlated with the presence of VLA ligands in the tissue microenvironment. Thymomas were classified as cortical (three), common (five), or medullary (two) type. Expression of VLA molecules and distribution of ECM in the three histological subtypes were reminiscent of those observed in the respective regions of the normal thymus. All cases of thymoma were characterized by overexpression of VLA molecules on neoplastic cells, which was associated with increased deposition of organized ECM rich in fibronectin, laminin, and collagen. Images Figure 1 Figure 3 PMID:8456937

Laminin Levels Regulate Tissue Migration and Anterior-Posterior Polarity during Egg Morphogenesis in Drosophila.

PubMed

Díaz de la Loza, María C; Díaz-Torres, Alfonsa; Zurita, Federico; Rosales-Nieves, Alicia E; Moeendarbary, Emad; Franze, Kristian; Martín-Bermudo, María D; González-Reyes, Acaimo

2017-07-05

Basement membranes (BMs) are specialized extracellular matrices required for tissue organization and organ formation. We study the role of laminin and its integrin receptor in the regulation of tissue migration during Drosophila oogenesis. Egg production in Drosophila involves the collective migration of follicle cells (FCs) over the BM to shape the mature egg. We show that laminin content in the BM increases with time, whereas integrin amounts in FCs do not vary significantly. Manipulation of integrin and laminin levels reveals that a dynamic balance of integrin-laminin amounts determines the onset and speed of FC migration. Thus, the interplay of ligand-receptor levels regulates tissue migration in vivo. Laminin depletion also affects the ultrastructure and biophysical properties of the BM and results in anterior-posterior misorientation of developing follicles. Laminin emerges as a key player in the regulation of collective cell migration, tissue stiffness, and the organization of anterior-posterior polarity in Drosophila. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The mechanism for keratinocyte detaching from pH-responsive chitosan.

PubMed

Chen, Yi-Hsin; Chang, Shao-Hsuan; Wang, I-Jong; Young, Tai-Horng

2014-11-01

In this study, we compared the detachment ratio of HaCaT and Hs68 cells from pH-responsive chitosan surface by raising medium pH from 7.20 to 7.65 for 60 min. The detachment ratio of elongated Hs68 cells was over 75%, but that of round-shaped HaCaT cells was less than 50%, even extending the incubation time to 6 h or enhancing the cytoskeletal contractile force with the Rho activator CN01. However, the addition of 2 mm of EDTA into the medium at pH 7.65 could effectively detach HaCaT cells (detachment ratio > 90%), indicating that the calcium ion played an important role in the detachment process. Therefore, the family of Ca(+2)-dependent integrin receptors was examined by RT-PCR, real-time PCR and immunocytochemistry. It was found the expression of integrin β4 (ITGb4) was HaCaT cell-specific and the mRNA level of ITGb4 in undetached HaCaT cells was significantly higher than that in detached ones. By modulating ITGb4 activity with specific functional blocking antibody ASC-8, the detachment ratio of HaCaT cells could be increased to be greater than 85%. Conversely, the addition of the ligand of ITGb4 laminin into the culture system decreased the medium pH-induced detachment ratio for HaCaT cells, but not for Hs68 cells. Further addition of ASC-8 could rescue the effect of laminin on preventing the detachment of HaCaT cells from pH-sensitive chitosan surface. Therefore, this study demonstrated the interaction of ITGb4 and laminin played an important role in controlling the detachment of HaCaT cells on pH-responsive chitosan. Copyright © 2014 Elsevier Ltd. All rights reserved.

Laminin-111 peptide C16 regulates invadopodia activity of malignant cells through β1 integrin, Src and ERK 1/2.

PubMed

Siqueira, Adriane S; Pinto, Monique P; Cruz, Mário C; Smuczek, Basilio; Cruz, Karen S P; Barbuto, José Alexandre M; Hoshino, Daisuke; Weaver, Alissa M; Freitas, Vanessa M; Jaeger, Ruy G

2016-07-26

Laminin peptides influence tumor behavior. In this study, we addressed whether laminin peptide C16 (KAFDITYVRLKF, γ1 chain) would increase invadopodia activity of cells from squamous cell carcinoma (CAL27) and fibrosarcoma (HT1080). We found that C16 stimulates invadopodia activity over time in both cell lines. Rhodamine-conjugated C16 decorates the edge of cells, suggesting a possible binding to membrane receptors. Flow cytometry showed that C16 increases activated β1 integrin, and β1 integrin miRNA-mediated depletion diminishes C16-induced invadopodia activity in both cell lines. C16 stimulates Src and ERK 1/2 phosphorylation, and ERK 1/2 inhibition decreases peptide-induced invadopodia activity. C16 also increases cortactin phosphorylation in both cells lines. Based on our findings, we propose that C16 regulates invadopodia activity over time of squamous carcinoma and fibrosarcoma cells, probably through β1 integrin, Src and ERK 1/2 signaling pathways.

Immobilization of Cell-Adhesive Laminin Peptides in Degradable PEGDA Hydrogels Influences Endothelial Cell Tubulogenesis

PubMed Central

Ali, Saniya; Saik, Jennifer E.; Gould, Dan J.; Dickinson, Mary E.

2013-01-01

Abstract Attachment, spreading, and organization of endothelial cells into tubule networks are mediated by interactions between cells in the extracellular microenvironment. Laminins are key extracellular matrix components and regulators of cell adhesion, migration, and proliferation. In this study, laminin-derived peptides were conjugated to poly(ethylene glycol) (PEG) monoacrylate and covalently incorporated into degradable PEG diacrylate (PEGDA) hydrogels to investigate the influence of these peptides on endothelial cellular adhesion and function in organizing into tubule networks. Degradable PEGDA hydrogels were synthesized by incorporating a matrix metalloproteinase (MMP)–sensitive peptide, GGGPQGIWGQGK (abbreviated PQ), into the polymer backbone. The secretion of MMP-2 and MMP-9 by endothelial cells promotes polymer degradation and consequently cell migration. We demonstrate the formation of extensive networks of tubule-like structures by encapsulated human umbilical vein endothelial cells in hydrogels with immobilized synthetic peptides. The resulting structures were stabilized by pericyte precursor cells (10T1/2s) in vitro. During tubule formation and stabilization, extracellular matrix proteins such as collagen IV and laminin were deposited. Tubules formed in the matrix of metalloproteinase sensitive hydrogels were visualized from 7 days to 4 weeks in response to different combination of peptides. Moreover, hydrogels functionalized with laminin peptides and transplanted in a mouse cornea supported the ingrowth and attachment of endothelial cells to the hydrogel during angiogenesis. Results of this study illustrate the use of laminin-derived peptides as potential candidates for modification of biomaterials to support angiogenesis. PMID:23914330

Novel Regulation of Integrin Trafficking by Rab11-FIP5 in Aggressive Prostate Cancer.

PubMed

Das, Lipsa; Gard, Jaime M C; Prekeris, Rytis; Nagle, Raymond B; Morrissey, Colm; Knudsen, Beatrice S; Miranti, Cindy K; Cress, Anne E

2018-05-14

The laminin-binding integrins, α3β1 and α6β1, are needed for tumor metastasis and their surface expression is regulated by endocytic recycling. β1 integrins share the Rab11 recycling machinery but the trafficking of α3β1 and α6β1 are distinct by an unknown mechanism. Using a mouse PDX tumor model containing human metastatic prostate cancer, Rab11 family interacting protein 5 (Rab11-FIP5) was identified as a lead candidate for α6β1 trafficking. Rab11-FIP5 and its membrane binding domain were required for α6β1 recycling, without affecting the other laminin-binding integrin (i.e., α3β1) or unrelated membrane receptors like CD44, transferrin receptor, or E-cadherin. Depletion of Rab11-FIP5 resulted in the intracellular accumulation of α6β1 in the Rab11 recycling compartment, loss of cell migration on laminin, and an unexpected loss of α6β1 recycling in cell-cell locations. Taken together, these data demonstrate that α6β1 is distinct from α3β1 via Rab11-FIP5 recycling and recycles in an unexpected cell-cell location. Rab11-FIP5 dependent a6b1 integrin recycling may be selectively targeted to limit migration of prostate cancer cells into laminin-rich tissues. Copyright ©2018, American Association for Cancer Research.

Dystroglycan modulates the ability of insulin-like growth factor-1 to promote oligodendrocyte differentiation.

PubMed

Galvin, Jason; Eyermann, Christopher; Colognato, Holly

2010-11-15

The adhesion receptor dystroglycan positively regulates terminal differentiation of oligodendrocytes, but the mechanism by which this occurs remains unclear. Using primary oligodendrocyte cultures, we identified and examined a connection between dystroglycan and the ability of insulin-like growth factor-1 (IGF-1) to promote oligodendrocyte differentiation. Consistent with previous reports, treatment with exogenous IGF-1 caused an increase in MBP protein that was preceded by activation of PI3K (AKT) and MAPK (ERK) signaling pathways. The extracellular matrix protein laminin was further shown to potentiate the effect of IGF-1 on oligodendrocyte differentiation. Depletion of the laminin receptor dystroglycan using siRNA, however, blocked the ability of IGF-1 to promote oligodendrocyte differentiation of cells grown on laminin, suggesting a role for dystroglycan in IGF-1-mediated differentiation. Indeed, loss of dystroglycan led to a reduction in the ability of IGF-1 to activate MAPK, but not PI3K, signaling pathways. Pharmacological inhibition of MAPK signaling also prevented IGF-1-induced increases in myelin basic protein (MBP), indicating that MAPK signaling was necessary to drive IGF-1-mediated enhancement of oligodendrocyte differentiation. Using immunoprecipitation, we found that dystroglycan, the adaptor protein Grb2, and insulin receptor substrate-1 (IRS-1), were associated in a protein complex. Taken together, our results suggest that the positive regulatory effect of laminin on oligodendrocyte differentiation may be attributed, at least in part, to dystroglycan's ability to promote IGF-1-induced differentiation.

Regional differences in the expression of laminin isoforms during mouse neural tube development

PubMed Central

Copp, Andrew J.; Carvalho, Rita; Wallace, Adam; Sorokin, Lydia; Sasaki, Takako; Greene, Nicholas D.E.; Ybot-Gonzalez, Patricia

2013-01-01

Many significant human birth defects originate around the time of neural tube closure or early during post-closure nervous system development. For example, failure of the neural tube to close generates anencephaly and spina bifida, faulty cell cycle progression is implicated in primary microcephaly, while defective migration of neuroblasts can lead to neuronal migration disorders such as lissencephaly. At the stage of neural tube closure, basement membranes are becoming organised around the neuroepithelium, and beneath the adjacent non-neural surface ectoderm. While there is circumstantial evidence to implicate basement membrane dynamics in neural tube and surface ectodermal development, we have an incomplete understanding of the molecular composition of basement membranes at this stage. In the present study, we examined the developing basement membranes of the mouse embryo at mid-gestation (embryonic day 9.5), with particular reference to laminin composition. We performed in situ hybridization to detect the mRNAs of all eleven individual laminin chains, and immunohistochemistry to identify which laminin chains are present in the basement membranes. From this information, we inferred the likely laminin variants and their tissues of origin: that is, whether a given basement membrane laminin is contributed by epithelium, mesenchyme, or both. Our findings reveal major differences in basement composition along the body axis, with the rostral neural tube (at mandibular arch and heart levels) exhibiting many distinct laminin variants, while the lumbar level where the neural tube is just closing shows a much simpler laminin profile. Moreover, there appears to be a marked difference in the extent to which the mesenchyme contributes laminin variants to the basement membrane, with potential contribution of several laminins rostrally, but no contribution caudally. This information paves the way towards a mechanistic analysis of basement membrane laminin function during early neural tube development in mammals. PMID:21524702

Matrix metalloproteinase-2 and its correlation with basal membrane components laminin-5 and collagen type IV in paediatric burn patients measured with Surface Plasmon Resonance Imaging (SPRI) biosensors.

PubMed

Weremijewicz, Artur; Matuszczak, Ewa; Sankiewicz, Anna; Tylicka, Marzena; Komarowska, Marta; Tokarzewicz, Anna; Debek, Wojciech; Gorodkiewicz, Ewa; Hermanowicz, Adam

2018-06-01

The purpose of this study was the determination of matrix metalloproteinase-2 and its correlation with basal membrane components laminin-5 and collagen type IV in the blood plasma of burn patients measured with Surface Plasmon Resonance Imaging (SPRI) biosensors. 31 children scalded by hot water who were managed at the Department of Paediatric Surgery between 2014-2015, after primarily presenting with burns in 4-20% TBSA were included into the study (age 9 months up to 14 years, mean age 2,5+1 years). There were 10 girls and 21 boys. Venous blood samples were drawn 2-6h, and 12-16h after the thermal injury, and on the subsequent days 3, 5 and 7. The matrix metalloproteinase-2, collagen type IV and laminin-5 concentrations were assessed using Surface Plasmon Resonance Imaging by the investigators blinded to the other data. The MMP-2, laminin-5 and collagen type IV concentrations in the blood plasma of patients with burns, were highest 12-16h after thermal injury, the difference was statistically significant. The MMP-2, laminin-5 and collagen type IV concentrations measured 3 days, 5 days and 7 days after the thermal injury, slowly decreased over time, and on the 7th day reached the normal range, when compared with the concentration measured in controls. Current work is the first follow-up study regarding MMP-2 in burns. MMP-2, laminin-5 and collagen type IV levels were elevated early after burn injury in the plasma of studied patients, and were highest 12-16h after the injury. MMP-2, laminin-5 and collagen type IV levels were not proportional to the severity of the burn. We believe in the possibility that the gradual decrease of MMP-2, collagen type IV and laminin-5 concentrations could be connected with the process of healing, but to prove it, more investigation is needed in this area. The SPR imaging biosensor is a good diagnostic tool for determination of MMP-2, laminin-5 and collagen type IV in blood plasma of patients with burns. Copyright © 2017 Elsevier Ltd and ISBI. All rights reserved.

Specific PET Imaging Probes for Early Detection of Prostate Cancer Metastases

DTIC Science & Technology

2011-05-01

K. S. (2005) 67-kDa laminin receptor promotes internalization of cytotoxic necrotizing factor 1-expressing Escherichia coli K1 into human brain...Chung JW, Kim KS (2005) 67-kDa laminin receptor promotes internalization of cytotoxic necrotizing factor 1-expressing Esch- erichia coli K1 into human...SUBTITLE 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER E -Mail: 5f

Microenvironmental changes in the progression from adenocarcinoma in situ to minimally invasive adenocarcinoma and invasive lepidic predominant adenocarcinoma of the lung.

PubMed

Naito, Masahito; Aokage, Keiju; Saruwatari, Kouichi; Hisakane, Kakeru; Miyoshi, Tomohiro; Hishida, Tomoyuki; Yoshida, Junji; Masato, Sugano; Kojima, Motohiro; Kuwata, Takeshi; Fujii, Satoshi; Ochiai, Atsushi; Sato, Yukitoshi; Tsuboi, Masahiro; Ishii, Genichiro

2016-10-01

Invasive lepidic predominant adenocarcinoma (LPA) of the lung is thought to progress in a stepwise fashion from adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA). The aim of this study was to investigate the microenvironmental changes during the development from AIS to LPA. Clinicopathological characteristics of AIS (n=51), MIA (n=59), LPA smaller than 3cm (LPA-S, n=113), and LPA larger than 3cm (LPA-L, n=47) were analyzed. We then evaluated the expression levels of epithelial-mesenchymal transition (EMT)-related molecules (E-cadherin, S100A4), invasion-related molecules (laminin-5, ezrin), stem-cell-related molecules (ALDH-1), and growth factor receptors (c-Met, EGFR) in cancer cells of each group (n=20). The number of tumor-promoting stromal cells, including podoplanin-positive cancer-associated fibroblasts (PDPN+ CAFs), CD204-positive tumor-associated macrophages (CD204+ TAMs), and CD34+ microvessel cells, were also analyzed. No significant difference in these characteristics was found between LPA-S and LPA-L. Laminin-5 expression in the non-invasive carcinoma component of MIA was significantly higher than that of AIS (p<0.001). During the progression from MIA to LPA-S, the expression level of laminin-5 in the invasive carcinoma component was significantly elevated (p<0.01). Moreover, tumor-promoting stromal cells were more frequently recruited in the invasive area of LPA-S (PDPN+ CAFs; p<0.05, CD204+ TAMs; p<0.001, CD34+ microvessel; p<0.05). Ezrin expression in the invasive carcinoma component of LPA-L was significantly increased (p<0.05) compared to LPA-S; however, the number of tumor-promoting stromal cells were not different between these two groups. Our current results indicated that microenvironmental molecular changes occur during the progression from MIA to LPA-S and suggested that this process may play an important role in disease progression from AIS to LPA. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Melatonin mitigates thioacetamide-induced hepatic fibrosis via antioxidant activity and modulation of proinflammatory cytokines and fibrogenic genes.

PubMed

Lebda, Mohamed A; Sadek, Kadry M; Abouzed, Tarek K; Tohamy, Hossam G; El-Sayed, Yasser S

2018-01-01

The potential antifibrotic effects of melatonin against induced hepatic fibrosis were explored. Rats were allocated into four groups: placebo; thioacetamide (TAA) (200mg/kg bwt, i.p twice weekly for two months); melatonin (5mg/kgbwt, i.p daily for a week before TAA and continued for an additional two months); and melatonin plus TAA. Hepatic fibrotic changes were evaluated biochemically and histopathologically. Hepatic oxidative/antioxidative indices were assessed. The expression of hepatic proinflammatory cytokines (tumor necrosis factor-α, and interleukin-1β), fibrogenic-related genes (transforming growth factor-1β, collagen I, collagen, III, laminin, and autotaxin) and an antioxidant-related gene (thioredoxin-1) were detected by qRT-PCR. In fibrotic rats, melatonin lowered serum aspartate aminotransferase, alanine aminotransferase, and autotaxin activities, bilirubin, hepatic hydroxyproline and plasma ammonia levels. Melatonin displayed hepatoprotective and antifibrotic potential as indicated by mild hydropic degeneration of some hepatocytes and mild fibroplasia. In addition, TAA induced the depletion of glutathione, glutathione s-transferase, glutathione peroxidase, superoxide dismutase, catalase, and paraoxonase-1 (PON-1), while inducing the accumulation of malondialdehyde, protein carbonyl (C=O) and nitric oxide (NO), and DNA fragmentation. These effects were restored by melatonin pretreatment. Furthermore, melatonin markedly attenuated the expression of proinflammatory cytokines and fibrogenic genes via the upregulation of thioredoxin-1 mRNA transcripts. Melatonin exhibits potent anti-inflammatory, antioxidant and fibrosuppressive activities against TAA-induced hepatic fibrogenesis via the suppression of oxidative stress, DNA damage, proinflammatory cytokines and fibrogenic gene transcripts. In addition, we demonstrate that the antifibrotic activity of melatonin is mediated by the induction of thioredoxin-1 with attenuation of autotaxin expressions. Copyright © 2017 Elsevier Inc. All rights reserved.

Distinct functions of the laminin β LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila

PubMed Central

2014-01-01

Background The Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles. Results Using an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos we obtained multiple mutants for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin β and collagen IV α1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV α2 chain is detected during late embryogenesis. Conclusions Assembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. Our data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational analysis suggests that the β6/β3/β8 interface of the laminin β LN domain is highly critical for formation of contiguous cardiac ECM layers. Certain mutations in the collagen IV triple helix-forming domain may exert a semi-dominant effect leading to an overall weakening of ECM structures as well as intracellular accumulation of collagen and other molecules, thus paralleling observations made in other organisms and in connection with collagen-related diseases. PMID:24935095

Modulatory Effects of Chemoradiation on Angiogenic Factors and Laminin in Cervical Cancer: Link with Treatment Response

PubMed

Sharma, Manoj; Khan, Rehan; Aggarwal, Mayank; Sharma, Alpana

2017-11-26

Objective: Carcinoma of the uterine cervix is either the first or second most common malignancy in Indian women, depending on the registry. Tumor growth and metastasis primarily are determined by angiogenesis and parameters of the molecular environment including extracellular matrix elements, growth factors and cytokines. Effects of chemo-irradiation on biomarkers like vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2) and laminin in patients with carcinoma cervix therefore need to be explored. Methods: Circulatory and mRNA levels of VEGF, Ang-2 and laminin in patients with stage III carcinoma cervix (n=40) were compared with those of normal healthy women (n=20). Measurement was prior to treatment, and after chemotherapy and teleradiation, using high sensitivity ELISA kits and Q-PCR. Clinical response was evaluated as per WHO criteria and was assessed for correlation with the biochemical markers. Results: Levels of all the studied molecules were significantly (p<0.001) higher in patients than in controls. After treatment significant decline (p<0.001) was noted. Out of 40 patients, 33 were complete responders and 7 were non-responders on clinical assessment. On comparison of before and after treatment levels of these molecules complete responders showed significant decline whereas non-responders showed non-significant decrease. Follow-up of the responders for 3 years, revealed 28 of 33 patients to still be disease free, the other 5 demonstrating recurrence. Conclusions: Higher levels of angiogenic factors along with laminin indicate roles played in disease progression aiding angiogenesis. These markers may serve as useful tools in post treatment disease mapping, for which available imaging methods may not provide a true picture. Creative Commons Attribution License

Effect of surface modification on protein retention and cell proliferation under strain.

PubMed

Dunkers, J P; Lee, H-J; Matos, M A; Pakstis, L M; Taboas, J M; Hudson, S D; Cicerone, M T

2011-07-01

When culturing cells on flexible surfaces, it is important to consider extracellular matrix treatments that will remain on the surface under mechanical strain. Here we investigate differences in laminin deposited on oxidized polydimethylsiloxane (PDMS) with plasma treatment (plasma-only) vs. plasma and aminopropyltrimethoxysilane treatment (silane-linked). We use specular X-ray reflectivity (SXR), transmission electron microscopy (TEM), and immunofluorescence to probe the quantity and uniformity of laminin. The surface coverage of laminin is approximately 45% for the plasma-only and 50% for the silane-linked treatment as determined by SXR. TEM and immunofluorescence reveal additional islands of laminin aggregates on the plasma-only PDMS compared with the relatively smooth and uniform silane-linked laminin surface. We also examine laminin retention under strain and vascular smooth muscle cell viability and proliferation under static and strain conditions. Equibiaxial stretching of the PDMS surfaces shows greatly improved retention of the silane-linked laminin over plasma-only. There are significantly more cells on the silane-linked surface after 4 days of equibiaxial strain. Published by Elsevier Ltd.

Preparation of laminin/nidogen adsorbed urinary bladder decellularized materials via a mussel-inspired polydopamine coating for pelvic reconstruction

PubMed Central

Ge, Liangpeng; Cao, Chuan; Chen, Shixuan; Shao, Bin; Li, Qianyong; Li, Qingtao; Liu, Lubin; Liang, Zhiqing; Huang, Yong

2017-01-01

Pelvic organ prolapse (POP) is a serious health issue that affects many adult women. A common strategy for POP reconstruction is to use scaffold materials to reconstruct the prolapsed pelvic organ. However, the existing materials for pelvic reconstruction do not meet the clinical requirements for biocompatibility, mechanics and immunological rejection. To address these concerns, urinary bladder decellularized materials (UBDM) was selected due to their good strain-stress resistance. To enhance its biocompatibility, laminin/nidogen was used to modify the UBDM with a mussel-inspired polydopamine coating. We found that the biocompatibility and mechanical properties of laminin/nidogen-Dopamine-UBDM were significantly enhanced and that the degradation rate of laminin/nidogen-Dopamine-UBDM was markedly reduced. Moreover, the expression of CD31 in the laminin/nidogen-Dopamine-UBDM group was higher than that in the normal UBDM group. The laminin/nidogen-Dopamine-UBDM treatment mainly guided M2 type macrophages and led to an inflammatory response. These results indicate that laminin/nidogen adsorbed urinary bladder decellularized materials are promising for use in pelvic reconstruction. PMID:29312483

Decrease in laminin content and protein excretion rate after five sixths nephrectomy and low-dose irradiation in the rat.

PubMed

Aunapuu, Marina; Arend, Andres; Kolts, Ivo; Egerbacher, Monika; Ots, Mai

2004-04-01

The effect of low-dose irradiation on laminin distribution and urine protein excretion in the remnant rat kidney has been studied. The rat remnant kidney formed after 5/6 nephrectomy is an experimental model of chronic renal failure. In the remnant kidney, focal segmental glomerulosclerosis is developed characterized by focal or segmental sclerosis in glomeruli, alterations in the tubules and thickening of the glomerular basement membrane. Low dose irradiation has been presumed to suppress sclerotic processes. In this study 24 male Wistar rats were subdivided into the nephrectomized group, nephrectomized and irradiated groups (1 or 3 Grey), and healthy control group. Animals were sacrificed at 2, 4 and 8 weeks after beginning the experiment. Laminin immunohistochemical staining was found along the tubular and glomerular basement membranes in all experimental groups, but with varying intensity. Laminin content in the basement membranes was decreased in early stages (week 2), especially after irradiation followed by increase during the later stages with relatively high levels at the end of the experiment (week 8). Irradiation at a dose of 3 Grey decreased protein excretion compared to the nephrectomized rats at all stages, while 1 Grey dose was ineffective. Based on decreased proteinuria we conclude that moderate low-dose irradiation has beneficial effects on the rat remnant kidney and that laminin in basement membranes is probably not the most crucial component in regulating membrane permeability.

The isolation and in vitro expansion of hepatic Sca-1 progenitor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clayton, Elizabeth, E-mail: Elizabeth.Clayton@ed.ac.uk; Forbes, Stuart J.

2009-04-17

The intra-hepatic population of liver progenitor cells expands during liver injury when hepatocyte proliferation is inhibited. These cells can be purified by density gradient centrifugation and cultured. Separated by size only this population contains small cells of hematopoietic, epithelial and endothelial lineages and is thought to contain liver stem cells. The identity of liver stem cells remains unknown although there is some evidence that tissue Sca1{sup +} CD45{sup -} cells display progenitor cell characteristics. We identified both intra-hepatic and gall bladder Sca1{sup +} cells following liver injury and expanded ex vivo Sca1 cells as part of heterogenous cell culture ormore » as a purified population. We found significant difference between the proliferation of Sca-1 cells when plated on laminin or collagen I while proliferation of heterogenous population was not affected by the extracellular matrix indicating the necessity for culture of Sca1{sup +} cells with laminin matrix or laminin producing cells in long term liver progenitor cell cultures.« less

Estradiol, tamoxifen and ICI 182,780 alter alpha3 and beta1 integrin expression and laminin-1 adhesion in oral squamous cell carcinoma cell cultures.

PubMed

Nelson, Katja; Helmstaedter, Victor; Moreau, Cynthia; Lage, Hermann

2008-01-01

Adhesion molecules such as integrins and extracellular matrix proteins like laminins have been identified to play an important role in cell proliferation, migration and invasion by regulating cell-extracellular matrix interaction in various cancers including oral squamous cell carcinoma (OSCC). In this study, the effect of estradiol (E2), and the E2 antagonists tamoxifen (TAM) and ICI 182,780 (ICI) on the expression of integrins and adhesion to laminin-1 in different OSCC in vitro models was analyzed. TAM and ICI inhibited growth in all OSCC cell lines. Dependent on estrogen receptor (ER) status E2 displayed a significant influence on growth after long-term administration. ICI reduced laminin-1 adhesion in all cell lines. beta1 Integrin transcription is reduced with TAM and E2 and alpha3 cell surface expression with TAM. This study shows that OSCC is estrogen and SERM sensitive and that these compounds can modulate cell-matrix interaction in part by modulating integrin expression and translation. The investigation also confirms that growth is significantly influenced by these adjuvant therapeutics. These data suggest that a greater understanding of basic biology and mechanisms of the ER and its ligands in oral squamous cells is needed to elucidate the use of specific pharmacological agents as therapeutics of anti-tumorigenic pathways.

Proteolytic Processing of Laminin-332 by Hepsin and Matriptase and Its Role in Prostate Cancer Progression

DTIC Science & Technology

2011-09-01

epithelial tumors, including breast, cervix , esophagus, liver, mesothelium, prostate, and colorectal cancers [36,38,61–69]. Interestingly, in the case of...Proteolytic Processing of Laminin-332 by Hepsin and Matriptase and Its Role in Prostate Cancer Progression Manisha Tripathi The Vanderbilt University...Nashville, TN 37203 Laminin-332 is lost in prostate cancer progression. Laminin-332 is known to be cleaved by various cell surface proteases

Distribution of Collagen I, III, and IV and Laminin in the Human Liver during Prenatal Development.

PubMed

Jović, Marko; Nikolić, Ivan; Todorović, Vera; Petrović, Aleksandar; Petrović, Vladimir; Mojsilović, Marijola; Denčić, Tijana

2018-06-27

In the absence of systematized data on the extracellular matrix components during prenatal liver development, the present study aimed to investigate the time of appearance and distribution of collagen types I, III, and IV and laminin. The study material included embryonic and fetal livers, aged 7-37 weeks, categorized into 3 trimesters. The material was stained using hematoxylin-eosin and immunohistochemistry methods for the identification of collagen I, III, and IV and laminin. Collagen I was detected near the end of the first trimester in the capsules and walls of interlobular veins. As the liver matures, collagen I is increasingly abundant in the capsules, portal area connective tissues, arterial walls, interlobular veins, sinusoids, and central veins. Collagen III and collagen IV appear in the middle of the first trimester in the capsules, portal areas, and walls of central veins, as well as the sinusoids particularly. In trimesters 2 and 3, these collagens are increasingly present in all the structures, but collagen IV is also present in nerve fibers. Laminin is sporadically present adjacent to the sinusoids in trimester 1, while in trimesters 2 and 3 this protein commonly appears in the walls of arteries and interlobular veins, in the basal membrane of bile ducts, and in nerve fibers. The contents of collagen I, III, and IV increase during prenatal development in the liver capsule, arterial and vein walls, sinusoids, and portal area. Laminin expression is consistent with that of the collagens with the exception that, within lobules, laminin disappears with liver maturation. © 2018 S. Karger AG, Basel.

Construction of a multi-functional extracellular matrix protein that increases number of N1E-115 neuroblast cells having neurites.

PubMed

Nakamura, Makiko; Mie, Masayasu; Mihara, Hisakazu; Nakamura, Makoto; Kobatake, Eiry

2009-10-01

An artificially designed fusion protein, which was designed to have strong cell adhesive activity and an active functional unit that enhances neuronal differentiation of mouse N1E-115 neuroblast cells, was developed. In this study, a laminin-1-derived IKVAV sequence, which stimulates neurite outgrowth in conditions of serum deprivation, was engineered and incorporated into an elastin-derived structural unit. The designed fusion protein also had a cell-adhesive RGD sequence derived from fibronectin. The resultant fusion protein could adsorb efficiently onto hydrophobic culture surfaces and showed cell adhesion activity similar to laminin. N1E-115 cells grown on the fusion protein exhibited more cells with neurites than cells grown on laminin-1. These results indicated that the constructed protein could retain properties of incorporated functional peptides and could provide effective signal transport. The strategy of designing multi-functional fusion proteins has the possibility for supporting current tissue engineering techniques. (c) 2009 Wiley Periodicals, Inc.

Avian pathogenic Escherichia coli bind fibronectin and laminin.

PubMed

Ramírez, Rosa María; Almanza, Yolanda; González, Rafael; García, Santos; Heredia, Norma

2009-04-01

Avian colisepticemia frequently occurs after respiratory tract damage, the primary site for infection allows bacteria to encounter an exposed basement membrane, where laminin and fibronectin are important components. We investigated the ability of an isolate of avian pathogenic Escherichia coli to bind fibronectin and laminin. Using Far-western dot blot analysis, we demonstrated the ability of this microorganism to bind basement membrane proteins fibronectin and laminin. Results from an ELISA-based approach indicate that the binding to these membrane proteins was bacterial-dose dependent. Furthermore, two specific E. coli polypeptides, of 32 kDa and 130 kDa, reacted with laminin and fibronectin, respectively. Further evaluation of these potential bacterial adhesins may provide insights into the pathogenesis of colibacillosis.

Identification of Cell Adhesive Sequences in the N-terminal Region of the Laminin α2 Chain*

PubMed Central

Hozumi, Kentaro; Ishikawa, Masaya; Hayashi, Takemitsu; Yamada, Yuji; Katagiri, Fumihiko; Kikkawa, Yamato; Nomizu, Motoyoshi

2012-01-01

The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117–128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin β1 and anti-integrin α2β1 antibodies. These results suggest that A2-8 promotes an integrin α2β1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2β1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2β1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2β1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions. PMID:22654118

A Novel In Vitro Method for Detecting Undifferentiated Human Pluripotent Stem Cells as Impurities in Cell Therapy Products Using a Highly Efficient Culture System

PubMed Central

Tano, Keiko; Yasuda, Satoshi; Kuroda, Takuya; Saito, Hirohisa; Umezawa, Akihiro; Sato, Yoji

2014-01-01

Innovative applications of cell therapy products (CTPs) derived from human pluripotent stem cells (hPSCs) in regenerative medicine are currently being developed. The presence of residual undifferentiated hPSCs in CTPs is a quality concern associated with tumorigencity. However, no simple in vitro method for direct detection of undifferentiated hPSCs that contaminate CTPs has been developed. Here, we show a novel approach for direct and sensitive detection of a trace amount of undifferentiated human induced pluripotent stem cells (hiPSCs) using a highly efficient amplification method in combination with laminin-521 and Essential 8 medium. Essential 8 medium better facilitated the growth of hiPSCs dissociated into single cells on laminin-521 than in mTeSR1 medium. hiPSCs cultured on laminin-521 in Essential 8 medium were maintained in an undifferentiated state and they maintained the ability to differentiate into various cell types. Essential 8 medium allowed robust hiPSC proliferation plated on laminin-521 at low cell density, whereas mTeSR1 did not enhance the cell growth. The highly efficient culture system using laminin-521 and Essential 8 medium detected hiPSCs spiked into primary human mesenchymal stem cells (hMSCs) or human neurons at the ratio of 0.001%–0.01% as formed colonies. Moreover, this assay method was demonstrated to detect residual undifferentiated hiPSCs in cell preparations during the process of hMSC differentiation from hiPSCs. These results indicate that our highly efficient amplification system using a combination of laminin-521 and Essential 8 medium is able to detect a trace amount of undifferentiated hPSCs contained as impurities in CTPs and would contribute to quality assessment of hPSC-derived CTPs during the manufacturing process. PMID:25347300

Distribution of Basement Membrane Molecules, Laminin and Collagen Type IV, in Normal and Degenerated Cartilage Tissues.

PubMed

Foldager, Casper Bindzus; Toh, Wei Seong; Gomoll, Andreas H; Olsen, Bjørn Reino; Spector, Myron

2014-04-01

The objective of the present study was to investigate the presence and distribution of 2 basement membrane (BM) molecules, laminin and collagen type IV, in healthy and degenerative cartilage tissues. Normal and degenerated tissues were obtained from goats and humans, including articular knee cartilage, the intervertebral disc, and meniscus. Normal tissue was also obtained from patella-tibial enthesis in goats. Immunohistochemical analysis was performed using anti-laminin and anti-collagen type IV antibodies. Human and goat skin were used as positive controls. The percentage of cells displaying the pericellular presence of the protein was graded semiquantitatively. When present, laminin and collagen type IV were exclusively found in the pericellular matrix, and in a discrete layer on the articulating surface of normal articular cartilage. In normal articular (hyaline) cartilage in the human and goat, the proteins were found co-localized pericellularly. In contrast, in human osteoarthritic articular cartilage, collagen type IV but not laminin was found in the pericellular region. Nonpathological fibrocartilaginous tissues from the goat, including the menisci and the enthesis, were also positive for both laminin and collagen type IV pericellularly. In degenerated fibrocartilage, including intervertebral disc, as in degenerated hyaline cartilage only collagen type IV was found pericellularly around chondrocytes but with less intense staining than in non-degenerated tissue. In calcified cartilage, some cells were positive for laminin but not type IV collagen. We report differences in expression of the BM molecules, laminin and collagen type IV, in normal and degenerative cartilaginous tissues from adult humans and goats. In degenerative tissues laminin is depleted from the pericellular matrix before collagen type IV. The findings may inform future studies of the processes underlying cartilage degeneration and the functional roles of these 2 extracellular matrix proteins, normally associated with BM.

Alterations in Pericyte Subpopulations are Associated with Elevated Blood-Tumor Barrier Permeability in Experimental Brain Metastasis of Breast Cancer

PubMed Central

Lyle, L. Tiffany; Lockman, Paul R.; Adkins, Chris E.; Mohammad, Afroz Shareef; Sechrest, Emily; Hua, Emily; Palmieri, Diane; Liewehr, David J.; Steinberg, Seth M.; Kloc, Wojciech; Izycka-Swieszewska, Ewa; Duchnowska, Renata; Nayyar, Naema; Brastianos, Priscilla K.; Steeg, Patricia S.; Gril, Brunilde

2016-01-01

Purpose The blood-brain barrier (BBB) is modified to a blood-tumor barrier (BTB) as a brain metastasis develops from breast or other cancers. We (a) quantified the permeability of experimental brain metastases; (b) determined the composition of the BTB; (c) identified which elements of the BTB distinguished metastases of lower permeability from those with higher permeability. Experimental Design A SUM190-BR3 experimental inflammatory breast cancer brain metastasis subline was established. Experimental brain metastases from this model system and two previously reported models (triple-negative MDA-231-BR6, HER2+ JIMT-1-BR3) were serially sectioned; low and high permeability lesions were identified with systemic 3kDa Texas Red dextran dye. Adjoining sections were used for quantitative immunofluorescence to known BBB and neuroinflammatory components. One-sample comparisons against a hypothesized value of one were performed with the Wilcoxon signed-rank test. Results When uninvolved brain was compared to any brain metastasis, alterations in endothelial, pericytic, astrocytic, and microglial components were observed. When metastases with relatively low- and high permeability were compared, increased expression of a desmin+ subpopulation of pericytes was associated with higher permeability (231-BR6 p=0.0002; JIMT-1-BR3 p=0.004; SUM190-BR3 p=0.008); desmin+ pericytes were also identified in human craniotomy specimens. Trends of reduced CD13+ pericytes (231-BR6 p=0.014; JIMT-1-BR3 p=0.002, SUM190-BR3, NS) and laminin α2 (231-BR6 p=0.001; JIMT-1-BR3 p=0.049; SUM190-BR3 p=0.023) were also observed with increased permeability. Conclusions We provide the first account of the composition of the BTB in experimental brain metastasis. Desmin+ pericytes and laminin α2 are potential targets for the development of novel approaches to increase chemotherapeutic efficacy. PMID:27245829

Alterations in Pericyte Subpopulations Are Associated with Elevated Blood-Tumor Barrier Permeability in Experimental Brain Metastasis of Breast Cancer.

PubMed

Lyle, L Tiffany; Lockman, Paul R; Adkins, Chris E; Mohammad, Afroz Shareef; Sechrest, Emily; Hua, Emily; Palmieri, Diane; Liewehr, David J; Steinberg, Seth M; Kloc, Wojciech; Izycka-Swieszewska, Ewa; Duchnowska, Renata; Nayyar, Naema; Brastianos, Priscilla K; Steeg, Patricia S; Gril, Brunilde

2016-11-01

The blood-brain barrier (BBB) is modified to a blood-tumor barrier (BTB) as a brain metastasis develops from breast or other cancers. We (i) quantified the permeability of experimental brain metastases, (ii) determined the composition of the BTB, and (iii) identified which elements of the BTB distinguished metastases of lower permeability from those with higher permeability. A SUM190-BR3 experimental inflammatory breast cancer brain metastasis subline was established. Experimental brain metastases from this model system and two previously reported models (triple-negative MDA-231-BR6, HER2 + JIMT-1-BR3) were serially sectioned; low- and high-permeability lesions were identified with systemic 3-kDa Texas Red dextran dye. Adjoining sections were used for quantitative immunofluorescence to known BBB and neuroinflammatory components. One-sample comparisons against a hypothesized value of one were performed with the Wilcoxon signed-rank test. When uninvolved brain was compared with any brain metastasis, alterations in endothelial, pericytic, astrocytic, and microglial components were observed. When metastases with relatively low and high permeability were compared, increased expression of a desmin + subpopulation of pericytes was associated with higher permeability (231-BR6 P = 0.0002; JIMT-1-BR3 P = 0.004; SUM190-BR3 P = 0.008); desmin + pericytes were also identified in human craniotomy specimens. Trends of reduced CD13 + pericytes (231-BR6 P = 0.014; JIMT-1-BR3 P = 0.002, SUM190-BR3, NS) and laminin α2 (231-BR6 P = 0.001; JIMT-1-BR3 P = 0.049; SUM190-BR3 P = 0.023) were also observed with increased permeability. We provide the first account of the composition of the BTB in experimental brain metastasis. Desmin + pericytes and laminin α2 are potential targets for the development of novel approaches to increase chemotherapeutic efficacy. Clin Cancer Res; 22(21); 5287-99. ©2016 AACR. ©2016 American Association for Cancer Research.

Laminin α2-Mediated Focal Adhesion Kinase Activation Triggers Alport Glomerular Pathogenesis

PubMed Central

Delimont, Duane; Dufek, Brianna M.; Meehan, Daniel T.; Zallocchi, Marisa; Gratton, Michael Anne; Phillips, Grady; Cosgrove, Dominic

2014-01-01

It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM) of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK) on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages. PMID:24915008

Pathologic Remodeling of Endoneurial Tubules in Human Neuromas.

PubMed

Karsy, Michael; Palmer, Cheryl A; Mahan, Mark A

2018-01-18

Laminins are extracellular matrix proteins that participate in endoneurial tubule formation and are important in the regeneration of nerves after injury. They act as scaffolds to guide nerves to distal targets and play a key role in neurite outgrowth. Because there is evidence that laminin architecture affects nerve regeneration, we evaluated endoneurial tubules by examining the laminin structure in clinical samples from patients with nerve injuries. In a retrospective review of eight nerve injury cases, we evaluated nerve histology in relation to clinical history and injury type. The immunohistochemical delineation of the laminin structure in relationship with the neuroma type was performed. Five cases of upper-trunk stretch injuries-four from childbirth injury and one from a motorcycle accident-and three cases of nerve laceration leading to neuroma formation were examined. In the upper-trunk stretch injuries, avulsed nerves demonstrated no neuroma formation with a linear laminin architecture and a regular Schwann cell arrangement, but increased fibrous tissue deposition. For neuromas-in-continuity after a stretch injury, laminin immunohistochemistry demonstrated a double-lumen laminin tubule, with encapsulation of the Schwann cells and axonal processes. Nerve laceration leading to stump neuroma formation had a similar double-lumen laminin tubule, but less severe fibrosis. In nerve injuries with regenerative capacity, endoneurial tubules become pathologically disorganized. A double-lumen endoneurial tubule of unclear significance develops. The consistency of this pattern potentially suggests a reproducible pathophysiologic process. Further exploration of this pathophysiologic healing may provide insight into the failure of programmed peripheral nerve regeneration after injury.

Laminin-111 improves skeletal muscle stem cell quantity and function following eccentric exercise.

PubMed

Zou, Kai; De Lisio, Michael; Huntsman, Heather D; Pincu, Yair; Mahmassani, Ziad; Miller, Matthew; Olatunbosun, Dami; Jensen, Tor; Boppart, Marni D

2014-09-01

Laminin-111 (α1, β1, γ1; LM-111) is an important component of the extracellular matrix that is required for formation of skeletal muscle during embryonic development. Recent studies suggest that LM-111 supplementation can enhance satellite cell proliferation and muscle function in mouse models of muscular dystrophy. The purpose of this study was to determine the extent to which LM-111 can alter satellite and nonsatellite stem cell quantity following eccentric exercise-induced damage in young adult, healthy mice. One week following injection of LM-111 or saline, mice either remained sedentary or were subjected to a single bout of downhill running (EX). While one muscle was preserved for evaluation of satellite cell number, the other muscle was processed for isolation of mesenchymal stem cells (MSCs; Sca-1+CD45-) via FACS at 24 hours postexercise. Satellite cell number was approximately twofold higher in LM-111/EX compared with all other groups (p<.05), and the number of satellite cells expressing the proliferation marker Ki67 was 50% to threefold higher in LM-111/EX compared with all other groups (p<.05). LM-111 also increased the quantity of embryonic myosin heavy chain-positive (eMHC+) fibers in young mice after eccentric exercise (p<.05). Although MSC percentage and number were not altered, MSC proinflammatory gene expression was decreased, and hepatocyte growth factor gene expression was increased in the presence of LM-111 (p<.05). Together, these data suggest that LM-111 supplementation provides a viable solution for increasing skeletal muscle stem cell number and/or function, ultimately allowing for improvements in the regenerative response to eccentric exercise. ©AlphaMed Press.

Bauhinia forficata lectin (BfL) induces cell death and inhibits integrin-mediated adhesion on MCF7 human breast cancer cells.

PubMed

Silva, Mariana C C; de Paula, Cláudia A A; Ferreira, Joana G; Paredes-Gamero, Edgar J; Vaz, Angela M S F; Sampaio, Misako U; Correia, Maria Tereza S; Oliva, Maria Luiza V

2014-07-01

Plant lectins have attracted great interest in cancer studies due to their antitumor activities. These proteins or glycoproteins specifically and reversibly bind to different types of carbohydrates or glycoproteins. Breast cancer, which presents altered glycosylation of cell surface glycoproteins, is one of the most frequent malignant diseases in women. In this work, we describe the effect of the lectin Bauhinia forficata lectin (BfL), which was purified from B. forficata Link subsp. forficata seeds, on the MCF7 human breast cancer cellular line, investigating the mechanisms involved in its antiproliferative activity. MCF7 cells were treated with BfL. Viability and adhesion alterations were evaluated using flow cytometry and western blotting. BfL inhibited the viability of the MCF7 cell line but was ineffective on MDA-MB-231 and MCF 10A cells. It inhibits MCF7 adhesion on laminin, collagen I and fibronectin, decreases α1, α6 and β1 integrin subunit expression, and increases α5 subunit expression. BfL triggers necrosis and secondary necrosis, with caspase-9 inhibition. It also causes deoxyribonucleic acid (DNA) fragmentation, which leads to cell cycle arrest in the G2/M phase and a decrease in the expression of the regulatory proteins pRb and p21. BfL shows selective cytotoxic effect and adhesion inhibition on MCF7 breast cancer cells. Cell death induction and inhibition of cell adhesion may contribute to understanding the action of lectins in breast cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture

PubMed Central

Nasu, Makoto; Takata, Nozomu; Danjo, Teruko; Sakaguchi, Hideya; Kadoshima, Taisuke; Futaki, Sugiko; Sekiguchi, Kiyotoshi; Eiraku, Mototsugu; Sasai, Yoshiki

2012-01-01

In the mammalian cortex, the dorsal telencephalon exhibits a characteristic stratified structure. We previously reported that three-dimensional (3D) culture of mouse ES cells (mESCs) can efficiently generate cortical neuroepithelium (NE) and layer-specific cortical neurons. However, the cortical NE generated in this mESC culture was structurally unstable and broke into small neural rosettes by culture day 7, suggesting that some factors for reinforcing the structural integrity were missing. Here we report substantial supporting effects of the extracellular matrix (ECM) protein laminin on the continuous formation of properly polarized cortical NE in floating aggregate culture of mESCs. The addition of purified laminin and entactin (a laminin-associated protein), even at low concentrations, stabilized the formation of continuous cortical NE as well as the maintenance of basement membrane and prevented rosette formation. Treatment with the neutralizing ß1-integrin antibody impaired the continuous NE formation. The stabilized cortical NE exhibited typical interkinetic nuclear migration of cortical progenitors, as seen in the embryonic cortex. The laminin-treated cortical NE maintained a continuous structure even on culture days 12 and 15, and contained ventricular, basal-progenitor, cortical-plate and Cajal-Retzius cell layers. The cortical NE in this culture was flanked by cortical hem-like tissue. Furthermore, when Shh was added, ventral telencephalic structures such as lateral ganglionic eminence–like tissue formed in the region adjacent to the cortical NE. Thus, our results indicate that laminin-entactin ECM promotes the formation of structurally stable telencephalic tissues in 3D ESC culture, and supports the morphogenetic recapitulation of cortical development. PMID:23300850

[Pathomechanism and therapeutic strategy of Fukuyama congenital muscular dystrophy and related disorders].

PubMed

Toda, Tatsushi

2009-11-01

Hypoglycosylation and reduced laminin-binding activity of alpha-dystroglycan are common characteristics of dystroglycanopathy, which is a group of congenital and limb-girdle muscular dystrophies. We previously identified the genes for Fukuyama congenital muscular dystrophy (FCMD) and muscle-eye-brain disease (MEB). FCMD, caused by a mutation in the fukutin gene, is a severe form of dystroglycanopathy. Knock-in mice carrying the founder retrotransposal insertion exhibited hypoglycosylated alpha-dystroglycan; however, no signs of muscular dystrophy were observed. More sensitive methods detected minor levels of intact alpha-dystroglycan, and solid-phase assays determined laminin binding levels to be approximately 50% of normal. In contrast, intact alpha-dystroglycan is undetectable in the dystrophic Large mouse, and laminin-binding activity is markedly reduced. These data indicate that a small amount of intact alpha-dystroglycan is sufficient to maintain muscle cell integrity in knock-in mice, suggesting that the treatment of dystroglycanopathies might not require the full recovery of glycosylation. Transfer of fukutin into knock-in mice restored glycosylation of alpha-dystroglycan. Transfer of LARGE produced laminin-binding forms of alpha-dystroglycan in both knock-in mice and the POMGnT1 mutant mouse. These data suggest that even partial restoration of alpha-dystroglycan glycosylation and laminin-binding activity by replacing or augmenting glycosylation-related genes might effectively deter dystroglycanopathy progression and thus provide therapeutic benefits.

Fabrication, characterization, and biological assessment of multilayer laminin γ2 DNA coatings on titanium surfaces.

PubMed

Yang, Guoli; Zhang, Jing; Dong, Wenjing; Liu, Li; Shi, Jue; Wang, Huiming

2016-03-21

The purpose of this work was to fabricate a multilayer laminin γ2 DNA coating on a titanium surface and evaluate its biological properties. A multilayer laminin γ2 DNA coating was fabricated on titanium using a layer-by-layer assembly technique. The rate of coating degradation was evaluated by detecting the amount of cDNA remaining. Surface analysis using X-ray photoelectron spectroscopy, atomic force microscopy, and surface contact angle measurements revealed the multilayer structure to consist of cationic lipid and confirmed that a laminin γ2 DNA layer could be fabricated on titanium via the layer-by-layer assembly process. The transfection efficiency was highest for five layers in the multilayer structure. HEK293 cells cultured on the multilayer films displayed significantly higher adhesion activity than the control group. The expression of laminin γ2 and the co-localization of integrin β4 and plectin were more obvious in HN4 cells cultured on the multilayer laminin γ2 DNA coating, while weak immunoreactivities were observed in the control group. We concluded that the DNA-loaded multilayer provided a surface with good biocompatibility and that the multilayer laminin γ2 DNA coating might be effective in improving cell adhesion and the formation of hemidesmosomes on titanium surfaces.

Fabrication, characterization, and biological assessment of multilayer laminin γ2 DNA coatings on titanium surfaces

PubMed Central

Yang, Guoli; Zhang, Jing; Dong, Wenjing; Liu, Li; Shi, Jue; Wang, Huiming

2016-01-01

The purpose of this work was to fabricate a multilayer laminin γ2 DNA coating on a titanium surface and evaluate its biological properties. A multilayer laminin γ2 DNA coating was fabricated on titanium using a layer-by-layer assembly technique. The rate of coating degradation was evaluated by detecting the amount of cDNA remaining. Surface analysis using X-ray photoelectron spectroscopy, atomic force microscopy, and surface contact angle measurements revealed the multilayer structure to consist of cationic lipid and confirmed that a laminin γ2 DNA layer could be fabricated on titanium via the layer-by-layer assembly process. The transfection efficiency was highest for five layers in the multilayer structure. HEK293 cells cultured on the multilayer films displayed significantly higher adhesion activity than the control group. The expression of laminin γ2 and the co-localization of integrin β4 and plectin were more obvious in HN4 cells cultured on the multilayer laminin γ2 DNA coating, while weak immunoreactivities were observed in the control group. We concluded that the DNA-loaded multilayer provided a surface with good biocompatibility and that the multilayer laminin γ2 DNA coating might be effective in improving cell adhesion and the formation of hemidesmosomes on titanium surfaces. PMID:26996815

Fabrication, characterization, and biological assessment of multilayer laminin γ2 DNA coatings on titanium surfaces

NASA Astrophysics Data System (ADS)

Yang, Guoli; Zhang, Jing; Dong, Wenjing; Liu, Li; Shi, Jue; Wang, Huiming

2016-03-01

The purpose of this work was to fabricate a multilayer laminin γ2 DNA coating on a titanium surface and evaluate its biological properties. A multilayer laminin γ2 DNA coating was fabricated on titanium using a layer-by-layer assembly technique. The rate of coating degradation was evaluated by detecting the amount of cDNA remaining. Surface analysis using X-ray photoelectron spectroscopy, atomic force microscopy, and surface contact angle measurements revealed the multilayer structure to consist of cationic lipid and confirmed that a laminin γ2 DNA layer could be fabricated on titanium via the layer-by-layer assembly process. The transfection efficiency was highest for five layers in the multilayer structure. HEK293 cells cultured on the multilayer films displayed significantly higher adhesion activity than the control group. The expression of laminin γ2 and the co-localization of integrin β4 and plectin were more obvious in HN4 cells cultured on the multilayer laminin γ2 DNA coating, while weak immunoreactivities were observed in the control group. We concluded that the DNA-loaded multilayer provided a surface with good biocompatibility and that the multilayer laminin γ2 DNA coating might be effective in improving cell adhesion and the formation of hemidesmosomes on titanium surfaces.

The lutheran/basal cell adhesion molecule promotes tumor cell migration by modulating integrin-mediated cell attachment to laminin-511 protein.

PubMed

Kikkawa, Yamato; Ogawa, Takaho; Sudo, Ryo; Yamada, Yuji; Katagiri, Fumihiko; Hozumi, Kentaro; Nomizu, Motoyoshi; Miner, Jeffrey H

2013-10-25

Cell-matrix interactions are critical for tumor cell migration. Lutheran (Lu), also known as basal cell adhesion molecule (B-CAM), competes with integrins for binding to laminin α5, a subunit of LM-511, a major component of basement membranes. Here we show that the preferential binding of Lu/B-CAM to laminin α5 promotes tumor cell migration. The attachment of Lu/B-CAM transfectants to LM-511 was slightly weaker than that of control cells, and this was because Lu/B-CAM disturbed integrin binding to laminin α5. Lu/B-CAM induced a spindle cell shape with pseudopods and promoted cell migration on LM-511. In addition, blocking with an anti-Lu/B-CAM antibody led to a flat cell shape and inhibited migration on LM-511, similar to the effects of an activating integrin β1 antibody. We conclude that tumor cell migration on LM-511 requires that Lu/B-CAM competitively modulates cell attachment through integrins. We suggest that this competitive interaction is involved in a balance between static and migratory cell behaviors.

The opposing roles of laminin-binding integrins in cancer.

PubMed

Ramovs, Veronika; Te Molder, Lisa; Sonnenberg, Arnoud

2017-01-01

Integrins play an important role in cell adhesion by linking the cytoskeleton of cells to components in the extracellular matrix. In this capacity, integrins cooperate with different cell surface receptors, including growth factor receptors and G-protein coupled receptors, to regulate intracellular signaling pathways that control cell polarization, spreading, migration, survival, and gene expression. A distinct subfamily of molecules in the integrin family of adhesion receptors is formed by receptors that mediate cell adhesion to laminins, major components of the basement membrane that lie under clusters of cells or surround them, separating them from other cells and/or adjacent connective tissue. During the past decades, many studies have provided evidence for a role of laminin-binding integrins in tumorigenesis, and both tumor-promoting and suppressive activities have been identified. In this review we discuss the dual role of the laminin-binding integrins α3β1 and α6β4 in tumor development and progression, and examine the factors and mechanisms involved in these opposing effects. Copyright © 2016 Elsevier B.V. All rights reserved.

Photodynamic treatment of epithelial tissue derived from patients with endometrial cancer: a contribution to the role of laminin and epidermal growth factor receptor in photodynamic therapy

NASA Astrophysics Data System (ADS)

Ziolkowski, Piotr P.; Symonowicz, Krzysztof; Osiecka, Beata J.; Rabczynski, Jerzy; Gerber, Jerzy

1999-07-01

Photodynamic therapy (PDT) was used to treat endometrial G1 cancer tissue derived from patients who had undergone a total hysterectomy and bilateral salpingo-oophorectomy. After surgical treatment the cancerous tissue was kept in a medium containing Dulbecco solution, fetal calf serum, and antibiotics. The tissue was then exposed to hematoporphyrin derivative (0.1 mg/l) and 24 h later exposed to light (total light dose--18 J/sq cm). Necrosis depth was evaluated 24 h later using a light microscope. In order to assess the possible role of the basal membrane component laminin, as well as epidermal growth factor receptor susceptibility to PDT, immunohistochemical studies were carried out. Additionally, nucleolar organizer regions evaluation was performed. Our experiment confirmed that PDT results in the necrosis in the treated endometrial cancer, while not affecting the laminin in the cancerous tissue. In contrast, PDT strongly affects the epidermal growth factor receptor and nucleolar organizer regions in cancer cells. We suggest that laminin may contribute to the prevention of cancer dissemination in the cases where PDT has to be repeated, and that after PDT the cells become less susceptible to a mitogen, like, e.g., epidermal growth factor.

Macrophage-derived microvesicles promote proliferation and migration of Schwann cell on peripheral nerve repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhan, Chuan, E-mail: zhchuansy@163.com; Ma, Cheng-bin; Yuan, Hong-mou

Background: Macrophages have been implicated in peripheral nerve regeneration. However, whether macrophages-derived microvesicles (MVs) are involved in this process remains unknown. In the present study, the effects of macrophages-derived MVs on proliferation and migration of Schwann cells (SCs) were evaluated in both in vitro and in vivo. Methods: Human monocytic leukaemia cell line (THP-1) was successfully driven to M1 and M2 phenotypes by delivery of either IFN-γ or IL-4, respectively. SCs incubated with M1 or M2 macrophages-derived MVs, the cell migration and proliferation were assessed, and expression levels of nerve growth factor (NGF) and Laminin were measured. A rat model of sciaticmore » nerve was established and the effects of macrophages-derived MVs on nerve regeneration were investigated. Results: M2-derived MVs elevated migration, proliferation, NFG and Laminin protein levels of SCs compared with M1-or M0-derived MVs. The relative expression levels of miR-223 were also increased in M2 macrophages and M2-derived MVs. Transfected M2 macrophages with miR-223 inhibitor then co-incubated with SCs, an inhibition of cell migration and proliferation and a down-regulated levels of NFG and Laminin protein expression were observed. In vivo, M2-derived MVs significantly increased the infiltration and axon number of SCs. Conclusion: M2-derived MVs promoted proliferation and migration of SCs in vitro and in vivo, which provided a therapeutic strategy for nerve regeneration. - Highlights: • M2 macrophages-derived MVs elevated migration and proliferation of SCs. • M2 macrophages-derived MVs up-regulated NFG and Laminin expression of SCs. • MiR-223 expression was increased in M2 macrophages-derived MVs. • MiR-223 inhibitor reduced migration and proliferation of SCs co-incubated with MVs. • MiR-223 inhibitor down-regulated NFG and Laminin levels of SCs co-incubated with MVs.« less

Isolation of Human Induced Pluripotent Stem Cell-Derived Dopaminergic Progenitors by Cell Sorting for Successful Transplantation

PubMed Central

Doi, Daisuke; Samata, Bumpei; Katsukawa, Mitsuko; Kikuchi, Tetsuhiro; Morizane, Asuka; Ono, Yuichi; Sekiguchi, Kiyotoshi; Nakagawa, Masato; Parmar, Malin; Takahashi, Jun

2014-01-01

Summary Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (DA) neurons for cell replacement therapy for Parkinson’s disease. However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. Here, we show that human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, CORIN. We induced DA neurons using scalable culture conditions on human laminin fragment, and the sorted CORIN+ cells expressed the midbrain DA progenitor markers, FOXA2 and LMX1A. When transplanted into 6-OHDA-lesioned rats, the CORIN+ cells survived and differentiated into midbrain DA neurons in vivo, resulting in significant improvement of the motor behavior, without tumor formation. In particular, the CORIN+ cells in a NURR1+ cell-dominant stage exhibited the best survival and function as DA neurons. Our method is a favorable strategy in terms of scalability, safety, and efficiency and may be advantageous for clinical application. PMID:24672756

Structure of Streptococcus agalactiae tip pilin GBS104: a model for GBS pili assembly and host interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krishnan, Vengadesan; Dwivedi, Prabhat; Kim, Brandon J.

The crystal structure of a 75 kDa central fragment of GBS104, a tip pilin from the 2063V/R strain of Streptococcus agalactiae (group B streptococcus; GBS), is reported. The crystal structure of a 75 kDa central fragment of GBS104, a tip pilin from the 2063V/R strain of Streptococcus agalactiae (group B streptococcus; GBS), is reported. In addition, a homology model of the remaining two domains of GBS104 was built and a model of full-length GBS104 was generated by combining the homology model (the N1 and N4 domains) and the crystal structure of the 75 kDa fragment (the N2 and N3 domains).more » This rod-shaped GBS104 model is constructed of three IgG-like domains (the N1, N2 and N4 domains) and one vWFA-like domain (the N3 domain). The N1 and N2 domains of GBS104 are assembled with distinct and remote segments contributed by the N- and C-termini. The metal-binding site in the N3 domain of GBS104 is in the closed/low-affinity conformation. Interestingly, this domain hosts two long arms that project away from the metal-binding site. Using site-directed mutagenesis, two cysteine residues that lock the N3 domain of GBS104 into the open/high-affinity conformation were introduced. Both wild-type and disulfide-locked recombinant proteins were tested for binding to extracellular matrix proteins such as collagen, fibronectin, fibrinogen and laminin, and an increase in fibronectin binding affinity was identified for the disulfide-locked N3 domain, suggesting that induced conformational changes may play a possible role in receptor binding.« less

Pathologic Remodeling of Endoneurial Tubules in Human Neuromas

PubMed Central

Karsy, Michael; Palmer, Cheryl A

2018-01-01

Background: Laminins are extracellular matrix proteins that participate in endoneurial tubule formation and are important in the regeneration of nerves after injury. They act as scaffolds to guide nerves to distal targets and play a key role in neurite outgrowth. Because there is evidence that laminin architecture affects nerve regeneration, we evaluated endoneurial tubules by examining the laminin structure in clinical samples from patients with nerve injuries. Methods: In a retrospective review of eight nerve injury cases, we evaluated nerve histology in relation to clinical history and injury type. The immunohistochemical delineation of the laminin structure in relationship with the neuroma type was performed. Results: Five cases of upper-trunk stretch injuries—four from childbirth injury and one from a motorcycle accident—and three cases of nerve laceration leading to neuroma formation were examined. In the upper-trunk stretch injuries, avulsed nerves demonstrated no neuroma formation with a linear laminin architecture and a regular Schwann cell arrangement, but increased fibrous tissue deposition. For neuromas-in-continuity after a stretch injury, laminin immunohistochemistry demonstrated a double-lumen laminin tubule, with encapsulation of the Schwann cells and axonal processes. Nerve laceration leading to stump neuroma formation had a similar double-lumen laminin tubule, but less severe fibrosis. Conclusions: In nerve injuries with regenerative capacity, endoneurial tubules become pathologically disorganized. A double-lumen endoneurial tubule of unclear significance develops. The consistency of this pattern potentially suggests a reproducible pathophysiologic process. Further exploration of this pathophysiologic healing may provide insight into the failure of programmed peripheral nerve regeneration after injury. PMID:29560300

Lung-Specific Loss of α3 Laminin Worsens Bleomycin-Induced Pulmonary Fibrosis

PubMed Central

Morales-Nebreda, Luisa I.; Rogel, Micah R.; Eisenberg, Jessica L.; Hamill, Kevin J.; Soberanes, Saul; Nigdelioglu, Recep; Chi, Monica; Cho, Takugo; Radigan, Kathryn A.; Ridge, Karen M.; Misharin, Alexander V.; Woychek, Alex; Hopkinson, Susan; Perlman, Harris; Mutlu, Gokhan M.; Pardo, Annie; Selman, Moises; Jones, Jonathan C. R.

2015-01-01

Laminins are heterotrimeric proteins that are secreted by the alveolar epithelium into the basement membrane, and their expression is altered in extracellular matrices from patients with pulmonary fibrosis. In a small number of patients with pulmonary fibrosis, we found that the normal basement membrane distribution of the α3 laminin subunit was lost in fibrotic regions of the lung. To determine if these changes play a causal role in the development of fibrosis, we generated mice lacking the α3 laminin subunit specifically in the lung epithelium by crossing mice expressing Cre recombinase driven by the surfactant protein C promoter (SPC-Cre) with mice expressing floxed alleles encoding the α3 laminin gene (Lama3fl/fl). These mice exhibited no developmental abnormalities in the lungs up to 6 months of age, but, compared with control mice, had worsened mortality, increased inflammation, and increased fibrosis after the intratracheal administration of bleomycin. Similarly, the severity of fibrosis induced by an adenovirus encoding an active form of transforming growth factor-β was worse in mice deficient in α3 laminin in the lung. Taken together, our results suggest that the loss of α3 laminin in the lung epithelium does not affect lung development, but plays a causal role in the development of fibrosis in response to bleomycin or adenovirally delivered transforming growth factor-β. Thus, we speculate that the loss of the normal basement membrane organization of α3 laminin that we observe in fibrotic regions from the lungs of patients with pulmonary fibrosis contributes to their disease progression. PMID:25188360

Lung-specific loss of α3 laminin worsens bleomycin-induced pulmonary fibrosis.

PubMed

Morales-Nebreda, Luisa I; Rogel, Micah R; Eisenberg, Jessica L; Hamill, Kevin J; Soberanes, Saul; Nigdelioglu, Recep; Chi, Monica; Cho, Takugo; Radigan, Kathryn A; Ridge, Karen M; Misharin, Alexander V; Woychek, Alex; Hopkinson, Susan; Perlman, Harris; Mutlu, Gokhan M; Pardo, Annie; Selman, Moises; Jones, Jonathan C R; Budinger, G R Scott

2015-04-01

Laminins are heterotrimeric proteins that are secreted by the alveolar epithelium into the basement membrane, and their expression is altered in extracellular matrices from patients with pulmonary fibrosis. In a small number of patients with pulmonary fibrosis, we found that the normal basement membrane distribution of the α3 laminin subunit was lost in fibrotic regions of the lung. To determine if these changes play a causal role in the development of fibrosis, we generated mice lacking the α3 laminin subunit specifically in the lung epithelium by crossing mice expressing Cre recombinase driven by the surfactant protein C promoter (SPC-Cre) with mice expressing floxed alleles encoding the α3 laminin gene (Lama3(fl/fl)). These mice exhibited no developmental abnormalities in the lungs up to 6 months of age, but, compared with control mice, had worsened mortality, increased inflammation, and increased fibrosis after the intratracheal administration of bleomycin. Similarly, the severity of fibrosis induced by an adenovirus encoding an active form of transforming growth factor-β was worse in mice deficient in α3 laminin in the lung. Taken together, our results suggest that the loss of α3 laminin in the lung epithelium does not affect lung development, but plays a causal role in the development of fibrosis in response to bleomycin or adenovirally delivered transforming growth factor-β. Thus, we speculate that the loss of the normal basement membrane organization of α3 laminin that we observe in fibrotic regions from the lungs of patients with pulmonary fibrosis contributes to their disease progression.

Quantitative Proteomic Analysis Reveals Metabolic Alterations, Calcium Dysregulation, and Increased Expression of Extracellular Matrix Proteins in Laminin α2 Chain–deficient Muscle*

PubMed Central

de Oliveira, Bruno Menezes; Matsumura, Cintia Y.; Fontes-Oliveira, Cibely C.; Gawlik, Kinga I.; Acosta, Helena; Wernhoff, Patrik; Durbeej, Madeleine

2014-01-01

Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain–deficient dy3K/dy3K mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain–deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978). PMID:24994560

A study on the effects of relativistic heavy charged particles on the cellular microenvironment

NASA Astrophysics Data System (ADS)

Costes, Sylvain Vincent

This study was done under the National Aeronautics Space Administration (NASA) effort to assess the effect of cosmic radiation on astronauts during a 3 year mission to Mars. Carcinogenesis is known to be induced more efficiently by cosmic radiation. Our attention was turned towards one of the most efficient cosmic particles in inducing cancer, relativistic Fe, and focused in assessing its effect on the cellular microenvironment (ECM). Previous observations on mammary glands were showing irregularities in the immunoreactivity of the ECM protein laminin one hour after whole body irradiation with 1GeV/amu Fe ions for a dose of 0.8 Gy. This effect was not observed after 5 Gy γ-rays exposure. The rapidity of such a change suggested that the effect might be due to a physical event specific to relativistic charged particles (HZE), rather than a biological event. Our study showed that this effect is actually a complex and rapid response of the microenvironment to highly ionizing radiation. It involves a fast disruption of the basement membrane of the ECM induced by the highly localized ionization and reactive oxygen formation around the track of the Fe ion. This disruption triggers further chemical and biological responses involved in the remodeling of the laminin network in the basement membrane. A metalloproteinase is suspected to be the intermediate protease affecting laminin. The HZE effect on the microenvironment was seen in both mouse mammary glands and skin, but the laminin isoforms sensitive to Fe ions were different for each organ, with a clear disruption of laminin-1 network in skin and of laminin-5 in mammary glands. In addition, the laminin receptor integrins seem to be involved in this mechanism, but its contribution is unclear at this point. Finally, such studies suggest a shift from the concept of relative biological effectiveness (RBE) used in classical radiation biology since the effect is only seen with HZE at viable whole body doses. In addition, this study shows that the use of an RBE for a microscopic biological endpoint, such as the disruption of the basement membrane, is irrelevant considering the complexity of such mechanism that is unique for very similar targets (i.e. basement membrane from the mammary glands versus skin of the same mouse). In conclusion, our studies show that HZE-irradiation elicits distinct microenvironment changes when compared to sparsely ionizing radiation. Laminin is an important mediator of epithelial integrity and serves as a barrier to invasive growth. A hallmark of cancer is the ability to destroy and traverse the basement membrane. Radiation induced changes in basement membrane integrity might thus promote neoplastic progression.

Integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} effectors p130Cas, Src and talin regulate carcinoma invasion and chemoresistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sansing, Hope A.; Sarkeshik, Ali; Yates, John R.

2011-03-11

Research highlights: {yields} Proteomics of clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} receptors in oral carcinoma. {yields} p130Cas, Dek, Src and talin regulate oral carcinoma invasion. {yields} p130Cas, talin, Src and zyxin regulate oral carcinoma resistance to cisplatin. -- Abstract: Ligand engagement by integrins induces receptor clustering and formation of complexes at the integrin cytoplasmic face that controls cell signaling and cytoskeletal dynamics critical for adhesion-dependent processes. This study searches for a subset of integrin effectors that coordinates both tumor cell invasion and resistance to the chemotherapeutic drug cisplatin in oral carcinomas. Candidate integrin effectors were identified in a proteomicsmore » screen of proteins recruited to clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta} or {alpha}{sub 6}{beta} receptors in oral carcinomas. Proteins with diverse functions including microtubule and actin binding proteins, and factors involved in trafficking, transcription and translation were identified in oral carcinoma integrin complexes. Knockdown of effectors in the oral carcinoma HN12 cells revealed that p130Cas, Dek, Src and talin were required for invasion through Matrigel. Disruption of talin or p130Cas by RNA interference increased resistance to cisplatin, whereas targeting Dek, Src or zyxin reduced HN12 resistance to cisplatin. Analysis of the spreading of HN12 cells on collagen I and laminin I revealed that a decrease in p130Cas or talin expression inhibited spreading on both matrices. Interestingly, a reduction in zyxin expression enhanced spreading on laminin I and inhibited spreading on collagen I. Reduction of Dek, Src, talin or zyxin expression reduced HN12 proliferation by 30%. Proliferation was not affected by a reduction in p130Cas expression. We conclude that p130Cas, Src and talin function in both oral carcinoma invasion and resistance to cisplatin.« less

Expression of hemidesmosomal and extracellular matrix proteins by normal and malignant human prostate tissue.

PubMed Central

Nagle, R. B.; Hao, J.; Knox, J. D.; Dalkin, B. L.; Clark, V.; Cress, A. E.

1995-01-01

The progression of prostate carcinoma may be influenced by the biochemical nature of the basal lamina surrounding the primary carcinoma cells. As a first step toward understanding this process, the composition and structure of the basal lamina in normal prostate, prostatic intraepithelial neoplasia, and human carcinoma were determined. In addition, a comparison was made between the attachments of the normal basal cell to its underlying basal lamina and those made by primary prostate carcinoma. The normal basal cells form both focal adhesions and hemidesmosomal-like structures as observed by transmission electron microscopy. The normal basal cells exhibited a polarized distribution of hemidesmosomal associated proteins including BP180, BP230, HD1, plectin, laminin-gamma 2(B2t), collagen VII, and the corresponding integrin laminin receptors alpha 6 beta 1 and alpha 6 beta 4. The expression and distribution pattern of these proteins were retained in the prostate intraepithelial neoplasia lesions. In contrast, the carcinoma cells uniformly lacked hemidesmosomal structures, the integrin alpha 6 beta 4, BP180, laminin-gamma 2 (B2t), and collagen VII but did express BP230 (30%), plectin, HD1 (15%), and the integrin laminin receptors alpha 3 beta 1 and alpha 6 beta 1. These results suggest that, although a detectable basal lamina structure is present in carcinoma, its composition and cellular attachments are abnormal. The loss of critical cellular attachments may play a role in influencing the progression potential of prostate carcinoma. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7778688

Laminins and cancer stem cells: Partners in crime?

PubMed

Qin, Yan; Rodin, Sergey; Simonson, Oscar E; Hollande, Frédéric

2017-08-01

As one of the predominant protein families within the extracellular matrix both structurally and functionally, laminins have been shown to be heavily involved in tumor progression and drug resistance. Laminins participate in key cellular events for tumor angiogenesis, cell invasion and metastasis development, including the regulation of epithelial-mesenchymal transition and basement membrane remodeling, which are tightly associated with the phenotypic characteristics of stem-like cells, particularly in the context of cancer. In addition, a great deal of studies and reports has highlighted the critical roles of laminins in modulating stem cell phenotype and differentiation, as part of the stem cell niche. Stemming from these discoveries a growing body of literature suggests that laminins may act as regulators of cancer stem cells, a tumor cell subpopulation that plays an instrumental role in long-term cancer maintenance, metastasis development and therapeutic resistance. The accumulating evidence in this emerging research area suggests that laminins represent potential therapeutic targets for anti-cancer treatments against cancer stem cells, and that they may be used as predictive and prognostic markers to inform clinical management and improve patient survival. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cross-linking reveals laminin coiled-coil architecture

PubMed Central

Armony, Gad; Jacob, Etai; Moran, Toot; Levin, Yishai; Mehlman, Tevie; Levy, Yaakov; Fass, Deborah

2016-01-01

Laminin, an ∼800-kDa heterotrimeric protein, is a major functional component of the extracellular matrix, contributing to tissue development and maintenance. The unique architecture of laminin is not currently amenable to determination at high resolution, as its flexible and narrow segments complicate both crystallization and single-particle reconstruction by electron microscopy. Therefore, we used cross-linking and MS, evaluated using computational methods, to address key questions regarding laminin quaternary structure. This approach was particularly well suited to the ∼750-Å coiled coil that mediates trimer assembly, and our results support revision of the subunit order typically presented in laminin schematics. Furthermore, information on the subunit register in the coiled coil and cross-links to downstream domains provide insights into the self-assembly required for interaction with other extracellular matrix and cell surface proteins. PMID:27815530

[Markers of stromal invasion during background and precancerous changes of the glandular epithelium and in adenocarcinoma of the cervix uteri].

PubMed

Danilova, N V; Andreeva, Iu Iu; Zavalishina, L É; Mal'kov, P G

2012-01-01

It is very difficult to identify stromal invasion when the glandular epithelium of the cervix uteri is involved. It is necessary to draw a clear distinction between its glandular structures and adenocarcinoma in situ, involving the preexisting crypts and invasive glands. An attempt was made to assess the possibilities of using as markers of invasion the following stromal proteins and adhesion molecules: CD44, E-cadherin, beta-catenin, tenascin, and laminin. Fifty-three cases of benign glandular changes, 66 cases of dysplasias and adenocarcinomas in situ, and 47 cases of invasive adenocarcinoma were examined. An immunohistochemical study was performed according to the standard protocol using the antibodies to CD44, laminin, tenascin, E-cadherin, and beta-catenin and a semiquantitative assessment of results was made. CD44 was found to be redistributed from the cells to the tumor stroma. CD44 was not detected in the stroma surrounding the intact glands, so were benign epithelial changes. In the tumor environment, there was, on the contrary, a reaction with CD44 in 74.5% of invasive adenocarcinomas cases (p < 0.05). The expression of tenascin in the invasive adenocarcinomas and around the foci of early stromal invasion significantly exceeded that in the stroma around the intact glands and dysplastic changes (p < 0.05). All the study groups showed a membrane reaction with E-cadherin and beta-catenin, which probably suggested that changes were absent in the Wnt signaling pathway. In 70.2% of invasive adenocarcinomas, laminin demonstrated a significant cytoplasmic expression in 5-30% of the tumor cells predominantly located along the tumor invasion area or in the deepest tumor complexes (p > 0.05). CD44 and tenascin are of great diagnostic value in examining invasive and microinvasive adenocarcinomas of the cervix uteri. E-cadherin and beta-catenin are of no diagnostic value in the study groups of pathological processes. Laminin is a potential marker of stromal invasion; however, its expression calls for further investigation.

Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hirako, Yoshiaki, E-mail: s47526a@cc.nagoya-u.ac.jp; Yonemoto, Yuki; Yamauchi, Tomoe

2014-06-10

Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and depositedmore » extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and β4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases. - Highlights: • A defined condition promoted accumulation of hemidesmosomes in human cultured cells. • A fraction isolated from the cells contained eight major polypeptides. • The polypeptides were the five major hemidesmosome proteins and laminin-332. • The cultured cells deposited laminin-332 in its unprocessed form under the condition. • We report a method to prepare a fraction highly enriched in hemidesmosome proteins.« less

Distribution of Basement Membrane Molecules, Laminin and Collagen Type IV, in Normal and Degenerated Cartilage Tissues

PubMed Central

Toh, Wei Seong; Gomoll, Andreas H.; Olsen, Bjørn Reino; Spector, Myron

2014-01-01

Objective: The objective of the present study was to investigate the presence and distribution of 2 basement membrane (BM) molecules, laminin and collagen type IV, in healthy and degenerative cartilage tissues. Design: Normal and degenerated tissues were obtained from goats and humans, including articular knee cartilage, the intervertebral disc, and meniscus. Normal tissue was also obtained from patella-tibial enthesis in goats. Immunohistochemical analysis was performed using anti-laminin and anti–collagen type IV antibodies. Human and goat skin were used as positive controls. The percentage of cells displaying the pericellular presence of the protein was graded semiquantitatively. Results: When present, laminin and collagen type IV were exclusively found in the pericellular matrix, and in a discrete layer on the articulating surface of normal articular cartilage. In normal articular (hyaline) cartilage in the human and goat, the proteins were found co-localized pericellularly. In contrast, in human osteoarthritic articular cartilage, collagen type IV but not laminin was found in the pericellular region. Nonpathological fibrocartilaginous tissues from the goat, including the menisci and the enthesis, were also positive for both laminin and collagen type IV pericellularly. In degenerated fibrocartilage, including intervertebral disc, as in degenerated hyaline cartilage only collagen type IV was found pericellularly around chondrocytes but with less intense staining than in non-degenerated tissue. In calcified cartilage, some cells were positive for laminin but not type IV collagen. Conclusions: We report differences in expression of the BM molecules, laminin and collagen type IV, in normal and degenerative cartilaginous tissues from adult humans and goats. In degenerative tissues laminin is depleted from the pericellular matrix before collagen type IV. The findings may inform future studies of the processes underlying cartilage degeneration and the functional roles of these 2 extracellular matrix proteins, normally associated with BM. PMID:26069692

Proteinases secreted by Fasciola hepatica degrade extracellular matrix and basement membrane components.

PubMed

Berasaín, P; Goñi, F; McGonigle, S; Dowd, A; Dalton, J P; Frangione, B; Carmona, C

1997-02-01

The invasive stages of the parasitic trematode Fasciola hepatica release proteinases into the medium in which they are maintained. In this study, we investigated the interaction of F. hepatica excretory/secretory (E/S) products and 2 cysteine proteinases (CL1 and CL2) purified from these products with extracellular matrix and basement membrane macromolecules. Fasciola hepatica E/S products contained collagenolytic activity on fibrillar types I and III collagen as well as basement membrane type IV collagen. CL1 and CL2 were capable of degrading acid-soluble type III and type IV collagen but not insoluble type I collagen. In contrast, neither the E/S products nor the purified CL1 and CL2 showed elastinolytic activity. Fibronectin and laminin were degraded by E/S products and by CL1 and CL2. Sequence analysis of fibronectin degradation products showed that the fragments obtained corresponded to complete biologically active domains. These results indicate that the cysteine proteinases secreted by F. hepatica may be involved in the process of tissue invasion by the parasite.

Topical Application of Liposomal Antioxidants for Protection Against CEES Induced Skin Damage

DTIC Science & Technology

2008-06-01

The DSM Nutritional Products (www.dsm.com ) supplied us with 100 g quantities of mixed tocopherols (with a very high content of γ-tocopherol) for...Studies on the Yucatan mini-pig have demonstrated that laminin in the dermo-epithelial junction is a target for partial protease degradation following HD...therapies. J Burns Wounds. 2005;4:e1. 14. Lindsay CD, Rice P. Changes in connective tissue macromolecular components of Yucatan mini-pig skin

Effects of Modifications in the Laminin-10 Basal Laminina on Prostate Cancer Invasion

DTIC Science & Technology

2006-10-01

factors. 2000. 5(6): p. 258-264. 5. Noel, A., et al., Emerging roles for proteinases in cancer. Invasion Metastasis, 1997. 17(5): p. 221-39. 6. Foda , H.D...May 2001 Diploma in Clinical Analysis Sophia College, Mumbai, India. June 1993 – May 1994 B.Sc. (Microbiology) Sophia College, Mumbai

Relationships between human sperm protamines, DNA damage and assisted reproduction outcomes.

PubMed

Simon, Luke; Castillo, Judit; Oliva, Rafael; Lewis, Sheena E M

2011-12-01

The exchange of histones with protamines in sperm DNA results in sperm chromatin compaction and protection. Variations in sperm protamine expression are associated with male infertility. The aim of this study was to investigate relationships between DNA fragmentation, sperm protamines and assisted reproduction treatment. Semen and spermatozoa prepared by density-gradient centrifugation (DGC) from 73 men undergoing IVF and 24 men undergoing intracytoplasmic sperm injection (ICSI) were included in the study. Nuclear DNA fragmentation was assessed using the alkaline Comet assay and protamines were separated by acid-urea polyacrylamide gels. Sperm DNA fragmentation and protamine content (P1-DNA, P2-DNA, P1+P2-DNA) decreased in spermatozoa after DGC. Abnormally high and low P1/P2 ratios were associated with increased sperm DNA fragmentation. Couples with idiopathic infertility had abnormally high P1/P2 ratios. Fertilization rates and embryo quality decreased as sperm DNA fragmentation or protamines increased. Sperm DNA fragmentation was lower in couples achieving pregnancies after IVF, but not after ICSI. There was no correlation between protamine content (P1-DNA, P2-DNA, P1+P2-DNA) or P1/P2 ratios and IVF or ICSI pregnancies. Increased sperm DNA fragmentation was associated with abnormal protamination and resulted in lower fertilization rates, poorer embryo quality and reduced pregnancy rates. Copyright © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Loss of cell invasiveness through PKC-mediated syndecan-1 downregulation in melanoma cells under anchorage independency.

PubMed

Wang, ChiaChen; Tseng, TingTing; Jhang, Yaoyun; Tseng, JenChih; Hsieh, ChiaoHui; Wu, Wen-guey; Lee, ShaoChen

2014-11-01

Anchorage-independent survival is one of the key features for malignant tumor cells. Whether specific gene alterations contributed by anchorage independency would further affect metastatic phenotypes of melanoma cells was unclear. We adapted suspension culture of melanoma cells to establish anchorage independency. The suspended melanoma cells lost their invasive abilities in vitro. Specific loss of laminin-binding ability in suspended melanoma cells was observed, which was correlated with downregulation of syndecan-1 as revealed by microarray and validated by qPCR and Western blot. Modulation of syndecan-1 expression level affected laminin binding, transwell migration and matrix metalloproteinase-2 secretion in melanoma cells. SDC1 expression and transwell migration were correlated with activity or level of protein kinase Cδ as evidence by specific inhibitors and shRNA transfection. In this study, we compared metastatic phenotypes and gene expressions of adherent and suspended melanoma cells. The anchorage independency led to protein kinase Cδ-mediated syndecan-1 downregulation, which contributed to loss of laminin-binding ability, reduced metalloproteinase-2 secretion and loss of invasiveness. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Omigapil treatment decreases fibrosis and improves respiratory rate in dy(2J) mouse model of congenital muscular dystrophy.

PubMed

Yu, Qing; Sali, Arpana; Van der Meulen, Jack; Creeden, Brittany K; Gordish-Dressman, Heather; Rutkowski, Anne; Rayavarapu, Sree; Uaesoontrachoon, Kitipong; Huynh, Tony; Nagaraju, Kanneboyina; Spurney, Christopher F

2013-01-01

Congenital muscular dystrophy is a distinct group of diseases presenting with weakness in infancy or childhood and no current therapy. One form, MDC1A, is the result of laminin alpha-2 deficiency and results in significant weakness, respiratory insufficiency and early death. Modification of apoptosis is one potential pathway for therapy in these patients. dy(2J) mice were treated with vehicle, 0.1 mg/kg or 1 mg/kg of omigapil daily via oral gavage over 17.5 weeks. Untreated age matched BL6 mice were used as controls. Functional, behavioral and histological measurements were collected. dy(2J) mice treated with omigapil showed improved respiratory rates compared to vehicle treated dy(2J) mice (396 to 402 vs. 371 breaths per minute, p<0.03) and similar to control mice. There were no statistical differences in normalized forelimb grip strength between dy(2J) and controls at baseline or after 17.5 weeks and no significant differences seen among the dy(2J) treatment groups. At 30-33 weeks of age, dy(2J) mice treated with 0.1 mg/kg omigapil showed significantly more movement time and less rest time compared to vehicle treated. dy(2J) mice showed normal cardiac systolic function throughout the trial. dy(2J) mice had significantly lower hindlimb maximal (p<0.001) and specific force (p<0.002) compared to the control group at the end of the trial. There were no statistically significant differences in maximal or specific force among treatments. dy(2J) mice treated with 0.1 mg/kg/day omigapil showed decreased percent fibrosis in both gastrocnemius (p<0.03) and diaphragm (p<0.001) compared to vehicle, and in diaphragm (p<0.013) when compared to 1 mg/kg/day omigapil treated mice. Omigapil treated dy(2J) mice demonstrated decreased apoptosis. Omigapil therapy (0.1 mg/kg) improved respiratory rate and decreased skeletal and respiratory muscle fibrosis in dy(2J) mice. These results support a putative role for the use of omigapil in laminin deficient congenital muscular dystrophy patients.

Effect of apoptosis and response of extracellular matrix proteins after chemotherapy application on human breast cancer cell spheroids.

PubMed

Oktem, G; Vatansever, S; Ayla, S; Uysal, A; Aktas, S; Karabulut, B; Bilir, A

2006-02-01

Multicellular tumor spheroid (MTS) represents a three-dimensional structural form of tumors in laboratory conditions, and it has the characteristics of avascular micrometastases or intervascular spaces of big tumors. Recent studies indicate that extracellular matrix (ECM) proteins play a critical role in tumor metastasis, therefore normal and cancer cells require an ECM for survival, proliferation and differentiation. Doxorubicin and Docetaxel are widely used in the therapy of breast cancer, as well as in in vivo and in vitro studies. In this study, we examined the effect of apoptosis and proliferation of cells on the human breast cancer cell line, MCF-7, by using p53, bcl-2 and Ki67 gene expression, and the tendency to metastasis with extracellular matrix proteins, laminin and type IV collagen after chemotherapy in the spheroid model. The apoptotic cell death in situ was detected by TUNEL method. TUNEL-positive cells and positive immunoreactivities of laminin, type IV collagen, p53 and, bcl-2 were detected in the control group. There was no laminin and type IV collagen immunoreactivities in spheroids of drug groups. While TUNEL-positive cells and p53 immunoreactivity were detected in Docetaxel, Doxorubicin and Docetaxel/Doxorubicin groups, p53 immunoreactivity was not observed in the Docetaxel group. There was no bcl-2 immunoreactivity in either drug group. In addition, we did not detect Ki67 immunoreactivity in both control and drug treatment groups. However, the absence of Ki67 protein in MCF-7 breast multicellular tumor spheroids is possibly related to the cells in G0 or S phase. These chemotherapeutic agents may affect the presence of ECM proteins in this in vitro model of micrometastasis of spheroids. These findings suggest that the possible mechanism of cell death in Doxorubicin and Docetaxel/Doxorubicin treatment groups is related to apoptosis through the p53 pathway. However, we considered the possibility that there is another control mechanism for the Docetaxel group.

Investigation of Polyurea-Crosslinked Silica Aerogels as a Neuronal Scaffold: A Pilot Study

PubMed Central

Sabri, Firouzeh; Cole, Judith A.; Scarbrough, Michael C.; Leventis, Nicholas

2012-01-01

Background Polymer crosslinked aerogels are an attractive class of materials for future implant applications particularly as a biomaterial for the support of nerve growth. The low density and nano-porous structure of this material combined with large surface area, high mechanical strength, and tunable surface properties, make aerogels materials with a high potential in aiding repair of injuries of the peripheral nervous system. However, the interaction of neurons with aerogels remains to be investigated. Methodology In this work the attachment and growth of neurons on clear polyurea crosslinked silica aerogels (PCSA) coated with: poly-L-lysine, basement membrane extract (BME), and laminin1 was investigated by means of optical and scanning electron microscopy. After comparing the attachment and growth capability of neurons on these different coatings, laminin1 and BME were chosen for nerve cell attachment and growth on PCSA surfaces. The behavior of neurons on treated petri dish surfaces was used as the control and behavior of neurons on treated PCSA discs was compared against it. Conclusions/Significance This study demonstrates that: 1) untreated PCSA surfaces do not support attachment and growth of nerve cells, 2) a thin application of laminin1 layer onto the PCSA discs adhered well to the PCSA surface while also supporting growth and differentiation of neurons as evidenced by the number of processes extended and b3-tubulin expression, 3) three dimensional porous structure of PCSA remains intact after fixing protocols necessary for preservation of biological samples and 4) laminin1 coating proved to be the most effective method for attaching neurons to the desired regions on PCSA discs. This work provides the basis for potential use of PCSA as a biomaterial scaffold for neural regeneration. PMID:22448239

Combination scaffolds of salmon fibrin, hyaluronic acid, and laminin for human neural stem cell and vascular tissue engineering

PubMed Central

Arulmoli, Janahan; Wright, Heather J.; Phan, Duc T.T.; Sheth, Urmi; Que, Richard A.; Botten, Giovanni A.; Keating, Mark; Botvinick, Elliot L.; Pathak, Medha M.; Zarembinski, Thomas I.; Yanni, Daniel S.; Razorenova, Olga V.; Hughes, Christopher C.W.; Flanagan, Lisa A.

2017-01-01

Human neural stem/progenitor cells (hNSPCs) are good candidates for treating central nervous system (CNS) trauma since they secrete beneficial trophic factors and differentiate into mature CNS cells; however, many cells die after transplantation. This cell death can be ameliorated by inclusion of a biomaterial scaffold, making identification of optimal scaffolds for hNSPCs a critical research focus. We investigated the properties of fibrin-based scaffolds and their effects on hNSPCs and found that fibrin generated from salmon fibrinogen and thrombin stimulates greater hNSPC proliferation than mammalian fibrin. Fibrin scaffolds degrade over the course of a few days in vivo, so we sought to develop a novel scaffold that would retain the beneficial properties of fibrin but degrade more slowly to provide longer support for hNSPCs. We found combination scaffolds of salmon fibrin with interpenetrating networks (IPNs) of hyaluronic acid (HA) with and without laminin polymerize more effectively than fibrin alone and generate compliant hydrogels matching the physical properties of brain tissue. Furthermore, combination scaffolds support hNSPC proliferation and differentiation while significantly attenuating the cell-mediated degradation seen with fibrin alone. HNSPCs express two fibrinogen-binding integrins, αVβ1 and α5β1, and several laminin binding integrins (α7β1, α6β1, α3β1) that can mediate interaction with the scaffold. Lastly, to test the ability of scaffolds to support vascularization, we analyzed human cord blood-derived endothelial cells alone and in co-culture with hNSPCs and found enhanced vessel formation and complexity in co-cultures within combination scaffolds. Overall, combination scaffolds of fibrin, HA, and laminin are excellent biomaterials for hNSPCs. PMID:27475528

Oxidation modifies the structure and function of the extracellular matrix generated by human coronary artery endothelial cells.

PubMed

Chuang, Christine Y; Degendorfer, Georg; Hammer, Astrid; Whitelock, John M; Malle, Ernst; Davies, Michael J

2014-04-15

ECM (extracellular matrix) materials, such as laminin, perlecan, type IV collagen and fibronectin, play a key role in determining the structure of the arterial wall and the properties of cells that interact with the ECM. The aim of the present study was to investigate the effect of peroxynitrous acid, an oxidant generated by activated macrophages, on the structure and function of the ECM laid down by HCAECs (human coronary artery endothelial cells) in vitro and in vivo. We show that exposure of HCAEC-derived native matrix components to peroxynitrous acid (but not decomposed oxidant) at concentrations >1 μM results in a loss of antibody recognition of perlecan, collagen IV, and cell-binding sites on laminin and fibronectin. Loss of recognition was accompanied by decreased HCAEC adhesion. Real-time PCR showed up-regulation of inflammation-associated genes, including MMP7 (matrix metalloproteinase 7) and MMP13, as well as down-regulation of the laminin α2 chain, in HCAECs cultured on peroxynitrous acid-treated matrix compared with native matrix. Immunohistochemical studies provided evidence of co-localization of laminin with 3-nitrotyrosine, a biomarker of peroxynitrous acid damage, in type II-III/IV human atherosclerotic lesions, consistent with matrix damage occurring during disease development in vivo. The results of the present study suggest a mechanism through which peroxynitrous acid modifies endothelial cell-derived native ECM proteins of the arterial basement membrane in atherosclerotic lesions. These changes to ECM and particularly perlecan and laminin may be important in inducing cellular dysfunction and contribute to atherogenesis.

Cranial neural crest recycle surface integrins in a substratum-dependent manner to promote rapid motility.

PubMed

Strachan, Lauren R; Condic, Maureen L

2004-11-08

Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although the migratory pathways of NCCs have been determined, the molecular mechanisms regulating NCC motility remain unclear. NCC migration is integrin dependent, and recent work has shown that surface expression levels of particular integrin alpha subunits are important determinants of NCC motility in vitro. Here, we provide evidence that rapid cranial NCC motility on laminin requires integrin recycling. NCCs showed both ligand- and receptor-specific integrin regulation in vitro. On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min. Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface. Blocking receptor recycling with bafilomycin A inhibited NCC motility on laminin, indicating that substratum-dependent integrin recycling is essential for rapid cranial neural crest migration.

Differential Effects of Tissue Culture Coating Substrates on Prostate Cancer Cell Adherence, Morphology and Behavior

PubMed Central

Liberio, Michelle S.; Sadowski, Martin C.; Soekmadji, Carolina; Davis, Rohan A.; Nelson, Colleen C.

2014-01-01

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-l-lysine, poly-l-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-l-lysine and poly-l-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement. PMID:25375165

Use of Epidermolysis Bullosa Biomarkers in Models of Vesicant Injury

DTIC Science & Technology

2006-09-01

on an epithelial cell “scatter factor ,” which was termed ladsin. This molecule is actually the same as laminin-332 and facilitates epithelial tumor ...blistering diseases, such as bullous pemphigoid, dermatitis herpetiformis, and pemphigus vulgaris22. Because proteases are likely to be involved in...exposure to cutaneous sulfur mustard (SM). 2. Molecular Studies: Biomarkers of epidermolysis bullosa (BEB), laminin alpha 3, laminin beta 3 and

Laminin-111-derived peptide conjugated fibrin hydrogel restores salivary gland function.

PubMed

Nam, Kihoon; Maruyama, Christina L; Wang, Ching-Shuen; Trump, Bryan G; Lei, Pedro; Andreadis, Stelios T; Baker, Olga J

2017-01-01

Hyposalivation reduces the patient quality of life, as saliva is important for maintaining oral health. Current treatments for hyposalivation are limited to medications such as the muscarinic receptor agonists, pilocarpine and cevimeline. However, these therapies only provide temporary relief. Therefore, alternative therapies are essential to restore salivary gland function. An option is to use bioengineered scaffolds to promote functional salivary gland regeneration. Previous studies demonstrated that the laminin-111 protein is critical for intact salivary gland cell cluster formation and organization. However, laminin-111 protein as a whole is not suitable for clinical applications as some protein domains may contribute to unwanted side effects such as degradation, tumorigenesis and immune responses. Conversely, the use of synthetic laminin-111 peptides makes it possible to minimize the immune reactivity or pathogen transfer. In addition, it is relatively simple and inexpensive as compared to animal-derived proteins. Therefore, the goal of this study was to demonstrate whether a 20 day treatment with laminin-111-derived peptide conjugated fibrin hydrogel promotes tissue regeneration in submandibular glands of a wound healing mouse model. In this study, laminin-111-derived peptide conjugated fibrin hydrogel significantly accelerated formation of salivary gland tissue. The regenerated gland tissues displayed not only structural but also functional restoration.

Laminin-111-derived peptide conjugated fibrin hydrogel restores salivary gland function

PubMed Central

Nam, Kihoon; Maruyama, Christina L.; Wang, Ching-Shuen; Trump, Bryan G.; Lei, Pedro; Andreadis, Stelios T.

2017-01-01

Hyposalivation reduces the patient quality of life, as saliva is important for maintaining oral health. Current treatments for hyposalivation are limited to medications such as the muscarinic receptor agonists, pilocarpine and cevimeline. However, these therapies only provide temporary relief. Therefore, alternative therapies are essential to restore salivary gland function. An option is to use bioengineered scaffolds to promote functional salivary gland regeneration. Previous studies demonstrated that the laminin-111 protein is critical for intact salivary gland cell cluster formation and organization. However, laminin-111 protein as a whole is not suitable for clinical applications as some protein domains may contribute to unwanted side effects such as degradation, tumorigenesis and immune responses. Conversely, the use of synthetic laminin-111 peptides makes it possible to minimize the immune reactivity or pathogen transfer. In addition, it is relatively simple and inexpensive as compared to animal-derived proteins. Therefore, the goal of this study was to demonstrate whether a 20 day treatment with laminin-111-derived peptide conjugated fibrin hydrogel promotes tissue regeneration in submandibular glands of a wound healing mouse model. In this study, laminin-111-derived peptide conjugated fibrin hydrogel significantly accelerated formation of salivary gland tissue. The regenerated gland tissues displayed not only structural but also functional restoration. PMID:29095857

Matrix metalloproteinase-9 expression in folliculostellate cells of rat anterior pituitary gland.

PubMed

Ilmiawati, Cimi; Horiguchi, Kotaro; Fujiwara, Ken; Yashiro, Takashi

2012-03-01

Folliculostellate (FS) cells of the anterior pituitary gland express a variety of regulatory molecules. Using transgenic rats that express green fluorescent protein specifically in FS cells, we recently demonstrated that FS cells in vitro showed marked changes in motility, proliferation, and that formation of cellular interconnections in the presence of laminin, a component of the extracellular matrix, closely resembled those observed in vivo. These findings suggested that FS cells express matrix metalloproteinase-9 (MMP-9), which assists their function on laminin. In the present study, we investigate MMP-9 expression in rat anterior pituitary gland and examine its role in motility and proliferation of FS cells on laminin. Immunohistochemistry, RT-PCR, immunoblotting, and gelatin zymography were performed to assess MMP-9 expression in the anterior pituitary gland and cultured FS cells. Real-time RT-PCR was used to quantify MMP-9 expression in cultured FS cells under different conditions and treatments. MMP-9 expression was inhibited by pharmacological inhibitor or downregulated by siRNA and time-lapse images were acquired. A 5-bromo-2'-deoxyuridine assay was performed to analyze the proliferation of FS cells. Our results showed that MMP-9 was expressed in FS cells, that this expression was upregulated by laminin, and that laminin induced MMP-9 secretion by FS cells. MMP-9 inhibition and downregulation did not impair FS motility; however, it did impair the capacity of FS cells to form interconnections and it significantly inhibited proliferation of FS cells on laminin. We conclude that MMP-9 is necessary in FS cell interconnection and proliferation in the presence of laminin.

Biosensor studies of collagen and laminin binding with immobilized Escherichia coli O157:H7 and inhibition with naturally occurring food additives

NASA Astrophysics Data System (ADS)

Medina, Marjorie B.

1999-01-01

Escherichia coli O157:H7 outbreaks were mostly due to consumption of undercooked contaminated beef which resulted in severe illness and several fatalities. Recalls of contaminated meat are costly for the meat industry. Our research attempts to understand the mechanisms of bacterial adhesion on animal carcass in order to eliminate or reduce pathogens in foods. We have reported the interactions of immobilized E. coli O157:H7 cells with extracellular matrix (ECM) components using a surface plasmon resonance biosensor (BIAcore). These studies showed that immobilized bacterial cells allowed the study of real-time binding interactions of bacterial surface with the ECM compounds, collagen I, laminin and fibronectin. Collagen I and laminin bound to the E. coli sensor surface with dissociation and association rates ranging from 106 to 109. Binding of collagen I and laminin mixture resulted in synergistic binding signals. An inhibition model was derived using collagen-laminin as the ligand which binds with E. coli sensor. A select group of naturally occurring food additives was evaluated by determining their effectivity in inhibiting the collagen-laminin binding to the bacterial sensor. Bound collagen-laminin was detached from the E. coli sensor surface with the aid of an organic acid. The biosensor results were verified with cell aggregation assays which were observed with optical and electron microscopes. These biosensor studies provided understanding of bacterial adhesion to connective tissue macromolecules. It also provided a model system for the rapid assessment of potential inhibitors that can be used in carcass treatment to inhibit or reduce bacterial contamination.

Laminin-5 gamma 2 chain as an invasivity marker for uni- and multifocal lesions in the lower anogenital tract.

PubMed

Nordström, Britta; Einhorn, N; Silfverswärd, C; Sjövall, K; Tryggvason, K; Auer, G

2002-01-01

During recent decades it has become apparent that there are two types of vulvar disease: the classic type found in elderly women with unicentric and unifocal lesions, and the type found in younger women, in which precancerous and invasive changes develop in the anogenital lower tract in a multicentric and multifocal fashion, often over a long period of observation. The laminin-5 gamma 2 chain is an extracellular protein that is a component of the basement membrane. Recently its expression has been recognized as a marker in cervical cancer that permits identification of invasive capacity. The aim of our study was to determine if laminin-5 gamma 2 chain antibody can act as a sensitivity marker of invasive capacity in precancerous and invasive carcinoma in women with uni- and multifocal changes in the anogenital tract. The result showed that all patients in the older group of women with invasive carcinoma of the vulva had moderate to high positive expression of the laminin-5 gamma 2 chain. In the group of younger patients with multifocal precancerous changes observed over long periods, most of the patients with vulva intraepithelial neoplasia (VIN) 3 showed laminin-5 gamma 2 chain positivity already in the precancerous changes, and all of them developed invasivity during the period of observation. Normal epithelium without atypia was mostly negative or of low immunoreactivity of laminin-5. In conclusion, positive laminin-5 gamma 2 chain expression seems to indicate the invasiveness potential of precancerous lesions and is also expressed in all investigated invasive carcinomas of the anogenital tract.

Tumor Cell Plasticity in Uveal Melanoma

PubMed Central

Folberg, Robert; Arbieva, Zarema; Moses, Jonas; Hayee, Amin; Sandal, Tone; Kadkol, ShriHari; Lin, Amy Y.; Valyi-Nagy, Klara; Setty, Suman; Leach, Lu; Chévez-Barrios, Patricia; Larsen, Peter; Majumdar, Dibyen; Pe’er, Jacob; Maniotis, Andrew J.

2006-01-01

The histological detection of laminin-rich vasculogenic mimicry patterns in human primary uveal melanomas is associated with death from metastases. We therefore hypothesized that highly invasive uveal melanoma cells forming vasculogenic mimicry patterns after exposure to a laminin-rich three-dimensional microenvironment would differentially express genes associated with invasive and metastatic behavior. However, we discovered that genes associated with differentiation (GDF15 and ATF3) and suppression of proliferation (CDKNa1/p21) were up-regulated in highly invasive uveal melanoma cells forming vasculogenic mimicry patterns, and genes associated with promotion of invasive and metastatic behavior such as CD44, CCNE2 (cyclin E2), THBS1 (thrombospondin 1), and CSPG2 (chondroitin sulfate proteoglycan; versican) were down-regulated. After forming vasculogenic mimicry patterns, uveal melanoma cells invaded only short distances, failed to replicate, and changed morphologically from the invasive epithelioid to the indolent spindle A phenotype. In human tissue samples, uveal melanoma cells within vasculogenic mimicry patterns assumed the spindle A morphology, and the expression of Ki67 was significantly reduced in adjacent melanoma cells. Thus, the generation of vasculogenic mimicry patterns is accompanied by dampening of the invasive and metastatic uveal melanoma genotype and phenotype and underscores the plasticity of these cells in response to cues from the microenvironment. PMID:17003493

CHLORHEXIDINE INHIBITS L1 CELL ADHESION MOLECULE MEDIATED NEURITE OUTGROWTH IN VITRO

PubMed Central

Milstone, Aaron M.; Bamford, Penny; Aucott, Susan W.; Tang, Ningfeng; White, Kimberly R.; Bearer, Cynthia F.

2013-01-01

Background Chlorhexidine is a skin disinfectant that reduces skin and mucous membrane bacterial colonization and inhibits organism growth. Despite numerous studies assessing chlorhexidine safety in term infants, residual concerns have limited its use in hospitalized neonates, especially low birth weight preterm infants. The aim of this study was to assess the potential neurotoxicity of chlorhexidine on the developing central nervous system using a well-established in vitro model of neurite outgrowth that includes laminin and L1 cell adhesion molecule (L1) as neurite outgrowth promoting substrates. Methods Cerebellar granule neurons are plated on either poly L-lysine, L1 or laminin. Chlorhexidine, hexachlorophene or their excipients are added to the media. Neurons are grown for 24 h, then fixed and neurite length measured. Results Chlorhexidine significantly reduced the length of neurites grown on L1 but not laminin. Chlorhexidine concentrations as low as 125 ng/ml statistically significantly reduced neurite length on L1. Hexachlorophene did not affect neurite length. Conclusion Chlorhexidine at concentrations detected in the blood following topical applications in preterm infants specifically inhibited L1 mediated neurite outgrowth of cerebellar granule neurons. It is now vital to determine whether the blood brain barrier is permeable to chlorhexidine in preterm infants. PMID:24126818

The Effect of Maternal Thyroid Disorders (Hypothyroidism and Hyperthyroidism) During Pregnancy and Lactation on Skin Development in Wistar Rat Newborns

PubMed Central

Amerion, Maryam; Tahajjodi, Somayye; Hushmand, Zahra; Mahdavi Shahri, Nasser; Nikravesh, Mohammad Reza; Jalali, Mahdi

2013-01-01

Objective(s): Previous studies have shown that thyroid hormones are necessary for normal development of many organs and because of the importance of skin as the largest and the most important organ in human body protection in spite of external environment, the study of thyroid hormones effects on skin development is considerable. In this survey we have tried to study the effects of maternal hypothyroidism on skin development in fetus during pregnancy and lactation by immunohistochemistry technique. Materials and Methods: Rats were divided into 4 groups, hypothyroids, hyperthyroids, hypothyroids are treated with levothyroxin and a control group. The rat mothers were exposed to PTU with 50 mg/lit dosage and levothyroxin with 1 mg/lit dosage and PTU and levothyroxin simultaneously and with the same dosage respectively in hypothyroid, hyperthyroid and treated hypothyroids with levothyroxin groups. After 14 days, blood sample was taken from mothers, and if thyroid hormones level had change well, mating was allowed. After pregnancy and delivery, 1th day dorsal skin (as the sample for pregnancy assay) and 10th day skin (as for lactation assay) was used for immunohystochemical and morphometric studies. Results: In this study it was observed that maternal hypothyroidism during pregnancy and lactation causes significant increase in laminin expression, in most areas of skin, and maternal hyperthyroidism during pregnancy and lactation causes significant decrease in laminin expression. Also significant decrease was observed in hair follicles number and epidermis thickness in hypothyroidism groups. Conclusion: This study showed maternal hypothyroidism causes significant decrease in epidermis thickness and hair follicles number and it causes less hair in fetus. Also maternal hypothyroidism causes large changes in laminin expression in different parts of skin. At the same time,maternal hyperthyroidism causes opposite results. In fact, thyroid hormones regulate laminin expression negatively which means increase in thyroid hormone level, decreases laminin expression. So changes in thyroid hormones level can influence skin development significantly. PMID:23826487

Synthesis and deposition of basement membrane proteins by primary brain capillary endothelial cells in a murine model of the blood-brain barrier.

PubMed

Thomsen, Maj Schneider; Birkelund, Svend; Burkhart, Annette; Stensballe, Allan; Moos, Torben

2017-03-01

The brain vascular basement membrane is important for both blood-brain barrier (BBB) development, stability, and barrier integrity and the contribution hereto from brain capillary endothelial cells (BCECs), pericytes, and astrocytes of the BBB is probably significant. The aim of this study was to analyse four different in vitro models of the murine BBB for expression and possible secretion of major basement membrane proteins from murine BCECs (mBCECs). mBCECs, pericytes and glial cells (mainly astrocytes and microglia) were prepared from brains of C57BL/6 mice. The mBCECs were grown as monoculture, in co-culture with pericytes or mixed glial cells, or as a triple-culture with both pericytes and mixed glial cells. The integrity of the BBB models was validated by measures of transendothelial electrical resistance (TEER) and passive permeability to mannitol. The expression of basement membrane proteins was analysed using RT-qPCR, mass spectrometry and immunocytochemistry. Co-culturing mBCECs with pericytes, mixed glial cells, or both significantly increased the TEER compared to the monoculture, and a low passive permeability was correlated with high TEER. The mBCECs expressed all major basement membrane proteins such as laminin-411, laminin-511, collagen [α1(IV)] 2 α2(IV), agrin, perlecan, and nidogen 1 and 2 in vitro. Increased expression of the laminin α5 subunit correlated with the addition of BBB-inducing factors (hydrocortisone, Ro 20-1724, and pCPT-cAMP), whereas increased expression of collagen IV α1 primarily correlated with increased levels of cAMP. In conclusion, BCECs cultured in vitro coherently form a BBB and express basement membrane proteins as a feature of maturation. Cover Image for this issue: doi: 10.1111/jnc.13789. © 2016 International Society for Neurochemistry.

Deficiency of merosin in dystrophic dy mouse homologue of congenital muscular dystrophy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sunada, Y.; Campbell, K.P.; Bernier, S.M.

1994-09-01

Merosin (laminin M chain) is the predominant laminin isoform in the basal lamina of striated muscle and peripheral nerve and is a native ligand for {alpha}-dystroglycan, a novel laminin receptor. Merosin is linked to the subsarcolemmal actin cytoskeleton via the dystrophin-glycoprotein complex (DGC), which plays an important role for maintenance of normal muscle function. We have mapped the mouse merosin gene, Lamm, to the region containing the dystrophia muscularis (dy) locus on chromosome 10. This suggested the possibility that a mutation in the merosin gene could be responsible for the dy mouse, an animal model for autosomal recessive muscular dystrophy,more » and prompted us to test this hypothesis. We analyzed the status of merosin expression in dy mouse by immunofluorescence and immunoblotting. In dy mouse skeletal and cardiac muscle and peripheral nerve, merosin was reduced greater than 90% as compared to control mice. However, the expression of laminin B1/B2 chains and collagen type IV was smaller to that in control mice. These findings strongly suggest that merosin deficiency may be the primary defect in the dy mouse. Furthermore, we have identified two patients afflicted with congenital muscular dystrophy with merosin deficiency, providing the basis for future studies of molecular pathogenesis and gene therapy.« less

Reticular basement membrane in asthma and COPD: Similar thickness, yet different composition

PubMed Central

Liesker, Jeroen JW; Hacken, Nick H Ten; Zeinstra-Smith, Mieke; Rutgers, Steven R; Postma, Dirkje S; Timens, Wim

2009-01-01

Background Reticular basement membrane (RBM) thickening has been variably associated with asthma and chronic obstructive pulmonary disease (COPD). Even if RBM thickness is similar in both diseases, its composition might still differ. Objective To assess whether RBM thickness and composition differ between asthma and COPD. Methods We investigated 24 allergic asthmatics (forced expiratory volume in one second [FEV1] 92% predicted), and 17 nonallergic COPD patients (FEV1 60% predicted), and for each group a control group of similar age and smoking habits (12 and 10 persons, respectively). Snap-frozen sections of bronchial biopsies were stained with hematoxylin/eosin and for collagen I, III, IV, V, laminin and tenascin. RBM thickening was assessed by digital image analysis. Relative staining intensity of each matrix component was determined. Results Mean (SD) RBM thickness was not significantly different between asthma and COPD 5.5 (1.3) vs 6.0 (1.8) μm, but significantly larger than in their healthy counterparts, ie, 4.7 (0.9) and 4.8 (1.2) μm, respectively. Collagen I and laminin stained significantly stronger in asthma than in COPD. Tenascin stained stronger in asthma than in healthy controls of similar age, and stronger in COPD controls than in asthma controls (p < 0.05). Conclusion RBM thickening occurs both in asthma and COPD. We provide supportive evidence that its composition differs in asthma and COPD. PMID:19436691

Sulfur mustard induces an endoplasmic reticulum stress response in the mouse ear vesicant model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Yoke-Chen; Wang, James D.; Svoboda, Kathy K.

The endoplasmic reticulum (ER) stress response is a cell survival pathway upregulated when cells are under severe stress. Severely damaged mouse ear skin exposed to the vesicant, sulfur mustard (bis-2-chloroethyl sulfide, SM), resulted in increased expression of ER chaperone proteins that accompany misfolded and incorrectly made proteins targeted for degradation. Time course studies with SM using the mouse ear vesicant model (MEVM) showed progressive histopathologic changes including edema, separation of the epidermis from the dermis, persistent inflammation, upregulation of laminin γ2 (one of the chains of laminin-332, a heterotrimeric skin glycoprotein required for wound repair), and delayed wound healing frommore » 24 h to 168 h post exposure. This was associated with time related increased expression of the cell survival ER stress marker, GRP78/BiP, and the ER stress apoptosis marker, GADD153/CHOP, suggesting simultaneous activation of both cell survival and non-mitochondrial apoptosis pathways. Dual immunofluorescence labeling of a keratinocyte migration promoting protein, laminin γ2 and GRP78/BIP, showed colocalization of the two molecules 72 h post exposure indicating that the laminin γ2 was misfolded after SM exposure and trapped within the ER. Taken together, these data show that ER stress is induced in mouse skin within 24 h of vesicant exposure in a defensive response to promote cell survival; however, it appears that this response is rapidly overwhelmed by the apoptotic pathway as a consequence of severe SM-induced injury. - Highlights: ► We demonstrated ER stress response in the mouse ear vesicant model. ► We described the asymmetrical nature of wound repair in the MEVM. ► We identified the distribution of various ER stress markers in the MEVM.« less

Evidence that a laminin-like insect protein mediates early events in the interaction of a Phytoparasite with its vector's salivary gland.

PubMed

de Almeida Dias, Felipe; Souza dos Santos, Andre Luis; Santos Lery, Letícia Miranda; Alves e Silva, Thiago Luiz; Oliveira, Mauricio Martins; Bisch, Paulo Mascarello; Saraiva, Elvira Maria; Souto-Padrón, Thaïs Cristina; Lopes, Angela Hampshire

2012-01-01

Phytomonas species are plant parasites of the family Trypanosomatidae, which are transmitted by phytophagous insects. Some Phytomonas species cause major agricultural damages. The hemipteran Oncopeltus fasciatus is natural and experimental host for several species of trypanosomatids, including Phytomonas spp. The invasion of the insect vectors' salivary glands is one of the most important events for the life cycle of Phytomonas species. In the present study, we show the binding of Phytomonas serpens at the external face of O. fasciatus salivary glands by means of scanning electron microscopy and the in vitro interaction of living parasites with total proteins from the salivary glands in ligand blotting assays. This binding occurs primarily through an interaction with a 130 kDa salivary gland protein. The mass spectrometry of the trypsin-digest of this protein matched 23% of human laminin-5 β3 chain precursor sequence by 16 digested peptides. A protein sequence search through the transcriptome of O. fasciatus embryo showed a partial sequence with 51% similarity to human laminin β3 subunit. Anti-human laminin-5 β3 chain polyclonal antibodies recognized the 130 kDa protein by immunoblotting. The association of parasites with the salivary glands was strongly inhibited by human laminin-5, by the purified 130 kDa insect protein, and by polyclonal antibodies raised against the human laminin-5 β3 chain. This is the first report demonstrating that a laminin-like molecule from the salivary gland of O. fasciatus acts as a receptor for Phytomonas binding. The results presented in this investigation are important findings that will support further studies that aim at developing new approaches to prevent the transmission of Phytomonas species from insects to plants and vice-versa.

Evidence That a Laminin-Like Insect Protein Mediates Early Events in the Interaction of a Phytoparasite with Its Vector's Salivary Gland

PubMed Central

Dias, Felipe de Almeida; dos Santos, Andre Luis Souza; Lery, Letícia Miranda Santos; Alves e Silva, Thiago Luiz; Oliveira, Mauricio Martins; Bisch, Paulo Mascarello; Saraiva, Elvira Maria; Souto-Padrón, Thaïs Cristina; Lopes, Angela Hampshire

2012-01-01

Phytomonas species are plant parasites of the family Trypanosomatidae, which are transmitted by phytophagous insects. Some Phytomonas species cause major agricultural damages. The hemipteran Oncopeltus fasciatus is natural and experimental host for several species of trypanosomatids, including Phytomonas spp. The invasion of the insect vectors' salivary glands is one of the most important events for the life cycle of Phytomonas species. In the present study, we show the binding of Phytomonas serpens at the external face of O. fasciatus salivary glands by means of scanning electron microscopy and the in vitro interaction of living parasites with total proteins from the salivary glands in ligand blotting assays. This binding occurs primarily through an interaction with a 130 kDa salivary gland protein. The mass spectrometry of the trypsin-digest of this protein matched 23% of human laminin-5 β3 chain precursor sequence by 16 digested peptides. A protein sequence search through the transcriptome of O. fasciatus embryo showed a partial sequence with 51% similarity to human laminin β3 subunit. Anti-human laminin-5 β3 chain polyclonal antibodies recognized the 130 kDa protein by immunoblotting. The association of parasites with the salivary glands was strongly inhibited by human laminin-5, by the purified 130 kDa insect protein, and by polyclonal antibodies raised against the human laminin-5 β3 chain. This is the first report demonstrating that a laminin-like molecule from the salivary gland of O. fasciatus acts as a receptor for Phytomonas binding. The results presented in this investigation are important findings that will support further studies that aim at developing new approaches to prevent the transmission of Phytomonas species from insects to plants and vice-versa. PMID:23118944

Functional regeneration of the transected recurrent laryngeal nerve using a collagen scaffold loaded with laminin and laminin-binding BDNF and GDNF

PubMed Central

Wang, Baoxin; Yuan, Junjie; Chen, Xinwei; Xu, Jiafeng; Li, Yu; Dong, Pin

2016-01-01

Recurrent laryngeal nerve (RLN) injury remains a challenge due to the lack of effective treatments. In this study, we established a new drug delivery system consisting of a tube of Heal-All Oral Cavity Repair Membrane loaded with laminin and neurotrophic factors and tested its ability to promote functional recovery following RLN injury. We created recombinant fusion proteins consisting of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) fused to laminin-binding domains (LBDs) in order to prevent neurotrophin diffusion. LBD-BDNF, LBD-GDNF, and laminin were injected into a collagen tube that was fitted to the ends of the transected RLN in rats. Functional recovery was assessed 4, 8, and 12 weeks after injury. Although vocal fold movement was not restored until 12 weeks after injury, animals treated with the collagen tube loaded with laminin, LBD-BDNF and LBD-GDNF showed improved recovery in vocalisation, arytenoid cartilage angles, compound muscle action potentials and regenerated fibre area compared to animals treated by autologous nerve grafting (p < 0.05). These results demonstrate the drug delivery system induced nerve regeneration following RLN transection that was superior to that induced by autologus nerve grafting. It may have potential applications in nerve regeneration of RLN transection injury. PMID:27558932

Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands

PubMed Central

1991-01-01

Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand. PMID:1848864

B4GALNT2 (GALGT2) Gene Therapy Reduces Skeletal Muscle Pathology in the FKRP P448L Mouse Model of Limb Girdle Muscular Dystrophy 2I.

PubMed

Thomas, Paul J; Xu, Rui; Martin, Paul T

2016-09-01

Overexpression of B4GALNT2 (previously GALGT2) inhibits the development of muscle pathology in mouse models of Duchenne muscular dystrophy, congenital muscular dystrophy 1A, and limb girdle muscular dystrophy 2D. In these models, muscle GALGT2 overexpression induces the glycosylation of α dystroglycan with the cytotoxic T cell glycan and increases the overexpression of dystrophin and laminin α2 surrogates known to inhibit disease. Here, we show that GALGT2 gene therapy significantly reduces muscle pathology in FKRP P448Lneo(-) mice, a model for limb girdle muscular dystrophy 2I. rAAVrh74.MCK.GALGT2-treated FKRP P448Lneo(-) muscles showed reduced levels of centrally nucleated myofibers, reduced variance, increased size of myofiber diameters, reduced myofiber immunoglobulin G uptake, and reduced muscle wasting at 3 and 6 months after treatment. GALGT2 overexpression in FKRP P448Lneo(-) muscles did not cause substantial glycosylation of α dystroglycan with the cytotoxic T cell glycan or increased expression of dystrophin and laminin α2 surrogates in mature skeletal myofibers, but it increased the number of embryonic myosin-positive regenerating myofibers. These data demonstrate that GALGT2 overexpression can reduce the extent of muscle pathology in FKRP mutant muscles, but that it may do so via a mechanism that differs from its ability to induce surrogate gene expression. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Mapping the Laminin Receptor Binding Domains of Neisseria meningitidis PorA and Haemophilus influenzae OmpP2

PubMed Central

Mahdavi, Jafar; Oldfield, Neil J.; Wheldon, Lee M.; Wooldridge, Karl G.; Ala'Aldeen, Dlawer A. A.

2012-01-01

Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae are major bacterial agents of meningitis. They each bind the 37/67-kDa laminin receptor (LamR) via the surface protein adhesins: meningococcal PilQ and PorA, H. influenzae OmpP2 and pneumococcal CbpA. We have previously reported that a surface-exposed loop of the R2 domain of CbpA mediates LamR-binding. Here we have identified the LamR-binding regions of PorA and OmpP2. Using truncated recombinant proteins we show that binding is dependent on amino acids 171–240 and 91–99 of PorA and OmpP2, respectively, which are predicted to localize to the fourth and second surface-exposed loops, respectively, of these proteins. Synthetic peptides corresponding to the loops bound LamR and could block LamR-binding to bacterial ligands in a dose dependant manner. Meningococci expressing PorA lacking the apex of loop 4 and H. influenzae expressing OmpP2 lacking the apex of loop 2 showed significantly reduced LamR binding. Since both loops are hyper-variable, our data may suggest a molecular basis for the range of LamR-binding capabilities previously reported among different meningococcal and H. influenzae strains. PMID:23049988

Mapping the laminin receptor binding domains of Neisseria meningitidis PorA and Haemophilus influenzae OmpP2.

PubMed

Abouseada, Noha M; Assafi, Mahde Saleh A; Mahdavi, Jafar; Oldfield, Neil J; Wheldon, Lee M; Wooldridge, Karl G; Ala'Aldeen, Dlawer A A

2012-01-01

Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae are major bacterial agents of meningitis. They each bind the 37/67-kDa laminin receptor (LamR) via the surface protein adhesins: meningococcal PilQ and PorA, H. influenzae OmpP2 and pneumococcal CbpA. We have previously reported that a surface-exposed loop of the R2 domain of CbpA mediates LamR-binding. Here we have identified the LamR-binding regions of PorA and OmpP2. Using truncated recombinant proteins we show that binding is dependent on amino acids 171-240 and 91-99 of PorA and OmpP2, respectively, which are predicted to localize to the fourth and second surface-exposed loops, respectively, of these proteins. Synthetic peptides corresponding to the loops bound LamR and could block LamR-binding to bacterial ligands in a dose dependant manner. Meningococci expressing PorA lacking the apex of loop 4 and H. influenzae expressing OmpP2 lacking the apex of loop 2 showed significantly reduced LamR binding. Since both loops are hyper-variable, our data may suggest a molecular basis for the range of LamR-binding capabilities previously reported among different meningococcal and H. influenzae strains.

Contribution of the 37-kDa laminin receptor precursor in the anti-metastatic PSP94-derived peptide PCK3145 cell surface binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Annabi, Borhane; Currie, Jean-Christophe; Bouzeghrane, Mounia

Purpose: PCK3145 is an anti-metastatic synthetic peptide with promising therapeutic efficacy against hormone-refractory prostate cancer. The characterization of the PCK3145 peptide cell surface binding/internalization mechanisms and of the receptors involved remained to be explored. Results: [{sup 14}C]PCK3145 cell surface binding assays showed rapid and transient kinetic profile, that was inhibited by RGD peptides, laminin, hyaluronan, and type-I collagen. RGD peptides were however unable to inhibit PCK3145 intracellular uptake. Far-Western ligand binding studies enabled the identification of the 37-kDa laminin receptor precursor (37LRP) as a potential ligand for PCK3145. Overexpression of the recombinant 37LRP indeed led to an increase in PCK3145more » binding but unexpectedly not to its uptake. Conclusions: Our data support the implication of laminin receptors in cell surface binding and in transducing PCK3145 anti-metastatic effects, and provide a rational for targeting cancers that express high levels of such laminin receptors.« less

Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo

Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin,more » respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the {beta}1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.« less

Fragmentation of tetrahydrofuran molecules by H(+), C(+), and O(+) collisions at the incident energy range of 25-1000 eV.

PubMed

Wasowicz, Tomasz J; Pranszke, Bogusław

2015-01-29

We have studied fragmentation processes of the gas-phase tetrahydrofuran (THF) molecules in collisions with the H(+), C(+), and O(+) cations. The collision energies have been varied between 25 and 1000 eV and thus covered a velocity range from 10 to 440 km/s. The following excited neutral fragments of THF have been observed: the atomic hydrogen H(n), n = 4-9, carbon atoms in the 2p3s (1)P1, 2p4p (1)D2, and 2p4p (3)P states and vibrationally and rotationally excited diatomic CH fragments in the A(2)Δ and B(2)Σ(-) states. Fragmentation yields of these excited fragments have been measured as functions of the projectile energy (velocity). Our results show that the fragmentation mechanism depends on the projectile cations and is dominated by electron transfer from tetrahydrofuran molecules to cations. It has been additionally hypothesized that in the C(+)+THF collisions a [C-C4H8O](+) complex is formed prior to dissociation. The possible reaction channels involved in fragmentation of THF under the H(+), C(+), and O(+) cations impact are also discussed.

The Fibronectin-Binding Protein Fnm Contributes to Adherence to Extracellular Matrix Components and Virulence of Enterococcus faecium

PubMed Central

Somarajan, Sudha R.; La Rosa, Sabina Leanti; Singh, Kavindra V.; Roh, Jung H.; Höök, Magnus

2015-01-01

The interaction between bacteria and fibronectin is believed to play an important role in the pathogenicity of clinically important Gram-positive cocci. In the present study, we identified a gene encoding a predicted fibronectin-binding protein of Enterococcus faecium (fnm), a homologue of Streptococcus pneumoniae pavA, in the genomes of E. faecium strain TX82 and all other sequenced E. faecium isolates. Full-length recombinant Fnm from strain TX82 bound to immobilized fibronectin in a concentration-dependent manner and also appeared to bind collagen type V and laminin, but not other proteins, such as transferrin, heparin, bovine serum albumin, mucin, or collagen IV. We demonstrated that the N-terminal fragment of Fnm is required for full fibronectin binding, since truncation of this region caused a 2.4-fold decrease (P < 0.05) in the adhesion of E. faecium TX82 to fibronectin. Deletion of fnm resulted in a significant reduction (P < 0.001) in the ability of the mutant, TX6128, to bind fibronectin relative to that of the wild-type strain; in situ reconstitution of fnm in the deletion mutant strain restored adherence. In addition, the Δfnm mutant was highly attenuated relative to TX82 (P ≤ 0.0001) in a mixed-inoculum rat endocarditis model. Taken together, these results demonstrate that Fnm affects the adherence of E. faecium to fibronectin and is important in the pathogenesis of experimental endocarditis. PMID:26371130

Study of the relationship between mononuclear inflammatory infiltrate and Ki-67 and basement membrane and extracellular matrix protein expression in radicular cysts.

PubMed

Mourão, R V C; Júnior, E C Pinheiro; Barros Silva, P G; Turatti, E; Mota, M R L; Alves, A P N N

2016-05-01

To evaluate the relationship between mononuclear inflammatory infiltrate and the expression of a proliferative immunomarker (Ki-67) as well as to evaluate basement membrane and extracellular matrix proteins (laminin and collagen type IV) in radicular cysts and dentigerous cysts (DC). Immunohistochemical analyses were performed in heavily inflamed radicular cysts (HIRC), slightly inflamed radicular cysts (SIRC) and DC (n = 20) using Ki-67 (Dako(®) , 1 : 50), anticollagen type IV (DBS(®) , 1 : 40) and antilaminin (DBS(®) , 1 : 20). The data were analysed using anova/Tukey's test (Ki-67) and Kruskal-Wallis/Dunn's test (collagen type IV and laminin) (P < 0.05). The immunoexpression of Ki-67 was significantly greater in the SIRC group compared with the HIRC and DC (P = 0.0040). Likewise, the immunoexpression of collagen type IV in the basement membrane of the SIRC group was significantly more continuous (P = 0.0475) than in the HIRC group. DC had significantly less collagen type IV in extracellular matrix immunoexpression than HIRC and SIRC (P = 0.0246). Laminin was absent in the basement membrane in the SIRC and DC groups, and the extracellular matrix of the HIRC was weak and punctate. The presence of inflammatory factors in the radicular cyst wall modified the expression of proliferation factors in the epithelial lining and the expression of collagen type IV and laminin in the basement membrane, but did not modify extracellular matrix behaviour in radicular cysts. © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Effects of repeated social defeat on adolescent mice on cocaine-induced CPP and self-administration in adulthood: integrity of the blood-brain barrier.

PubMed

Rodríguez-Arias, Marta; Montagud-Romero, Sandra; Rubio-Araiz, Ana; Aguilar, María A; Martín-García, Elena; Cabrera, Roberto; Maldonado, Rafael; Porcu, Francesca; Colado, María Isabel; Miñarro, José

2017-01-01

Social stress in adulthood enhances cocaine self-administration, an effect that has been related with an increase in extracellular signal-regulated kinase and p38α mitogen-activated protein kinase phosphorylation. A detrimental effect of cocaine on blood-brain barrier (BBB) integrity has also been reported. This study evaluates the effects of repeated social defeat (RSD) during adolescence on the reinforcing and motivational effects of cocaine in adult mice and the changes induced by RSD on BBB permeability. Cocaine self-administration, conditioned place preference and quantitative analysis of claudin-5, laminin, collagen-IV and IgG immunoreactivity took place 3 weeks after RSD. Mice socially defeated during adolescence developed conditioned place preference and exhibited reinstated preference with a non-effective dose of cocaine (1 mg/kg). RSD mice needed significantly more sessions than control animals for the preference induced by 25 mg/kg of cocaine to be extinguished. However, acquisition of cocaine self-administration (0.5 mg/kg per injection) was delayed in the RSD group. Mice exposed to RSD displayed significant changes in BBB structure in adulthood, with a marked reduction in expression of the tight junction protein claudin-5 and an increase in basal laminin degradation (reflected by a decrease in laminin and collagen-IV expression) in the nucleus accumbens and hippocampus. The detrimental effect induced by cocaine (25 mg/kg) on collagen-IV expression in the hippocampus was more pronounced in RSD mice. In summary, our findings suggest that stress and cocaine can increase the long-term vulnerability of the brain to subsequent environmental insults as a consequence of a sustained disruption of the BBB. © 2015 Society for the Study of Addiction.

Epigallocatechin gallate (EGCG) suppresses lipopolysaccharide-induced Toll-like receptor 4 (TLR4) activity via 67 kDa laminin receptor (67LR) in 3T3-L1 adipocytes.

PubMed

Bao, Suqing; Cao, Yanli; Zhou, Haicheng; Sun, Xin; Shan, Zhongyan; Teng, Weiping

2015-03-18

Obesity-related insulin resistance is associated with chronic systemic low-grade inflammation, and toll-like receptor 4 (TLR4) regulates inflammation. We investigated the pathways involved in epigallocatechin gallate (EGCG) modulation of insulin and TLR4 signaling in adipocytes. Inflammation was induced in adipocytes by lipopolysaccharide (LPS). An antibody against the 67 kDa laminin receptor (67LR, to which EGCG exclusively binds) was used to examine the effect of EGCG on TLR4 signaling, and a TLR4/MD-2 antibody was used to inhibit TLR4 activity and to determine the insulin sensitivity of differentiated 3T3-L1 adipocytes. We found that EGCG dose-dependently inhibited LPS stimulation of adipocyte inflammation by reducing inflammatory mediator and cytokine levels (IKKβ, p-NF-κB, TNF-α, and IL-6). Pretreatment with the 67LR antibody prevented EGCG inhibition of inflammatory cytokines, decreased glucose transporter isoform 4 (GLUT4) expression, and inhibited insulin-stimulated glucose uptake. TLR4 inhibition attenuated inflammatory cytokine levels and increased glucose uptake by reversing GLUT4 levels. These data suggest that EGCG suppresses TLR4 signaling in LPS-stimulated adipocytes via 67LR and attenuates insulin-stimulated glucose uptake associated with decreased GLUT4 expression.

Sequenced response of extracellular matrix deadhesion and fibrotic regulators after muscle damage is involved in protection against future injury in human skeletal muscle

PubMed Central

Mackey, Abigail L.; Brandstetter, Simon; Schjerling, Peter; Bojsen-Moller, Jens; Qvortrup, Klaus; Pedersen, Mette M.; Doessing, Simon; Kjaer, Michael; Magnusson, S. Peter; Langberg, Henning

2011-01-01

The purpose of this study was to test the hypothesis that remodeling of skeletal muscle extracellular matrix (ECM) is involved in protecting human muscle against injury. Biopsies were obtained from medial gastrocnemius muscles after a single bout of electrical stimulation (B) or a repeated bout (RB) 30 d later, or 30 d after a single stimulation bout (RBc). A muscle biopsy was collected from the control leg for comparison with the stimulated leg. Satellite cell content, tenascin C, and muscle regeneration were assessed by immunohistochemistry; real-time PCR was used to measure mRNA levels of collagens, laminins, heat-shock proteins (HSPs), inflammation, and related growth factors. The large responses of HSPs, CCL2, and tenascin C detected 48 h after a single bout were attenuated in the RB trial, indicative of protection against injury. Satellite cell content and 12 target genes, including IGF-1, were elevated 30 d after a single bout. Among those displaying the greatest difference vs. control muscle, ECM laminin-β1 and collagen types I and III were elevated ∼6- to 9-fold (P<0.001). The findings indicate that the sequenced events of load-induced early deadhesion and later strengthening of skeletal muscle ECM play a role in protecting human muscle against future injury.—Mackey, A. L., Brandstetter, S., Schjerling, P., Bojsen-Moller, J., Qvortrup, K., Pedersen, M. M., Doessing, S. Kjaer, M., Magnusson, S. P., Langberg, H. Sequenced response of extracellular matrix deadhesion and fibrotic regulators after muscle damage is involved in protection against future injury in human skeletal muscle. PMID:21368102

Laminin and Matrix metalloproteinase 11 regulate Fibronectin levels in the zebrafish myotendinous junction.

PubMed

Jenkins, Molly H; Alrowaished, Sarah S; Goody, Michelle F; Crawford, Bryan D; Henry, Clarissa A

2016-01-01

Remodeling of the extracellular matrix (ECM) regulates cell adhesion as well as signaling between cells and their microenvironment. Despite the importance of tightly regulated ECM remodeling for normal muscle development and function, mechanisms underlying ECM remodeling in vivo remain elusive. One excellent paradigm in which to study ECM remodeling in vivo is morphogenesis of the myotendinous junction (MTJ) during zebrafish skeletal muscle development. During MTJ development, there are dramatic shifts in the primary components comprising the MTJ matrix. One such shift involves the replacement of Fibronectin (Fn)-rich matrix, which is essential for both somite and early muscle development, with laminin-rich matrix essential for normal function of the myotome. Here, we investigate the mechanism underlying this transition. We show that laminin polymerization indirectly promotes Fn downregulation at the MTJ, via a matrix metalloproteinase 11 (Mmp11)-dependent mechanism. Laminin deposition and organization is required for localization of Mmp11 to the MTJ, where Mmp11 is both necessary and sufficient for Fn downregulation in vivo. Furthermore, reduction of residual Mmp11 in laminin mutants promotes a Fn-rich MTJ that partially rescues skeletal muscle architecture. These results identify a mechanism for Fn downregulation at the MTJ, highlight crosstalk between laminin and Fn, and identify a new in vivo function for Mmp11. Taken together, our data demonstrate a novel signaling pathway mediating Fn downregulation. Our data revealing new regulatory mechanisms that guide ECM remodeling during morphogenesis in vivo may inform pathological conditions in which Fn is dysregulated.

Laminin and integrin expression in the ventral ectodermal ridge of the mouse embryo: implications for regulation of BMP signalling.

PubMed

Lopez-Escobar, Beatriz; De Felipe, Beatriz; Sanchez-Alcazar, Jose Antonio; Sasaki, Takako; Copp, Andrew J; Ybot-Gonzalez, Patricia

2012-11-01

The ventral ectodermal ridge (VER) is an important signalling centre in the mouse tail-bud following completion of gastrulation. BMP regulation is essential for VER function, but how these signals are transmitted between adjacent tissues is unclear. We investigated the idea that extracellular matrix components might be involved, using immunohistochemistry and in situ hybridisation to detect all known α, β, and γ laminin chains and their mRNAs in the early tail bud. We identified an apparently novel laminin variant, comprising α5, β3 and γ2 chains, as a major component of the VER basement membrane at E9.5. Strikingly, only the mRNAs for these chains were co-expressed in VER cells, suggesting that lamin532 may be the sole basement membrane laminin at this stage. Since α6 integrin was also expressed in VER cells, this raises the possibility of cell-matrix interactions regulating BMP signalling at this site of caudal morphogenesis. Laminin532 could interact with α6-containing integrin to direct differentiation of the specialised VER cells from surface ectoderm. Copyright © 2012 Wiley Periodicals, Inc.

Laminin and integrin expression in the ventral ectodermal ridge of the mouse embryo: implications for regulation of BMP signalling

PubMed Central

Lopez-Escobar, Beatriz; de Felipe, Beatriz; Sanchez-Alcazar, Jose Antonio; Sasaki, Takako; Copp, Andrew J.; Ybot-Gonzalez, Patricia

2013-01-01

Background The ventral ectodermal ridge (VER) is an important signalling centre in the mouse tail-bud following completion of gastrulation. BMP regulation is essential for VER function, but how these signals are transmitted between adjacent tissues is unclear. Results We investigated the idea that extracellular matrix components might be involved, using immunohistochemistry and in situ hybridisation to detect all known α, β and γ laminin chains and their mRNAs in the early tail bud. We identified an apparently novel laminin variant, comprising α5, β3 and γ2 chains, as a major component of the VER basement membrane at E9.5. Strikingly, only the mRNAs for these chains were co-expressed in VER cells, suggesting that lamin532 may be the sole basement membrane laminin at this stage. Since α6 integrin was also expressed in VER cells, this raises the possibility of cell-matrix interactions regulating BMP signalling at this site of caudal morphogenesis. Conclusions Laminin532 could interact with α6-containing integrin to direct differentiation of the specialised VER cells from surface ectoderm. PMID:22911573

Collagen VII plays a dual role in wound healing

PubMed Central

Nyström, Alexander; Velati, Daniela; Mittapalli, Venugopal R.; Fritsch, Anja; Kern, Johannes S.; Bruckner-Tuderman, Leena

2013-01-01

Although a host of intracellular signals is known to contribute to wound healing, the role of the cell microenvironment in tissue repair remains elusive. Here we employed 2 different mouse models of genetic skin fragility to assess the role of the basement membrane protein collagen VII (COL7A1) in wound healing. COL7A1 secures the attachment of the epidermis to the dermis, and its mutations cause a human skin fragility disorder coined recessive dystrophic epidermolysis bullosa (RDEB) that is associated with a constant wound burden. We show that COL7A1 is instrumental for skin wound closure by 2 interconnected mechanisms. First, COL7A1 was required for re-epithelialization through organization of laminin-332 at the dermal-epidermal junction. Its loss perturbs laminin-332 organization during wound healing, which in turn abrogates strictly polarized expression of integrin α6β4 in basal keratinocytes and negatively impacts the laminin-332/integrin α6β4 signaling axis guiding keratinocyte migration. Second, COL7A1 supported dermal fibroblast migration and regulates their cytokine production in the granulation tissue. These findings, which were validated in human wounds, identify COL7A1 as a critical player in physiological wound healing in humans and mice and may facilitate development of therapeutic strategies not only for RDEB, but also for other chronic wounds. PMID:23867500

Genetics Home Reference: laryngo-onycho-cutaneous syndrome

MedlinePlus

... This red, bumpy tissue is normally produced during wound healing and is usually replaced by skin cells as ... suggest that laminin 332 is also involved in wound healing. Additionally, researchers have proposed roles for laminin 332 ...

Overexpression of α3, β3 and γ2 chains of laminin-332 is associated with poor prognosis in pancreatic ductal adenocarcinoma

PubMed Central

Chen, Jun; Zhang, Hao; Luo, Jiansheng; Wu, Xiaokang; Li, Xueming; Zhao, Xinyi; Zhou, Dongkai; Yu, Shian

2018-01-01

Pancreatic ductal adenocarcinoma (PDA) is a worldwide health problem. Early diagnosis and assessment may enhance the quality of life and survival of patients. The present study investigated the potential correlations between the gene and protein expression of laminin-332 (LM-332 or laminin-5) and clinicopathological factors as well as evaluating its influence on the survival of patients with PDA. The expression of LM-332 subunit mRNAs in pancreatic carcinoma specimens from 37 patients was investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Using immunohistochemical methods, the protein expressions of the three chains of LM-322 (LNα3, LNβ3 and LNγ2) were determined in 96 pancreatic carcinoma specimens, for association analysis with clinicopathological characteristics from patient data. The results of the prognosis analysis of three mRNAs expression datasets were validated in The Cancer Genome Atlas datasets. RT-qPCR results indicated that the overall relative values of LNα3 and LNγ2 mRNAs were increased in pancreatic carcinoma compared with the control. In immunostaining analyses LNα3 and LNγ2 expression was observed in all tumor tissues from the 96 patient samples. The expression levels of LNα3, LNβ3 and LNγ2 were associated with each other. LNα3 and LNγ2 positivity was significantly associated with differentiation, depth of invasion and advanced stage (P<0.05). The samples were classified into three groups: Basement membrane (B) type, cytoplasmic (C) type and mixed (M) type, according to their LNγ2 immunohistochemical expression patterns. The B type correlated significantly with differentiation (P=0.010) and the M type was significantly associated with hepatic metastasis (P=0.031). Patients with B-type LNγ2 demonstrated significantly better outcomes than patients with the C or M type (P=0.012 and P=0.003, respectively). Overexpression of the α3, β3 and γ2 chains of LM-332 may serve an important role in the progression and prognosis of PDA. PMID:29928402

The caspase-generated cleavage product of Ets-1 p51 and Ets-1 p27, Cp17, induces apoptosis.

PubMed

Choul-Li, Souhaila; Tulasne, David; Aumercier, Marc

2016-11-04

The transcription factor Ets-1 is involved in various physiological processes and invasive pathologies. Human Ets-1 exists under three isoforms: p51, the predominant full-length isoform, p42 and p27, shorter alternatively spliced isoforms. We have previously demonstrated that Ets-1 p51, but not the spliced variant Ets-1 p42, is processed by caspases in vitro and during apoptosis. However, the caspase cleavage of the second spliced variant Ets-1 p27 remains to investigate. In the present study, we demonstrate that Ets-1 p27 is a cleavage substrate of caspases. We show that Ets-1 p27 is processed in vitro by caspase-3, resulting in three C-terminal fragments Cp20, Cp17 and Cp14. Similarly, Ets-1 p27 was cleaved during apoptotic cell death induced by anisomycin, producing fragments consistent with those observed in in vitro cleavage assay. These fragments are generated by cleavage at three sites located in the exon VII-encoded region of Ets-1 p27. As a functional consequences, Cp17 fragment, the major cleavage product generated during apoptosis, induced itself apoptosis when transfected into cells. Our results show that Ets-1 p27 is cleaved in the same manner as Ets-1 p51 within the exon VII-encoded region, thus generating a stable C-terminal fragment that induces cell death by initiating apoptosis. Copyright © 2016 Elsevier Inc. All rights reserved.

HGF potentiates extracellular matrix-driven migration of human myoblasts: involvement of matrix metalloproteinases and MAPK/ERK pathway.

PubMed

González, Mariela Natacha; de Mello, Wallace; Butler-Browne, Gillian S; Silva-Barbosa, Suse Dayse; Mouly, Vincent; Savino, Wilson; Riederer, Ingo

2017-10-10

The hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration. We evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting. We demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin. We demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving the migration of donor cells within the host tissue, a main issue regarding this approach.

Cell attachment functionality of bioactive conducting polymers for neural interfaces.

PubMed

Green, Rylie A; Lovell, Nigel H; Poole-Warren, Laura A

2009-08-01

Bioactive coatings for neural electrodes that are tailored for cell interactions have the potential to produce superior implants with improved charge transfer capabilities. In this study synthetically produced anionically modified laminin peptides DEDEDYFQRYLI and DCDPGYIGSR were used to dope poly(3,4-ethylenedioxythiophene) (PEDOT) electrodeposited on platinum (Pt) electrodes. Performance of peptide doped films was compared to conventional polymer PEDOT/paratoluene sulfonate (pTS) films using SEM, XPS, cyclic voltammetry, impedance spectroscopy, mechanical hardness and adherence. Bioactivity of incorporated peptides and their affect on cell growth was assessed using a PC12 neurite outgrowth assay. It was demonstrated that large peptide dopants produced softer PEDOT films with a minimal decrease in electrochemical stability, compared to the conventional dopant, pTS. Cell studies revealed that the YFQRYLI ligand retained neurite outgrowth bioactivity when DEDEDYFQRYLI was used as a dopant, but the effect was strongly dependant on initial cell attachment. Alternate peptide dopant, DCDPGYIGSR was found to impart superior cell attachment properties when compared to DEDEDYFQRYLI, but attachment on both peptide doped polymers could be enhanced by coating with whole native laminin.

In vitro evaluation of gel-encapsulated adipose derived stem cells: Biochemical cues for in vivo peripheral nerve repair.

PubMed

de Luca, A C; Fonta, C M; Raffoul, W; di Summa, P G; Lacour, S P

2018-03-01

Adipose-derived stem cells (ASC) are becoming one of the most exploited cells in peripheral nerve repair. They are fast-growing and able to protect neurons from apoptosis; they can reduce post-injury latency and the risk of muscle atrophy. This study evaluates laminin-loaded fibrin gel as an ASC-carrying scaffold for nerve repair. In vitro, ASC retained their proliferative activity but showed significant increase in proliferation rate when encapsulated in gels with low laminin concentrations (i.e., 1 μg/mL). We observed a linear decrease of ASC proliferation rate with increasing laminin concentration from 1 to 100 μg/mL. We next examined the effect of the ASC-carrying fibrin gels on in vitro dorsal root ganglia (DRG) neurite extension, then in vivo sciatic nerve regeneration in adult rats. The ASC-carrying gel was embedded in 15-mm-long, 1.5-mm-diameter polydimethylsiloxane regenerative conduits for in vivo evaluation. At 8-week post implantation, robust regeneration was observed across the long gap. Taken together, these results suggest ASC-carrying gels are a potential path to improve the efficacy of nerve regeneration through artificial guidance conduits and electrode nerve interfaces. Copyright © 2017 John Wiley & Sons, Ltd.

Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines.

PubMed

Rahman, Maryam; Reyner, Karina; Deleyrolle, Loic; Millette, Sebastien; Azari, Hassan; Day, Bryan W; Stringer, Brett W; Boyd, Andrew W; Johns, Terrance G; Blot, Vincent; Duggal, Rohit; Reynolds, Brent A

2015-03-01

Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture.

Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines

PubMed Central

Reyner, Karina; Deleyrolle, Loic; Millette, Sebastien; Azari, Hassan; Day, Bryan W.; Stringer, Brett W.; Boyd, Andrew W.; Johns, Terrance G.; Blot, Vincent; Duggal, Rohit; Reynolds, Brent A.

2015-01-01

Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture. PMID:25806119

Polymerized laminin-332 matrix supports rapid and tight adhesion of keratinocytes, suppressing cell migration.

PubMed

Kariya, Yoshinobu; Sato, Hiroki; Katou, Naoko; Kariya, Yukiko; Miyazaki, Kaoru

2012-01-01

Laminin-332 (α3ß3γ2) (Lm332) supports the stable anchoring of basal keratinocytes to the epidermal basement membrane, while it functions as a motility factor for wound healing and cancer invasion. To understand these contrasting activities of Lm332, we investigated Lm332 matrices deposited by normal human keratinocytes and other Lm332-expressing cell lines. All types of the cells efficiently deposited Lm332 on the culture plates in specific patterns. On the contrary, laminins containing laminin ß1 and/or γ1 chains, such as Lm511 and Lm311, were not deposited on the culture plates even if secreted into culture medium. The Lm332 deposition was not inhibited by function-blocking antibodies to the α3 and α6 integrins but was inhibited by sodium selenate, suggesting that sulfated glycosaminoglycans on cell surface, e.g. heparan sulfate proteoglycans, might be involved in the process. HEK293 cells overexpressing exogenous Lm332 (Lm332-HEK) almost exclusively deposited Lm332 on the plates. The deposited Lm332 matrix showed a mesh-like network structure as analyzed by electron microscopy, suggesting that Lm332 was highly polymerized. When biological activity was analyzed, the Lm332 matrix rather suppressed the migration of keratinocytes as compared with purified Lm332, which highly promoted the cell migration. The Lm332 matrix supported adhesion of keratinocytes much more strongly and stably than purified Lm332. Integrin α3ß1 bound to the Lm332 matrix at a three times higher level than purified Lm332. Normal keratinocytes prominently showed integrin α6ß4-containing, hemidesmosome-like structures on the Lm332 matrix but not on the purified one. These results indicate that the polymerized Lm332 matrix supports stable cell adhesion by interacting with both integrin α6ß4 and α3ß1, whereas unassembled soluble Lm332 supports cell migration.

Bridging defects in chronic spinal cord injury using peripheral nerve grafts combined with a chitosan-laminin scaffold and enhancing regeneration through them by co-transplantation with bone-marrow-derived mesenchymal stem cells: Case series of 14 patients

PubMed Central

Amr, Sherif M.; Gouda, Ashraf; Koptan, Wael T.; Galal, Ahmad A.; Abdel-Fattah, Dina Sabry; Rashed, Laila A.; Atta, Hazem M.; Abdel-Aziz, Mohammad T.

2014-01-01

Objective To investigate the effect of bridging defects in chronic spinal cord injury using peripheral nerve grafts combined with a chitosan-laminin scaffold and enhancing regeneration through them by co-transplantation with bone-marrow-derived mesenchymal stem cells. Methods In 14 patients with chronic paraplegia caused by spinal cord injury, cord defects were grafted and stem cells injected into the whole construct and contained using a chitosan-laminin paste. Patients were evaluated using the International Standards for Classification of Spinal Cord Injuries. Results Chitosan disintegration leading to post-operative seroma formation was a complication. Motor level improved four levels in 2 cases and two levels in 12 cases. Sensory-level improved six levels in two cases, five levels in five cases, four levels in three cases, and three levels in four cases. A four-level neurological improvement was recorded in 2 cases and a two-level neurological improvement occurred in 12 cases. The American Spinal Impairment Association (ASIA) impairment scale improved from A to C in 12 cases and from A to B in 2 cases. Although motor power improvement was recorded in the abdominal muscles (2 grades), hip flexors (3 grades), hip adductors (3 grades), knee extensors (2–3 grades), ankle dorsiflexors (1–2 grades), long toe extensors (1–2 grades), and plantar flexors (0–2 grades), this improvement was too low to enable them to stand erect and hold their knees extended while walking unaided. Conclusion Mesenchymal stem cell-derived neural stem cell-like cell transplantation enhances recovery in chronic spinal cord injuries with defects bridged by sural nerve grafts combined with a chitosan-laminin scaffold. PMID:24090088

Adhesive interactions of human multiple myeloma cell lines with different extracellular matrix molecules.

PubMed

Kibler, C; Schermutzki, F; Waller, H D; Timpl, R; Müller, C A; Klein, G

1998-06-01

Multiple myeloma represents a human B cell malignancy which is characterized by a predominant localization of the malignant cell clone within the bone marrow. With the exception of the terminal stage of the disease the myeloma tumor cells do not circulate in the peripheral blood. The bone marrow microenvironment is believed to play an important role in homing, proliferation and terminal differentiation of myeloma cells. Here we have studied the expression of several extracellular matrix (ECM) molecules in the bone marrow of multiple myeloma patients and analyzed their adhesive capacities with four different human myeloma-derived cell lines. All ECM molecules analyzed (tenascin, laminin, fibronectin, collagen types I, III, V and VI) could be detected in bone marrow cryostat sections of multiple myeloma patients. Adhesion assays showed that only laminin, the microfibrillar collagen type VI and fibronectin were strong adhesive components for the myeloma cell lines U266, IM-9, OPM-2 and NCI-H929. Tenascin and collagen type I were only weak adhesive substrates for these myeloma cells. Adhesion to laminin and fibronectin was beta 1-integrin-mediated since addition of anti-beta 1-integrin antibodies could inhibit the binding of the four different cell types to both matrix molecules. In contrast, integrins do not seem to be involved in binding of the myeloma cells to collagen type VI. Instead, inhibition of binding by heparin suggested that membrane-bound heparan sulfate proteoglycans are responsible ligands for binding to collagen type VI. Adhesion assays with several B-cell lines resembling earlier differentiation stages revealed only weak interactions with tenascin and no interactions with collagen type VI, laminin or fibronectin. In summary, the interactions of human myeloma cells with the extracellular matrix may explain the specific retention of the plasma cells within the bone marrow.

Thrombospondin-induced adhesion of human keratinocytes.

PubMed Central

Varani, J; Nickoloff, B J; Riser, B L; Mitra, R S; O'Rourke, K; Dixit, V M

1988-01-01

Human epidermal keratinocytes obtained from normal skin attached and spread on thrombospondin (TSP)-coated plastic dishes but failed to attach and spread on untreated plastic culture dishes or dishes coated with fibronectin or laminin. These cells produced minimal amounts of immunoreactive TSP. Keratinocytes established in culture on MCDB 153 medium and maintained for one to three passages in an undifferentiated state by continued cultivation in this low Ca2+-containing medium attached and spread on plastic dishes as well as on TSP-coated dishes. These cells also secreted significant amounts of TSP into the culture medium. When the keratinocytes were incubated for one day in MCDB 153 medium supplemented with high Ca2+ or in MEM (which also contains high Ca2+), there was decreased secretion of TSP into the culture medium concomitant with a reduction in attachment and spreading on plastic culture dishes. Proteolytic fragments of TSP were examined for stimulation of keratinocyte attachment and spreading. A 140-kd fragment produced by removal of the 25-kd heparin-binding domain had similar activity to the intact molecule while the 25-kd fragment was without effect. Further proteolytic treatment of the 140-kd fragment gave rise to a fragment consisting of 120 kd and 18-D moieties held together in disulphide linkage. This fragment did not support attachment or spreading. This study reveals that normal epidermal keratinocytes grown under conditions that maintain the undifferentiated state are able to produce TSP and utilize it as an attachment factor. When keratinocytes are grown under conditions that promote differentiation, ability to produce and utilize TSP is diminished. Since TSP is present at the dermal-epidermal junction and because TSP promotes keratinocyte attachment and spreading, this molecule may play an important role in maintaining normal growth of the basal cell layer and may also participate in reepithelialization during wound repair. Images PMID:2452837

Podocyte-Specific VEGF-A Gain of Function Induces Nodular Glomerulosclerosis in eNOS Null Mice

PubMed Central

Veron, Delma; Aggarwal, Pardeep K.; Velazquez, Heino; Kashgarian, Michael; Moeckel, Gilbert

2014-01-01

VEGF-A and nitric oxide are essential for glomerular filtration barrier homeostasis and are dysregulated in diabetic nephropathy. Here, we examined the effect of excess podocyte VEGF-A on the renal phenotype of endothelial nitric oxide synthase (eNOS) knockout mice. Podocyte-specific VEGF164 gain of function in eNOS−/− mice resulted in nodular glomerulosclerosis, mesangiolysis, microaneurysms, and arteriolar hyalinosis associated with massive proteinuria and renal failure in the absence of diabetic milieu or hypertension. In contrast, podocyte-specific VEGF164 gain of function in wild-type mice resulted in less pronounced albuminuria and increased creatinine clearance. Transmission electron microscopy revealed glomerular basement membrane thickening and podocyte effacement in eNOS−/− mice with podocyte-specific VEGF164 gain of function. Furthermore, glomerular nodules overexpressed collagen IV and laminin extensively. Biotin-switch and proximity ligation assays demonstrated that podocyte-specific VEGF164 gain of function decreased glomerular S-nitrosylation of laminin in eNOS−/− mice. In addition, treatment with VEGF-A decreased S-nitrosylated laminin in cultured podocytes. Collectively, these data indicate that excess glomerular VEGF-A and eNOS deficiency is necessary and sufficient to induce Kimmelstiel-Wilson–like nodular glomerulosclerosis in mice through a process that involves deposition of laminin and collagen IV and de-nitrosylation of laminin. PMID:24578128

Potent pro-inflammatory and pro-fibrotic molecules, osteopontin and galectin-3, are not major disease modulators of laminin α2 chain-deficient muscular dystrophy

PubMed Central

Gawlik, Kinga I.; Holmberg, Johan; Svensson, Martina; Einerborg, Mikaela; Oliveira, Bernardo M. S.; Deierborg, Tomas; Durbeej, Madeleine

2017-01-01

A large number of human diseases are caused by chronic tissue injury with fibrosis potentially leading to organ failure. There is a need for more effective anti-fibrotic therapies. Congenital muscular dystrophy type 1A (MDC1A) is a devastating form of muscular dystrophy caused by laminin α2 chain-deficiency. It is characterized with early inflammation and build-up of fibrotic lesions, both in patients and MDC1A mouse models (e.g. dy3K/dy3K). Despite the enormous impact of inflammation on tissue remodelling in disease, the inflammatory response in MDC1A has been poorly described. Consequently, a comprehensive understanding of secondary mechanisms (impaired regeneration, enhanced fibrosis) leading to deterioration of muscle phenotype in MDC1A is missing. We have monitored inflammatory processes in dy3K/dy3K muscle and created mice deficient in laminin α2 chain and osteopontin or galectin-3, two pro-inflammatory and pro-fibrotic molecules drastically increased in dystrophic muscle. Surprisingly, deletion of osteopontin worsened the phenotype of dy3K/dy3K mice and loss of galectin-3 did not reduce muscle pathology. Our results indicate that osteopontin could even be a beneficial immunomodulator in MDC1A. This knowledge is essential for the design of future therapeutic interventions for muscular dystrophies that aim at targeting inflammation, especially that osteopontin inhibition has been suggested for Duchenne muscular dystrophy therapy. PMID:28281577

Pairing-energy coefficients of neutron-rich fragments in spallation reactions

NASA Astrophysics Data System (ADS)

Niu, Fei; Ma, Chun-Wang

2018-02-01

The ratio of pairing-energy coefficient to temperature (a p/T) of neutron-rich fragments produced in spallation reactions has been investigated by adopting an isobaric yield ratio method deduced in the framework of a modified Fisher model. A series of spallation reactions, 0.5A and 1A GeV 208Pb + p, 1A GeV 238U + p, 0.5A GeV 136Xe + d, 0.2A, 0.5A and 1A GeV 136Xe + p, and 56Fe + p with incident energy ranging from 0.3A to 1.5A GeV, has been analysed. An obvious odd-even staggering is shown in the fragments with small neutron excess (I ≡ N-Z), and in the relatively small-A fragments which have large I. The values of a p/T for the fragments, with I from 0 to 36, have been found to be in a range from -4 to 4, and most values of a p/T fall in the range from -1 to 1. It is suggested that a small pairing-energy coefficient should be considered in predicting the cross sections of fragments in spallation reactions. It is also concluded that the method proposed in this article is not good for fragments with A/A s > 85% (where A s is the mass number of the spallation system). Supported by National Natural Science Foundation of China (U1732135), Natural Science Foundation of Henan Province (162300410179) and Henan Normal University for the Excellent Youth (154100510007)

Identification of beta-2 as a key cell adhesion molecule in PCa cell neurotropic behavior: a novel ex vivo and biophysical approach.

PubMed

Jansson, Keith H; Castillo, Deborah G; Morris, Joseph W; Boggs, Mary E; Czymmek, Kirk J; Adams, Elizabeth L; Schramm, Lawrence P; Sikes, Robert A

2014-01-01

Prostate cancer (PCa) is believed to metastasize through the blood/lymphatics systems; however, PCa may utilize the extensive innervation of the prostate for glandular egress. The interaction of PCa and its nerve fibers is observed in 80% of PCa and is termed perineural invasion (PNI). PCa cells have been observed traveling through the endoneurium of nerves, although the underlying mechanisms have not been elucidated. Voltage sensitive sodium channels (VSSC) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two auxiliary beta (β) subunits with inherent cell adhesion molecule (CAM) functions. The beta-2 isoform (gene SCN2B) interacts with several neural CAMs, while interacting putatively with other prominent neural CAMs. Furthermore, beta-2 exhibits elevated mRNA and protein levels in highly metastatic and castrate-resistant PCa. When overexpressed in weakly aggressive LNCaP cells (2BECFP), beta-2 alters LNCaP cell morphology and enhances LNCaP cell metastasis associated behavior in vitro. We hypothesize that PCa cells use beta-2 as a CAM during PNI and subsequent PCa metastasis. The objective of this study was to determine the effect of beta-2 expression on PCa cell neurotropic metastasis associated behavior. We overexpressed beta-2 as a fusion protein with enhanced cyan fluorescence protein (ECFP) in weakly aggressive LNCaP cells and observed neurotropic effects utilizing our novel ex vivo organotypic spinal cord co-culture model, and performed functional assays with neural matrices and atomic force microscopy. With increased beta-2 expression, PCa cells display a trend of enhanced association with nerve axons. On laminin, a neural CAM, overexpression of beta-2 enhances PCa cell migration, invasion, and growth. 2BECFP cells exhibit marked binding affinity to laminin relative to LNECFP controls, and recombinant beta-2 ectodomain elicits more binding events to laminin than BSA control. Functional overexpression of VSSC beta subunits in PCa may mediate PCa metastatic behavior through association with neural matrices.

"Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions"

PubMed Central

2012-01-01

Background Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (KD) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a KD of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro. PMID:22463075

Alport syndrome and Pierson syndrome: Diseases of the glomerular basement membrane.

PubMed

Funk, Steven D; Lin, Meei-Hua; Miner, Jeffrey H

2018-04-16

The glomerular basement membrane (GBM) is an important component of the kidney's glomerular filtration barrier. Like all basement membranes, the GBM contains type IV collagen, laminin, nidogen, and heparan sulfate proteoglycan. It is flanked by the podocytes and glomerular endothelial cells that both synthesize it and adhere to it. Mutations that affect the GBM's collagen α3α4α5(IV) components cause Alport syndrome (kidney disease with variable ear and eye defects) and its variants, including thin basement membrane nephropathy. Mutations in LAMB2 that impact the synthesis or function of laminin α5β2γ1 (LM-521) cause Pierson syndrome (congenital nephrotic syndrome with eye and neurological defects) and its less severe variants, including isolated congenital nephrotic syndrome. The very different types of kidney diseases that result from mutations in collagen IV vs. laminin are likely due to very different pathogenic mechanisms. A better understanding of these mechanisms should lead to targeted therapeutic approaches that can help people with these rare but important diseases. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

ILK modulates epithelial polarity and matrix formation in hair follicles.

PubMed

Rudkouskaya, Alena; Welch, Ian; Dagnino, Lina

2014-03-01

Hair follicle morphogenesis requires coordination of multiple signals and communication between its epithelial and mesenchymal constituents. Cell adhesion protein platforms, which include integrins and integrin-linked kinase (ILK), are critical for hair follicle formation. However, their precise contribution to this process is poorly understood. We show that in the absence of ILK, the hair follicle matrix lineage fails to develop, likely due to abnormalities in development of apical-basal cell polarity, as well as in laminin-511 and basement membrane assembly at the tip of the hair bud. These defects also result in impaired specification of hair matrix and absence of precortex and inner sheath root cell lineages. The molecular pathways affected in ILK-deficient follicles are similar to those in the absence of epidermal integrin β1 and include Wnt, but not sonic hedgehog, signaling. ILK-deficient hair buds also show abnormalities in the dermal papilla. Addition of exogenous laminin-511 restores morphological and molecular markers associated with hair matrix formation, indicating that ILK regulates hair bud cell polarity and functions upstream from laminin-511 assembly to regulate the developmental progression of hair follicles beyond the germ stage.

Sustained phenotypic reversion of junctional epidermolysis bullosa dog keratinocytes: Establishment of an immunocompetent animal model for cutaneous gene therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spirito, Flavia; Capt, Annabelle; Rio, Marcela Del

2006-01-20

Gene transfer represents the unique therapeutic issue for a number of inherited skin disorders including junctional epidermolysis bullosa (JEB), an untreatable genodermatose caused by mutations in the adhesion ligand laminin 5 ({alpha}3{beta}3{gamma}2) that is secreted in the extracellular matrix by the epidermal basal keratinocytes. Because gene therapy protocols require validation in animal models, we have phenotypically reverted by oncoretroviral transfer of the curative gene the keratinocytes isolated from dogs with a spontaneous form of JEB associated with a genetic mutation in the {alpha}3 chain of laminin 5. We show that the transduced dog JEB keratinocytes: (1) display a sustained secretionmore » of laminin 5 in the extracellular matrix; (2) recover the adhesion, proliferation, and clonogenic capacity of wild-type keratinocytes; (3) generate fully differentiated stratified epithelia that after grafting on immunocompromised mice produce phenotypically normal skin and sustain permanent expression of the transgene. We validate an animal model that appears particularly suitable to demonstrate feasibility, efficacy, and safety of genetic therapeutic strategies for cutaneous disorders before undertaking human clinical trials.« less

Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

NASA Astrophysics Data System (ADS)

Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

2011-07-01

Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Å resolution.

The Progressive Fragmentation of 332P/Ikeya-Murakami

NASA Astrophysics Data System (ADS)

Kleyna, J. T.; Ye, Q.-Z.; Hui, M.-T.; Meech, K. J.; Wainscoat, R.; Micheli, M.; Keane, J. V.; Weaver, H. A.; Weryk, R.

2016-08-01

We describe 2016 January-April observations of the fragments of 332P/Ikeya-Murakami, a comet earlier observed in a 2010 October outburst. We present photometry of the fragments and perform simulations to infer the time of breakup. We argue that the eastern-most rapidly brightening fragment (F4) best corresponds to the original nucleus, rather than the initial bright fragment F1. We compute radial and tangential nongravitational parameters, A 1 = (1.5 ± 0.4) × 10-8 au day-2 and (7.2 ± 1.9) × 10-9 au day-2 both are consistent with zero at the 4σ level. Monte Carlo simulations indicate that the fragments were emitted on the outbound journey well after the 2010 outburst, with bright fragment F1 splitting in early 2014 and the fainter fragments within months of the 2016 January recovery. Western fragment F7 is the oldest, dating from 2011. We suggest that the delayed onset of the splitting is consistent with a self-propagating crystallization of water ice.

Immunohistochemical expression of basement membrane proteins of verrucous carcinoma of the oral mucosa.

PubMed

Arduino, Paolo G; Carrozzo, Marco; Pagano, Marco; Broccoletti, Roberto; Scully, Crispian; Gandolfo, Sergio

2010-06-01

Squamous cell carcinoma (SCC) of the oral cavity is an extremely invasive tumour of stratified squamous epithelium that spreads throughout degradation of the basement membrane (BM) and extra-cellular matrix. Oral verrucous carcinoma (VC) is a rare low-grade variant of oral SCC that penetrates into the subepithelial connective tissue. It also has a different clinical behaviour from classical oral SCC. We investigated the immunohistochemical expression of laminin, laminin-5, collagen IV and fibronectin in VC, severe epithelial dysplasia (SED) and SCC in order to analyse if the pattern of these molecules expression contributes to the differences in the biological behaviour of these diseases. The staining pattern of laminin was less intensive in SCC compared with SED and VC, and collagen IV expression was increased in VC compared with SED. Discontinuities of laminin, collagen IV and fibronectin were more evident in SED than in VC. This study indicates that VC has a biological behaviour different from SED or SCC, observable by immunohistochemistry in the BM zone.

MID-INFRARED SPECTROPHOTOMETRIC OBSERVATIONS OF FRAGMENTS B AND C OF COMET 73P/SCHWASSMANN-WACHMANN 3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harker, David E.; Woodward, Charles E.; Kelley, Michael S.

2011-01-15

We present mid-infrared spectra and images from the Gemini-N (+ Michelle) observations of fragments SW3-[B] and SW3-[C] of the ecliptic (Jupiter family) comet 73P/Schwassmann-Wachmann 3 pre-perihelion. We observed fragment B soon after an outburst event (between 2006 April 16-26 UT) and detected crystalline silicates. The mineralogy of both fragments was dominated by amorphous carbon and amorphous pyroxene. The grain size distribution (assuming a Hanner-modified power law) for fragment SW3-[B] has a peak grain radius of a{sub p} {approx} 0.5 {mu}m, and for fragment SW3-[C], a{sub p} {approx} 0.3 {mu}m; both values are larger than the peak grain radius of themore » size distribution for the dust ejected from ecliptic comet 9P/Tempel 1 during the Deep Impact event (a{sub p} = 0.2 {mu}m). The silicate-to-carbon ratio and the silicate crystalline mass fraction for the submicron to micron-sized portion of the grain size distribution on the nucleus of fragment SW3-[B] were 1.341{sup +0.250}{sub -0.253} and 0.335{sup +0.089}{sub -0.112}, respectively, while on the nucleus of fragment SW3-[C] they were 0.671{sup +0.076}{sub -0.076} and 0.257{sup +0.039}{sub -0.043}, respectively. The similarity in mineralogy and grain properties between the two fragments implies that 73P/Schwassmann-Wachmann 3 is homogeneous in composition. The slight differences in grain size distribution and silicate-to-carbon ratio between the two fragments likely arise because SW3-[B] was actively fragmenting throughout its passage while the activity in SW3-[C] was primarily driven by jets. The lack of diverse mineralogy in the fragments SW3-[B] and SW3-[C] of 73P/Schwassmann-Wachmann 3 along with the relatively larger peak in the coma grain size distribution suggests that the parent body of this comet may have formed in a region of the solar nebula with different environmental properties than the natal sites where comet C/1995 O1 (Hale-Bopp) and 9P/Tempel 1 nuclei aggregated.« less

Wound Healing Is Defective in Mice Lacking Tetraspanin CD151

PubMed Central

Cowin, Allison J.; Adams, Damian; Geary, Sean M.; Wright, Mark D.; Jones, Jonathan C.R.; Ashman, Leonie K.

2010-01-01

The tetraspanin CD151 forms complexes in epithelial cell membranes with laminin-binding integrins α6 β4, α3 β1, and α6 β1, and modifies integrin-mediated cell migration in vitro. We demonstrate in this study that CD151 expression is upregulated in a distinct temporal and spatial pattern during wound healing, particularly in the migrating epidermal tongue at the wound edge, suggesting a role for CD151 in keratinocyte migration. We show that healing is significantly impaired in CD151-null mice, with wounds gaping wider at 7 days post-injury. The rate of re-epithelialization of the CD151-null wounds is adversely affected, with significantly less wound area being covered by migrating epidermal cells. Our studies reveal that although laminin levels are similar in wild-type and CD151-null wounds, the organization of the laminin in the basement membrane is impaired. Furthermore, upregulation of α6 and β4 integrin expression is adversely affected in CD151-null mice wounds. In contrast, we find no significant effect of CD151 gene knockout on α3 and β1 integrin expression in wound repair. We suggest that mice lacking the CD151 gene are defective in wound healing, primarily owing to impairment of the re-epithelialization process. This may be due to defective basement membrane formation and epithelial cell adhesion and migration. PMID:16410781

Laminin and collagen modulate expression of the small leucine-rich proteoglycan fibromodulin in rat anterior pituitary gland.

PubMed

Syaidah, Rahimi; Horiguchi, Kotaro; Fujiwara, Ken; Tsukada, Takehiro; Kikuchi, Motoshi; Yashiro, Takashi

2013-11-01

The anterior pituitary is a complex organ consisting of five types of hormone-producing cells, non–hormone-producing cells such as folliculostellate (FS) cells and vascular cells (endothelial cells and pericytes). We have previously shown that FS cells and pericytes produce fibromodulin, a small leucine-rich proteoglycan (SLRP). SLRPs are major proteoglycans of the extracellular matrix (ECM) and are important in regulating cell signaling pathways and ECM assembly. However, the mechanism regulating fibromodulin expression in the anterior pituitary has not been elucidated. Here, we investigate whether fibromodulin expression is modulated by major anterior pituitary ECM components such as laminin and type I collagen. Using transgenic rats expressing green fluorescent protein (GFP) specifically in FS cells, we examine fibromodulin expression in GFP-positive (FS cells) and GFP-negative cells (e.g., pericytes, endocrine cells and endothelial cells). Immunostaining and Western blot analysis were used to assess protein expression in the presence and absence of laminin or type I collagen. We confirmed fibromodulin expression in the pituitary and observed the up-regulation of fibromodulin in FS cells in the presence of ECM components. However, neither laminin nor type I collagen affected expression in GFP-negative cells. This suggests that laminin and type I collagen support the function of FS cells by increasing fibromodulin protein expression in the anterior pituitary.

The C-terminal fragment of big endothelin-1 does not potentiate the vasoactive effects of endothelin-1.

PubMed

Ottosson-Seeberger, A; Hemsén, A; Lundberg, J M; Ahlborg, G

1998-01-01

The aim was to study the cardiovascular effects of the C-terminal (22-38) fragment of big endothelin-1, which is produced by the cleavage of big endothelin-1 (big ET-1) to endothelin-1 (ET-1). An intravenous infusion of the (22-38) fragment (4, 8 and 12 pmol kg-1 min-1, each dose for 10 min) was given to 10 healthy subjects. Four control subjects received 0.9% saline. Two additional subjects received ET-1 1 (0.2 and 4 pmol kg-1 min-1, each dose for 20 min) alone or combined with an equimolar infusion of the (22-38) fragment on two separate occasions. The fragment infusion did not alter heart rate, mean arterial blood pressure, cardiac output, systemic or pulmonary vascular resistance, splanchnic, cerebral or forearm blood flow. Renal blood flow showed a slight fall (11%, P < 0.001) in the fragment group of the same magnitude as in a previous control study. After the fragment infusion, a decrease in mean pulmonary arterial pressure (MPAP) by 12% (P < 0.01) and in pulmonary capillary wedge pressure (PCWP) by 31% (P < 0.001) was noted, which did not differ from the pulmonary pressures in the saline-infused control group. The (22-38) fragment, when combined with ET-1, was not able to modify the effects of ET-1 on heart rate, mean arterial blood pressure, splanchnic and renal blood flow. Consequently, the exogenous (22-38) fragment does not seem to cause any significant cardiovascular effects in healthy humans.

Short-term effects of splenectomy on serum fibrosis indexes in liver cirrhosis patients.

PubMed

Kong, Degang; Chen, Xiuli; Lu, Shichun; Guo, Qingliang; Lai, Wei; Wu, Jushan; Lin, Dongdong; Zeng, Daobing; Duan, Binwei; Jiang, Tao; Cao, Jilei

2015-01-01

To determine the changing patterns of 4 liver fibrosis markers pre and post splenectomy (combined with pericardial devascularization [PCDV]) and to examine the short-term effects of splenectomy on liver fibrosis. Four liver fibrosis markers of 39 liver cirrhosis patients were examined pre, immediately post, 2 days post, and 1 week post (15 cases) splenectomy (combined with PCDV). The laminin (LN) level decreased immediately post surgery compared with the preoperative LN level (P < 0.05). The type IV collagen level decreased immediately post surgery compared with that pre surgery (P < 0.05), it significantly increased (P < 0.05) 2 days post surgery and significantly decreased 1 week post surgery (P < 0.05). Hyaluronic acid and the procollagen III N-terminal peptide levels increased significantly 2 days post surgery compared with that pre and immediately post surgery, they significantly decreased 1 week post surgery compared to 2 days post surgery (P < 0.05). In the short-term, the 4 liver fibrosis markers and the FibroScans post splenectomy showed characteristic changes, splenectomy may transiently initiate the degradation process of liver fibrosis.

Advanced Medical Countermeasures Consortium

DTIC Science & Technology

2010-10-01

acid, gamma-linolenic acid, and antioxidants in patients with acute respiratory distress syndrome. Enteral Nutrition in ARDS Study Group. Crit Care Med...molecular mechanism of action, and (3) efficacy of countermeasures for HD injury. Studies on the Yucatan mini-pig have demonstrated that laminin in...Lindsay CD, Rice P. Changes in connective tissue macromolecular components of Yucatan mini-pig skin following application of sulphur mustard vapour. Hum

Application and expression of HSV gG1 protein from a recombinant strain.

PubMed

Yan, Hua; Yan, Huishen; Huang, Tao; Li, Guocai; Gong, Weijuan; Jiao, Hongmei; Chen, Hongju; Ji, Mingchun

2010-11-01

According to the homologous sequence of glycoprotein G1 (gG1) genes from different strains of herpes simplex virus type 1 (HSV-1), a pair of primers was designed to amplify the gG1 gene fragment by PCR. Both the PCR product and the pGEX-4T-1 vector were digested with EcoR I and Sal I. The gG1 gene fragment was subcloned into the digested pGEX-4T-1 vector to construct a recombinant plasmid (pGEX-4T-1-gG1). The resultant plasmid was identified by dual-enzyme digestion and sequence analysis, and then transformed into Escherichia coli BL21 for expression under the induction of isopropyl β-D-1-thiogalactoside (IPTG). The expressed GST-gG1 fragment was detected by SDS-PAGE and purified by affinity chromatography. The properties of GST-gG1 fragment were evaluated by immunoblot analysis. Enzyme-linked immunosorbent assays (ELISAs) based on the GST-gG1 fragment were used for determining IgG or IgM to HSV-1. The GST-gG1 fragment-specific ELISA was also compared with ELISA with whole-HSV-1 antigen and commercial ELISA kits. The gG1-specific IgG and IFN-γ producing CD8+ T cells were induced in mice immunized with the GST-gG1 fragment. These results indicated that the GST-gG1 fragment could be used for replacing whole-virus antigen to detect IgM and IgG to HSV-1 in human sera, which provided a strategy for developing vaccines to protect HSV-1 infection using gG1 fragment. Copyright © 2010 Elsevier B.V. All rights reserved.

Macrophage Depletion Attenuates Extracellular Matrix Deposition and Ductular Reaction in a Mouse Model of Chronic Cholangiopathies

PubMed Central

Syn, Wing-Kin; Lagaisse, Kimberly; van Hul, Noemi; Heindryckx, Femke; Sowa, Jan-Peter; Peeters, Liesbeth; Van Vlierberghe, Hans; Leclercq, Isabelle A.; Canbay, Ali

2016-01-01

Chronic cholangiopathies, such as primary and secondary sclerosing cholangitis, are progressive disease entities, associated with periportal accumulation of inflammatory cells, encompassing monocytes and macrophages, peribiliary extracellular matrix (ECM) deposition and ductular reaction (DR). This study aimed to elucidate the relevance of macrophages in the progression of chronic cholangiopathies through macrophage depletion in a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) mouse model. One group of mice received a single i.p. injection of Clodronate encapsulated liposomes (CLOLipo) at day 7 of a 14 day DDC treatment, while control animals were co-treated with PBSLipo instead. Mice were sacrificed after 7 or respectively 14 days of treatment for immunohistochemical assessment of macrophage recruitment (F4/80), ECM deposition (Sirius Red, Laminin) and DR (CK19). Macrophage depletion during a 14 day DDC treatment resulted in a significant inhibition of ECM deposition. Porto-lobular migration patterns of laminin-rich ECM and ductular structures were significantly attenuated and a progression of DR was effectively inhibited by macrophage depletion. CLOLipo co-treatment resulted in a confined DR to portal regions without amorphous cell clusters. This study suggests that therapeutic options selectively directed towards macrophages might represent a feasible treatment for chronic cholestatic liver diseases. PMID:27618307

Suppression of lysyl-tRNA synthetase, KRS, causes incomplete epithelial-mesenchymal transition and ineffective cell‑extracellular matrix adhesion for migration.

PubMed

Nam, Seo Hee; Kang, Minkyung; Ryu, Jihye; Kim, Hye-Jin; Kim, Doyeun; Kim, Dae Gyu; Kwon, Nam Hoon; Kim, Sunghoon; Lee, Jung Weon

2016-04-01

The cell-adhesion properties of cancer cells can be targeted to block cancer metastasis. Although cytosolic lysyl-tRNA synthetase (KRS) functions in protein synthesis, KRS on the plasma membrane is involved in cancer metastasis. We hypothesized that KRS is involved in cell adhesion-related signal transduction for cellular migration. To test this hypothesis, colon cancer cells with modulated KRS protein levels were analyzed for cell-cell contact and cell-substrate adhesion properties and cellular behavior. Although KRS suppression decreased expression of cell-cell adhesion molecules, cells still formed colonies without being scattered, supporting an incomplete epithelial mesenchymal transition. Noteworthy, KRS-suppressed cells still exhibited focal adhesions on laminin, with Tyr397-phopshorylated focal adhesion kinase (FAK), but they lacked laminin-adhesion-mediated extracellular signal-regulated kinase (ERK) and paxillin activation. KRS, p67LR and integrin α6β1 were found to interact, presumably to activate ERK for paxillin expression and Tyr118 phosphorylation even without involvement of FAK, so that specific inhibition of ERK or KRS in parental HCT116 cells blocked cell-cell adhesion and cell-substrate properties for focal adhesion formation and signaling activity. Together, these results indicate that KRS can promote cell-cell and cell-ECM adhesion for migration.

SU9516 Increases α7β1 Integrin and Ameliorates Disease Progression in the mdx Mouse Model of Duchenne Muscular Dystrophy.

PubMed

Sarathy, Apurva; Wuebbles, Ryan D; Fontelonga, Tatiana M; Tarchione, Ashley R; Mathews Griner, Lesley A; Heredia, Dante J; Nunes, Andreia M; Duan, Suzann; Brewer, Paul D; Van Ry, Tyler; Hennig, Grant W; Gould, Thomas W; Dulcey, Andrés E; Wang, Amy; Xu, Xin; Chen, Catherine Z; Hu, Xin; Zheng, Wei; Southall, Noel; Ferrer, Marc; Marugan, Juan; Burkin, Dean J

2017-06-07

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by mutations in the dystrophin gene, resulting in a complete loss of the dystrophin protein. Dystrophin is a critical component of the dystrophin glycoprotein complex (DGC), which links laminin in the extracellular matrix to the actin cytoskeleton within myofibers and provides resistance to shear stresses during muscle activity. Loss of dystrophin in DMD patients results in a fragile sarcolemma prone to contraction-induced muscle damage. The α7β1 integrin is a laminin receptor protein complex in skeletal and cardiac muscle and a major modifier of disease progression in DMD. In a muscle cell-based screen for α7 integrin transcriptional enhancers, we identified a small molecule, SU9516, that promoted increased α7β1 integrin expression. Here we show that SU9516 leads to increased α7B integrin in murine C2C12 and human DMD patient myogenic cell lines. Oral administration of SU9516 in the mdx mouse model of DMD increased α7β1 integrin in skeletal muscle, ameliorated pathology, and improved muscle function. We show that these improvements are mediated through SU9516 inhibitory actions on the p65-NF-κB pro-inflammatory and Ste20-related proline alanine rich kinase (SPAK)/OSR1 signaling pathways. This study identifies a first in-class α7 integrin-enhancing small-molecule compound with potential for the treatment of DMD. Copyright © 2017 The American Society of Gene and Cell Therapy. All rights reserved.

Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin.

PubMed

Maciaszek, Jamie L; Partola, Kostyantyn; Zhang, Jing; Andemariam, Biree; Lykotrafitis, George

2014-12-18

Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding. Copyright © 2014 Elsevier Ltd. All rights reserved.

Quinapril decreases myocardial accumulation of extracellular matrix components in spontaneously hypertensive rats.

PubMed

Panizo, A; Pardo, J; Hernández, M; Galindo, M F; Cenarruzabeitia, E; Díez, J

1995-08-01

In genetic and acquired hypertension, a structural remodeling of the nonmyocyte compartment of myocardium, including the accumulation of fibrillar collagen and other components of the extracellular matrix (ECM) within the interstitium, represents a determinant of pathologic hypertrophy that leads to ventricular dysfunction. Therefore, to evaluate the potential benefit of the angiotensin converting enzyme (ACE) inhibitor quinapril in reversing the interstitial remodeling in spontaneously hypertensive rats (SHR) with established left ventricular hypertrophy (LVH), we treated 16-week-old male SHR with oral quinapril (average dose, 10 mg/kg body weight/day) for 20 weeks. Interstitial fibrosis was determined morphometrically using an automatic image analyzer. The amount of collagen was evaluated by measuring myocardial hydroxyproline concentration. Myocardial deposition of collagen molecules (types I, III, and IV) and other ECM components (fibronectin, laminin) was analyzed by immunohistochemical techniques using specific monoclonal antibodies. The activity of ACE was measured in left ventricular tissue by a fluorometric assay. In quinapril-treated SHR compared with 36-week-old untreated SHR and age- and sex-matched Wistar-Kyoto (WKY) controls, we found 1) a lesser degree of LVH and a lesser level of blood pressure, 2) a lesser degree of interstitial fibrosis, represented by less interstitial collagen volume fraction (5.73 +/- 0.45% v 3.42 +/- 0.28%, P < .05; WKY, 3.44 +/- 0.66%), 3) a lower hydroxyproline concentration (1.09 +/- 0.05 mumol/L/g dry weight/100 g body weight to 0.81 +/- 0.05 mumol/L/g dry weight/100 g body weight, P < .05; WKY, 0.96 +/- 0.06 mumol/L/g dry weight/100 g body weight), 4) a lesser presence of collagen fibers, and 5) a lesser presence of collagen IV, fibronectin, and laminin.(ABSTRACT TRUNCATED AT 250 WORDS)

IgG1-iS18 impedes the adhesive and invasive potential of early and late stage malignant melanoma cells.

PubMed

Munien, Carmelle; Rebelo, Thalia M; Ferreira, Eloise; Weiss, Stefan F T

2017-02-15

The 37kDa/67kDa laminin receptor (LRP/LR) is a non-integrin laminin receptor which is overexpressed in tumorigenic cells and supports progression of cancer via promoting metastasis, angiogenesis and telomerase activity and impediment of apoptosis. The present study investigates the role of LRP/LR on the metastatic potential of early (A375) and late (A375SM) stage malignant melanoma cells. Flow cytometry revealed that both early and late stage malignant melanoma cells display high levels of LRP/LR on their cell surface. Flow cytometry and western blot analysis showed that late stage malignant melanoma cells display significantly higher total and cell surface LRP/LR levels in comparison to early stage malignant melanoma cells and the poorly invasive breast cancer (MCF-7) control cell line. Targeting LRP/LR using the LRP/LR specific antibody IgG1-iS18 resulted in a significant reduction of the adhesive potential to laminin-1 and the invasive potential through the 'ECM-simulating' Matrigel™ of both early and late stage malignant melanoma cells. Furthermore, Pearson's correlation coefficient confirmed that increased LRP levels correlate with the increased invasive and adhesive potential in early and late stage melanoma cells. Thus, blocking LRP/LR using the IgG1-iS18 antibody may therefore be a promising therapeutic strategy for early and late stage malignant melanoma treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Template Based Design of Anti-Metastatic Drugs from the Active Conformation of Laminin Peptide II

DTIC Science & Technology

2001-01-01

p40 (LBP/p40) gene Maeda, M., Kawasaki, K., Mu, Y., Kamada, H., during sea urchin development. Exp. Cell Res. 221, Tsutsumi, Y., Smith, T. J. & Mayumi...represents the average of six replicates + SEM . minance of putative heparin-binding phage recov- ered from elution with peptide 11. Putative heparin...scrambled sequence peptide, WAQADSTPE, was used as a sequence specificity control. The data shown is the average of six replicate wells ± SEM . Statistics were

Addition of sphingosine-1-phosphate to human oocyte culture medium decreases embryo fragmentation.

PubMed

Hannoun, Antoine; Ghaziri, Ghina; Abu Musa, Antoine; Zreik, Tony G; Hajameh, Fatiha; Awwad, Johnny

2010-03-01

Apoptosis is implicated in the fragmentation of preimplantation mammalian embryos, yet the extent of this association remains controversial. The aim of this study was to assess the ability of sphingosine-1-phosphate (S1P), a known anti-apoptotic substance, to reduce the fragmentation rate of human preimplantation embryos when added to their culture microenvironment. Mature human oocytes were inseminated using intracytoplasmic sperm injection, incubated for 3 days and evaluated for embryo quality and fragmentation by the same embryologist. Oocytes in the study group were manipulated and cultured in culture medium supplemented with S1P to a 20 micromol/l concentration. A total of 46 patients donated 177 mature oocytes for the study group and 546 oocytes for the control group. The fertilization rate was significantly lower in the S1P-supplemented group (52.4% versus 67.3%; P=0.002) and the proportion of grade I embryos with less than 15% fragmentation was significantly higher in the same group (79.5% versus 53.9%; P<0.0001). Sphingosine-1-phosphate added to the culture medium of human preimplantation embryos is associated with a significantly lower fragmentation rate and hence better quality embryos. The clinical significance of these findings on reproductive outcome remains highly speculative awaiting further studies to translate this improvement in embryo quality into better pregnancy rates. Copyright 2009 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Laminin active peptide/agarose matrices as multifunctional biomaterials for tissue engineering.

PubMed

Yamada, Yuji; Hozumi, Kentaro; Aso, Akihiro; Hotta, Atsushi; Toma, Kazunori; Katagiri, Fumihiko; Kikkawa, Yamato; Nomizu, Motoyoshi

2012-06-01

Cell adhesive peptides derived from extracellular matrix components are potential candidates to afford bio-adhesiveness to cell culture scaffolds for tissue engineering. Previously, we covalently conjugated bioactive laminin peptides to polysaccharides, such as chitosan and alginate, and demonstrated their advantages as biomaterials. Here, we prepared functional polysaccharide matrices by mixing laminin active peptides and agarose gel. Several laminin peptide/agarose matrices showed cell attachment activity. In particular, peptide AG73 (RKRLQVQLSIRT)/agarose matrices promoted strong cell attachment and the cell behavior depended on the stiffness of agarose matrices. Fibroblasts formed spheroid structures on the soft AG73/agarose matrices while the cells formed a monolayer with elongated morphologies on the stiff matrices. On the stiff AG73/agarose matrices, neuronal cells extended neuritic processes and endothelial cells formed capillary-like networks. In addition, salivary gland cells formed acini-like structures on the soft matrices. These results suggest that the peptide/agarose matrices are useful for both two- and three-dimensional cell culture systems as a multifunctional biomaterial for tissue engineering. Copyright © 2012 Elsevier Ltd. All rights reserved.

Biodegradable neural cell culture sheet made of poly(lactic-co-glycolic acid) thin film with micropatterns of Dulbecco’s phosphate-buffered saline (-) containing laminin layers

NASA Astrophysics Data System (ADS)

Nakamura, Yuki; Horiuchi, Shunpu; Nishioka, Yasushiro

2018-02-01

In the regenerative medicine field of nervous systems, techniques used to fabricate microstructures of neurons on flexible and biodegradable substrates have attracted attention. In this research, biodegradable and flexible neuron culture thin films that enable the selective axonal outgrowth of neurons were fabricated using poly(lactic-co-glycolic acid) (PLGA) thin films with micropatterns of Dulbecco’s phosphate-buffered saline (D-PBS) (-) containing laminin layers. The 100-µm-thick PLGA thin films were fabricated by diluting PLGA in acetone (5% w/w) and the solution was distributed onto a poly(dimethylsiloxane) (PDMS) mold. D-PBS (-) micropatterns containing laminin layers with widths of 10-150 µm were fabricated by micromolding in capillaries (MIMIC) and the microstencil method. Rat neurons were selectively cultured for 3 d on the laminin micropatterns; using the MIMIC method, the cells properly adhered to a pattern wider than 30 µm, while with the microstencil method, the necessary pattern width for proper adhesion was more than 50 µm.

Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alcaraz, Jordi; Xu, Ren; Mori, Hidetoshi

2008-10-20

In the mammary gland, epithelial cells are embedded in a 'soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for {beta}-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of {beta}-casein expression required both laminin signalling and a 'soft' extracellular matrix, as is the case in normal tissuesmore » in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of {beta}-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.« less

An adhesome comprising laminin, dystroglycan and myosin IIA is required during notochord development in Xenopus laevis.

PubMed

Buisson, Nicolas; Sirour, Cathy; Moreau, Nicole; Denker, Elsa; Le Bouffant, Ronan; Goullancourt, Aline; Darribère, Thierry; Bello, Valérie

2014-12-01

Dystroglycan (Dg) is a transmembrane receptor for laminin that must be expressed at the right time and place in order to be involved in notochord morphogenesis. The function of Dg was examined in Xenopus laevis embryos by knockdown of Dg and overexpression and replacement of the endogenous Dg with a mutated form of the protein. This analysis revealed that Dg is required for correct laminin assembly, for cell polarization during mediolateral intercalation and for proper differentiation of vacuoles. Using mutations in the cytoplasmic domain, we identified two sites that are involved in cell polarization and are required for mediolateral cell intercalation, and a site that is required for vacuolation. Furthermore, using a proteomic analysis, the cytoskeletal non-muscle myosin IIA has been identified for the first time as a molecular link between the Dg-cytoplasmic domain and cortical actin. The data allowed us to identify the adhesome laminin-Dg-myosin IIA as being required to maintain the cortical actin cytoskeleton network during vacuolation, which is crucial to maintain the shape of notochordal cells. © 2014. Published by The Company of Biologists Ltd.

Expression of the proteoglycan syndecan-4 and the mechanism by which it mediates stress fiber formation in folliculostellate cells in the rat anterior pituitary gland.

PubMed

Horiguchi, Kotaro; Kouki, Tom; Fujiwara, Ken; Tsukada, Takehiro; Ly, Floren; Kikuchi, Motoshi; Yashiro, Takashi

2012-08-01

Folliculostellate (FS) cells in the anterior pituitary gland appear to have multifunctional properties. FS cells connect to each other at gap junctions and thereby form a histological and functional network. We have performed a series of studies on network formation in FS cells and recently reported that FS cells markedly prolong their cytoplasmic processes and form numerous interconnections with neighboring FS cells in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. In this study, we investigated the mechanism of this extension of FS cell cytoplasmic processes under the influence of laminin and found that laminin promoted stress fiber formation within FS cells. Next, we noted that formation of stress fibers in FS cells was mediated by syndecan-4, a transmembrane proteoglycan that binds ECM and soluble factors via their extracellular glycosaminoglycan chain. We then observed that expressions of syndecan-4 and α-actinin (a microfilament bundling protein that cross-links actin stress fibers in FS cells) were upregulated by laminin. Using specific siRNA of syndecan-4, actin polymerization of FS cells was inhibited. Our findings suggest that FS cells received a signal from laminin-syndecan-4 interaction, which resulted in morphological changes, and that the formation of a morphological and functional network in FS cells was transduced by a syndecan-4-dependent mechanism in the presence of ECM.

Active zones of mammalian neuromuscular junctions: formation, density, and aging.

PubMed

Nishimune, Hiroshi

2012-12-01

Presynaptic active zones are synaptic vesicle release sites that play essential roles in the function and pathology of mammalian neuromuscular junctions (NMJs). The molecular mechanisms of active zone organization use presynaptic voltage-dependent calcium channels (VDCCs) in NMJs as scaffolding proteins. VDCCs interact extracellularly with the muscle-derived synapse organizer, laminin β2 and interact intracellularly with active zone-specific proteins, such as Bassoon, CAST/Erc2/ELKS2alpha, ELKS, Piccolo, and RIMs. These molecular mechanisms are supported by studies in P/Q- and N-type VDCCs double-knockout mice, and they are consistent with the pathological conditions of Lambert-Eaton myasthenic syndrome and Pierson syndrome, which are caused by autoantibodies against VDCCs or by a laminin β2 mutation. During normal postnatal maturation, NMJs maintain the density of active zones, while NMJs triple their size. However, active zones become impaired during aging. Propitiously, muscle exercise ameliorates the active zone impairment in aged NMJs, which suggests the potential for therapeutic strategies. © 2012 New York Academy of Sciences.

Endothelin and sex hormones modulate the fibronectin synthesis by cultured human skin scleroderma fibroblasts

PubMed Central

Soldano, S; Montagna, P; Villaggio, B; Parodi, A; Gianotti, G; Sulli, A; Seriolo, B; Secchi, M E; Cutolo, M

2009-01-01

Objective: To evaluate the influence of endothelin-1 (ET-1) and sex hormones on cell proliferation and extracellular matrix (ECM) synthesis (ie, fibronectin, laminin) by cultured normal and scleroderma (SSc) human skin fibroblasts (FBs). Methods: Primary cultures of FBs were treated with ET-1 and sex hormones (17β-oestradiol or testosterone) for 24 h. Cell growth was analysed by methiltetrazolium salt test, ECM synthesis was evaluated by immunocytochemistry and western blot, both at 24 h. Results: In normal FBs, ET-1 and 17β-oestradiol, as well as their combination, increased cell growth (p<0.001, p<0.001, p<0.01 vs untreated cells (control), respectively) and fibronectin synthesis (p<0.05, p<0.05, p<0.01 vs control, respectively). By contrast, testosterone either alone or in combination with ET-1 did not influence cell proliferation, but decreased fibronectin synthesis (p<0.05, testosterone vs control). In SSc FBs, ET-1 and 17β-oestradiol alone or their combination induced an increased fibronectin synthesis (p<0.05, p<0.05, p<0.01 vs control, respectively). Unexpectedly, testosterone induced an increase of fibronectin synthesis (p<0.05 vs control). Conclusions: ET-1 and 17β-oestradiol seem to exert a profibrotic effect in normal and SSc culture FBs and might suggest their synergistic effect in the pathogenesis of the fibrotic process in SSc. PMID:18952637

Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation

PubMed Central

Seeger, Tanja; Hart, Melanie; Patarroyo, Manuel; Rolauffs, Bernd; Aicher, Wilhelm K.; Klein, Gerd

2015-01-01

Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells. PMID:26406476

ILK modulates epithelial polarity and matrix formation in hair follicles

PubMed Central

Rudkouskaya, Alena; Welch, Ian; Dagnino, Lina

2014-01-01

Hair follicle morphogenesis requires coordination of multiple signals and communication between its epithelial and mesenchymal constituents. Cell adhesion protein platforms, which include integrins and integrin-linked kinase (ILK), are critical for hair follicle formation. However, their precise contribution to this process is poorly understood. We show that in the absence of ILK, the hair follicle matrix lineage fails to develop, likely due to abnormalities in development of apical–basal cell polarity, as well as in laminin-511 and basement membrane assembly at the tip of the hair bud. These defects also result in impaired specification of hair matrix and absence of precortex and inner sheath root cell lineages. The molecular pathways affected in ILK-deficient follicles are similar to those in the absence of epidermal integrin β1 and include Wnt, but not sonic hedgehog, signaling. ILK-deficient hair buds also show abnormalities in the dermal papilla. Addition of exogenous laminin-511 restores morphological and molecular markers associated with hair matrix formation, indicating that ILK regulates hair bud cell polarity and functions upstream from laminin-511 assembly to regulate the developmental progression of hair follicles beyond the germ stage. PMID:24371086

β1 integrin signaling promotes neuronal migration along vascular scaffolds in the post-stroke brain.

PubMed

Fujioka, Teppei; Kaneko, Naoko; Ajioka, Itsuki; Nakaguchi, Kanako; Omata, Taichi; Ohba, Honoka; Fässler, Reinhard; García-Verdugo, José Manuel; Sekiguchi, Kiyotoshi; Matsukawa, Noriyuki; Sawamoto, Kazunobu

2017-02-01

Cerebral ischemic stroke is a main cause of chronic disability. However, there is currently no effective treatment to promote recovery from stroke-induced neurological symptoms. Recent studies suggest that after stroke, immature neurons, referred to as neuroblasts, generated in a neurogenic niche, the ventricular-subventricular zone, migrate toward the injured area, where they differentiate into mature neurons. Interventions that increase the number of neuroblasts distributed at and around the lesion facilitate neuronal repair in rodent models for ischemic stroke, suggesting that promoting neuroblast migration in the post-stroke brain could improve efficient neuronal regeneration. To move toward the lesion, neuroblasts form chain-like aggregates and migrate along blood vessels, which are thought to increase their migration efficiency. However, the molecular mechanisms regulating these migration processes are largely unknown. Here we studied the role of β1-class integrins, transmembrane receptors for extracellular matrix proteins, in these migrating neuroblasts. We found that the neuroblast chain formation and blood vessel-guided migration critically depend on β1 integrin signaling. β1 integrin facilitated the adhesion of neuroblasts to laminin and the efficient translocation of their soma during migration. Moreover, artificial laminin-containing scaffolds promoted neuroblast chain formation and migration toward the injured area. These data suggest that laminin signaling via β1 integrin supports vasculature-guided neuronal migration to efficiently supply neuroblasts to injured areas. This study also highlights the importance of vascular scaffolds for cell migration in development and regeneration. Copyright © 2017 3-V Biosciences. Published by Elsevier B.V. All rights reserved.

Anti-LRP/LR Specific Antibody IgG1-iS18 Impedes Adhesion and Invasion of Liver Cancer Cells

PubMed Central

Chetty, Carryn; Khumalo, Thandokuhle; Da Costa Dias, Bianca; Reusch, Uwe; Knackmuss, Stefan; Little, Melvyn; Weiss, Stefan F. T.

2014-01-01

Two key events, namely adhesion and invasion, are pivotal to the occurrence of metastasis. Importantly, the 37 kDa/67 kDa laminin receptor (LRP/LR) has been implicated in enhancing these two events thus facilitating cancer progression. In the current study, the role of LRP/LR in the adhesion and invasion of liver cancer (HUH-7) and leukaemia (K562) cells was investigated. Flow cytometry revealed that the HUH-7 cells displayed significantly higher cell surface LRP/LR levels compared to the poorly-invasive breast cancer (MCF-7) control cells, whilst the K562 cells displayed significantly lower cell surface LRP/LR levels in comparison to the MCF-7 control cells. However, Western blotting and densitometric analysis revealed that all three tumorigenic cell lines did not differ significantly with regards to total LRP/LR levels. Furthermore, treatment of liver cancer cells with anti-LRP/LR specific antibody IgG1-iS18 (0.2 mg/ml) significantly reduced the adhesive potential of cells to laminin-1 and the invasive potential of cells through the ECM-like Matrigel, whilst leukaemia cells showed no significant differences in both instances. Additionally, Pearson's correlation coefficients suggested direct proportionality between cell surface LRP/LR levels and the adhesive and invasive potential of liver cancer and leukaemia cells. These findings suggest the potential use of anti-LRP/LR specific antibody IgG1-iS18 as an alternative therapeutic tool for metastatic liver cancer through impediment of the LRP/LR- laminin-1 interaction. PMID:24798101

Inhibition of EMMPRIN and MMP-9 Expression by Epigallocatechin-3-Gallate through 67-kDa Laminin Receptor in PMA-Induced Macrophages.

PubMed

Wang, Qi-Ming; Wang, Hao; Li, Ya-Fei; Xie, Zhi-Yong; Ma, Yao; Yan, Jian-Jun; Gao, Yi Fan Wei; Wang, Ze-Mu; Wang, Lian-Sheng

2016-01-01

It is well documented that overexpression of EMMPRIN (extracellular matrix metalloproteinase inducer) and MMPs (matrix metalloproteinases) by monocytes/macrophages plays an important role in atherosclerotic plaque rupture. Green tea polyphenol epigallocatechin-3-gallate (EGCG) has a variety of pharmacological properties and exerts cardiovascular protective effects. Recently, the 67-kD laminin receptor (67LR) has been identified as a cell surface receptor of EGCG. The aim of the present study was to evaluate the effects of EGCG on the expression of EMMPRIN and MMP-9 in PMA-induced macrophages, and the potential mechanisms underlying its effects. Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA). Protein expression and MMP-9 activity were assayed by Western blot and Gelatin zymography, respectively. Real-time PCR was used to examine EMMPRIN and MMP-9 mRNA expression. We showed that EGCG (10-50µmol/L) significantly inhibited the expression of EMMPRIN and MMP-9 and activation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and c-Jun N-terminal kinase (JNK) in PMA-induced macrophages. Downregulation of EMMPRIN by gene silencing hindered PMA-induced MMP-9 secretion and expression, indicating an important role of EMMPRIN in the inhibition of MMP-9 by EGCG. Moreover, 67LR was involved in EGCG-mediated suppression of EMMPRIN and MMP-9 expression. Anti-67LR antibody treatment led to abrogation of the inhibitory action of EGCG on the expression of EMMPRIN and MMP-9 and activation of ERK1/2, p38, and JNK. Our results indicate that EGCG restrains EMMPRIN and MMP-9 expression via 67LR in PMA-induced macrophages, which also suggests that EGCG may be a possible therapeutic agent for stabilizing atherosclerotic plaque. © 2016 The Author(s) Published by S. Karger AG, Basel.

Effects of third fragment size and displacement on non-union of femoral shaft fractures after locking for intramedullary nailing.

PubMed

Lee, J R; Kim, H-J; Lee, K-B

2016-04-01

The femoral shaft fractures with large fragments makes anatomical reduction challenging and often results in non-union. In some studies, the degree of fragment displacement was reported to have affected non-union, but the association between the one fragment size and degree of displacement has not been fully clarified. Therefore we performed a retrospective study to assess: (1) the more influential factor of non-union: the degree of fragment displacement, or the fragment size? (2) the non-union rates according to different sizes and degrees of displacement. The degree of displacement is the more potent factor of non-union than the third fragment size in femoral shaft fractures. We assessed retrospectively 64 cases, which could be followed up for longer than one year. Fragments were divided according to the length of their long axis into three groups: group A (0-3.9cm), (n=21); group B (4-7.9cm), (n=22); group C (8cm or more), (n=21). Fragment displacement was also assessed in the proximal (P) or distal (D) end to the nearest cortex of the femoral shaft, and divided into the following groups: group P1 (n=44) or D1 (n=47), (0-9mm); group P2 (n=10) or D2 (n=11), (10-19mm); group P3 (n=7) or D3 (n=3), (20-29mm); and group P4 (n=3) or D4 (n=3), (30mm or more). The bone union rate was 86% in the small (less than 8cm) fragment groups and 71% in the large (8cm or more) fragment group (P=0.046). With respect to the degree of displacement, the union rate was lower (P=0.001) and the average union time was longer (P=0.012) in the 20mm or more group for both the proximal fragment part and the distal fragment part (P=0.002, P=0.014). A logistic regression analysis underlined the displacement in the proximal site (OR: 0.298, 95% CI: 0.118-0.750) as in the distal site (OR: 0.359, 95% CI: 0.162-0.793) as a larger effect on union rate than the fragment size that as no effect in logistic regression (OR 3.8, 95% CI: 0.669-21.6). Non-union develops significantly more frequently in femoral shaft fractures with fragments 8cm or longer or when the displacement in the proximal area is 20mm or greater and 10mm or greater in the distal area during the intramedullary nailing procedure. Regarding union rate, the degree of displacement has more influence than the third fragment size in femoral shaft fractures. IV, retrospective cohort study. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

The activity of the anti-apoptotic fragment generated by the caspase-3/p120 RasGAP stress-sensing module displays strict Akt isoform specificity.

PubMed

Vanli, Güliz; Peltzer, Nieves; Dubuis, Gilles; Widmann, Christian

2014-12-01

The caspase-3/p120 RasGAP module acts as a stress sensor that promotes pro-survival or pro-death signaling depending on the intensity and the duration of the stressful stimuli. Partial cleavage of p120 RasGAP generates a fragment, called fragment N, which protects stressed cells by activating Akt signaling. Akt family members regulate many cellular processes including proliferation, inhibition of apoptosis and metabolism. These cellular processes are regulated by three distinct Akt isoforms: Akt1, Akt2 and Akt3. However, which of these isoforms are required for fragment N mediated protection have not been defined. In this study, we investigated the individual contribution of each isoform in fragment N-mediated cell protection against Fas ligand induced cell death. To this end, DLD1 and HCT116 isogenic cell lines lacking specific Akt isoforms were used. It was found that fragment N could activate Akt1 and Akt2 but that only the former could mediate the protective activity of the RasGAP-derived fragment. Even overexpression of Akt2 or Akt3 could not rescue the inability of fragment N to protect cells lacking Akt1. These results demonstrate a strict Akt isoform requirement for the anti-apoptotic activity of fragment N. Copyright © 2014 Elsevier Inc. All rights reserved.

Extracellular matrix molecules and cell adhesion molecules induce neurites through different mechanisms

PubMed Central

1990-01-01

It has recently become clear that both extracellular matrix (ECM) glycoproteins and various cell adhesion molecules (CAMs) can promote neurite outgrowth from primary neurons, though little is known of the intracellular mechanisms through which these signals are transduced. We have previously obtained evidence that protein kinase C function is an important part of the neuronal response to laminin (Bixby, J.L. 1989. Neuron. 3:287-297). Because such CAMs as L1 (Lagenauer, C., and V. Lemmon. 1987. Proc. Natl. Acad. Sci. USA. 84:7753-7757) and N-cadherin (Bixby, J.L. and R. Zhang. 1990. J. Cell Biol. 110:1253-1260) can be purified and used as substrates to promote neurite growth, we have now tested whether the response to CAMs is similarly dependent on protein kinase C. We find that inhibition of protein kinase C inhibits growth on fibronectin or collagen as well as on laminin. In contrast, C kinase inhibition actually potentiates the initial growth response to L1 or N- cadherin. The later "phase" of outgrowth on both of these CAMs is inhibited, however. Additionally, phorbol esters, which have no effect on neurite growth when optimal laminin concentrations are used, potentiate growth even on optimal concentrations of L1 or N-cadherin. The results indicate that different intracellular mechanisms operate during initial process outgrowth on ECM substrates as compared to CAM substrates, and suggest that protein kinase C function is required for continued neurite growth on each of these glycoproteins. PMID:2277083

Quantitative experimental modelling of fragmentation during explosive volcanism

NASA Astrophysics Data System (ADS)

Thordén Haug, Ø.; Galland, O.; Gisler, G.

2012-04-01

Phreatomagmatic eruptions results from the violent interaction between magma and an external source of water, such as ground water or a lake. This interaction causes fragmentation of the magma and/or the host rock, resulting in coarse-grained (lapilli) to very fine-grained (ash) material. The products of phreatomagmatic explosions are classically described by their fragment size distribution, which commonly follows power laws of exponent D. Such descriptive approach, however, considers the final products only and do not provide information on the dynamics of fragmentation. The aim of this contribution is thus to address the following fundamental questions. What are the physics that govern fragmentation processes? How fragmentation occurs through time? What are the mechanisms that produce power law fragment size distributions? And what are the scaling laws that control the exponent D? To address these questions, we performed a quantitative experimental study. The setup consists of a Hele-Shaw cell filled with a layer of cohesive silica flour, at the base of which a pulse of pressurized air is injected, leading to fragmentation of the layer of flour. The fragmentation process is monitored through time using a high-speed camera. By varying systematically the air pressure (P) and the thickness of the flour layer (h) we observed two morphologies of fragmentation: "lift off" where the silica flour above the injection inlet is ejected upwards, and "channeling" where the air pierces through the layer along sub-vertical conduit. By building a phase diagram, we show that the morphology is controlled by P/dgh, where d is the density of the flour and g is the gravitational acceleration. To quantify the fragmentation process, we developed a Matlab image analysis program, which calculates the number and sizes of the fragments, and so the fragment size distribution, during the experiments. The fragment size distributions are in general described by power law distributions of exponents D. This procedure allows, for the first time, to determine the scaling laws that govern the number of fragments (N), the average size of the fragments (A) and D. We show that (1) N scales with P^(1/2), (2) A scales with P^(-2/3), (3) D scales with P^(1/5). Our experimental procedure thus appears as a unique tool to unravel the complex physics of fragmentation during phreatomagmatic explosions.

Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line.

PubMed

Hirako, Yoshiaki; Yonemoto, Yuki; Yamauchi, Tomoe; Nishizawa, Yuji; Kawamoto, Yoshiyuki; Owaribe, Katsushi

2014-06-10

Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and β4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases. Copyright © 2014 Elsevier Inc. All rights reserved.

Effect of Chinese traditional compound, Gan-fu-kang, on CCl(4)-induced liver fibrosis in rats and its probable molecular mechanisms.

PubMed

Xu, Ting-Ting; Jiang, Miao-Na; Li, Cong; Che, Ying; Jia, Yu-Jie

2007-03-01

To explore the antifibrotic effect of traditional Chinese medicine compound Gan-fu-kang (GFK) on CCl(4)-induced liver fibrosis in rats and its probable mechanisms. The effects of GFK on CCl(4)-induced liver fibrosis were tested in rats. The liver histopathology was examined by light microscope, polaring microscope and electron microscope. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assayed and the content of albumin (ALB) and hydroxyproline in the liver was measured. The expression of transforming growth factor-beta(1) (TGF-beta(1)) and laminin (LN) was determined by immunohistochemistry. Semi-quantitive computation of collagen types I and III and laminin was done. The expression of MMP-2 and TIMP-1 was assayed by reverse transcription polymerase chain reaction (RT-PCR). Upon pathological examination, GFK treatment had significantly reversed liver fibrosis. Hepatic extracellular matrix (ECM) deposition was significantly reduced, as evidenced by the reduction of the content of hydroxyproline, collagen types I and III, and laminin. Hepatic function was improved by GFK treatment, as evidenced by the increase of plasma ALB and A/G, and by the decrease of serum ALT and AST. TGF-beta(1) in liver was significantly reduced. A significant expression of MMP-2 and TIMP-1 mRNA in liver were downregulated after GFK treatment. The traditional Chinese medicine compound recipe GFK has an antifibrotic effect on CCl(4)-induced liver fibrosis in rats, which improves hepatic function and lessens the deposition of collagen in the liver. The probable antifibrotic mechanisms were: inhibiting the expression of TGF-beta(1) and decreasing expressions of MMP-2 and TIMP-1.

Lsa21, a novel leptospiral protein binding adhesive matrix molecules and present during human infection

PubMed Central

Atzingen, Marina V; Barbosa, Angela S; De Brito, Thales; Vasconcellos, Silvio A; de Morais, Zenáide M; Lima, Dirce MC; Abreu, Patricia AE; Nascimento, Ana LTO

2008-01-01

Background It has been well documented over past decades that interaction of pathogens with the extracellular matrix (ECM) plays a primary role in host cell attachment and invasion. Adherence to host tissues is mediated by surface-exposed proteins expressed by the microorganisms during infection. The mechanisms by which pathogenic leptospires invade and colonize the host remain poorly understood since few virulence factors contributing to the pathogenesis of the disease have been identified. Whole-genome sequencing analysis of L. interrogans allowed identification of a repertoire of putative leptospiral surface proteins. Results Here, we report the identification and characterization of a new leptospiral protein that exhibits extracellular matrix-binding properties, called as Lsa21 (leptospiral surface adhesin, 21 kDa). Compatible with its role in adhesion, the protein was shown to be surface-exposed by indirect immunofluorescence. Attachment of Lsa21 to laminin, collagen IV, and plasma fibronectin was specific and dose dependent. Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction. The gene coding for Lsa21 is present in pathogenic strains belonging to the L. interrogans species but was not found in the saprophytic L. biflexa serovar Patoc strain Patoc 1. Loss of gene expression occurs upon culture attenuation of pathogenic strains. Environmental factors such as osmolarity and temperature affect Lsa21 expression at the transcriptional level. Moreover, anti-Lsa21 serum labeled liver and kidney tissues of human fatal cases of leptospirosis. Conclusion Our data suggest a role of Lsa21 in the pathogenesis of leptospirosis. PMID:18445272

Role for cell adhesion and glycosyl (HNK-1 and oligomannoside) recognition in the sharpening of the regenerating retinotectal projection in goldfish.

PubMed

Schmidt, J T; Schachner, M

1998-12-01

Cell-adhesion molecules (CAMs) are thought to play crucial roles in development and plasticity in the nervous system. This study tested for a role for cell adhesion and in particular, the recognition of two glycosyl epitopes (HNK-1 and oligomannoside) in the activity-driven sharpening of the retinotopic map formed by the regenerating retinal fibers of goldfish. HNK-1 is a prominent glycosyl epitope on many CAMs and extracellular matrix (ECM) molecules, including NCAM, L1, ependymin, and integrins, which have all been implicated in synaptic plasticity. To test for a role of HNK-1 in the sharpening process, we used osmotic minipumps to infuse HNK-1 antibodies for 7-21 days into the tectal ventricle starting at 18 days after optic nerve crush. Retinotopic maps recorded at 76-86 days postcrush showed a lack of sharpening similar to that seen previously with two antibodies to ependymin, an HNK-1-positive ECM component present in cerebrospinal fluid. The multiunit receptive fields at each point averaged 26 degrees versus 11-12 degrees in regenerates infused with control antibodies or Ringer's alone. The HNK-1 epitope also binds to the G2 domain of laminin to mediate neuron-ECM adhesion. To test for a role for laminin, a polyclonal antibody was similarly infused and also prevented sharpening to approximately the same degree. The results support a role for the HNK-1 epitope and laminin in retinotectal sharpening. The oligomannoside epitope (recognized by monoclonal antibody L3) on the CAM L1 interacts with NCAM on the same cell to promote stronger L1 homophilic interactions between cells. Both an L1-like molecule and NCAM are prominently reexpressed in the regenerating retinotectal system of fish. Infusion of oligomannosidic glycopeptides resulted in decreased sharpening, with multiunit receptive fields that averaged 22.7 degrees. Infusions of mannose-poor glycopeptides less prominently disrupted sharpening, with average multiunit receptive fields of 18 degrees. Thus, oligomannosidic glycans in particular may play a role in retinotopic sharpening. Blocking glycan-mediated interactions between CAMs and ECM molecules could decrease the extent of exploratory growth of retinal axon collaterals, preventing them from finding their retinotopic sites, or could interfere with L1 or NCAM and laminin binding at the synaptic densities preventing stabilization of retinotopically appropriate synapses. Together, these results support a prominent role for cell adhesion and glycan epitopes in visual synaptic plasticity.

[Protection and bidirectional effect of rhubarb anthraquinone and tannins for rats' liver].

PubMed

Qin, Lu-shan; Zhao, Hai-ping; Zhao, Yan-ling; Ma, Zhi-jiel; Zeng, Ling-na; Zhang, Ya-ming; Zhang, Ping; Yan, Dan; Bai, Zhao-fang; Li, Yue; Hao, Qing-xiu; Zhao, Kui-jun; Wang, Jia-bo; Xiao, Xiao-he

2014-06-01

To compare the bidirectional effect of rhubarb total anthraquinone (TA) and total tannins (TT) on rats' liver. One hundred rats were randomly divided into 10 groups, i.e., the blank group, the model group, the blank + high dose TA group, the blank +low dose TA group, the blank + high dose TT group, the blank + low dose TT group, the model + high dose TA group, the model + low dose TA group, the model +high dose TT group, and the model + low dose TT group, 10 in each group. The carbon tetrachloride (CCI4) was used to prepare the acute liver injury rat model. TA and TT of rhubarb (at 5.40 g crude drugs/kg and 14.69 g crude drugs/kg) were intragastrically administrated to rats in all groups except the blank group and the model group, once daily for 6 successive days.The general state of rats, biochemical indices such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), laminin (LN), hyaluronic acid (HA), transforming growth factor beta1 (TGF-beta1), as well pathological results of rat liver tissues. Finally the protection laws of TA and TT for rats' liver were analyzed using factor analysis. Compared with the blank control group, all biochemical indices increased in the blank group (P < 0.05, P < 0.01). HA also increased in the blank + high dose TA group; AST, ALT, and HA also increased in the blank +high dose TT group (P < 0.05). Compared with the model group, AST, ALT, ALP, HA, and TGF-beta1 significantly decreased in the model + low dose TA group, the model + high dose TA group, the model + low dose TT group (P < 0.05, P < 0.01). Serum AST, ALT, and ALP also decreased in the model + high dose TT group (P < 0.05, P < 0.01). Pathological results showed that mild swollen liver cells in the model + high dose TA group. Fatty degeneration and fragmental necrosis around the central veins occurred in the blank + high dose TA group. The pathological injury was inproved in the model +low dose TA group. Two common factors, liver fibrosis and liver cell injury, were extracted by using factor analysis. TA showed stronger improvement of the two common factors than TT. Rhubarb TA and TT showed protective and harmful effects on rats' liver. At an equivalent dosage, TA had better liver protection than TT. High dose TT played a role in liver injury to some extent.

[Construction of thr461 --> Asn461 and Ile462 --> Val462 mutation vector of P4501A1 gene].

PubMed

Wei, Qing; Liu, Yi-Min; Wang, Hui; Zhao, Xiao-Lin; Ren, Tie-ling; Xiao, Yong-mei

2006-09-01

To construct Thr461 --> Asn461 and Ile462 --> Val462 mutation vector of P4501A1 gene and to provide scientific base for deeply researching on the function of cytochrome 1A1 gene (CYP1A1) and the mechanism of carcinogenesis. According to cDNA sequence of human CYP1A1 gene, universal primers (Pm3/Pm4) and mutant primers (Pt15/Pt16 and Pt17/Pt18) containing restriction enzyme site and mutation site were designed. The first set of primers involving Pm3/Pt16 and Pm3/Pt18 amplified a forward 1.5kb fragment from pGEM-T-CYP1A1 plasmid. The second set of primers involving Pt15/Pm4 and Pt17/Pm4 amplified a reverse 177-bp fragment from 10ng pGEM-T-CYP1A1 plasmid. The third set of primers involving Pm3/Pm4 amplified a 1.5kb fragment from the fomer PCR amplifications. The third PCR products were separated, purified and recovered from 1% agarose gel, then inserted into pMD-T vector. Subsequently the conjunct products were transformed into E. coil strain DH-5alpha., then the single clone was screened out and plasmids were extracted from such clone finally verified by restriction endonuclease analysis and sequencing. A 1.5kb fragment of tricycle PCR amplifications were digested by restriction endonucleases (BamHI and SailI) and sequenced bidirectionally by universal primers(T7p and SP6). The results verified that the cloned fragment including Asn461 and Val462 mutant site had 99.9% homology with the human cDNA of CYP1A1 gene in Genebank. The objective fragment containing Asn461 and Va462 mutant site with cDNA of the CYP1A1 gene has been successfully constructed in this experiment.

A dystonia-like movement disorder with brain and spinal neuronal defects is caused by mutation of the mouse laminin β1 subunit, Lamb1

PubMed Central

Liu, Yi Bessie; Tewari, Ambika; Salameh, Johnny; Arystarkhova, Elena; Hampton, Thomas G; Brashear, Allison; Ozelius, Laurie J; Khodakhah, Kamran; Sweadner, Kathleen J

2015-01-01

A new mutant mouse (lamb1t) exhibits intermittent dystonic hindlimb movements and postures when awake, and hyperextension when asleep. Experiments showed co-contraction of opposing muscle groups, and indicated that symptoms depended on the interaction of brain and spinal cord. SNP mapping and exome sequencing identified the dominant causative mutation in the Lamb1 gene. Laminins are extracellular matrix proteins, widely expressed but also known to be important in synapse structure and plasticity. In accordance, awake recording in the cerebellum detected abnormal output from a circuit of two Lamb1-expressing neurons, Purkinje cells and their deep cerebellar nucleus targets, during abnormal postures. We propose that dystonia-like symptoms result from lapses in descending inhibition, exposing excess activity in intrinsic spinal circuits that coordinate muscles. The mouse is a new model for testing how dysfunction in the CNS causes specific abnormal movements and postures. DOI: http://dx.doi.org/10.7554/eLife.11102.001 PMID:26705335

Mechanical stabilization of proteolytically degradable polyethylene glycol dimethacrylate hydrogels through peptide interaction.

PubMed

Lim, Hyun Ju; Khan, Zara; Lu, Xi; Perera, T Hiran; Wilems, Thomas S; Ravivarapu, Krishna T; Smith Callahan, Laura A

2018-04-15

Balancing enhancement of neurite extension against loss of matrix support in synthetic hydrogels containing proteolytically degradable and bioactive signaling peptides to optimize tissue formation is difficult. Using a systematic approach, polyethylene glycol hydrogels containing concurrent continuous concentration gradients of the laminin derived bioactive signaling peptide, Ile-Lys-Val-Ala-Val (IKVAV), and collagen derived matrix metalloprotease degradable peptide, GPQGIWGQ, were fabricated and characterized. During proteolytic degradation of the concentration gradient hydrogels, the IKVAV and IWGQ cleavage fragment from GPQGIWGQ were found to interact and stabilize the bulk Young's Modulus of the hydrogel. Further testing of discrete samples containing GPQGIWGQ or its cleavage fragments, GPQG and IWGQ, indicates hydrophobic interactions between the peptides are not necessary for mechanical stabilization of the hydrogel, but changes in the concentration ratio between the peptides tethered in the hydrogel and salts and ions in the swelling solution can affect the stabilization. Encapsulation of human induced pluripotent stem cell derived neural stem cells did not reduce the mechanical properties of the hydrogel over a 14 day neural differentiation culture period, and IKVAV was found to maintain concentration dependent effects on neurite extension and mRNA gene expression of neural cytoskeletal markers, similar to previous studies. As a result, this work has significant implications for the analysis of biological studies in matrices, as the material and mechanical properties of the hydrogel may be unexpectedly temporally changing during culture due to interactions between peptide signaling elements, underscoring the need for greater matrix characterization during the degradation and cell culture. Greater emulation of the native extracellular matrix is necessary for tissue formation. To achieve this, matrices are becoming more complex, often including multiple bioactive signaling elements. However, peptide signaling in polyethylene glycol matrices and amino acids interactions between peptides can affect hydrogel material and mechanical properties, but are rarely studied. The current study identifies such an interaction between laminin derived peptide, IKVAV, and collagen derived matrix metalloprotease degradable peptide, GPQGIWGQ. Previous studies using these peptides did not identify their interactions' ability to mechanically stabilize the hydrogel during degradation. This work underscores the need for greater matrix characterization and consideration of bioactive signaling element effects temporally on the matrix's material and mechanical properties, as they can contribute to cellular response. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo.

PubMed

Stenzel, Denise; Franco, Claudio A; Estrach, Soline; Mettouchi, Amel; Sauvaget, Dominique; Rosewell, Ian; Schertel, Andreas; Armer, Hannah; Domogatskaya, Anna; Rodin, Sergey; Tryggvason, Karl; Collinson, Lucy; Sorokin, Lydia; Gerhardt, Holger

2011-10-28

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo

PubMed Central

Stenzel, Denise; Franco, Claudio A; Estrach, Soline; Mettouchi, Amel; Sauvaget, Dominique; Rosewell, Ian; Schertel, Andreas; Armer, Hannah; Domogatskaya, Anna; Rodin, Sergey; Tryggvason, Karl; Collinson, Lucy; Sorokin, Lydia; Gerhardt, Holger

2011-01-01

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo. PMID:21979816

The use of autologous adult, allogenic juvenile, and combined juvenile-adult cartilage fragments for the repair of chondral defects.

PubMed

Bonasia, Davide Edoardo; Martin, James A; Marmotti, Antonio; Kurriger, Gail L; Lehman, Abigail D; Rossi, Roberto; Amendola, Annunziato

2016-12-01

The goal of the study was to evaluate the repair of chondral lesions treated with combined autologous adult/allogenic juvenile cartilage fragments, compared with isolated adult and isolated juvenile cartilage fragments. Fifty-eight adult (>16 week old) and five juvenile (<6 week old) New Zealand White female rabbits were used. A large osteochondral defect was created in the center of the femoral trochlea of adult rabbits. The rabbits were divided in four groups: Group 1 = untreated defects (controls); Group 2 = adult cartilage fragments; Group 3 = juvenile cartilage fragments; and Group 4 = adult + juvenile cartilage fragments. Killings were performed at 3 and 6 months. The defects were evaluated with ICRS macroscopic score, modified O'Driscoll score, and Collagen type II immunostaining. At 3 months, Group 4 performed better than Group 1, in terms of modified O'Driscoll score (p = 0.001) and Collagen type II immunostaining (p = 0.015). At 6 months, Group 4 showed higher modified O'Driscoll score (p = 0.003) and Collagen type II immunostaining score (p < 0.001) than Group 1. Histologically, also Group 3 performed better than Group 1 (p = 0.03), and Group 4 performed better than Group 2 (p = 0.004). Mixing adult and juvenile cartilage fragments improved cartilage repair in a rabbit model. In the clinical setting, a new "one-stage" procedure combining the two cartilage sources can be hypothesized, with the advantages of improved chondral repair and large defect coverage, because of the use of an off-the-shelf juvenile allograft. Further studies on larger animals and clinical trials are required to confirm these results.

Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Ren; Nelson, Celeste M.; Muschler, John L.

2009-06-03

Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. We showed previously that in addition to prolactin, signaling by laminin-111 was necessary to restore functional differentiation of mammary epithelia. Here, we elucidate two additional aspects of laminin-111 action. We show that in 2D cultures, the prolactin receptor is basolaterally localized and physically segregated from its apically placed ligand. Detachment of the cells exposes the receptor to ligation by prolactin leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We showmore » that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the prolactin receptor and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and {beta}-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression.« less

Severe congenital muscular dystrophy in a Mexican family with a new nonsense mutation (R2578X) in the laminin alpha-2 gene.

PubMed

Coral-Vazquez, Ramon M; Rosas-Vargas, Haydee; Meza-Espinosa, Pedro; Mendoza, Irma; Huicochea, Juan C; Ramon, Guillermo; Salamanca, Fabio

2003-01-01

The congenital muscular dystrophies (CMDs) are a heterogeneous group of autosomal recessive disorders. Approximately one half of cases diagnosed with classic CMD show primary deficiency of the laminin alpha2 chain of merosin. Complete absence of this protein is usually associated with a severe phenotype characterized by drastic muscle weakness and characteristic changes in white matter in cerebral magnetic resonance imaging (MRI). Here we report an 8-month-old Mexican female infant, from a consanguineous family, with classical CMD. Serum creatine kinase was elevated, muscle biopsy showed dystrophic changes, and there were abnormalities in brain MRI. Immunofluorescence analysis demonstrated the complete absence of laminin alpha2. In contrast, expression of alpha-, beta-, gamma-, and delta-sarcoglycans and dystrophin, all components of the dystrophin-glycoprotein complex, appeared normal. A homozygous C long right arrow T substitution at position 7781 that generated a stop codon in the G domain of the protein was identified by mutation analysis of the laminin alpha2 gene ( LAMA2). Sequence analysis on available DNA samples of the family showed that parents and other relatives were carriers of the mutation.

Carcinogen-Induced Microenvironment in Breast Cancer

DTIC Science & Technology

2000-04-01

unknown, the ability of radiation to induce TGF-P3 activation may gens , such as ionizing radiation, is to modify paracrine interactions indeed play a...radiation of the basement membrane proteins laminin and colla- induced proteases. In particular, tissue-type plasmino- gen IV were unchanged during the week...following gen activator is induced in a variety of irradiated cells radiation, marked changes in the periepithelial and and tissues (33) while plasmin

Osteoblast adhesion to orthopaedic implant alloys: Effects of cell adhesion molecules and diamond-like carbon coating

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kornu, R.; Kelly, M.A.; Smith, R.L.

1996-11-01

In total joint arthroplasty, long-term outcomes depend in part on the biocompatibility of implant alloys. This study analyzed effects of surface finish and diamond-like carbon coating on osteoblast cell adhesion to polished titanium-aluminum-vanadium and polished or grit-blasted cobalt-chromium-molybdenum alloys. Osteoblast binding was tested in the presence and absence of the cell adhesion proteins fibronectin, laminin, fibrinogen, and vitronectin and was quantified by measurement of DNA content. Although adherence occurred in serum-free medium, maximal osteoblast binding required serum and was similar for titanium and cobalt alloys at 2 and 12 hours. With the grit-blasted cobalt alloy, cell binding was reduced 48%more » (p < 0.05) by 24 hours. Coating the alloys with diamond-like carbon did not alter osteoblast adhesion, whereas fibronectin pretreatment increased cell binding 2.6-fold (p < 0.05). In contrast, fibrinogen, vitronectin, and laminin did not enhance cell adhesion. These results support the hypothesis that cell adhesion proteins can modify cell binding to orthopaedic alloys. Although osteoblast binding was not affected by the presence of diamond-like carbon, this coating substance may influence other longer term processes, such as bone formation, and deserves further study. 40 refs., 4 figs.« less

Impact of co-incorporating laminin peptide dopants and neurotrophic growth factors on conducting polymer properties.

PubMed

Green, Rylie A; Lovell, Nigel H; Poole-Warren, Laura A

2010-01-01

Conductive neural interfaces tailored for cell interaction by incorporation of bioactive factors are hypothesized to produce superior neuroprostheses with improved charge transfer capabilities. This study examined the effect of entrapping nerve growth factor (NGF) within the conducting polymer poly(ethylene dioxythiophene) (PEDOT) during electrodeposition to create a polymer capable of stimulating neurite outgrowth from proximal neural tissue. NGF entrapment was performed on polymers doped with laminin peptides DEDEDYFQRYLI and DCDPGYIGSR and, additionally, a conventional dopant, paratoluene sulphonate (pTS). All polymer coatings were analysed for a range of physical, electrical and mechanical properties, with the biological activity of ligands examined using a PC12 neurite outgrowth assay. NGF was successfully entrapped in PEDOT during electrodeposition and was shown to produce a softer interface than conventional conducting polymers and films without the NGF modification. However, it was found that the use of a peptide dopant combined with NGF entrapment resulted in polymers with diminished electrical and mechanical stability. Entrapped NGF was determined to be biologically active, with PEDOT/pTS/NGF producing neurite outgrowth comparable with control films where NGF was supplied via the medium. Future studies will determine the effect of typical neural prosthetic stimulation regimes on the release of neurotrophins and subsequent cell response.

Measurement of substance P metabolites in rat CNS.

PubMed

Sakurada, T; Le Grevés, P; Stewart, J; Terenius, L

1985-03-01

A procedure based on ion-exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P (SP), the SP(1-7), and C-terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50- to 100-fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1-7) comigrated with the authentic standard whereas practically all activity isolated as C-terminal fragments comigrated with SP(5-11). The levels of C-terminal fragments in rat brain areas rich in SP and in spinal cord were 1-2% of those of parent compound. The levels of SP(1-7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1-7) levels fell more rapidly than those of SP or C-terminal fragments.

Morphology of the posteromedial fragment in pertrochanteric fractures: A three-dimensional computed tomography analysis.

PubMed

Sharma, Gaurav; Gn, Kiran Kumar; Khatri, Kavin; Singh, Ravijot; Gamanagatti, Shivanand; Sharma, Vijay

2017-02-01

In this study we describe the morphology of the posteromedial fragment in pertrochanteric fractures using 3D CT scans and answer two questions 1) Do differences exist between the 3D CT appearances of posteromedial fragments and the depictions made in the AO classification 2) Does the posteromedial fragment affect stability in pertrochanteric fractures, in terms of fracture collapse? Preoperative CT scans of eight 31-A1 and fifty 31-A2 fractures were analysed. The presence of PM fragment, its fragmentation, greater trochanter (GT) involvement, lesser trochanter (LT) fragment size (in terms of its posterior and medial extent as well as LT length), LT fragment displacement (in terms of medial displacement and rotation) were determined. All fractures were treated with a DHS. Fracture collapse was determined on postoperative radiographs. The relationship between fracture collapse and patient factors including age, gender, fracture type (A1 versus A2), characteristics of the posteromedial fragment, and the presence of a lateral wall fracture were determined. Three out of eight 31-A1 fractures demonstrated a separate GT fragment (three part fracture). Out of the 50 31-A2 fractures, 12 had a single PM fragment, which included the LT and GT in continuity. The more common four part fractures seem to form by further fragmentation of this basic form. In A2 fractures, the GT was almost always broken and the broken fragment comprised a mean 56% of normal GT. The LT fragment involved an average of 74% of the posterior wall, and an average of 36% of the medial wall of the proximal femur. Larger LT fragments were less displaced as compared to smaller fragments. Univariate regression analyses revealed that fracture collapse was significantly correlated with fracture type (A1 versus A2, p 0.036), GT size (p 0.002) and the presence of a lateral wall fracture (p<0.001). This study revealed some important differences between the 3D CT appearances and AO classification of pertrochanteric fractures. Further, neither fragmentation of the posteromedial fragment, nor the size of the lesser trochanter fragment was found to predict stability in pertrochanteric fractures. A perioperative lateral wall fracture is the main determinant of stability in these fractures. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparison of particle production in quark and gluon fragmentation at s˜10GeV

NASA Astrophysics Data System (ADS)

Briere, R. A.; Ferguson, T.; Tatishvili, G.; Vogel, H.; Watkins, M. E.; Rosner, J. L.; Adam, N. E.; Alexander, J. P.; Cassel, D. G.; Duboscq, J. E.; Ehrlich, R.; Fields, L.; Galik, R. S.; Gibbons, L.; Gray, R.; Gray, S. W.; Hartill, D. L.; Heltsley, B. K.; Hertz, D.; Jones, C. D.; Kandaswamy, J.; Kreinick, D. L.; Kuznetsov, V. E.; Mahlke-Krüger, H.; Onyisi, P. U. E.; Patterson, J. R.; Peterson, D.; Pivarski, J.; Riley, D.; Ryd, A.; Sadoff, A. J.; Schwarthoff, H.; Shi, X.; Stroiney, S.; Sun, W. M.; Wilksen, T.; Weinberger, M.; Athar, S. B.; Patel, R.; Potlia, V.; Yelton, J.; Rubin, P.; Cawlfield, C.; Eisenstein, B. I.; Karliner, I.; Kim, D.; Lowrey, N.; Naik, P.; Selen, M.; White, E. J.; Wiss, J.; Mitchell, R. E.; Shepherd, M. R.; Besson, D.; Swift, H. K.; Pedlar, T. K.; Cronin-Hennessy, D.; Gao, K. Y.; Hietala, J.; Kubota, Y.; Klein, T.; Lang, B. W.; Poling, R.; Scott, A. W.; Smith, A.; Zweber, P.; Dobbs, S.; Metreveli, Z.; Seth, K. K.; Tomaradze, A.; Ernst, J.; Ecklund, K. M.; Severini, H.; Love, W.; Savinov, V.; Aquines, O.; Li, Z.; Lopez, A.; Mehrabyan, S.; Mendez, H.; Ramirez, J.; Huang, G. S.; Miller, D. H.; Pavlunin, V.; Sanghi, B.; Shipsey, I. P. J.; Xin, B.; Adams, G. S.; Anderson, M.; Cummings, J. P.; Danko, I.; Hu, D.; Moziak, B.; Napolitano, J.; He, Q.; Insler, J.; Muramatsu, H.; Park, C. S.; Thorndike, E. H.; Yang, F.; Coan, T. E.; Gao, Y. S.; Artuso, M.; Blusk, S.; Butt, J.; Li, J.; Menaa, N.; Moneti, G. C.; Mountain, R.; Nisar, S.; Randrianarivony, K.; Sia, R.; Skwarnicki, T.; Stone, S.; Wang, J. C.; Zhang, K.; Bonvicini, G.; Cinabro, D.; Dubrovin, M.; Lincoln, A.; Asner, D. M.; Edwards, K. W.

2007-07-01

Using e+e-→hadrons data collected with the CLEO-III detector at the Cornell Electron Storage Ring, we study the inclusive production of baryons/antibaryons (p,Λ) and mesons (ϕ and f2(1270)) in gluon-fragmentation and quark-fragmentation processes. We first corroborate previous per-event total particle yields in Υ(1S)→ggg compared with nearby continuum (e+e-→qq¯) indicating greater (˜×2) per-event yields of baryons in 3-gluon fragmentation. We find similar results when we extend that comparison to include the Υ(2S) and Υ(3S) resonances. With higher statistics, we now also probe the momentum dependence of these per-event particle yields. Next, we compare particle production in the photon-tagged process Υ(1S)→ggγ with that in e+e-→qq¯γ events, to allow comparison of two-parton with three-parton particle-specific fragmentation. For each particle, we determine the “enhancement” ratio, defined as the ratio of particle yields per gluon-fragmentation event compared to quark-fragmentation event. Thus defined, an enhancement of 1.0 implies equal per-event production in gluon and quark fragmentation. In the photon-tagged analysis (Υ(1S)→ggγ compared to e+e-→qq¯γ), we find almost no enhancement for protons (˜1.2±0.1), but a significant enhancement (˜1.9±0.3) for Λ’s. This small measured proton enhancement rate is supported by a study of baryon production in χb2→gg→p+X relative to χb1→qq¯g→p+X. Overall, per-event baryon production in radiative two-gluon fragmentation is somewhat smaller than that observed in three-gluon decays of the Υ(1S). Our results for baryon production are inconsistent with the predictions of the JETSET (7.3) fragmentation model.

C-terminal substance P fragments elicit histamine release from a murine mast cell line.

PubMed

Krumins, S A; Broomfield, C A

1993-01-01

Incubation of mouse mast cells with C-terminal substance P fragments in the micromolar range caused a release of histamine. Maximum release was observed with the tetrapeptide SP(8-11), followed by the tripeptide SP(9-11). SP(6-11) and SP(5-11) were nearly equipotent, while SP(4-11) caused only a slight histamine release. The substance P parent molecule and the N-terminal substance P fragments SP(1-4), SP(1-6) and SP(1-7) evoked no release of histamine. In confirmation of our previous findings, incubation with neurokinin A caused a release comparable to that of SP(8-11). Whereas neurokinin A-induced release was partially preventable by pretreating the cells with the NK2 receptor-selective antagonist cyclo(Gln-Trp-Phe-(R)Gly[ANC-2]Leu-Met), SP(8-11)-induced release was completely abolished by such treatment. The results provide the first evidence for the involvement of NK2 tachykinin receptors in the release of histamine by C-terminal substance P fragments.

Secondary Fragmentation of Comet Shoemaker-Levy 9 and the Ramifications for the Progenitor's Breakup in July 1992

NASA Technical Reports Server (NTRS)

Sekanina, Z.; Chodas, P. W.; Yeomans, D. K.

1998-01-01

Comprehensive analysis of discrete events of secondary fragmentation leads to a conceptually new understanding of the process of disintegration of comet Shoemaker-Levy 9. We submit that the jovian tidal forces inflicted extensive cracks throughout the interior of the original nucleus but did not split it apart. The initial disruption was apparently accomplished by stresses exerted on the cracked object by its fast rotation during the early post-perijove period of time. We argue that this disruption was in fact a rapid sequence of episodes during July 1992 that gave birth to the 12 on-train, or primary, fragments: A, C, D, E, G, H, K, L, Q (later Q(sub 1)), R, S, and W. The discrete events of secondary fragmentation, which gave birth to the off-train fragments, are understood in this scenario as stochastic manifestations of the continuing process of progressive disintegration. Of the 13 off-train fragments considered, nine were secondary--B, F, G(sub 2), M, N, P (later P(sub 2) or P(sub 2a)), Q(sub 2), U, and V--and four tertiary (J, P(sub 1), P(sub 2b), and T). The separation parameters of 11 off-train fragments were determined. The vectorial distribution of separation velocities of these fragments shows a strong concentration toward a great circle, unquestionably an effect of the approximately conserved angular momentum of the progenitor comet since the time of its initial disruption. Also apparent is their clumping (except for P(sub 1)) to a segment along the great circle, implying that the fragments were consistently released from one side of their parents, thus explaining for the first time why the off-train fragments preferentially appeared on one side of the nuclear train. In order to obtain a consistent solution, our model requires that the points of separation be on the antisolar side of the parent fragments, where thermal stresses are likely to enhance the effect of rotation. The episodes of secondary fragmentation are found to have nine months after the close encounter with Jupiter in early July 1992, and the separation velocities ranged between 0.36 and 1.7 m/s. The spin-axis position is determined to have been nearly in the jovicentric orbit plane, which rules out the Asphaug-Benz-Solem strengthless aggregate model as a plausible breakup hypothesis. Since the separation velocities are rotational in nature, they cannot substantially exceed the critical limit for centrifugal breakup and offer an estimate for the original nuclear dimensions. The comet's nucleus is found to have been approximately 10 km in diameter and spinning rapidly. With the exception of P(sub 1) and apparently also P(sub 2) and F, no nongravitational deceleration was detected in the motions of the off-train fragments. Serious doubts are cast on continuing appreciable activity of any of these fragments. Indeed, when it was necessary to introduce a deceleration into the equations of motion, the effect appears to have been due to the action of solar radiation pressure on the centroid of centimeter-sized particulates in the disintegrating condensations.

Peripheral nerve pathology, including aberrant Schwann cell differentiation, is ameliorated by doxycycline in a laminin-α2-deficient mouse model of congenital muscular dystrophy

PubMed Central

Homma, Sachiko; Beermann, Mary Lou; Miller, Jeffrey Boone

2011-01-01

The most common form of childhood congenital muscular dystrophy, Type 1A (MDC1A), is caused by mutations in the human LAMA2 gene that encodes the laminin-α2 subunit. In addition to skeletal muscle deficits, MDC1A patients typically show a loss of peripheral nerve function. To identify the mechanisms underlying this loss of nerve function, we have examined pathology and cell differentiation in sciatic nerves and ventral roots of the laminin-α2-deficient (Lama2−/−) mice, which are models for MDC1A. We found that, compared with wild-type, sciatic nerves of Lama2−/− mice had a significant increase in both proliferating (Ki67+) cells and premyelinating (Oct6+) Schwann cells, but also had a significant decrease in both immature/non-myelinating [glial fibrillary acidic protein (GFAP)+] and myelinating (Krox20+) Schwann cells. To extend our previous work in which we found that doxycycline, which has multiple effects on mammalian cells, improves motor behavior and more than doubles the median life-span of Lama2−/− mice, we also determined how nerve pathology was affected by doxycycline treatment. We found that myelinating (Krox20+) Schwann cells were significantly increased in doxycycline-treated compared with untreated sciatic nerves. In addition, doxycycline-treated peripheral nerves had significantly less pathology as measured by assays such as amount of unmyelinated or disorganized axons. This study thus identified aberrant proliferation and differentiation of Schwann cells as key components of pathogenesis in peripheral nerves and provided proof-of-concept that pharmaceutical therapy can be of potential benefit for peripheral nerve dysfunction in MDC1A. PMID:21505075

Plasmid P1 replication: negative control by repeated DNA sequences.

PubMed Central

Chattoraj, D; Cordes, K; Abeles, A

1984-01-01

The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pieces of the incA fragment that each contain only three repeats destabilize P1 plasmids efficiently. This result makes it unlikely that incA specifies a regulatory product. Our in vivo results suggest that the repeating DNA sequence itself negatively controls replication by titrating a P1-determined protein, RepA, that is essential for replication. Consistent with this hypothesis is the observation that the RepA protein binds to the incA fragment in vitro. Images PMID:6387706

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jamieson,K.; Wu, J.; Hubbard, S.

The human laminin receptor (LamR) interacts with many ligands, including laminin, prions, Sindbis virus, and the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and has been implicated in a number of diseases. LamR is overexpressed on tumor cells, and targeting LamR elicits anti-cancer effects. Here, we report the crystal structure of human LamR, which provides insights into its function and should facilitate the design of novel therapeutics targeting LamR.

Altered expression of extracellular matrix molecules and their receptors in chronic pancreatitis and pancreatic adenocarcinoma in comparison with normal pancreas.

PubMed

Shimoyama, S; Gansauge, F; Gansauge, S; Oohara, T; Beger, H G

1995-12-01

The aim of this study was to elucidate the expression and distribution patterns of both integrins and extracellular matrix (ECM) molecules in chronic pancreatitis (CP) and pancreatic adenocarcinoma (PC) compared with normal pancreas (NP). Expression of nine alpha-subunits (alpha 2-alpha 6, alpha V, alpha L, alpha M, and alpha X), four beta-subunits (beta 1, beta 3-beta 5), and four ECM molecules (type IV collagen, laminin, fibronectin, and vitronectin) was investigated immunohistochemically. In CP, all integrins except alpha V showed nearly the same staining patterns compared with NP. Some acinar cells in CP expressed alpha V. Whereas alpha 2, alpha 3, and alpha 6 expression was stronger and diffuse, no alpha 5 expression was seen in PC. Basement membrane (BM) showed continuous staining in CP, whereas it showed discontinuous/absent staining in PC with antitype IV collagen, laminin, and vitronectin antibodies. Some carcinoma cells showed reverse correlation between alpha 2, alpha 3, and alpha 6 expression and type IV collagen and laminin expression. Fibronectin showed diffuse stromal expression in CP and PC. Some acinar cells or duct cells in CP carcinoma cells in PC showed intracellular VN expression. These results suggest that these integrins and ECM molecules are involved in inflammatory and malignant processes in pancreas.

Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy

PubMed Central

Tang, Wenxing; Bhatt, Avni; Smith, Adam N.; Crowley, Paula J.; Brady, L. Jeannine; Long, Joanna R.

2016-01-01

The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ~57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to 1) globally characterize cell walls isolated from a Gram-positive bacterium and 2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin. PMID:26837620

Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy.

PubMed

Tang, Wenxing; Bhatt, Avni; Smith, Adam N; Crowley, Paula J; Brady, L Jeannine; Long, Joanna R

2016-02-01

The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ~57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

Anti-apoptotic Role of Caspase-cleaved GAB1 Adaptor Protein in Hepatocyte Growth Factor/Scatter Factor-MET Receptor Protein Signaling*

PubMed Central

Le Goff, Arnaud; Ji, Zongling; Leclercq, Bérénice; Bourette, Roland P.; Mougel, Alexandra; Guerardel, Cateline; de Launoit, Yvan; Vicogne, Jérôme; Goormachtigh, Gautier; Fafeur, Véronique

2012-01-01

The GRB2-associated binder 1 (GAB1) docking/scaffold protein is a key mediator of the MET-tyrosine kinase receptor activated by hepatocyte growth factor/scatter factor (HGF/SF). Activated MET promotes recruitment and tyrosine phosphorylation of GAB1, which in turn recruits multiple proteins and mediates MET signaling leading to cell survival, motility, and morphogenesis. We previously reported that, without its ligand, MET is a functional caspase target during apoptosis, allowing the generation of a p40-MET fragment that amplifies apoptosis. In this study we established that GAB1 is also a functional caspase target by evidencing a caspase-cleaved p35-GAB1 fragment that contains the MET binding domain. GAB1 is cleaved by caspases before MET, and the resulting p35-GAB1 fragment is phosphorylated by MET upon HGF/SF binding and can interact with a subset of GAB1 partners, PI3K, and GRB2 but not with SHP2. This p35-GAB1 fragment favors cell survival by maintaining HGF/SF-induced MET activation of AKT and by hindering p40-MET pro-apoptotic function. These data demonstrate an anti-apoptotic role of caspase-cleaved GAB1 in HGF/SF-MET signaling. PMID:22915589

Release of Matrix Metalloproteinases-2 and 9 by S-Nitrosylated Caveolin-1 Contributes to Degradation of Extracellular Matrix in tPA-Treated Hypoxic Endothelial Cells

PubMed Central

Bi, Gang; Zhu, Yihui; Jun, Wei; Ma, Wenlin; Wu, Huimin

2016-01-01

Intracranial hemorrhage remains the most feared complication in tissue plasminogen activator (tPA) thrombolysis for ischemic stroke. However, the underlying molecular mechanisms are still poorly elucidated. In this study, we reported an important role of caveolin-1 (Cav-1) s-nitrosylation in matrix metalloproteinase (MMP)-2 and 9 secretion from tPA-treated ischemic endothelial cells. Brain vascular endothelial cells (bEND3) were exposed to oxygen-glucose deprivation (OGD) for 2 h before adding recombinant human tPA for 6 h. This treatment induced a significant increase of MMP2 and 9 in the media of bEND3 cells and a simultaneous degradation of fibronectin and laminin β-1, the two main components of extracellular matrix (ECM). Inhibition of MMP2 and 9 with SB-3CT completely blocked the degradation of fibronectin and laminin β-1. ODG+tPA treatment led to Cav-1 shedding from bEND3 cells into the media. Notably, OGD triggered nitric oxide (NO) production and S-nitrosylationof Cav-1 (SNCav-1). Meanwhile tPA induced activation of ERK signal pathway and stimulates the secretion of SNCav-1. Pretreatment of bEND3 cells with C-PTIO (a NO scavenger) or U0126 (a specific ERK inhibitor) significantly reduced OGD-induced S-nitrosylation of Cav-1 in cells and blocked the secretion of Cav-1 and MMP2 and 9 into the media as well as the degradation of fibronectin and laminin β-1 in OGD and tPA-treated cells. These data indicate that OGD-triggered Cav-1 S-nitrosylation interacts with tPA-induced ERK activation to augment MMP2 and 9 secretion and subsequent ECM degradation, which may account for the exacerbation of ischemic blood brain barrier damage following tPA thrombolysis for ischemic stroke. PMID:26881424

Release of Matrix Metalloproteinases-2 and 9 by S-Nitrosylated Caveolin-1 Contributes to Degradation of Extracellular Matrix in tPA-Treated Hypoxic Endothelial Cells.

PubMed

Song, Haoming; Cheng, Youjun; Bi, Gang; Zhu, Yihui; Jun, Wei; Ma, Wenlin; Wu, Huimin

2016-01-01

Intracranial hemorrhage remains the most feared complication in tissue plasminogen activator (tPA) thrombolysis for ischemic stroke. However, the underlying molecular mechanisms are still poorly elucidated. In this study, we reported an important role of caveolin-1 (Cav-1) s-nitrosylation in matrix metalloproteinase (MMP)-2 and 9 secretion from tPA-treated ischemic endothelial cells. Brain vascular endothelial cells (bEND3) were exposed to oxygen-glucose deprivation (OGD) for 2 h before adding recombinant human tPA for 6 h. This treatment induced a significant increase of MMP2 and 9 in the media of bEND3 cells and a simultaneous degradation of fibronectin and laminin β-1, the two main components of extracellular matrix (ECM). Inhibition of MMP2 and 9 with SB-3CT completely blocked the degradation of fibronectin and laminin β-1. ODG+tPA treatment led to Cav-1 shedding from bEND3 cells into the media. Notably, OGD triggered nitric oxide (NO) production and S-nitrosylationof Cav-1 (SNCav-1). Meanwhile tPA induced activation of ERK signal pathway and stimulates the secretion of SNCav-1. Pretreatment of bEND3 cells with C-PTIO (a NO scavenger) or U0126 (a specific ERK inhibitor) significantly reduced OGD-induced S-nitrosylation of Cav-1 in cells and blocked the secretion of Cav-1 and MMP2 and 9 into the media as well as the degradation of fibronectin and laminin β-1 in OGD and tPA-treated cells. These data indicate that OGD-triggered Cav-1 S-nitrosylation interacts with tPA-induced ERK activation to augment MMP2 and 9 secretion and subsequent ECM degradation, which may account for the exacerbation of ischemic blood brain barrier damage following tPA thrombolysis for ischemic stroke.

N- and C-terminal substance P fragments: differential effects on striatal [3H]substance P binding and NK1 receptor internalization.

PubMed

Michael-Titus, A T; Blackburn, D; Connolly, Y; Priestley, J V; Whelpton, R

1999-07-13

N- and C-terminal substance P (SP) fragments increase striatal dopamine outflow at nanomolar concentrations. This contrasts with their low affinity for NK1 receptors. To explore this discrepancy, we investigated the interaction of SP and SP fragments with NK1 sites in fresh striatal slices, the same model used in the functional studies on dopamine outflow. [3H]SP bound specifically to one site (Kd = 6.6 +/- 0.9 nM; Bmax = 12.6 +/- 0.7 fmol/mg protein). [3H]SP binding was displaced by SP (IC50 = 11.8 nM), but not by SP(1-7) or SP(5-11), up to 10 microM. In contrast, 10 nM SP(1-7) or SP(5-11) induced significant internalization of the NK1 receptor, similar to that induced by SP. We suggest that SP fragments have high affinity for an NK1 receptor conformer which is different from that labelled by [3H]SP.

Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo

PubMed Central

Goody, Michelle F.; Kelly, Meghan W.; Lessard, Kevin N.; Khalil, Andre; Henry, Clarissa A.

2010-01-01

Cell-matrix adhesion complexes (CMACs) play fundamental roles during morphogenesis. Given the ubiquitous nature of CMACs and their roles in many cellular processes, one question is how specificity of CMAC function is modulated. The clearly defined cell behaviors that generate segmentally reiterated axial skeletal muscle during zebrafish development comprise an ideal system with which to investigate CMAC function during morphogenesis. We found that Nicotinamide riboside kinase 2b (Nrk2b) cell autonomously modulates the molecular composition of CMACs in vivo. Nrk2b is required for normal Laminin polymerization at the myotendinous junction (MTJ). In Nrk2b-deficient embryos, at MTJ loci where Laminin is not properly polymerized, muscle fibers elongate into adjacent myotomes and are abnormally long. In yeast and human cells, Nrk2 phosphorylates Nicotinamide Riboside and generates NAD+ through an alternative salvage pathway. Exogenous NAD+ treatment rescues MTJ development in Nrk2b-deficient embryos, but not in laminin mutant embryos. Both Nrk2b and Laminin are required for localization of Paxillin, but not β-Dystroglycan, to CMACs at the MTJ. Overexpression of Paxillin in Nrk2b-deficient embryos is sufficient to rescue MTJ integrity. Taken together, these data show that Nrk2b plays a specific role in modulating subcellular localization of discrete CMAC components that in turn play roles in musculoskeletal development. Furthermore, these data suggest that Nrk2b-mediated synthesis of NAD+ is functionally upstream of Laminin adhesion and Paxillin subcellular localization during MTJ development. These results indicate a previously unrecognized complexity to CMAC assembly in vivo and also elucidate a novel role for NAD+ during morphogenesis. PMID:20566368

Pathogenicity of a Human Laminin β2 Mutation Revealed in Models of Alport Syndrome.

PubMed

Funk, Steven D; Bayer, Raymond H; Malone, Andrew F; McKee, Karen K; Yurchenco, Peter D; Miner, Jeffrey H

2018-03-01

Pierson syndrome is a congenital nephrotic syndrome with eye and neurologic defects caused by mutations in laminin β 2 ( LAMB2 ), a major component of the glomerular basement membrane (GBM). Pathogenic missense mutations in human LAMB2 cluster in or near the laminin amino-terminal (LN) domain, a domain required for extracellular polymerization of laminin trimers and basement membrane scaffolding. Here, we investigated an LN domain missense mutation, LAMB2-S80R, which was discovered in a patient with Pierson syndrome and unusually late onset of proteinuria. Biochemical data indicated that this mutation impairs laminin polymerization, which we hypothesized to be the cause of the patient's nephrotic syndrome. Testing this hypothesis in genetically altered mice showed that the corresponding amino acid change (LAMB2-S83R) alone is not pathogenic. However, expression of LAMB2-S83R significantly increased the rate of progression to kidney failure in a Col4a3 -/- mouse model of autosomal recessive Alport syndrome and increased proteinuria in Col4a5 +/- females that exhibit a mild form of X-linked Alport syndrome due to mosaic deposition of collagen α 3 α 4 α 5(IV) in the GBM. Collectively, these data show the pathogenicity of LAMB2-S80R and provide the first evidence of genetic modification of Alport phenotypes by variation in another GBM component. This finding could help explain the wide range of Alport syndrome onset and severity observed in patients with Alport syndrome, even for family members who share the same COL4 mutation. Our results also show the complexities of using model organisms to investigate genetic variants suspected of being pathogenic in humans. Copyright © 2018 by the American Society of Nephrology.

Isolation of dendritic-cell-like S100β-positive cells in rat anterior pituitary gland.

PubMed

Horiguchi, Kotaro; Fujiwara, Ken; Yoshida, Saishu; Higuchi, Masashi; Tsukada, Takehiro; Kanno, Naoko; Yashiro, Takashi; Tateno, Kozue; Osako, Shunji; Kato, Takako; Kato, Yukio

2014-07-01

S100β-protein-positive cells in the anterior pituitary gland appear to possess multifunctional properties. Because of their pleiotropic features, S100β-positive cells are assumed to be of a heterogeneous or even a non-pituitary origin. The observation of various markers has allowed these cells to be classified into populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. The isolation and characterization of each heterogeneous population is a prerequisite for clarifying the functional character and origin of the cells. We attempt to isolate two of the subpopulations of S100β-positive cells from the anterior lobe. First, from transgenic rats that express green fluorescent protein (GFP) driven by the S100β protein promoter, we fractionate GFP-positive cells with a cell sorter and culture them so that they can interact with laminin, a component of the extracellular matrix. We observe that one morphological type of GFP-positive cells possesses extended cytoplasmic processes and shows high adhesiveness to laminin (process type), whereas the other is round in shape and exhibits low adherence to laminin (round type). We successfully isolate cells of the round type from the cultured GFP-positive cells by taking advantage of their low affinity to laminin and then measure mRNA levels of the two cell types by real-time polymerase chain reaction. The resultant data show that the process type expresses vimentin (mesenchymal cell marker) and glial fibrillary acidic protein (astrocyte marker). The round type expresses dendritic cell markers, CD11b and interleukin-6. Thus, we found a method for isolating dendritic-cell-like S100β-positive cells by means of their property of adhering to laminin.

High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding*

PubMed Central

Cabrera, Paula V.; Pang, Mabel; Marshall, Jamie L.; Kung, Raymond; Nelson, Stanley F.; Stalnaker, Stephanie H.; Wells, Lance; Crosbie-Watson, Rachelle H.; Baum, Linda G.

2012-01-01

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function. PMID:22570487

Expression of the plasminogen activator system and the inhibitors PAI-1 and PAI-2 in posttraumatic lesions of the CNS and brain injuries following dramatic circulatory arrests: an immunohistochemical study.

PubMed

Dietzmann, K; von Bossanyi, P; Krause, D; Wittig, H; Mawrin, C; Kirches, E

2000-01-01

Plasminogen activators as inducible extracellular serine proteases are involved in a variety of processes, such as the degradation of brain structures. In regions of brain degradation, an increase in the expression of genes encoding cytokines and proteinases has recently been demonstrated. We tested the hypothesis, whether the plasminogen activator system as well as the plasminogen activator inhibitors are expressed and possibly involved in a proteolytic cascade that breaks down the extracellular matrix as a result of ischemic or posttraumatic brain destructions. To study this supposition, we investigated immunohistochemically the expression of tPA, uPA and its receptor, the plasminogen activator inhibitors PAI-1 and PAI-2, tetranectin as well as the laminin breakdown as an event of secondary brain injury. Brain tissue from 21 autopsy cases with severe brain injuries, material from 14 ischemic infarcts and 11 controls with acute hypoxia were used. All components of the plasminogen activator system studied were over-expressed immunohistochemically in reactive astrocytes, microglia and endothelial cells around the lesion zone. Tetranectin showed an analogous distribution to the plasminogen activator system. A reduced immunoreactivity of laminin within the identical region of destruction was detected concomitant with laminin remnants in perivascular macrophages, so that a remarkable role of the plasmin cascade in the degradation of extracellular matrix proteins in the brain is taken into consideration.

Basement Membrane Defects in Genetic Kidney Diseases

PubMed Central

Chew, Christine; Lennon, Rachel

2018-01-01

The glomerular basement membrane (GBM) is a specialized structure with a significant role in maintaining the glomerular filtration barrier. This GBM is formed from the fusion of two basement membranes during development and its function in the filtration barrier is achieved by key extracellular matrix components including type IV collagen, laminins, nidogens, and heparan sulfate proteoglycans. The characteristics of specific matrix isoforms such as laminin-521 (α5β2γ1) and the α3α4α5 chain of type IV collagen are essential for the formation of a mature GBM and the restricted tissue distribution of these isoforms makes the GBM a unique structure. Detailed investigation of the GBM has been driven by the identification of inherited abnormalities in matrix proteins and the need to understand pathogenic mechanisms causing severe glomerular disease. A well-described hereditary GBM disease is Alport syndrome, associated with a progressive glomerular disease, hearing loss, and lens defects due to mutations in the genes COL4A3, COL4A4, or COL4A5. Other proteins associated with inherited diseases of the GBM include laminin β2 in Pierson syndrome and LMX1B in nail patella syndrome. The knowledge of these genetic mutations associated with GBM defects has enhanced our understanding of cell–matrix signaling pathways affected in glomerular disease. This review will address current knowledge of GBM-associated abnormalities and related signaling pathways, as well as discussing the advances toward disease-targeted therapies for patients with glomerular disease. PMID:29435440

Increasing α7β1-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis without changing gene expression

PubMed Central

Liu, Jianming; Burkin, Dean J.; Kaufman, Stephen J.

2008-01-01

The dystrophin-glycoprotein complex maintains the integrity of skeletal muscle by associating laminin in the extracellular matrix with the actin cytoskeleton. Several human muscular dystrophies arise from defects in the components of this complex. The α7β1-integrin also binds laminin and links the extracellular matrix with the cytoskeleton. Enhancement of α7-integrin levels alleviates pathology in mdx/utrn−/− mice, a model of Duchenne muscular dystrophy, and thus the integrin may functionally compensate for the absence of dystrophin. To test whether increasing α7-integrin levels affects transcription and cellular functions, we generated α7-integrin-inducible C2C12 cells and transgenic mice that overexpress the integrin in skeletal muscle. C2C12 myoblasts with elevated levels of integrin exhibited increased adhesion to laminin, faster proliferation when serum was limited, resistance to staurosporine-induced apoptosis, and normal differentiation. Transgenic expression of eightfold more integrin in skeletal muscle did not result in notable toxic effects in vivo. Moreover, high levels of α7-integrin in both myoblasts and in skeletal muscle did not disrupt global gene expression profiles. Thus increasing integrin levels can compensate for defects in the extracellular matrix and cytoskeleton linkage caused by compromises in the dystrophin-glycoprotein complex without triggering apparent overt negative side effects. These results support the use of integrin enhancement as a therapy for muscular dystrophy. PMID:18045857

Usefulness of a Simple Immunohistochemical Staining Technique to Differentiate Anti-p200 Pemphigoid From Other Autoimmune Blistering Diseases: A Report of 2 Cases.

PubMed

García-Díez, I; Martínez-Escala, M E; Ishii, N; Hashimoto, T; Mascaró Galy, J M; Pujol, R M; Herrero-González, J E

Anti-p200 pemphigoid is a rare autoimmune subepidermal blistering disease characterized by the presence of circulating immunoglobulin G antibodies directed against laminin gamma-1, a 200-kDa protein located in the lamina lucida of the basement membrane. We review the clinical, histopathological and immunological characteristics of the first 2 cases described in Spain. Anti-p200 pemphigoid shares histopathological and immunopathological findings with epidermolysis bullosa acquisita, the main entity in the differential diagnosis. However, its management follows the same guidelines as those used for bullous pemphigoid. The diagnosis is confirmed by immunoblotting, which is a complex technique available in few centers. We propose the immunohistochemical detection of collagen type IV on the floor of the blister, combined with standard immunofluorescence techniques, as a simple, accessible alternative to differentiate anti-p200 pemphigoid from epidermolysis bullosa acquisita. Copyright © 2016 AEDV. Publicado por Elsevier España, S.L.U. All rights reserved.

Fragment Bb in Amniotic Fluid: Evidence for complement activation by the alternative pathway in women with Intra-amniotic Infection/Inflammation

PubMed Central

Vaisbuch, Edi; Romero, Roberto; Erez, Offer; Tovi, Shali Mazaki; Pedro, Kusanovic Juan; Soto, Eleazar; Gotsch, Francesca; Dong, Zhong; Chaiworapongsa, Tinnakorn; Kim, Sun Kwon; Mittal, Pooja; Pacora, Percy; Yeo, Lami; Hassan, Sonia S.

2013-01-01

Objective Fragment Bb is an activator of the alternative pathway of the complement system. Recently, increased first trimester maternal plasma concentrations of this fragment were reported in patients destined to have a spontaneous preterm delivery before 34 weeks of gestation. The aim of this study was to determine whether the amniotic fluid (AF) concentrations of fragment Bb change with gestational age, spontaneous labor (term and preterm), and in the presence of intra-amniotic infection/ inflammation (IAI). Study design This cross-sectional study included patients in the following groups: 1) midtrimester (n=64); 2) term in spontaneous labor (n=70); 3) term not in labor (n=43); 4) spontaneous preterm labor (PTL) who delivered at term (n=76); 5) PTL without IAI who delivered preterm (n=73); 6) PTL with IAI (n=76); 7) prelabor rupture of the membranes (preterm PROM) without IAI (n=71); and 8) preterm PROM with IAI (n=71). Fragment Bb concentration in amniotic fluid was determined by an enzyme-linked immunoassay. Non-parametric statistics were used for analyses. Results 1) Fragment Bb was detected in all AF samples (n=544); 2) The median AF concentration of fragment Bb in patients at term not in labor was significantly higher than that of those in the mid-trimester [2.42 μg/mL, interquartile range (IQR) 1.78-3.22 vs. 1.64 μg/mL, IQR 1.06-3.49; p<0.001]; 3) Among patients with PTL, those with IAI had a higher median AF fragment Bb concentration than that of woman without IAI who delivered preterm (4.82 μg/mL, IQR 3.32-6.08 vs. 3.67 μg/mL, IQR 2.35-4.57; p<0.001) and than that of women with an episode of PTL who delivered at term (3.21 μg/mL, IQR 2.39-4.16; p<0.001); 4) Similarly, among patients with preterm PROM, the median AF fragment Bb concentration was higher in individuals with IAI than in those without IAI (4.24 μg/mL, IQR 2.58-5.79 vs. 2.79 μg/mL, IQR 2.09-3.89; p<0.001). 5) Among patients at term, the median AF fragment Bb concentration did not differ between women with spontaneous labor and those without labor (term in labor: 2.47 μg/mL, IQR 1.86-3.22; p=0.97). Conclusions 1) Fragment Bb, an activator of the alternative complement pathway, is a physiologic constituent of the amniotic fluid, and its concentration increases with advancing gestational age; 2) Amniotic fluid concentrations of fragment Bb are higher in pregnancies complicated with IAI; and 3) Labor at term is not associated with changes in the amniotic fluid concentrations of fragment Bb. These findings suggest a role for fragment Bb in the host immune response against IAI. PMID:19603351

Dynamic monitoring of p53 translocation to mitochondria for the analysis of specific inhibitors using luciferase-fragment complementation.

PubMed

Noda, Natsumi; Awais, Raheela; Sutton, Robert; Awais, Muhammad; Ozawa, Takeaki

2017-12-01

Intracellular protein translocation plays a pivotal role in regulating complex biological processes, including cell death. The tumor suppressor p53 is a transcription factor activated by DNA damage and oxidative stress that also translocates from the cytosol into the mitochondrial matrix to facilitate necrotic cell death. However, specific inhibitors of p53 mitochondrial translocation are largely unknown. To explore the inhibitors of p53, we developed a bioluminescent probe to monitor p53 translocation from cytosol to mitochondria using luciferase fragment complementation assays. The probe is composed of a novel pair of luciferase fragments, the N-terminus of green click beetle luciferase CBG68 (CBGN) and multiple-complement luciferase fragment (McLuc1). The combination of luciferase fragments showed significant luminescence intensity and high signal-to-background ratio. When the p53 connected with McLuc1 translocates from cytosol into mitochondrial matrix, CBGN in mitochondrial matrix enables to complement with McLuc1, resulting in the restoration of the luminescence. The luminescence intensity was significantly increased under hydrogen peroxide-induced oxidative stress following the complementation of CBGN and McLuc1. Pifithrin-μ, a selective inhibitor of p53 mitochondrial translocation, prevented the mitochondrial translocation of the p53 probe in a concentration-dependent manner. Furthermore, the high luminescence intensity made it easier to visualize the p53 translocation at a single cell level under a bioluminescence microscope. This p53 mitochondrial translocation assay is a new tool for high-throughput screening to identify novel p53 inhibitors, which could be developed as drugs to treat diseases in which necrotic cell death is a major contributor. © 2017 Wiley Periodicals, Inc.

Merozoite surface protein-1 genetic diversity in Plasmodium malariae and Plasmodium brasilianum from Brazil.

PubMed

Guimarães, Lilian O; Wunderlich, Gerhard; Alves, João M P; Bueno, Marina G; Röhe, Fabio; Catão-Dias, José L; Neves, Amanda; Malafronte, Rosely S; Curado, Izilda; Domingues, Wilson; Kirchgatter, Karin

2015-11-16

The merozoite surface protein 1 (MSP1) gene encodes the major surface antigen of invasive forms of the Plasmodium erythrocytic stages and is considered a candidate vaccine antigen against malaria. Due to its polymorphisms, MSP1 is also useful for strain discrimination and consists of a good genetic marker. Sequence diversity in MSP1 has been analyzed in field isolates of three human parasites: P. falciparum, P. vivax, and P. ovale. However, the extent of variation in another human parasite, P. malariae, remains unknown. This parasite shows widespread, uneven distribution in tropical and subtropical regions throughout South America, Asia, and Africa. Interestingly, it is genetically indistinguishable from P. brasilianum, a parasite known to infect New World monkeys in Central and South America. Specific fragments (1 to 5) covering 60 % of the MSP1 gene (mainly the putatively polymorphic regions), were amplified by PCR in isolates of P. malariae and P. brasilianum from different geographic origin and hosts. Sequencing of the PCR-amplified products or cloned PCR fragments was performed and the sequences were used to construct a phylogenetic tree by the maximum likelihood method. Data were computed to give insights into the evolutionary and phylogenetic relationships of these parasites. Except for fragment 4, sequences from all other fragments consisted of unpublished sequences. The most polymorphic gene region was fragment 2, and in samples where this region lacks polymorphism, all other regions are also identical. The low variability of the P. malariae msp1 sequences of these isolates and the identification of the same haplotype in those collected many years apart at different locations is compatible with a low transmission rate. We also found greater diversity among P. brasilianum isolates compared with P. malariae ones. Lastly, the sequences were segregated according to their geographic origins and hosts, showing a strong genetic and geographic structure. Our data show that there is a low level of sequence diversity and a possible absence of allelic dimorphism of MSP1 in these parasites as opposed to other Plasmodium species. P. brasilianum strains apparently show greater divergence in comparison to P. malariae, thus P. malariae could derive from P. brasilianum, as it has been proposed.

Mutations in the Human Laminin β2 (LAMB2) Gene and the Associated Phenotypic Spectrum

PubMed Central

Matejas, Verena; Hinkes, Bernward; Alkandari, Faisal; Al-Gazali, Lihadh; Annexstad, Ellen; Aytac, Mehmet B.; Barrow, Margaret; Bláhová, Kvĕta; Bockenhauer, Detlef; Cheong, Hae Il; Maruniak-Chudek, Iwona; Cochat, Pierre; Dötsch, Jörg; Gajjar, Priya; Hennekam, Raoul C.; Janssen, Françoise; Kagan, Mikhail; Kariminejad, Ariana; Kemper, Markus J.; Koenig, Jens; Kogan, Jillene; Kroes, Hester Y.; Kuwertz-Bröking, Eberhard; Lewanda, Amy F.; Medeira, Ana; Muscheites, Jutta; Niaudet, Patrick; Pierson, Michel; Saggar, Anand; Seaver, Laurie; Suri, Mohnish; Tsygin, Alexey; Wühl, Elke; Zurowska, Aleksandra; Uebe, Steffen; Hildebrandt, Friedhelm; Antignac, Corinne; Zenker, Martin

2010-01-01

Mutations of LAMB2 typically cause autosomal recessive Pierson syndrome, a disorder characterized by congenital nephrotic syndrome, ocular and neurologic abnormalities, but may occasionally be associated with milder or oligosymptomatic disease variants. LAMB2 encodes the basement membrane protein laminin β2 which is incorporated in specific heterotrimeric laminin isoforms and has an expression pattern corresponding to the pattern of organ manifestations in Pierson syndrome. Herein we review all previously reported and several novel LAMB2 mutations in relation to the associated phenotype in patients from 39 unrelated families. The majority of disease-causing LAMB2 mutations are truncating, consistent with the hypothesis that loss of laminin β2 function is the molecular basis of Pierson syndrome. While truncating mutations are distributed across the entire gene, missense mutations are clearly clustered in the N-terminal LN domain, which is important for intermolecular interactions. There is an association of missense mutations and small in frame deletions with a higher mean age at onset of renal disease and with absence of neurologic abnormalities, thus suggesting that at least some of these may represent hypomorphic alleles. Nevertheless, genotype alone does not appear to explain the full range of clinical variability, and therefore hitherto unidentified modifiers are likely to exist. PMID:20556798

A Laminin G-EGF-Laminin G module in Neurexin IV is essential for the apico-lateral localization of Contactin and organization of septate junctions.

PubMed

Banerjee, Swati; Paik, Raehum; Mino, Rosa E; Blauth, Kevin; Fisher, Elizabeth S; Madden, Victoria J; Fanning, Alan S; Bhat, Manzoor A

2011-01-01

Septate junctions (SJs) display a unique ultrastructural morphology with ladder-like electron densities that are conserved through evolution. Genetic and molecular analyses have identified a highly conserved core complex of SJ proteins consisting of three cell adhesion molecules Neurexin IV, Contactin, and Neuroglian, which interact with the cytoskeletal FERM domain protein Coracle. How these individual proteins interact to form the septal arrays that create the paracellular barrier is poorly understood. Here, we show that point mutations that map to specific domains of neurexin IV lead to formation of fewer septae and disorganization of SJs. Consistent with these observations, our in vivo domain deletion analyses identified the first Laminin G-EGF-Laminin G module in the extracellular region of Neurexin IV as necessary for the localization of and association with Contactin. Neurexin IV protein that is devoid of its cytoplasmic region is able to create septae, but fails to form a full complement of SJs. These data provide the first in vivo evidence that specific domains in Neurexin IV are required for protein-protein interactions and organization of SJs. Given the molecular conservation of SJ proteins across species, our studies may provide insights into how vertebrate axo-glial SJs are organized in myelinated axons.

A Laminin G-EGF-Laminin G Module in Neurexin IV Is Essential for the Apico-Lateral Localization of Contactin and Organization of Septate Junctions

PubMed Central

Banerjee, Swati; Paik, Raehum; Mino, Rosa E.; Blauth, Kevin; Fisher, Elizabeth S.; Madden, Victoria J.; Fanning, Alan S.; Bhat, Manzoor A.

2011-01-01

Septate junctions (SJs) display a unique ultrastructural morphology with ladder-like electron densities that are conserved through evolution. Genetic and molecular analyses have identified a highly conserved core complex of SJ proteins consisting of three cell adhesion molecules Neurexin IV, Contactin, and Neuroglian, which interact with the cytoskeletal FERM domain protein Coracle. How these individual proteins interact to form the septal arrays that create the paracellular barrier is poorly understood. Here, we show that point mutations that map to specific domains of neurexin IV lead to formation of fewer septae and disorganization of SJs. Consistent with these observations, our in vivo domain deletion analyses identified the first Laminin G-EGF-Laminin G module in the extracellular region of Neurexin IV as necessary for the localization of and association with Contactin. Neurexin IV protein that is devoid of its cytoplasmic region is able to create septae, but fails to form a full complement of SJs. These data provide the first in vivo evidence that specific domains in Neurexin IV are required for protein-protein interactions and organization of SJs. Given the molecular conservation of SJ proteins across species, our studies may provide insights into how vertebrate axo-glial SJs are organized in myelinated axons. PMID:22022470

Care fragmentation, quality, and costs among chronically ill patients.

PubMed

Frandsen, Brigham R; Joynt, Karen E; Rebitzer, James B; Jha, Ashish K

2015-05-01

To assess the relationship between care fragmentation and both quality and costs of care for commercially insured, chronically ill patients. We used claims data from 2004 to 2008 for 506,376 chronically ill, privately insured enrollees of a large commercial insurance company to construct measures of fragmentation. We included patients in the sample if they had chronic conditions in any of the following categories: cardiovascular disease, diabetes, asthma, arthritis, or migraine. We assigned each patient a fragmentation index based on the patterns of care of their primary care provider (PCP), with care patterns spread across a higher number of providers considered to be more fragmented. We used regression analysis to examine the relationship between fragmentation and both quality and cost outcomes. Patients of PCPs in the highest quartile of fragmentation had a higher chance of having a departure from clinical best practice (32.8%, vs 25.9% among patients of PCPs in the lowest quartile of fragmentation; P < .001). Similarly, patients of PCPs with high fragmentation had higher rates of preventable hospitalizations (9.1% in highest quartile vs 7.1% in lowest quartile; P < .001). High fragmentation was associated with $4542 higher healthcare spending ($10,396 in the highest quartile vs $5854 in the lowest quartile; P < .001). We found similar or larger effects on quality and costs among patients when we examined the most frequently occurring disease groups individually. Chronically ill patients whose primary care providers offer highly fragmented care more often experience lapses in care quality and incur greater healthcare costs.

Prothrombin fragment 1+2 in urine as a marker on coagulation activity in patients with suspected pulmonary embolism.

PubMed

Wexels, Fredrik; Dahl, Ola E; Pripp, Are H; Seljeflot, Ingebjørg; Borris, Lars C; Haslund, Anniken; Gudmundsen, Tor E; Lauritzen, Trine; Lassen, Michael R

2014-07-01

We have recently reported that increased levels of urine prothrombin fragment 1+2 reflected radiologically verified deep vein thrombosis. In this study we evaluated whether urine prothrombin fragment 1+2 was associated with pulmonary embolism in non-selected patients. Patients with clinical suspected pulmonary embolism were interviewed on comorbidities and medications. Urine was collected from each patient before radiological examination and snap frozen until analysed on urine prothrombin fragment 1+2 with an ELISA kit. Imaging of the pulmonary arteries were conducted with contrast enhanced computer tomography. Pulmonary embolism was diagnosed in 44/197 patients. Non-significantly higher urine prothrombin fragment 1+2 levels were found in non-selected patients with pulmonary embolism vs. those without (p=0.324). Significantly higher urine prothrombin fragment 1+2 levels were found in the pulmonary embolism positive patients without comorbidities (n=13) compared to the control group (n=28) (p=0.009). The calculated sensitivity, specificity and negative predictive value using the lowest detectable urine prothrombin fragment 1+2 level was 82%, 34% and 87%, respectively. There was no significant urine prothrombin fragment 1+2 level difference in patients with and without pulmonary embolism. In non-comorbide pulmonary embolism positive patients the urine prothrombin fragment 1+2 levels were significantly higher compared to the control group. The negative predictive value found in this study indicates that uF1+2 has the potential to identify patients with a low risk of PE. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization of the replication region of the Bacillus subtilis plasmid pLS20: a novel type of replicon.

PubMed Central

Meijer, W J; de Boer, A J; van Tongeren, S; Venema, G; Bron, S

1995-01-01

A 3.1 kb fragment of the large (approximately 55 kb) Bacillus subtilis plasmid pLS20 containing all the information for autonomous replication was cloned and sequenced. In contrast to the parental plasmid, derived minireplicons were unstably maintained. Using deletion analysis the fragment essential and sufficient for replication was delineated to 1.1 kb. This 1.1 kb fragment is located between two divergently transcribed genes, denoted orfA and orfB, neither of which is required for replication. orfA shows homology to the B.subtilis chromosomal genes rapA (spoOL, gsiA) and rapB (spoOP). The 1.1 kb fragment, which is characterized by the presence of several regions of dyad symmetry, contains no open reading frames of more than 85 codons and shows no similarity with other known plasmid replicons. The structural organization of the pLS20 minimal replicon is entirely different from that of typical rolling circle plasmids from Gram-positive bacteria. The pLS20 minireplicons replicate in polA5 and recA4 B.subtilis strains. Taken together, these results strongly suggest that pLS20 belongs to a new class of theta replicons. PMID:7667098

A Saccharomyces cerevisiae mitochondrial DNA fragment activates Reg1p-dependent glucose-repressible transcription in the nucleus.

PubMed

Santangelo, G M; Tornow, J

1997-12-01

As part of an effort to identify random carbon-source-regulated promoters in the Saccharomyces cerevisiae genome, we discovered that a mitochondrial DNA fragment is capable of directing glucose-repressible expression of a reporter gene. This fragment (CR24) originated from the mitochondrial genome adjacent to a transcription initiation site. Mutational analyses identified a GC cluster within the fragment that is required for transcriptional induction. Repression of nuclear CR24-driven transcription required Reg1p, indicating that this mitochondrially derived promoter is a member of a large group of glucose-repressible nuclear promoters that are similarly regulated by Reg1p. In vivo and in vitro binding assays indicated the presence of factors, located within the nucleus and the mitochondria, that bind to the GC cluster. One or more of these factors may provide a regulatory link between the nucleus and mitochondria.

Estrogen and progesterone receptors have distinct roles in the establishment of the hyperplastic phenotype in PR-A transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simian, Marina; Bissell, Mina J.; Barcellos-Hoff, Mary Helen

2009-05-11

Expression of the A and B forms of progesterone receptor (PR) in an appropriate ratio is critical for mammary development. Mammary glands of PR-A transgenic mice, carrying an additional A form of PR as a transgene, exhibit morphological features associated with the development of mammary tumors. Our objective was to determine the roles of estrogen (E) and progesterone (P) in the genesis of mammary hyperplasias/preneoplasias in PR-A transgenics. We subjected PR-A mice to hormonal treatments and analyzed mammary glands for the presence of hyperplasias and used BrdU incorporation to measure proliferation. Quantitative image analysis was carried out to compare levelsmore » of latency-associated peptide and transforming growth factor beta 1 (TGF{beta}1) between PR-A and PR-B transgenics. Basement membrane disruption was examined by immunofluorescence and proteolytic activity by zymography. The hyperplastic phenotype of PR-A transgenics is inhibited by ovariectomy, and is reversed by treatment with E + P. Studies using the antiestrogen ICI 182,780 or antiprogestins RU486 or ZK 98,299 show that the increase in proliferation requires signaling through E/estrogen receptor alpha but is not sufficient to give rise to hyperplasias, whereas signaling through P/PR has little impact on proliferation but is essential for the manifestation of hyperplasias. Increased proliferation is correlated with decreased TGF{beta}1 activation in the PR-A transgenics. Analysis of basement membrane integrity showed loss of laminin-5, collagen III and collagen IV in mammary glands of PR-A mice, which is restored by ovariectomy. Examination of matrix metalloproteases (MMPs) showed that total levels of MMP-2 correlate with the steady-state levels of PR, and that areas of laminin-5 loss coincide with those of activation of MMP-2 in PR-A transgenics. Activation of MMP-2 is dependent on treatment with E and P in ovariectomized wild-type mice, but is achieved only by treatment with P in PR-A mice. These data establish a link between hormonal response, proliferation, modulation of MMP activity and maintenance of basement membrane integrity that depend on a balance in the expression levels of PR-A and PR-B isoforms. Notably, concomitant increased proliferation, due to inhibition of TGF{beta}1 activation, and loss of basement membrane integrity, via increased MMP-2 activity, appear to be prerequisites for the PR-A hyperplastic phenotype.« less

Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli.

PubMed

Bu, Dawei; Zhou, Yuwei; Tang, Jian; Jing, Fang; Zhang, Wei

2013-12-01

Abnormal brain natriuretic peptide (BNP) secretion is regarded as the dominating mechanism of cerebral salt wasting syndrome (CSW), which results from a renal loss of sodium and water during intracranial disease leading to hyponatremia. Scale preparation of therapeutic single-chain variable fragment (scFv) that can neutralize elevated circulating BNP may have potential value for clinical use. In this report, we used a recently isolated humanized anti-BNP scFv fragment (3C1) as model antibody (Ab) to evaluate the potential of scale production of this therapeutic protein. The truncated gene encoding for scFv fragment cloned in pET22b (+) was mainly overexpressed as inclusion bodies in Escherichia coli (E. coli) Rosetta (DE3) pLysS cells. The insoluble fragment was solubilized and purified by Ni-NTA agarose resin under denaturation conditions, and recovered via an effective refolding buffer containing 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.5 M arginine, 2 mM GSH, 1 mM GSSG, and 5% glycerol. The refolded scFv fragment was concentrated by PEG20000, and dialyzed in PBS (containing 5% glycerol, pH 7.4). The final yield was approximately 10.2 mg active scFv fragment per liter of culture (3.4 g wet weight cells). The scFv fragment was more than 95% pure assessed by SDS-PAGE assay. Recombinant scFv fragment with His tag displayed its immunoreactivity with anti-His tag Ab by western blotting. ELISA showed the scFv fragment specifically bound to BNP, and it displayed similar activity as the traditional anti-BNP monoclonal Ab (mAb). Thus, the current strategy allows convenient small-scale production of this therapeutic protein. Copyright © 2013 Elsevier Inc. All rights reserved.

Neurotrophic factors switch between two signaling pathways that trigger axonal growth.

PubMed

Paveliev, Mikhail; Lume, Maria; Velthut, Agne; Phillips, Matthew; Arumäe, Urmas; Saarma, Mart

2007-08-01

Integration of multiple inputs from the extracellular environment, such as extracellular matrix molecules and growth factors, is a crucial process for cell function and information processing in multicellular organisms. Here we demonstrate that co-stimulation of dorsal root ganglion neurons with neurotrophic factors (NTFs) - glial-cell-line-derived neurotrophic factor, neurturin or nerve growth factor - and laminin leads to axonal growth that requires activation of Src family kinases (SFKs). A different, SFK-independent signaling pathway evokes axonal growth on laminin in the absence of the NTFs. By contrast, axonal branching is regulated by SFKs both in the presence and in the absence of NGF. We propose and experimentally verify a Boolean model of the signaling network triggered by NTFs and laminin. Our results demonstrate that NTFs provide an environmental cue that triggers a switch between separate pathways in the cell signaling network.

Primary cell culture and morphological characterization of canine dermal papilla cells and dermal fibroblasts.

PubMed

Bratka-Robia, Christine B; Mitteregger, Gerda; Aichinger, Amanda; Egerbacher, Monika; Helmreich, Magdalena; Bamberg, Elmar

2002-02-01

Skin biopsies were taken from female dogs, the primary hair follicles isolated and the dermal papilla dissected. After incubation in supplemented Amniomax complete C100 medium in 24-well culture plates, the dermal papilla cells (DPC) grew to confluence within 3 weeks. Thereafter, they were subcultivated every 7 days. Dermal fibroblast (DFB) cultures were established by explant culture of interfollicular dermis in serum-free medium, where they reached confluence in 10 days. They were subcultivated every 5 days. For immunohistochemistry, cells were grown on cover slips for 24 h, fixed and stained with antibodies against collagen IV and laminin. DPC showed an aggregative growth pattern and formation of pseudopapillae. Intensive staining for collagen IV and laminin could be observed until the sixth passage. DFB grew as branching, parallel lines and showed only weak staining for collagen IV and laminin.

Contact lens materials, mucin fragmentation and relation to symptoms.

PubMed

Berry, Monica; Purslow, Chris; Murphy, Paul J; Pult, Heiko

2012-07-01

Mucins adhere to contact lenses (CLs), reflecting the renewal of the preocular fluid and enzymatic activity at the ocular surface. In this study, we aimed to analyze mucin fragmentation on materials new to the ocular surface and investigate whether this correlates with wearing comfort. Lenses were obtained from new CL wearers after 2 weeks each of wearing vifilcon A, followed by senofilcon A, and then by vifilcon A lenses. Symptoms were evaluated using the Ocular Surface Disease Index (OSDI). CLs were extracted in a mixture of guanidinium hydrochloride and radioimmunoprecipitation assay buffer. Mucin mobility was analyzed after electrophoresis, Western blotting, and visualization with antibodies against mucin peptide core. Mobilities, normalized to total reactivity in the lane, were compared between visits for each subject and were expressed as shifts. Mucin (MUC)5AC polymers exceeding 260 kDa were observed in agarose gels; NuPAGE resolved polymers from 260 to 3.5 kDa: when large mucins were detected, the smallest fragments were missing. Fragmentation patterns were significantly different between lens types for MUC1 (analysis of variance, P = 0.006) and MUC4 (P < 0.001) but not for MUC5AC or MUC16 (P > 0.293). Mobility shifts of MUC1 and MUC4 were significantly negatively correlated (Pearson, r = -0.908; P = 0.002). For OSDI scores >15, mucin fragmentation was unchanged, whereas for OSDI scores <15, MUC4 and MUC5AC fragments were longer on vifilcon A than on senofilcon lenses (unpaired t test, P = 0.046), irrespective of the direction of change (analysis of variance, P > 0.366). Changes in MUC1 breakdown were significantly negatively correlated to the overall OSDI score (r = -0.891, P = 0.001). In asymptomatic CL wearers, only changes in mucin fragmentation in response to a new material were consistent and fast, irrespective of CL order. Lack of change seems, therefore, to be connected with discomfort during CL wear.

Cryobiology of coral fragments.

PubMed

Hagedorn, Mary; Farrell, Ann; Carter, Virginia L

2013-02-01

Around the world, coral reefs are dying due to human influences, and saving habitat alone may not stop this destruction. This investigation focused on the biological processes that will provide the first steps in understanding the cryobiology of whole coral fragments. Coral fragments are a partnership of coral tissue and endosymbiotic algae, Symbiodinium sp., commonly called zooxanthellae. These data reflected their separate sensitivities to chilling and a cryoprotectant (dimethyl sulfoxide) for the coral Pocillopora damicornis, as measured by tissue loss and Pulse Amplitude Modulated fluorometry 3weeks post-treatment. Five cryoprotectant treatments maintained the viability of the coral tissue and zooxanthellae at control values (1M dimethyl sulfoxide at 1.0, 1.5 and 2.0h exposures, and 1.5M dimethyl sulfoxide at 1.0 and 1.5h exposures, P>0.05, ANOVA), whereas 2M concentrations did not (P<0.05, ANOVA). A seasonal response to chilling was observed in the coral tissue, but not in the zooxanthellae. During the winter when the fragments were chilled, the coral tissue remained relatively intact (∼25% loss) post-treatment, but the zooxanthellae numbers in the tissue declined after 5min of chilling (P<0.05, ANOVA). However, in the late spring, coral tissue (∼75% loss) and zooxanthellae numbers declined in response to chilling alone (P<0.05, ANOVA). When a cryoprotectant (1M dimethyl sulfoxide) was used in concert with chilling it protected the coral against tissue loss after 45min of cryoprotectant exposure (P>0.05, ANOVA), but it did not protect against the loss of zooxanthellae (P<0.05, ANOVA). The zooxanthellae are the most sensitive element in the coral fragment complex and future cryopreservation protocols must be guided by their greater sensitivity. Copyright © 2012 Elsevier Inc. All rights reserved.

The NH2-terminus of substance P modulates NMDA-induced activity in the mouse spinal cord.

PubMed

Hornfeldt, C S; Sun, X; Larson, A A

1994-05-01

Excitatory amino acids (EAAs) and substance P are believed to transmit nociceptive information in the spinal cord. As substance P NH2-terminal fragments can modulate non-NMDA EAA-mediated activity, we examined the effects of substance P fragments to ascertain whether the COOH- or NH2-terminus of substance P modulates the actions of NMDA in the spinal cord. NMDA activity was measured by the intensity of behaviors produced by NMDA (0.2 nmol) administered intrathecally in the mouse. The NMDA response was attenuated after pretreatment with either substance P (22.5 pmol, 30 min) or the NH2-terminal fragment of substance P, SP-(1-7). Pretreatment with the COOH-terminal fragment SP-(5-11) (22.5 pmol, 30 min), a neurokinin ligand, had no effect on NMDA-induced behaviors, suggesting that the inhibitory effect of substance P is caused by the NH2-terminus. Pretreatment with D-Pro2,D-Phe7 substance P-(1-7), a SP-(1-7) antagonist, potentiated NMDA activity, suggesting a tonic inhibitory effect of the substance P NH2-terminus. Desensitization to NMDA typically develops when NMDA is injected at 2 min intervals. While pretreatment with SP-(1-7) inhibited NMDA, coadministration of SP-(1-7) (22.5 pmol), with the first of four injections of NMDA, first inhibited but then potentiated responses to each challenge with NMDA. Coadministration of the same dose of SP-(1-7) with the fourth injection of NMDA immediately potentiated the response to NMDA.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells

PubMed Central

Nakagawa, Masato; Taniguchi, Yukimasa; Senda, Sho; Takizawa, Nanako; Ichisaka, Tomoko; Asano, Kanako; Morizane, Asuka; Doi, Daisuke; Takahashi, Jun; Nishizawa, Masatoshi; Yoshida, Yoshinori; Toyoda, Taro; Osafune, Kenji; Sekiguchi, Kiyotoshi; Yamanaka, Shinya

2014-01-01

In order to apply human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to regenerative medicine, the cells should be produced under restricted conditions conforming to GMP guidelines. Since the conventional culture system has some issues that need to be addressed to achieve this goal, we developed a novel culture system. We found that recombinant laminin-511 E8 fragments are useful matrices for maintaining hESCs and hiPSCs when used in combination with a completely xeno-free (Xf) medium, StemFit™. Using this system, hESCs and hiPSCs can be easily and stably passaged by dissociating the cells into single cells for long periods, without any karyotype abnormalities. Human iPSCs could be generated under feeder-free (Ff) and Xf culture systems from human primary fibroblasts and blood cells, and they possessed differentiation abilities. These results indicate that hiPSCs can be generated and maintained under this novel Ff and Xf culture system. PMID:24399248

The Development of Therapeutic and Diagnostic Countermeasures to WMD by the Advanced Medical Countermeasures Consortium

DTIC Science & Technology

2008-09-01

and antioxidants in patients with acute respiratory distress syndrome. Enteral Nutrition in ARDS Study Group. Crit Care Med 27: 1409–1420, 1999. 30...mechanism of action, and (3) efficacy of countermeasures for HD injury. Studies on the Yucatan mini-pig have demonstrated that laminin in the dermo...Rice P. Changes in connective tissue macromolecular components of Yucatan mini-pig skin following application of sulphur mustard vapour. Hum Exp Toxicol

The Development of Therapeutic and Diagnostic Countermeasures to WMD by the Advanced Medical Countermeasures Consortium

DTIC Science & Technology

2007-09-01

pantothenate mixture. J. Ocul. Pharmacol. Ther. 1998; 14: 423–428. 14. Brown RFR, Rice P. Histopathological changes in Yucatan minipig skin following...antioxidants in patients with acute respiratory distress syndrome. Enteral Nutrition in ARDS Study Group. Crit Care Med 27: 1409–1420, 1999. 30. Galley HF...countermeasures for HD injury. Studies on the Yucatan mini-pig have demonstrated that laminin in the dermo-epithelial junction is a target for partial

A newly identified protein of Leptospira interrogans mediates binding to laminin.

PubMed

Longhi, Mariana T; Oliveira, Tatiane R; Romero, Eliete C; Gonçales, Amane P; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2009-10-01

Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. The search for novel antigens that could be relevant in host-pathogen interactions is being pursued. These antigens have the potential to elicit several activities, including adhesion. This study focused on a hypothetical predicted lipoprotein of Leptospira, encoded by the gene LIC12895, thought to mediate attachment to extracellular matrix (ECM) components. The gene was cloned and expressed in Escherichia coli BL21 Star (DE3)pLys by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. The capacity of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC12895, named Lsa27 (leptospiral surface adhesin, 27 kDa), bound strongly to laminin in a dose-dependent and saturable fashion. Moreover, Lsa27 was recognized by antibodies from serum samples of confirmed leptospirosis specimens in both the initial and the convalescent phases of the disease. Lsa27 is most likely a surface protein of Leptospira as revealed in liquid-phase immunofluorescence assays with living organisms. Taken together, these data indicate that this newly identified membrane protein is expressed during natural infection and may play a role in mediating adhesion of L. interrogans to its host.

Isolation of the endosperm-specific LPAAT gene promoter from coconut (Cocos nucifera L.) and its functional analysis in transgenic rice plants.

PubMed

Xu, Li; Ye, Rongjian; Zheng, Yusheng; Wang, Zhekui; Zhou, Peng; Lin, Yongjun; Li, Dongdong

2010-09-01

As one of the key tropical crops, coconut (Cocos nucifera L.) is a member of the monocotyledonous family Aracaceae (Palmaceae). In this study, we amplified the upstream region of an endosperm-specific expression gene, Lysophosphatidyl acyltransferase (LPAAT), from the coconut genomic DNA by chromosome walking. In this sequence, we found several types of promoter-related elements including TATA-box, CAAT-box and Skn1-motif. In order to further examine its function, three different 5'-deletion fragments were inserted into pBI101.3, a plant expression vector harboring the LPAAT upstream sequence, leading to pBI101.3-L1, pBI101.3-L2 and pBI101.3-L3, respectively. We obtained transgenic plants of rice by Agrobacterium-mediated callus transformation and plant regeneration and detected the expression of gus gene by histochemical staining and fluorometric determination. We found that gus gene driven by the three deletion fragments was specifically expressed in the endosperm of rice seeds, but not in the empty vector of pBI101.3 and other tissues. The highest expression level of GUS was at 15 DAF in pBI101.3-L3 and pBI101.3-L2 transgenic lines, while the same level was detected at 10 DAF in pBI101.3-L1. The expression driven by the whole fragment was up to 1.76- and 2.8-fold higher than those driven by the -817 bp and -453 bp upstream fragments, and 10.7-fold higher than that driven by the vector without the promoter. Taken together, our results strongly suggest that these promoter fragments from coconut have a significant potential in genetically improving endosperm in main crops.

Spatiotemporal distribution of extracellular matrix changes during mouse duodenojejunal flexure formation.

PubMed

Onouchi, Sawa; Ichii, Osamu; Nakamura, Teppei; Elewa, Yaser Hosny Ali; Kon, Yasuhiro

2016-08-01

Although gut flexures characterize gut morphology, the mechanisms underlying flexure formation remain obscure. Previously, we analyzed the mouse duodenojejunal flexure (DJF) as a model for its formation and reported asymmetric morphologies between the inner and outer bending sides of the fetal mouse DJF, implying their contribution to DJF formation. We now present the extracellular matrix (ECM) as an important factor for gut morphogenesis. We investigate ECM distribution during mouse DJF formation by histological techniques. In the intercellular space of the gut wall, high Alcian-Blue positivity for proteoglycans shifted from the outer to the inner side of the gut wall during DJF formation. Immunopositivity for fibronectin, collagen I, or pan-tenascin was higher at the inner than at the outer side. Collagen IV and laminins localized to the epithelial basement membrane. Beneath the mesothelium at the pre-formation stage, collagen IV and laminin immunopositivity showed inverse results, corresponding to the different cellular characteristics at this site. At the post-formation stage, however, laminin positivity beneath the mesothelium was the reverse of that observed during the pre-formation stage. High immunopositivity for collagen IV and laminins at the inner gut wall mesenchyme of the post-formation DJF implied a different blood vessel distribution. We conclude that ECM distribution changes spatiotemporally during mouse DJF formation, indicating ECM association with the establishment of asymmetric morphologies during this process.

Molecular cytogenetic anomalies and phenotype alterations in a newly established cell line from Wilms tumor with diffuse anaplasia.

PubMed

Faussillon, Marine; Murakami, Ichiro; Bichat, Magalie; Telvi, Louise; Jeanpierre, Cécile; Nezelof, Christian; Jaubert, Francis; Gogusev, Jean

2008-07-01

The novel continuous cell line WT-Pe.1 was established in vitro from Wilms tumor with histological features of diffuse anaplasia. The cultures grew as poorly differentiated epithelial-like cells with pleomorphic polygonal shapes and formation of typical monolayers. WT-Pe.1 cells were immunoreactive for cytokeratin, vimentin, laminin, villin, CD10, and CD24 proteins. Conventional cytogenetic analysis by RHG-banding revealed a hypotriploid karyotype with numerous abnormalities including ring chromosomes, double-minutes, homogeneous staining regions, radial structures, dicentrics, and several marker chromosomes. Comparative genomic hybridization analysis revealed DNA copy numbers losses on chromosome segments 1p, 3p, 6q, 9q34.1 approximately q34.3, 11q24 approximately q25, 14q12 approximately qter, 16q, 18q, and 22q11 approximately q13; gain of genomic material was localized on chromosome arms 1q, 4p, 6q, and 7p and the entire chromosome 12. With DNA from the original tumor, copy number losses were detected on chromosomes 1p, 14q, 16q, 17q, and 22q and gains were observed on 1q, 4p, 8q, 12p, 12q, and chromosome 14p. Copy number amplifications of distinct loci were found on 1q21.1 and 4p15.3, as well as an elevated copy number of cyclin D2 (CCND2) and cyclin D associated kinase (CDK4) genes on chromosome 12 (confirmed by fluorescence in situ hybridization).

Peptide docking of HIV-1 p24 with single chain fragment variable (scFv) by CDOCKER algorithm

NASA Astrophysics Data System (ADS)

Karim, Hana Atiqah Abdul; Tayapiwatana, Chatchai; Nimmanpipug, Piyarat; Zain, Sharifuddin M.; Rahman, Noorsaadah Abdul; Lee, Vannajan Sanghiran

2014-10-01

In search for the important residues that might have involve in the binding interaction between the p24 caspid protein of HIV-1 fragment (MET68 - PRO90) with the single chain fragment variable (scFv) of FAB23.5, modern computational chemistry approach has been conducted and applied. The p24 fragment was initially taken out from the 1AFV protein molecule consisting of both light (VL) and heavy (VH) chains of FAB23.5 as well as the HIV-1 caspid protein. From there, the p24 (antigen) fragment was made to dock back into the protein pocket receptor (antibody) by using the CDOCKER algorithm to conduct the molecular docking process. The score calculated from the CDOCKER gave 15 possible docked poses with various docked ligand's positions, the interaction energy as well as the binding energy. The best docked pose that imitates the original antigen's position was determined and further processed to the In Situ minimization to obtain the residues interaction energy as well as to observe the hydrogen bonds interaction in the protein-peptide complex. Based on the results demonstrated, the specific residues in the complex that have shown immense lower interaction energies in the 5Å vicinity region from the peptide are from the heavy chain (VH:TYR105) and light chain (VL: ASN31, TYR32, and GLU97). Those residues play vital roles in the binding mechanism of Antibody-Antigen (Ab-Ag) complex of p24 with FAB23.5.

Laminin-111 and the Level of Nuclear Actin Regulate Epithelial Quiescence via Exportin-6.

PubMed

Fiore, Ana Paula Zen Petisco; Spencer, Virginia A; Mori, Hidetoshi; Carvalho, Hernandes F; Bissell, Mina J; Bruni-Cardoso, Alexandre

2017-06-06

Nuclear actin (N-actin) is known to participate in the regulation of gene expression. We showed previously that N-actin levels mediate the growth and quiescence of mouse epithelial cells in response to laminin-111 (LN1), a component of the mammary basement membrane (BM). We know that BM is defective in malignant cells, and we show here that it is the LN1/N-actin pathway that is aberrant in human breast cancer cells, leading to continuous growth. Photobleaching assays revealed that N-actin exit in nonmalignant cells begins as early as 30 min after LN1 treatment. LN1 attenuates the PI3K pathway leading to upregulation of exportin-6 (XPO6) activity and shuttles actin out of the nucleus. Silencing XPO6 prevents quiescence. Malignant cells are impervious to LN1 signaling. These results shed light on the crucial role of LN1 in quiescence and differentiation and how defects in the LN1/PI3K/XPO6/N-actin axis explain the loss of tissue homeostasis and growth control that contributes to malignant progression. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Influence of fragment size and postoperative joint congruency on long-term outcome of posterior malleolar fractures.

PubMed

Drijfhout van Hooff, Cornelis Christiaan; Verhage, Samuel Marinus; Hoogendoorn, Jochem Maarten

2015-06-01

One of the factors contributing to long-term outcome of posterior malleolar fractures is the development of osteoarthritis. Based on biomechanical, cadaveric, and small population studies, fixation of posterior malleolar fracture fragments (PMFFs) is usually performed when fragment size exceeds 25-33%. However, the influence of fragment size on long-term clinical and radiological outcome size remains unclear. A retrospective cohort study of 131 patients treated for an isolated ankle fracture with involvement of the posterior malleolus was performed. Mean follow-up was 6.9 (range, 2.5-15.9) years. Patients were divided into groups depending on size of the fragment, small (<5%, n = 20), medium (5-25%, n = 86), or large (>25%, n = 25), and presence of step-off after operative treatment. We have compared functional outcome measures (AOFAS, AAOS), pain (VAS), and dorsiflexion restriction compared to the contralateral ankle and the incidence of osteoarthritis on X-ray. There were no nonunions, 56% of patients had no radiographic osteoarthritis, VAS was 10 of 100, and median clinical score was 90 of 100. More osteoarthritis occurred in ankle fractures with medium and large PMFFs compared to small fragments (small 16%, medium 48%, large 54%; P = .006). Also when comparing small with medium-sized fragments (P = .02), larger fragment size did not lead to a significantly decreased function (median AOFAS 95 vs 88, P = .16). If the PMFF size was >5%, osteoarthritis occurred more frequently when there was a postoperative step-off ≥1 mm in the tibiotalar joint surface (41% vs 61%, P = .02) (whether the posterior fragment had been fixed or not). In this group, fixing the PMFF did not influence development of osteoarthritis. However, in 42% of the cases with fixation of the fragment a postoperative step-off remained (vs 45% in the group without fixation). Osteoarthritis is 1 component of long-term outcome of malleolar fractures, and the results of this study demonstrate that there was more radiographic osteoarthritis in patients with medium and large posterior fragments than in those with small fragments. Radiographic osteoarthritis also occurred more frequently when postoperative step-off was 1 mm or more, whether the posterior fragment was fixed or not. However, clinical scores were not different for these groups. Level IV, retrospective case series. © The Author(s) 2015.

New Observational Evidence of Active Asteroid P/2010 A2: Slow Rotation of the Largest Fragment

NASA Astrophysics Data System (ADS)

Kim, Yoonyoung; Ishiguro, Masateru; Lee, Myung Gyoon

2017-06-01

We report new observations of the active asteroid P/2010 A2 taken when it made its closest approach to Earth (1.06 au in 2017 January) after its first discovery in 2010. Despite a crucial role of the rotational period in clarifying its ejection mechanism, the rotational property of P/2010 A2 has not yet been studied due to the extreme faintness of this tiny object (∼120 m in diameter). Taking advantage of the best observing geometry since the discovery, we succeed in obtaining the rotational light curve of the largest fragment with Gemini/GMOS-N. We find that (1) the largest fragment has a double-peaked period of 11.36 ± 0.02 hr spinning much slower than its critical spin period; (2) the largest fragment is a highly elongated object (a/b ≥ 1.94) with an effective radius of {61.9}-9.2+16.8 m; (3) the size distribution of the ejecta follows a broken power law (the power indices of the cumulative size distributions of the dust and fragments are 2.5 ± 0.1 and 5.2 ± 0.1, respectively); (4) the mass ratio of the largest fragment to the total ejecta is around 0.8; and (5) the dust cloud morphology is in agreement with the anisotropic ejection model in Kim et al. These new characteristics of the ejecta obtained in this work are favorable to the impact shattering hypothesis.

SRC-induced disintegration of adherens junctions of madin-darby canine kidney cells is dependent on endocytosis of cadherin and antagonized by Tiam-1.

PubMed

Palovuori, Riitta; Sormunen, Raija; Eskelinen, Sinikka

2003-12-01

The effects of Src tyrosine kinase activation in subconfluent temperature sensitive (ts)-Src-transformed Madin-Darby canine kidney (MDCK) cells were analyzed by shifting them from nonpermissive (40.5 degrees C) to permissive (35 degrees C) temperature. Already, in 15 minutes, adherens junction components were released from the lateral walls and accumulated to basal surfaces. Simultaneously, membranous actin staining vanished, actin bundles appeared at the basal surface, and the cells flattened. The only component phosphorylated and translocated after the shift to 35 degrees C was p120ctn. The epithelial-mesenchymal transition could be inhibited by a specific inhibitor of Src kinase, PP2, or by inhibiting endocytosis. Therefore, Src activation was responsible for the transition, but not because of phosphorylation of adherens junction components but by way of activation of endocytic machinery and RhoGTPase. Expression of an RacGEF, Tiam-1 (T-lymphoma invasion and metastasis gene 1), prevented flattening of Src-transformed MDCK cells at 35 degrees C and resulted in accumulation of cadherin to lateral membranes. In the case where the Src-MDCK cells were cultivated at 35 degrees C and shifted for short time periods to 40.5 degrees C, cadherin rapidly returned to lateral membranes, whereas actin and p120ctn followed hours afterward. This further supports the view that cadherin internalization is the primary target of Src kinase. We also looked at the cell morphology and distribution of cadherin and Tiam-1 in cells grown in three-dimensional gels composed of collagen and laminin or in Matrigel. At nonpermissive temperature, both Src-MDCK and Tiam-1-transfected Src-MDCK cells exhibited nonpolarized morphology in collagen I, a loose cluster in the mixture of collagen I and laminin, and a differentiated cyst in Matrigel. In growth factor-depleted Matrigel, the Src-MDCK cells grew in nondifferentiated clusters, whereas Tiam-1-transfected cells went to apoptosis. The differentiated phenotype of both cell lines could be rescued by Matrigel-conditioned medium, platelet-derived growth factor, or cholera toxin. Concomitantly, both cadherin and Tiam-1 were recruited to lateral membranes. Therefore, cadherin and Tiam-1 seem to be the key players in the differentiation process of MDCK cells.

Oxygen partial pressure modulates 67-kDa laminin receptor expression, leading to altered activity of the green tea polyphenol, EGCG.

PubMed

Tsukamoto, Shuntaro; Yamashita, Shuya; Kim, Yoon Hee; Kumazoe, Motofumi; Huang, Yuhui; Yamada, Koji; Tachibana, Hirofumi

2012-09-21

(-)-Epigallocatechin-3-O-gallate (EGCG) exhibits anti-tumor activity mediated via the 67-kDa laminin receptor (67LR). In this study, we found that 67LR protein levels are reduced by exposure to low O(2) levels (5%), without affecting the expression of HIF-1α. We also found that EGCG-induced anti-cancer activity is abrogated under low O(2) levels (5%) in various cancer cells. Notably, treatment with the proteasome inhibitor, prevented down-regulation of 67LR and restored sensitivity to EGCG under 5% O(2). In summary, 67LR expression is highly sensitive to O(2) partial pressure, and the activity of EGCG can be regulated in cancer cells by O(2) partial pressure. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Relative transverse momentum distributions of bottom hadrons produced in 1.96 TeV proton-antiproton collisions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roy, Philippe; /McGill U.

2008-05-01

Fragmentation is the process by which bare quarks dress themselves up as hadrons. Since we cannot get reliable calculations of this process using perturbative quantum chromodynamics, the fragmentation properties of quarks must be obtained empirically. We report on the signal extraction and relative transverse momentum p{sub T} spectrum determination that will lead to a high precision measurement of relative fragmentation fractions of b quarks into B hadrons, in 1.96 TeV p{bar p} collisions. Using 1.9 fb{sup -1} of data taken with the CDF-II detector, we fully reconstruct 473 {+-} 42 B{sub s}{sup 0} {yields} D{sub s}{sup -} {pi}{sup +}, 15206more » {+-} 203 B{sup 0} {yields} D{sup -} {pi}{sup +}, 1483 {+-} 45 B{sup 0} {yields} D*{sup -}{pi}{sup +} and 4444 {+-} 297 {Lambda}{sub b}{sup 0} {yields} {Lambda}{sub c}{sup +}{pi}{sup -} candidate decays. In order to reduce systematic uncertainties, ratios of p{sub T} spectra are reported. We find that B{sub s} and B{sup 0} mesons are produced with similar p{sub T} and {Lambda}{sub b} baryons are produced with lower p{sub T} than B{sup 0} mesons. Our results are consistent with previous CDF measurements suggesting a difference between fragmentation processes observed at lepton and hadron colliders.« less

Modified citrus pectin stops progression of liver fibrosis by inhibiting galectin-3 and inducing apoptosis of stellate cells.

PubMed

Abu-Elsaad, Nashwa M; Elkashef, Wagdi Fawzi

2016-05-01

Modified citrus pectin (MCP) is a pH modified form of the dietary soluble citrus peel fiber known as pectin. The current study aims at testing its effect on liver fibrosis progression. Rats were injected with CCl4 (1 mL/kg, 40% v/v, i.p., twice a week for 8 weeks). Concurrently, MCP (400 or 1200 mg/kg) was administered daily in drinking water from the first week in groups I and II (prophylactic model) and in the beginning of week 5 in groups III and IV (therapeutic model). Liver function biomarkers (ATL, AST, and ALP), fibrosis markers (laminin and hyaluronic acid), and antioxidant biomarkers (reduced glutathione (GSH) and superoxide dismutase (SOD)) were measured. Stained liver sections were scored for fibrosis and necroinflammation. Additionally, expression of galectin-3 (Gal-3), α-smooth muscle actin (SMA), tissue inhibitor metalloproteinase (TIMP)-1, collagen (Col)1A1, caspase (Cas)-3, and apoptosis related factor (FAS) were assigned. Modified pectin late administration significantly (p < 0.05) decreased malondialdehyde (MDA), TIMP-1, Col1A1, α-SMA, and Gal-3 levels and increased levels of FAS, Cas-3, GSH, and SOD. It also decreased percentage of fibrosis and necroinflammation significantly (p < 0.05). It can be concluded that MCP can attenuate liver fibrosis through an antioxidant effect, inhibition of Gal-3 mediated hepatic stellate cells activation, and induction of apoptosis.

The PDGFRα-laminin B1-keratin 19 cascade drives tumor progression at the invasive front of human hepatocellular carcinoma

PubMed Central

Govaere, O; Petz, M; Wouters, J; Vandewynckel, Y-P; Scott, E J; Topal, B; Nevens, F; Verslype, C; Anstee, Q M; Van Vlierberghe, H; Mikulits, W; Roskams, T

2017-01-01

Human hepatocellular carcinomas (HCCs) expressing the biliary/hepatic progenitor cell marker keratin 19 (K19) have been linked with a poor prognosis and exhibit an increase in platelet-derived growth factor receptor α (PDGFRα) and laminin beta 1 (LAMB1) expression. PDGFRα has been reported to induce de novo synthesis of LAMB1 protein in a Sjogren syndrome antigen B (La/SSB)-dependent manner in a murine metastasis model. However, the role of this cascade in human HCC remains unclear. This study focused on the functional role of the PDGFRα-La/SSB-LAMB1 pathway and its molecular link to K19 expression in human HCC. In surgical HCC specimens from a cohort of 136 patients, PDGFRα expression correlated with K19 expression, microvascular invasion and metastatic spread. In addition, PDGFRα expression in pre-operative needle biopsy specimens predicted poor overall survival during a 5-year follow-up period. Consecutive histological staining demonstrated that the signaling components of the PDGFRα-La/SSB-LAMB1 pathway were strongly expressed at the invasive front. K19-positive HCC cells displayed high levels of α2β1 integrin (ITG) receptor, both in vitro and in vivo. In vitro activation of PDGFRα signaling triggered the translocation of nuclear La/SSB into the cytoplasm, enhanced the protein synthesis of LAMB1 by activating its internal ribosome entry site, which in turn led to increased secretion of laminin-111. This effect was abrogated by the PDGFRα-specific inhibitor crenolanib. Importantly LAMB1 stimulated ITG-dependent focal adhesion kinase/Src proto-oncogene non-receptor tyrosine kinase signaling. It also promoted the ITG-specific downstream target Rho-associated coiled-coil containing protein kinase 2, induced K19 expression in an autocrine manner, invadopodia formation and cell invasion. Finally, we showed that the knockdown of LAMB1 or K19 in subcutaneous xenograft mouse models resulted in significant loss of cells invading the surrounding stromal tissue and reduced HepG2 colonization into lung and liver after tail vein injection. The PDGFRα-LAMB1 pathway supports tumor progression at the invasive front of human HCC through K19 expression. PMID:28783171

Structural Insights into Ail-Mediated Adhesion in Yersinia pestis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamashita, Satoshi; Lukacik, Petra; Barnard, Travis J.

2012-01-30

Ail is an outer membrane protein from Yersinia pestis that is highly expressed in a rodent model of bubonic plague, making it a good candidate for vaccine development. Ail is important for attaching to host cells and evading host immune responses, facilitating rapid progression of a plague infection. Binding to host cells is important for injection of cytotoxic Yersinia outer proteins. To learn more about how Ail mediates adhesion, we solved two high-resolution crystal structures of Ail, with no ligand bound and in complex with a heparin analog called sucrose octasulfate. We identified multiple adhesion targets, including laminin and heparin,more » and showed that a 40 kDa domain of laminin called LG4-5 specifically binds to Ail. We also evaluated the contribution of laminin to delivery of Yops to HEp-2 cells. This work constitutes a structural description of how a bacterial outer membrane protein uses a multivalent approach to bind host cells.« less

Chemical inhibition of prometastatic lysyl-tRNA synthetase–laminin receptor interaction

PubMed Central

Kim, Dae Gyu; Lee, Jin Young; Kwon, Nam Hoon; Fang, Pengfei; Zhang, Qian; Wang, Jing; Young, Nicolas L.; Guo, Min; Cho, Hye Young; Mushtaq, AmeeqUl; Jeon, Young Ho; Choi, Jin Woo; Han, Jung Min; Kang, Ho Woong; Joo, Jae Eun; Hur, Youn; Kang, Wonyoung; Yang, Heekyoung; Nam, Do-Hyun; Lee, Mi-Sook; Lee, Jung Weon; Kim, Eun-Sook; Moon, Aree; Kim, Kibom; Kim, Doyeun; Kang, Eun Joo; Moon, Youngji; Rhee, Kyung Hee; Han, Byung Woo; Yang, Jee Sun; Han, Gyoonhee; Yang, Won Suk; Lee, Cheolju; Wang, Ming-Wei; Kim, Sunghoon

2014-01-01

Lysyl-tRNA synthetase (KRS), a protein synthesis enzyme in the cytosol, relocates to the plasma membrane after a laminin signal and stabilizes a 67-kDa laminin receptor (67LR) that is implicated in cancer metastasis; however, its potential as an antimetastatic therapeutic target has not been explored. We found that the small compound BC-K-YH16899, which binds to KRS, impinged on interaction of KRS with 67LR and suppressed metastasis in 3 different mouse models. The compound inhibited KRS–67LR interaction in two ways. First, it directly blocked the association between KRS and 67LR. Second, it suppressed the dynamic movement of the N-terminal extension of KRS and reduced membrane localization of KRS. However, it did not affect the catalytic activity of KRS. Our results suggest that specific modulation of a cancer-related KRS–67LR interaction may offer a way to control metastasis while avoiding the toxicities associated with inhibition of the normal functions of KRS. PMID:24212136

Biological Effects Induced by Specific Advanced Glycation End Products in the Reconstructed Skin Model of Aging

PubMed Central

Pageon, Hervé; Zucchi, Hélène; Dai, Zhenyu; Sell, David R.; Strauch, Christopher M.; Monnier, Vincent M.; Asselineau, Daniel

2015-01-01

Abstract Advanced glycation end products (AGEs) accumulate in the aging skin. To understand the biological effects of individual AGEs, skin reconstructed with collagen selectively enriched with Nɛ-(carboxymethyl)-lysine (CML), Nɛ-(carboxyethyl)-lysine (CEL), methylglyoxal hydroimidazolone (MG-H1), or pentosidine was studied. Immunohistochemistry revealed increased expression of α6 integrin at the dermal epidermal junction by CEL and CML (p<0.01). Laminin 5 was diminished by CEL and MG-H1 (p<0.05). Both CML and CEL induced a robust increase (p<0.01) in procollagen I. In the culture medium, IL-6, VEGF, and MMP1 secretion were significantly decreased (p<0.05) by MG-H1. While both CEL and CML decreased MMP3, only CEL decreased IL-6 and TIMP1, while CML stimulated TIMP1 synthesis significantly (p<0.05). mRNA expression studies using qPCR in the epidermis layer showed that CEL increased type 7 collagen (COL7A1), β1, and α6 integrin, while CML increased only COL7A1 (p<0.05). MG-H1-modified collagen had no effect. Importantly, in the dermis layer, MMP3 mRNA expression was increased by both CML and MG-H1. CML also significantly increased the mRNAs of MMP1, TIMP1, keratinocyte growth factor (KGF), IL-6, and monocyte chemoattractant protein 1 (MCP1) (p<0.05). Mixed effects were present in CEL-rich matrix. Minimally glycoxidized pentosidine-rich collagen suppressed most mRNAs of the genes studied (p<0.05) and decreased VEGF and increased MCP1 protein expression. Taken together, this model of the aging skin suggests that a combination of AGEs tends to counterbalance and thus minimizes the detrimental biological effects of individual AGEs. PMID:26309782

Serum Liver Fibrosis Markers in the Prognosis of Liver Cirrhosis: A Prospective Observational Study.

PubMed

Qi, Xingshun; Liu, Xu; Zhang, Yongguo; Hou, Yue; Ren, Linan; Wu, Chunyan; Chen, Jiang; Xia, Chunlian; Zhao, Jiajun; Wang, Di; Zhang, Yanlin; Zhang, Xia; Lin, Hao; Wang, Hezhi; Wang, Jinling; Cui, Zhongmin; Li, Xueyan; Deng, Han; Hou, Feifei; Peng, Ying; Wang, Xueying; Shao, Xiaodong; Li, Hongyu; Guo, Xiaozhong

2016-08-02

BACKGROUND The prognostic role of serum liver fibrosis markers in cirrhotic patients remains unclear. We performed a prospective observational study to evaluate the effect of amino-terminal pro-peptide of type III pro-collagen (PIIINP), collagen IV (CIV), laminin (LN), and hyaluronic acid (HA) on the prognosis of liver cirrhosis. MATERIAL AND METHODS All patients who were diagnosed with liver cirrhosis and admitted to our department were prospectively enrolled. PIIINP, CIV, LN, and HA levels were tested. RESULTS Overall, 108 cirrhotic patients were included. Correlation analysis demonstrated that CIV (coefficient r: 0.658, p<0.001; coefficient r: 0.368, p<0.001), LN (coefficient r: 0.450, p<0.001; coefficient r: 0.343, p<0.001), and HA (coefficient r: 0.325, p=0.001; coefficient r: 0.282, p=0.004) levels, but not PIIINP level (coefficient r: 0.081, p=0.414; coefficient r: 0.090, p=0.363), significantly correlated with Child-Pugh and MELD scores. Logistic regression analysis demonstrated that HA (odds ratio=1.00003, 95% confidence interval [CI]=1.000004-1.000056, p=0.022) was significantly associated with the 6-month mortality. Receiver operating characteristics analysis demonstrated that the area under the curve (AUC) of HA for predicting the 6-month mortality was 0.612 (95%CI=0.508-0.709, p=0.1531). CONCLUSIONS CIV, LN, and HA levels were significantly associated with the severity of liver dysfunction, but might be inappropriate for the prognostic assessment of liver cirrhosis.

Integrin alpha 3 beta 1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells.

PubMed

Coopman, P J; Thomas, D M; Gehlsen, K R; Mueller, S C

1996-11-01

The mechanisms and receptors involved in phagocytosis by nonhematopoietic cells are not well understood. The involvement of the alpha 3 beta 1 integrin in phagocytosis of the extracellular matrix by human breast cancer cells was studied. The possible role of this integrin was suggested since alpha 3 and beta 1 but not alpha 2 subunits are concentrated at membrane sites where local degradation of fluorescently labeled gelatin occurs. Strikingly, anti-alpha 3 integrin monoclonal antibodies (mAbs) stimulate the phagocytosis of fluorescently labeled gelatin films, gelatin beads, and Matrigel films in a quantitative phagocytosis assay. Stimulation of the gelatin uptake by the anti-alpha 3 mAb is dose responsive, saturable, and time dependent. Antibodies against other integrin subunits have a lower stimulatory effect (anti-beta 1) or no significant effect (anti-alpha 2, -alpha 5, -alpha 6, and -alpha v) on gelatin phagocytosis. The synthetic HGD-6 human laminin peptide that binds specifically the alpha 3 beta 1 integrin, but not the scrambled HSGD-6 control peptide, also markedly stimulates gelatin uptake in a dose-responsive way. Furthermore, the stimulatory effects of the HGD-6 peptide and the anti-alpha 3 mAb are additive, suggesting that they might promote phagocytosis in different ways. Other laminin (YIGSR, IKVAV) and fibronectin (GRGDS) peptides have no effect on gelatin phagocytosis. Immunofluorescence shows that the alpha 3 and the beta 1, but not the alpha 2 integrin subunit, concentrate into patches on the cell surface after treatment with their respective mAbs. And, both gelatin and the alpha 3 beta 1 but not the alpha 2 beta 1 integrin are cointernalized and routed to acidic vesicles such as lysosomes. In conclusion, we demonstrate that human breast cancer cells locally degrade and phagocytose the extracellular matrix and show for the first time that the alpha 3 beta 1 integrin participates in this phagocytosis. We hypothesize that the anti-alpha 3 antibodies and the laminin peptide HGD-6 activate the alpha 3 beta 1 integrin, which results in a downstream signaling cascade stimulating phagocytosis.

The extracellular matrix component laminin promotes gap junction formation in the rat anterior pituitary gland.

PubMed

Horiguchi, Kotaro; Kouki, Tom; Fujiwara, Ken; Kikuchi, Motoshi; Yashiro, Takashi

2011-03-01

Folliculo-stellate (FS) cells in the anterior pituitary gland are believed to have multifunctional properties. FS cells connect to each other not only by mechanical means, but also by gap junctional cell-to-cell communication. Using transgenic rats that express green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary gland (S100b-GFP rats), we recently revealed that FS cells in primary culture markedly change their shape, and form numerous interconnections with neighboring FS cells in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. Morphological and functional changes in cells are believed to be partly modified by matricrine signaling, by which ECM components function as cellular signals. In the present study, we examined whether gap junction formation between FS cells is affected by matricrine cues. A cell sorter was used to isolate FS cells from male S100b-GFP rat anterior pituitary for primary culture. We observed that mRNA and protein levels of connexin 43 in gap junction channels were clearly higher in the presence of laminin. In addition, we confirmed the formation of gap junctions between FS cells in primary culture by electron microscopy. Interestingly, we also observed that FS cells in the presence of laminin displayed well-developed rough endoplasmic reticulum and Golgi apparatus. Our findings suggest that, in anterior pituitary gland, FS cells may facilitate functional roles such as gap junctional cell-to-cell communication by matricrine signaling.

Extracellular matrix components direct porcine muscle stem cell behavior

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilschut, Karlijn J.; Haagsman, Henk P.; Roelen, Bernard A.J., E-mail: b.a.j.roelen@uu.nl

2010-02-01

In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatinmore » and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.« less

Synthesis and properties of 1,3-dioxo-1H-inden-2(3H)-ylidene fragment and (3-(dicyanomethylene)-5,5-dimethylcyclohex-1-enyl)vinyl fragment containing derivatives of azobenzene for holographic recording materials

NASA Astrophysics Data System (ADS)

Zarins, Elmars; Kokars, Valdis; Ozols, Andris; Augustovs, Peteris

2011-06-01

New glassy 1,3-dioxo-1H-inden-2(3H)-ylidene fragment und (3-(dicyanomethylene)-5,5-dimethylcyclohex-1-enyl)vinyl fragment containing push-pull type derivatives of azobenzene able to create thin layers have been synthesized. Thin films of synthesized glasses for holographic recording were prepared using spin coating technique from saturated chloroform solution. Holographic grating recording in films of 6a-b, 7 and 12 has been experimentally studied at 633 and 532 nm in both transmission and reflection modes with p-p recording beam polarizations. The film 12 was found to be the most efficient at both wavelengths in transmission mode exhibiting the maximum self-diffraction efficiency of 9.9% at 633 nm, and 15.3% at 532 nm. The film of 6a was the most efficient in reflection mode with the maximum selfdiffraction efficiency of about 3%.

Highly efficient in vivo delivery of PMO into regenerating myotubes and rescue in laminin-α2 chain-null congenital muscular dystrophy mice.

PubMed

Aoki, Yoshitsugu; Nagata, Tetsuya; Yokota, Toshifumi; Nakamura, Akinori; Wood, Matthew J A; Partridge, Terence; Takeda, Shin'ichi

2013-12-15

Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is among the more promising approaches to the treatment of several neuromuscular disorders including Duchenne muscular dystrophy. The main weakness of this approach arises from the low efficiency and sporadic nature of the delivery of charge-neutral PMO into muscle fibers, the mechanism of which is unknown. In this study, to test our hypothesis that muscle fibers take up PMO more efficiently during myotube formation, we induced synchronous muscle regeneration by injection of cardiotoxin into the tibialis anterior muscle of Dmd exon 52-deficient mdx52 and wild-type mice. Interestingly, by in situ hybridization, we detected PMO mainly in embryonic myosin heavy chain-positive regenerating fibers. In addition, we showed that PMO or 2'-O-methyl phosphorothioate is taken up efficiently into C2C12 myotubes when transfected 24-72 h after the induction of differentiation but is poorly taken up into undifferentiated C2C12 myoblasts suggesting efficient uptake of PMO in the early stages of C2C12 myotube formation. Next, we tested the therapeutic potential of PMO for laminin-α2 chain-null dy(3K)/dy(3K) mice: a model of merosin-deficient congenital muscular dystrophy (MDC1A) with active muscle regeneration. We confirmed the recovery of laminin-α2 chain and slightly prolonged life span following skipping of the mutated exon 4 in dy(3K)/dy(3K) mice. These findings support the idea that PMO entry into fibers is dependent on a developmental stage in myogenesis rather than on dystrophinless muscle membranes and provide a platform for developing PMO-mediated therapies for a variety of muscular disorders, such as MDC1A, that involve active muscle regeneration.

Impact of renal anatomy on shock wave lithotripsy outcomes for lower pole kidney stones: results of a prospective multifactorial analysis controlled by computerized tomography.

PubMed

Torricelli, Fabio C M; Marchini, Giovanni S; Yamauchi, Fernando I; Danilovic, Alexandre; Vicentini, Fabio C; Srougi, Miguel; Monga, Manoj; Mazzucchi, Eduardo

2015-06-01

We evaluated which variables impact fragmentation and clearance of lower pole calculi after shock wave lithotripsy. We prospectively evaluated patients undergoing shock wave lithotripsy for a solitary 5 to 20 mm lower pole kidney stone between June 2012 and August 2014. Patient body mass index and abdominal waist circumference were recorded. One radiologist blinded to shock wave lithotripsy outcomes measured stone size, area and density, stone-to-skin distance, infundibular length, width and height, and infundibulopelvic angle based on baseline noncontrast computerized tomography. Fragmentation, success (defined as residual fragments less than 4 mm in asymptomatic patients) and the stone-free rate were evaluated by noncontrast computerized tomography 12 weeks postoperatively. Univariate and multivariate analysis was performed. A total of 100 patients were enrolled in the study. Mean stone size was 9.1 mm. Overall fragmentation, success and stone-free rates were 76%, 54% and 37%, respectively. On logistic regression body mass index (OR 1.27, 95% CI 1.11-1.49, p = 0.004) and stone density (OR 1.0026, 95% CI 1.0008-1.0046, p = 0.005) significantly impacted fragmentation. Stone size (OR 1.24, 95% CI 1.07-1.48, p = 0.039) and stone density (OR 1.0021, 95% CI 1.0007-1.0037, p = 0.012) impacted the success rate while stone size (OR 1.24, 95% CI 1.04-1.50, p = 0.029), stone density (OR 1.0015, 95% CI 1.0001-1.0032, p = 0.046) and infundibular length (OR 1.1035, 95% CI 1.015-1.217, p = 0.015) impacted the stone-free rate. The best outcomes were found in patients with a body mass index of 30 kg/m(2) or less, stones 10 mm or less and 900 HU or less, and an infundibular length of 25 mm or less. The coexistence of significant unfavorable variables led to a stone-free rate of less than 20%. Obese patients with higher than 10 mm density stones (greater than 900 HU) in the lower pole of the kidney with an infundibular length of greater than 25 mm should be discouraged from undergoing shock wave lithotripsy. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

VEGFR-1 Expressed by Malignant Melanoma-Initiating Cells Is Required for Tumor Growth

PubMed Central

Frank, Natasha Y.; Schatton, Tobias; Kim, Soo; Zhan, Qian; Wilson, Brian J.; Ma, Jie; Saab, Karim R.; Osherov, Veronika; Widlund, Hans R.; Gasser, Martin; Waaga-Gasser, Ana-Maria; Kupper, Thomas S.; Murphy, George F.; Frank, Markus H.

2011-01-01

Melanoma growth is driven by malignant melanoma-initiating cells (MMIC) identified by expression of the ATP-binding cassette (ABC) member ABCB5. ABCB5+ melanoma subpopulations have been shown to overexpress the vasculogenic differentiation markers CD144 (VE-cadherin) and TIE1 and are associated with CD31− vasculogenic mimicry (VM), an established biomarker associated with increased patient mortality. Here we identify a critical role for VEGFR-1 signaling in ABCB5+ MMIC-dependent VM and tumor growth. Global gene expression analyses, validated by mRNA and protein determinations, revealed preferential expression of VEGFR-1 on ABCB5+ tumor cells purified from clinical melanomas and established melanoma lines. In vitro, VEGF induced the expression of CD144 in ABCB5+ subpopulations that constitutively expressed VEGFR-1 but not in ABCB5− bulk populations that were predominantly VEGFR-1−. In vivo, melanoma-specific shRNA-mediated knockdown of VEGFR-1 blocked the development of ABCB5+ VM morphology and inhibited ABCB5+ VM-associated production of the secreted melanoma mitogen laminin. Moreover, melanoma-specific VEGFR-1 knockdown markedly inhibited tumor growth (by >90%). Our results show that VEGFR-1 function in MMIC regulates VM and associated laminin production and show that this function represents one mechanism through which MMICs promote tumor growth. PMID:21212411

VEGFR-1 expressed by malignant melanoma-initiating cells is required for tumor growth.

PubMed

Frank, Natasha Y; Schatton, Tobias; Kim, Soo; Zhan, Qian; Wilson, Brian J; Ma, Jie; Saab, Karim R; Osherov, Veronika; Widlund, Hans R; Gasser, Martin; Waaga-Gasser, Ana-Maria; Kupper, Thomas S; Murphy, George F; Frank, Markus H

2011-02-15

Melanoma growth is driven by malignant melanoma-initiating cells (MMIC) identified by expression of the ATP-binding cassette (ABC) member ABCB5. ABCB5(+) melanoma subpopulations have been shown to overexpress the vasculogenic differentiation markers CD144 (VE-cadherin) and TIE1 and are associated with CD31(-) vasculogenic mimicry (VM), an established biomarker associated with increased patient mortality. Here we identify a critical role for VEGFR-1 signaling in ABCB5(+) MMIC-dependent VM and tumor growth. Global gene expression analyses, validated by mRNA and protein determinations, revealed preferential expression of VEGFR-1 on ABCB5(+) tumor cells purified from clinical melanomas and established melanoma lines. In vitro, VEGF induced the expression of CD144 in ABCB5(+) subpopulations that constitutively expressed VEGFR-1 but not in ABCB5(-) bulk populations that were predominantly VEGFR-1(-). In vivo, melanoma-specific shRNA-mediated knockdown of VEGFR-1 blocked the development of ABCB5(+) VM morphology and inhibited ABCB5(+) VM-associated production of the secreted melanoma mitogen laminin. Moreover, melanoma-specific VEGFR-1 knockdown markedly inhibited tumor growth (by > 90%). Our results show that VEGFR-1 function in MMIC regulates VM and associated laminin production and show that this function represents one mechanism through which MMICs promote tumor growth. ©2011 AACR.

Epstein-Barr virus recombinants from overlapping cosmid fragments.

PubMed

Tomkinson, B; Robertson, E; Yalamanchili, R; Longnecker, R; Kieff, E

1993-12-01

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection of restriction fragment length polymorphisms in clinical isolates and serially passaged Pseudomonas aeruginosa strains.

PubMed Central

Hjelm, L N; Branstrom, A A; Warren, R L

1990-01-01

An 800-base-pair HindIII-PstI fragment that flanks a hot spot for Tn7 insertion was isolated from the chromosome of Pseudomonas aeruginosa and cloned into pUC12. The fragment was used to probe XhoI digests of genomic DNA from 18 P. aeruginosa isolates collected from sputum samples of seven cystic fibrosis patients. Only two XhoI restriction fragment length polymorphisms (RFLPs), of 3.7 and 7.7 kilobases (kb), were detected. Isolate WSU3531-1 (3.7-kb XhoI fragment) and WSU3860 (7.7-kb XhoI fragment), while isolated from the same patient, showed different RFLPs. Serial passages of isolate WSU3531-1 demonstrated that this strain was phenotypically stable. In contrast, colony and pigment variants were readily isolated at a frequency of 1% from serial passages of isolate WSU3860. When XhoI-digested genomic DNA from phenotypic variants of serially passaged WSU3860 were probed with the 800-base-pair HindIII-PstI fragment, the probe hybridized to a 10.4-kb XhoI fragment from three isolates. Restriction analysis of the genomic DNA digested with a variety of restriction enzymes showed that a 2.7-kb insertion occurred in the same region for all three isolates. There appeared to be no correlation between changes in the RFLP and changes in colony morphology. Images PMID:1977762

Insulin-like growth factor-1 (IGF-1) promotes primordial follicle growth and reduces DNA fragmentation through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signalling pathway.

PubMed

Bezerra, Maria É S; Barberino, Ricássio S; Menezes, Vanúzia G; Gouveia, Bruna B; Macedo, Taís J S; Santos, Jamile M S; Monte, Alane P O; Barros, Vanessa R P; Matos, Maria H T

2018-05-30

We investigated the effects of insulin-like growth factor 1 (IGF-1) on the morphology and follicular activation of ovine preantral follicles cultured in situ and whether the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway is involved in IGF-1 action in the sheep ovary. Ovine ovarian fragments were fixed for histological and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) analyses (fresh control) or cultured in supplemented alpha-minimum essential medium (α-MEM+; control) or α-MEM+ with IGF-1 (1, 10, 50, 100 or 200ngmL-1) for 7 days. Follicles were classified as normal or atretic, primordial or growing and the oocyte and follicle diameters were measured. DNA fragmentation was evaluated by TUNEL assay. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was performed on the fresh control, α-MEM+ and 100ngmL-1 IGF-1 samples. Inhibition of PI3K activity was performed through pretreatment with the PI3K inhibitor LY294002 and phosphorylated AKT (pAKT) expression was analysed after culture in the absence or presence of LY294002. IGF-1 at 100ngmL-1 increased (P<0.05) follicular activation compared with α-MEM+ and decreased TUNEL-positive cells (P<0.05) compared with other treatments. PCNA-positive cells also increased (P<0.05) in 100ngmL-1 IGF-1. LY294002 significantly inhibited follicular activation stimulated by α-MEM+ and 100ngmL-1 IGF-1 and reduced pAKT expression in follicles. Overall, IGF-1 at 100ngmL-1 promoted primordial follicle activation, cell proliferation and reduced DNA fragmentation after in situ culture through the PI3K/AKT pathway.

[Expression of matrix metalloproteinase-19 in the human cornea. Wound healing in the MMP-19 knock-out mouse model].

PubMed

Treumer, F; Flöhr, C; Klettner, A; Nölle, B; Roider, J

2010-07-01

At present there are no data in the literature on the expression of matrix metalloprotein-19 in the human cornea. The aim of this study was to analyze the expression of matrix metalloproteinase-19 in the human cornea and to investigate its potential role in corneal wound healing using a MMP-19 knock-out mouse model. A method with Western blotting and immunohistological staining for MMP-19 was performed using paraffin embedded human corneas. Excimer laser keratectomy was performed in wild type (wt) and MMP-19 knock-out (ko) mice and the rate of re-epithelialization was analyzed after 8 h and 18 h. MMP-19 was strongly expressed in the human corneal epithelium mainly in the basal cell layer. MMP-19 was not expressed in the corneal stroma. In the mouse model the size of the corneal lesion after 8 h was 83% (wt) and 89.9% (ko) of the initial area (p=0.09). After 18 h the lesion was 17% (wt) and 13.3% (ko) of the initial area (p=0.01). Laminin-5 was expressed in the migrating epithelial cells with no differences between wild type and knock-out mouse. MMP-19 showed a strong expression in the basal cells of the human corneal epithelium. Corneal re-epithelialization was slightly faster in the MMP-19 knock-out mouse. No differences in the expression of laminin-5 could be detected.

Comparison of (99m)Tc-labeled PR81 and its F(ab')₂ fragments as radioimmunoscintigraphy agents for breast cancer imaging.

PubMed

Salouti, Mojtaba; Babaei, Mohammad Hossein; Rajabi, Hossein; Foroutan, Haleh; Rasaee, Mohammad Javad; Rajabi, Ahmad Bitarafan; Mohammadnejad, Javad; Shafiee, Mohammad; Mazidi, Mohammad; Daha, Fariba Johari

2011-02-01

We digested anti-MUC1 monoclonal antibody PR81 to produce F(ab')₂ fragments. A comparison was performed between the two radiolabeled PR81 and F(ab')₂ fragments for breast tumor imaging in a mouse model. The optimum conditions for pepsin digestion of PR81 were investigated in terms of enzymes: antibody ratio, digestion time duration and preserved immunoreactivity of the produced fragments. The F(ab')₂ fragments were labeled with Technetium-99m using HYNIC as a chelator and tricine as a co-ligand. The immunoreactivity of the complexes was assessed by radioimmunoassay using MCF7 cells. Biodistribution and imaging studies were performed in female BALB/c mice with breast tumor xenograft at 4, 8 and 24 h post-administration. The PR81 was labeled with technetium-99m in the same way for comparison. The optimum time duration for PR81 digestion was found to be 28 h at an enzyme:antibody weight ratio of 1:20 that resulted in 95.2 ± 4.7% purity. The labeling of intact PR81 and its F(ab')₂ fragments were 87.6 ± 4.2 and 76.1 ± 3.3% after 1 h, respectively (p value <0.05). The percentage of immunoreactivity of F(ab')₂ fragments and intact PR81 were 75.4 ± 2.1% and 85.7 ± 2.9%, respectively (p value <0.05). The biodistribution and imaging studies demonstrated localization of the fragments at 4 h post-administration with high sensitivity and specificity. The results showed that F(ab')₂ fragment of PR81 is more suitable than intact PR81 for safer and more rapid detection of human breast cancer.

A double-labeling procedure for sequence analysis of picomole amounts of nonradioactive RNA fragments.

PubMed Central

Gupta, R C; Randerath, E; Randerath, K

1976-01-01

A double-labeling procedure for sequence analysis of nonradioactive polyribonucleotides is detailed, which is based on controlled endonucleolytic degradation of 3'-terminally (3H)-labeled oligonucleotide-(3') dialcohols and 5"-terminal analysis of the partial (3H)-labeled fragments following their separation according to chain length by polyethyleneimine- (PEI-)cellulose TLC and detection by fluorography. Undesired nonradioactive partial digestion products are eliminated by periodate oxidation. The 5'-termini are assayed by enzymic incorporation of (32p)-label into the isolated fragments, enzymic release of (32p)-labeled nucleoside-(5') monophosphates, two-dimensional PEI-cellulose chromatography, and autoradiography. Using this procedure, as little as 0.1 - 0.3 A260 unit of tRNA is needed to sequence all fragments in complete ribonuclease T1 and A digests, whereas radioactive derivative methods previously described by us1-4 required 4 - 6 A260 units. Images PMID:826884

A Fragment-Based Ligand Screen Against Part of a Large Protein Machine: The ND1 Domains of the AAA+ ATPase p97/VCP.

PubMed

Chimenti, Michael S; Bulfer, Stacie L; Neitz, R Jeffrey; Renslo, Adam R; Jacobson, Matthew P; James, Thomas L; Arkin, Michelle R; Kelly, Mark J S

2015-07-01

The ubiquitous AAA+ ATPase p97 functions as a dynamic molecular machine driving several cellular processes. It is essential in regulating protein homeostasis, and it represents a potential drug target for cancer, particularly when there is a greater reliance on the endoplasmic reticulum-associated protein degradation pathway and ubiquitin-proteasome pathway to degrade an overabundance of secreted proteins. Here, we report a case study for using fragment-based ligand design approaches against this large and dynamic hexamer, which has multiple potential binding sites for small molecules. A screen of a fragment library was conducted by surface plasmon resonance (SPR) and followed up by nuclear magnetic resonance (NMR), two complementary biophysical techniques. Virtual screening was also carried out to examine possible binding sites for the experimental hits and evaluate the potential utility of fragment docking for this target. Out of this effort, 13 fragments were discovered that showed reversible binding with affinities between 140 µM and 1 mM, binding stoichiometries of 1:1 or 2:1, and good ligand efficiencies. Structural data for fragment-protein interactions were obtained with residue-specific [U-(2)H] (13)CH3-methyl-labeling NMR strategies, and these data were compared to poses from docking. The combination of virtual screening, SPR, and NMR enabled us to find and validate a number of interesting fragment hits and allowed us to gain an understanding of the structural nature of fragment binding. © 2015 Society for Laboratory Automation and Screening.

Defects in subventricular zone pigmented epithelium-derived factor niche signaling in the senescence-accelerated mouse prone-8.

PubMed

Castro-Garcia, Paola; Díaz-Moreno, María; Gil-Gas, Carmen; Fernández-Gómez, Francisco J; Honrubia-Gómez, Paloma; Álvarez-Simón, Carmen Belén; Sánchez-Sánchez, Francisco; Cano, Juan Carlos Castillo; Almeida, Francisco; Blanco, Vicente; Jordán, Joaquín; Mira, Helena; Ramírez-Castillejo, Carmen

2015-04-01

We studied potential changes in the subventricular zone (SVZ) stem cell niche of the senescence-accelerated mouse prone-8 (SAM-P8) aging model. Bromodeoxyuridine (BrdU) assays with longtime survival revealed a lower number of label-retaining stem cells in the SAM-P8 SVZ compared with the SAM-Resistant 1 (SAM-R1) control strain. We also found that in SAM-P8 niche signaling is attenuated and the stem cell pool is less responsive to the self-renewal niche factor pigmented epithelium-derived factor (PEDF). Protein analysis demonstrated stable amounts of the PEDF ligand in the SAM-P8 SVZ niche; however, SAM-P8 stem cells present a significant expression decrease of patatin-like phospholipase domain containing 2, a receptor for PEDF (PNPLA2-PEDF) receptor, but not of laminin receptor (LR), a receptor for PEDF (LR-PEDF) receptor. We observed changes in self-renewal related genes (hairy and enhancer of split 1 (Hes1), hairy and enhancer of split 1 (Hes5), Sox2] and report that although these genes are down-regulated in SAM-P8, differentiation genes (Pax6) are up-regulated and neurogenesis is increased. Finally, sheltering mammalian telomere complexes might be also involved given a down-regulation of telomeric repeat binding factor 1 (Terf1) expression was observed in SAM-P8 at young age periods. Differences between these 2 models, SAM-P8 and SAM-R1 controls, have been previously detected at more advanced ages. We now describe alterations in the PEDF signaling pathway and stem cell self-renewal at a very young age, which could be involved in the premature senescence observed in the SAM-P8 model. © FASEB.

A clinicopathological study of the expression of extracellular matrix components in urothelial carcinoma.

PubMed

Ioachim, Elli; Michael, Michalis; Stavropoulos, Nicolaos E; Kitsiou, Evangelia; Salmas, Marios; Malamou-Mitsi, Vasiliki

2005-03-01

To measure the immunohistochemical expression of the extracellular matrix (ECM) components tenascin, fibronectin, collagen type IV and laminin in urothelial carcinomas, and to correlate their expression with clinicopathological features to clarify the prognostic value of these molecules and their role in tumour progression. Tumour specimens obtained during transurethral resection of bladder tumour (TURBT) from 103 patients (82 men and 2 1 women, mean age 66.7 years, range 27-89) were studied retrospectively. The expression of tenascin, fibronectin, collagen type IV and laminin was correlated with clinicopathological features (tumour grade and stage, multiplicity, simultaneous in situ component, the proliferative activity as estimated by the two proliferation associated indices, Ki-67 and proliferating cell nuclear antigen, the recurrence rate, and the progression of invading tumour). Specimens investigated for tenascin expression from patients with superficial bladder cancers were categorized into 28 treated by TURBT only and 53 who had TURBT followed by intravesical instillations of interferon. Cytoplasmic tenascin expression was detected in tumour cells in 20% of specimens. Tenascin was expressed in the tumour stroma in 76% of specimens, and was positively correlated with tumour grade and stage. Stromal tenascin expression was positively correlated with proliferative activity, and with the expression of fibronectin and collagen type IV. Fibronectin was expressed in the tumour stroma in 89% of specimens and was positively correlated with tumour stage, proliferative activity, and expression of collagen type IV and laminin. Collagen type IV was expressed in 93% of specimens, and was positively correlated with tumour grade and stage. Laminin was expressed in 78% of specimens and had no significant correlation with the clinicopathological features. Patients treated with TURBT alone and who had low levels of tenascin had a longer tumour-free interval than those with high levels of tenascin. Levels of tenascin might be valuable for predicting the risk of early recurrence. The expression of tenascin, fibronectin and collagen type IV seems to be correlated with more aggressive tumour behaviour. Furthermore, their interrelationships could indicate that they are involved in the remodelling of bladder cancer tissue, probably influencing tumour progression.

Corticocapsular cleavage during phacoemulsification: Viscodissection versus hydrodissection. Miyake-Apple view analysis.

PubMed

Vasavada, Vaishali; Vasavada, Viraj A; Werner, Liliana; Mamalis, Nick; Vasavada, Abhay R; Crandall, Alan S

2008-07-01

To compare the corticocapsular separation created by viscodissection and by hydrodissection using Miyake-Apple video-photographic analysis. University-based center. Fourteen cadaver eyes were randomized to hydrodissection (Group 1, n=7) or viscodissection (Group 2, n=7). Twelve eyes were prepared for Miyake-Apple viewing. One eye in each group was prepared to simulate a closed chamber. On Miyake-Apple viewing, corticocapsular separation was graded from 0 to 3 after hydrodissection/viscodissection and at the sculpting, nuclear division, and early and late fragment removal stages. The surgeon's subjective impression of the mechanical cushion effect was noted. Histopathology of the capsular bag was performed in 4 eyes in each group to determine the amount of residual lens epithelial cells (LECs). More space was created and maintained between the capsule and cortex in Group 2 than in Group 1. The mean scores in Groups 1 and 2 were endpoint of hydrodissection/viscodissection 1.92+/-0.38 and 2.58+/-0.38, respectively (P=.05); sculpting, 1.6+/-0.42 and 2.3+/-0.45, respectively (P=.05); nuclear division, 0.9+/-0.42 and 1.5+/-0.35, respectively (P=.13); early fragment removal, 0.6+/-0.22 and 1.0+/-0, respectively (P=.04); and late fragment removal, 0.1+/-0.22 and 0.4+/-0.22, respectively (P=.17). Although only early fragment removal achieved statistical significance, the surgeon's impression confirmed greater cushion effect with viscodissection. Residual LECs in the 2 groups were comparable. Viscodissection as an adjunct to hydrodissection created and maintained greater corticocapsular separation than hydrodissection alone during phacoemulsification.

Flanking sequence determination and specific PCR identification of transgenic wheat B102-1-2.

PubMed

Cao, Jijuan; Xu, Junyi; Zhao, Tongtong; Cao, Dongmei; Huang, Xin; Zhang, Piqiao; Luan, Fengxia

2014-01-01

The exogenous fragment sequence and flanking sequence between the exogenous fragment and recombinant chromosome of transgenic wheat B102-1-2 were successfully acquired using genome walking technology. The newly acquired exogenous fragment encoded the full-length sequence of transformed genes with transformed plasmid and corresponding functional genes including ubi, vector pBANF-bar, vector pUbiGUSPlus, vector HSP, reporter vector pUbiGUSPlus, promoter ubiquitin, and coli DH1. A specific polymerase chain reaction (PCR) identification method for transgenic wheat B102-1-2 was established on the basis of designed primers according to flanking sequence. This established specific PCR strategy was validated by using transgenic wheat, transgenic corn, transgenic soybean, transgenic rice, and non-transgenic wheat. A specifically amplified target band was observed only in transgenic wheat B102-1-2. Therefore, this method is characterized by high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of transgenic wheat B102-1-2.

Preantral follicle density in ovarian biopsy fragments and effects of mare age.

PubMed

Alves, K A; Alves, B G; Gastal, G D A; Haag, K T; Gastal, M O; Figueiredo, J R; Gambarini, M L; Gastal, E L

2017-04-01

The aims of the present study were to: (1) evaluate preantral follicle density in ovarian biopsy fragments within and among mares; (2) assess the effects of mare age on the density and quality of preantral follicles; and (3) determine the minimum number of ovarian fragments and histological sections needed to estimate equine follicle density using a mathematical model. The ovarian biopsy pick-up method was used in three groups of mares separated according to age (5-6, 7-10 and 11-16 years). Overall, 336 preantral follicles were recorded with a mean follicle density of 3.7 follicles per cm 2 . Follicle density differed (P<0.05) among animals, ovarian fragments from the same animal, histological sections and age groups. More (P<0.05) normal follicles were observed in the 5-6 years (97%) than the 11-16 years (84%) age group. Monte Carlo simulations showed a higher probability (90%; P<0.05) of detecting follicle density using two experimental designs with 65 histological sections and three to four ovarian fragments. In summary, equine follicle density differed among animals and within ovarian fragments from the same animal, and follicle density and morphology were negatively affected by aging. Moreover, three to four ovarian fragments with 65 histological sections were required to accurately estimate follicle density in equine ovarian biopsy fragments.

Fragment-based virtual screening approach and molecular dynamics simulation studies for identification of BACE1 inhibitor leads.

PubMed

Manoharan, Prabu; Ghoshal, Nanda

2018-05-01

Traditional structure-based virtual screening method to identify drug-like small molecules for BACE1 is so far unsuccessful. Location of BACE1, poor Blood Brain Barrier permeability and P-glycoprotein (Pgp) susceptibility of the inhibitors make it even more difficult. Fragment-based drug design method is suitable for efficient optimization of initial hit molecules for target like BACE1. We have developed a fragment-based virtual screening approach to identify/optimize the fragment molecules as a starting point. This method combines the shape, electrostatic, and pharmacophoric features of known fragment molecules, bound to protein conjugate crystal structure, and aims to identify both chemically and energetically feasible small fragment ligands that bind to BACE1 active site. The two top-ranked fragment hits were subjected for a 53 ns MD simulation. Principle component analysis and free energy landscape analysis reveal that the new ligands show the characteristic features of established BACE1 inhibitors. The potent method employed in this study may serve for the development of potential lead molecules for BACE1-directed Alzheimer's disease therapeutics.

Effects of Modification in the Laminin-10 Basal Lamina on Prostate Cancer Invasion

DTIC Science & Technology

2007-10-01

assays of ERK phosphorylations were performed by Western blotting with specific antibodies (14). 4. For immunohistochemistry analysis of MT1-MMP...Maquoi, E., Munaut, C., Remacle, A., and Foidart, J. M. (1997) Invasion Metastasis 17(5), 221-239 6. Foda , H. D., and Zucker, S. (2001) Drug Discov

Cre-loxP–mediated Inactivation of the α6A Integrin Splice Variant In Vivo: Evidence for a Specific Functional Role of α6A in Lymphocyte Migration but Not in Heart Development

PubMed Central

Gimond, Clotilde; Baudoin, Christian; van der Neut, Ronald; Kramer, Duco; Calafat, Jero; Sonnenberg, Arnoud

1998-01-01

Two splice variants of the α6 integrin subunit, α6A and α6B, with different cytoplasmic domains, have previously been described. While α6B is expressed throughout the development of the mouse, the expression of α6A begins at 8.5 days post coitum and is initially restricted to the myocardium. Later in ontogeny, α6A is found in various epithelia and in certain cells of the immune system. In this study, we have investigated the function of α6A in vivo by generating knockout mice deficient for this splice variant. The Cre- loxP system of the bacteriophage P1 was used to specifically remove the exon encoding the cytoplasmic domain of α6A in embryonic stem cells, and the deletion resulted in the expression of α6B in all tissues that normally express α6A. We show that α6A−/− mice develop normally and are fertile. The substitution of α6A by α6B does not impair the development and function of the heart, hemidesmosome formation in the epidermis, or keratinocyte migration. Furthermore, T cells differentiated normally in α6A−/− mice. However, the substitution of α6A by α6B leads to a decrease in the migration of lymphocytes through laminin-coated Transwell filters and to a reduction of the number of T cells isolated from the peripheral and mesenteric lymph nodes. Lymphocyte homing to the lymph nodes, which involves various types of integrin–ligand interactions, was not affected in the α6A knockout mice, indicating that the reduced number of lymph node cells could not be directly attributed to defects in lymphocyte trafficking. Nevertheless, the expression of α6A might be necessary for optimal lymphocyte migration on laminin in certain pathological conditions. PMID:9763436

Effect of 3D-scaffold formation on differentiation and survival in human neural progenitor cells.

PubMed

Ortinau, Stefanie; Schmich, Jürgen; Block, Stephan; Liedmann, Andrea; Jonas, Ludwig; Weiss, Dieter G; Helm, Christiane A; Rolfs, Arndt; Frech, Moritz J

2010-11-11

3D-scaffolds have been shown to direct cell growth and differentiation in many different cell types, with the formation and functionalisation of the 3D-microenviroment being important in determining the fate of the embedded cells. Here we used a hydrogel-based scaffold to investigate the influences of matrix concentration and functionalisation with laminin on the formation of the scaffolds, and the effect of these scaffolds on human neural progenitor cells cultured within them. In this study we used different concentrations of the hydrogel-based matrix PuraMatrix. In some experiments we functionalised the matrix with laminin I. The impact of concentration and treatment with laminin on the formation of the scaffold was examined with atomic force microscopy. Cells from a human fetal neural progenitor cell line were cultured in the different matrices, as well as in a 2D culture system, and were subsequently analysed with antibody stainings against neuronal markers. In parallel, the survival rate of the cells was determined by a live/dead assay. Atomic force microscopy measurements demonstrated that the matrices are formed by networks of isolated PuraMatrix fibres and aggregates of fibres. An increase of the hydrogel concentration led to a decrease in the mesh size of the scaffolds and functionalisation with laminin promoted aggregation of the fibres (bundle formation), which further reduces the density of isolated fibres. We showed that laminin-functionalisation is essential for human neural progenitor cells to build up 3D-growth patterns, and that proliferation of the cells is also affected by the concentration of matrix. In addition we found that 3D-cultures enhanced neuronal differentiation and the survival rate of the cells compared to 2D-cultures. Taken together, we have demonstrated a direct influence of the 3D-scaffold formation on the survival and neuronal differentiation of human neural progenitor cells. These findings emphasize the importance of optimizing 3D-scaffolds protocols prior to in vivo engraftment of stem and progenitor cells in the context of regenerative medicine.

Dual mini-fragment plating for midshaft clavicle fractures: a clinical and biomechanical investigation.

PubMed

Prasarn, Mark L; Meyers, Kathleen N; Wilkin, Geoffrey; Wellman, David S; Chan, Daniel B; Ahn, Jaimo; Lorich, Dean G; Helfet, David L

2015-12-01

We sought to evaluate clinical and biomechanical outcomes of dual mini-fragment plate fixation for clavicle fractures. We hypothesized that this technique would produce an anatomical reduction with good clinical outcomes, be well tolerated by patients, and demonstrate equivalent biomechanics to single plating. Dual mini-fragment plating was performed for 17 isolated, displaced midshaft clavicle fractures. Functional outcomes and complications were retrospectively reviewed. A sawbones model compared dual plating biomechanics to a (1) superior 3.5-mm locking reconstruction plate, or (2) antero-inferior 3.5-mm locking reconstruction plate. On biomechanical testing, with anterior loading, dual plating was significantly more rigid than single locked anterior-plating (p = 0.02) but less rigid than single locked superior-plating (p = 0.001). With superior loading, dual plating trended toward higher rigidity versus single locked superior-plating (p = 0.07) but was less rigid than single locked anterior-plating (p = 0.03). No statistically significant differences in axial loading (p = 0.27) or torsion (p = 0.23) were detected. Average patient follow-up was 16.1 months (12-38). Anatomic reduction was achieved and maintained through final healing (average 14.7 weeks). No patient underwent hardware removal. Average 1-year DASH score was 4.0 (completed in 88 %). Displaced midshaft clavicle fractures can be effectively managed with dual mini-fragment plating. This technique results in high union rates and excellent clinical outcomes. Compared to single plating, dual plating is biomechanically equivalent in axial loading and torsion, yet offers better multi-planar bending stiffness despite the use of smaller plates. This technique may decrease the need for secondary surgery due to implant prominence and may aid in fracture reduction by buttressing butterfly fragments in two planes.

Detection and localisation of protein-protein interactions in Saccharomyces cerevisiae using a split-GFP method.

PubMed

Barnard, Emma; McFerran, Neil V; Trudgett, Alan; Nelson, John; Timson, David J

2008-05-01

An alternative method for monitoring protein-protein interactions in Saccharomyces cerevisiae has been developed. It relies on the ability of two fragments of enhanced green fluorescent protein (EGFP) to reassemble and fluoresce when fused to interacting proteins. Since this fluorescence can be detected in living cells, simultaneous detection and localisation of interacting pairs is possible. DNA sequences encoding N- and C-terminal EGFP fragments flanked by sequences from the genes of interest were transformed into S. cerevisiae JPY5 cells and homologous recombination into the genome verified by PCR. The system was evaluated by testing known interacting proteins: labelling of the phosphofructokinase subunits, Pfk1p and Pfk2p, with N- and C-terminal EGFP fragments, respectively, resulted in green fluorescence in the cytoplasm. The system works in other cellular compartments: labelling of Idh1p and Idh2p (mitochondrial matrix), Sdh3p and Sdh4p (mitochondrial membrane) and Pap2p and Mtr4p (nucleus) all resulted in fluorescence in the appropriate cellular compartment.

Study of streptococcal hemoprotein receptor (Shr) in iron acquisition and virulence of M1T1 group A streptococcus.

PubMed

Dahesh, Samira; Nizet, Victor; Cole, Jason N

2012-11-15

Streptococcus pyogenes (group A streptococcus, GAS) is a human bacterial pathogen of global significance, causing severe invasive diseases associated with serious morbidity and mortality. To survive within the host and establish an infection, GAS requires essential nutrients, including iron. The streptococcal hemoprotein receptor (Shr) is a surface-localized GAS protein that binds heme-containing proteins and extracellular matrix components. In this study, we employ targeted allelic exchange mutagenesis to investigate the role of Shr in the pathogenesis of the globally disseminated serotype M1T1 GAS. The shr mutant exhibited a growth defect in iron-restricted medium supplemented with ferric chloride, but no significant differences were observed in neutrophil survival, antimicrobial peptide resistance, cell surface charge, fibronectin-binding or adherence to human epithelial cells and keratinocytes, compared with wild-type. However, the shr mutant displayed a reduction in human blood proliferation, laminin-binding capacity and was attenuated for virulence in in vivo models of skin and systemic infection. We conclude that Shr augments GAS adherence to laminin, an important extracellular matrix attachment component. Furthermore, Shr-mediated iron uptake contributes to GAS growth in human blood, and is required for full virulence of serotype M1T1 GAS in mouse models of invasive disease.

Inhibition of mitochondrial fragmentation diminishes Huntington’s disease–associated neurodegeneration

PubMed Central

Guo, Xing; Disatnik, Marie-Helene; Monbureau, Marie; Shamloo, Mehrdad; Mochly-Rosen, Daria; Qi, Xin

2013-01-01

Huntington’s disease (HD) is the result of expression of a mutated Huntingtin protein (mtHtt), and is associated with a variety of cellular dysfunctions including excessive mitochondrial fission. Here, we tested whether inhibition of excessive mitochondrial fission prevents mtHtt-induced pathology. We developed a selective inhibitor (P110-TAT) of the mitochondrial fission protein dynamin-related protein 1 (DRP1). We found that P110-TAT inhibited mtHtt-induced excessive mitochondrial fragmentation, improved mitochondrial function, and increased cell viability in HD cell culture models. P110-TAT treatment of fibroblasts from patients with HD and patients with HD with iPS cell–derived neurons reduced mitochondrial fragmentation and corrected mitochondrial dysfunction. P110-TAT treatment also reduced the extent of neurite shortening and cell death in iPS cell–derived neurons in patients with HD. Moreover, treatment of HD transgenic mice with P110-TAT reduced mitochondrial dysfunction, motor deficits, neuropathology, and mortality. We found that p53, a stress gene involved in HD pathogenesis, binds to DRP1 and mediates DRP1-induced mitochondrial and neuronal damage. Furthermore, P110-TAT treatment suppressed mtHtt-induced association of p53 with mitochondria in multiple HD models. These data indicate that inhibition of DRP1-dependent excessive mitochondrial fission with a P110-TAT–like inhibitor may prevent or slow the progression of HD. PMID:24231356

Variation in ribosomal and mitochondrial DNA sequences demonstrates the existence of intraspecific groups in Paramecium multimicronucleatum (Ciliophora, Oligohymenophorea).

PubMed

Tarcz, Sebastian; Potekhin, Alexey; Rautian, Maria; Przyboś, Ewa

2012-05-01

This is the first phylogenetic study of the intraspecific variability within Paramecium multimicronucleatum with the application of two-loci analysis (ITS1-5.8S-ITS2-5'LSU rDNA and COI mtDNA) carried out on numerous strains originated from different continents. The species has been shown to have a complex structure of several sibling species within taxonomic species. Our analysis revealed the existence of 10 haplotypes for the rDNA fragment and 15 haplotypes for the COI fragment in the studied material. The mean distance for all of the studied P. multimicronucleatum sequence pairs was p=0.025/0.082 (rDNA/COI). Despite the greater variation of the COI fragment, the COI-derived tree topology is similar to the tree topology constructed on the basis of the rDNA fragment. P. multimicronucleatum strains are divided into three main clades. The tree based on COI fragment analysis presents a greater resolution of the studied P. multimicronucleatum strains. Our results indicate that the strains of P. multimicronucleatum that appear in different clades on the trees could belong to different syngens. Copyright © 2012 Elsevier Inc. All rights reserved.

Integrative modules for efficient genome engineering in yeast

PubMed Central

Amen, Triana; Kaganovich, Daniel

2017-01-01

We present a set of vectors containing integrative modules for efficient genome integration into the commonly used selection marker loci of the yeast Saccharomyces cerevisiae. A fragment for genome integration is generated via PCR with a unique set of short primers and integrated into HIS3, URA3, ADE2, and TRP1 loci. The desired level of expression can be achieved by using constitutive (TEF1p, GPD1p), inducible (CUP1p, GAL1/10p), and daughter-specific (DSE4p) promoters available in the modules. The reduced size of the integrative module compared to conventional integrative plasmids allows efficient integration of multiple fragments. We demonstrate the efficiency of this tool by simultaneously tagging markers of the nucleus, vacuole, actin, and peroxisomes with genomically integrated fluorophores. Improved integration of our new pDK plasmid series allows stable introduction of several genes and can be used for multi-color imaging. New bidirectional promoters (TEF1p-GPD1p, TEF1p-CUP1p, and TEF1p-DSE4p) allow tractable metabolic engineering. PMID:28660202

Immunohistochemical characterisation of the hepatic stem cell niche in feline hepatic lipidosis: a preliminary morphological study.

PubMed

Valtolina, Chiara; Robben, Joris H; Favier, Robert P; Rothuizen, Jan; Grinwis, Guy Cm; Schotanus, Baukje A; Penning, Louis C

2018-05-01

Objectives The aim of this study was to describe the cellular and stromal components of the hepatic progenitor cell niche in feline hepatic lipidosis (FHL). Methods Immunohistochemical staining for the progenitor/bile duct marker (K19), activated Kupffer cells (MAC387), myofibroblasts (alpha-smooth muscle actin [α-SMA]) and the extracellular matrix component laminin were used on seven liver biopsies of cats with FHL and three healthy cats. Double immunofluorescence stainings were performed to investigate co-localisation of different cell types in the hepatic progenitor cell (HPC) niche. Results HPCs, Kupffer cells, myofibroblasts and laminin deposition were observed in the liver samples of FHL, although with variability in the expression and positivity of the different immunostainings between different samples. When compared with the unaffected cats where K19 positivity and minimal α-SMA and laminin positivity were seen mainly in the portal area, in the majority of FHL samples K19 and α-SMA-positive cells and laminin positivity were seen also in the periportal and parenchymatous area. MAC387-positive cells were present throughout the parenchyma. Conclusions and relevance This is a preliminary morphological study to describe the activation and co-localisation of components of the HPC niche in FHL. Although the HPC niche in FHL resembles that described in hepatopathies in dogs and in feline lymphocytic cholangitis, the expression of K19, α-SMA, MAC387 and lamin is more variable in FHL, and a common pattern of activation could not be established. Nevertheless, when HPCs were activated, a spatial association between HPCs and their niche could be demonstrated.

Ablation of lens epithelial cells with a laser photolysis system: Histopathology, ultrastructure, and immunochemistry

PubMed Central

Mamalis, Nick; Grossniklaus, Hans E.; Waring, George O.; Werner, Liliana; Brubaker, Jacob; Davis, Don; Espandar, Ladan; Walker, Rudolf; Thyzel, Reinhardt

2010-01-01

PURPOSE To evaluate efficacy of a neodymium:YAG (Nd:YAG) laser photolysis system in removing lens epithelial cells (LECs) and characterize the effect of the laser on laminin and fibronectin involved in LEC adhesion and migration. METHODS Cadaver eyes were evaluated using the Miyake technique. The lenses were removed with phacoemulsification. The modified Nd:YAG laser was used to clean the LECs from the capsule. Only the fornix was cleaned in some eyes and the anterior subcapsular area in other eyes. Some areas were not treated and acted as controls. Standard irrigation/aspiration (I/A) removal of LECs was performed in additional eyes. The eyes were analyzed using light microscopy and immunohistochemical staining. RESULTS Histopathologic evaluation showed that the laser removed the LECs from the anterior lens capsule and from the fornix. Immunohistochemical staining showed fibronectin and laminin staining in the untreated areas that was absent in the treated areas. Standard I/A removal of the LECs showed absence of cells but persistent laminin and fibronectin. Electron microscopy showed epithelial cells in untreated areas with an absence of the LECs and debris in treated areas. CONCLUSIONS The laser photolysis system removed LECs from the anterior lens capsule and capsule fornix. Along with the cells, laminin, fibronectin, and cell debris remained in the untreated areas but were removed by the treatment. This treatment may be useful in preventing posterior capsule opacification. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned. Additional disclosures are found in the footnotes. PMID:20494774

Genetic diversity of Plasmodium Vivax revealed by the merozoite surface protein-1 icb5-6 fragment.

PubMed

Ruan, Wei; Zhang, Ling-Ling; Feng, Yan; Zhang, Xuan; Chen, Hua-Liang; Lu, Qiao-Yi; Yao, Li-Nong; Hu, Wei

2017-06-05

Plasmodium vivax remains a potential cause of morbidity and mortality for people living in its endemic areas. Understanding the genetic diversity of P. vivax from different regions is valuable for studying population dynamics and tracing the origins of parasites. The PvMSP-1 gene is highly polymorphic and has been used as a marker in many P. vivax population studies. The aim of this study was to investigate the genetic diversity of the PvMSP-1 gene icb5-6 fragment and to provide more genetic polymorphism data for further studies on P. vivax population structure and tracking of the origin of clinical cases. Nested PCR and sequencing of the PvMSP-1 icb5-6 marker were performed to obtain the nucleotide sequences of 95 P. vivax isolates collected from Zhejiang province, China. To investigate the genetic diversity of PvMSP-1, the 95 nucleotide sequences of the PvMSP-1 icb5-6 fragment were genotyped and analyzed using DnaSP v5, MEGA software. The 95 P. vivax isolates collected from Zhejiang province were either indigenous cases or imported cases from different regions around the world. A total of 95 sequences ranging from 390 to 460 bp were obtained. The 95 sequences were genotyped into four allele-types (Sal I, Belem, R-III and R-IV) and 17 unique haplotypes. R-III and Sal I were the predominant allele-types. The haplotype diversity (Hd) and nucleotide diversity (Pi) were estimated to be 0.729 and 0.062, indicating that the PvMSP-1 icb5-6 fragment had the highest level of polymorphism due to frequent recombination processes and single nucleotide polymorphism. The values of dN/dS and Tajima's D both suggested neutral selection for the PvMSP-1icb5-6 fragment. In addition, a rare recombinant style of R-IV type was identified. This study presented high genetic diversity in the PvMSP-1 marker among P. vivax strains from around the world. The genetic data is valuable for expanding the polymorphism information on P. vivax, which could be helpful for further study on population dynamics and tracking the origin of P. vivax.

1,10-phenanthroline inhibits the metallopeptidase secreted by Phialophora verrucosa and modulates its growth, morphology and differentiation.

PubMed

Granato, Marcela Queiroz; Massapust, Priscila de Araújo; Rozental, Sonia; Alviano, Celuta Sales; dos Santos, André Luis Souza; Kneipp, Lucimar Ferreira

2015-04-01

Phialophora verrucosa is one of the etiologic agents of chromoblastomycosis, a fungal infection that affects cutaneous and subcutaneous tissues. This disease is chronic, recurrent and difficult to treat. Several studies have shown that secreted peptidases by fungi are associated with important pathophysiological processes. Herein, we have identified and partially characterized the peptidase activity secreted by P. verrucosa conidial cells. Using human serum albumin as substrate, the best hydrolysis profile was detected at extreme acidic pH (3.0) and at 37 °C. The enzymatic activity was completely blocked by classical metallopeptidase inhibitors/chelating agents as 1,10-phenanthroline and EGTA. Zinc ions stimulated the metallo-type peptidase activity in a dose-dependent manner. Several proteinaceous substrates were cleaved, in different extension, by the P. verrucosa metallopeptidase activity, including immunoglobulin G, fibrinogen, collagen types I and IV, fibronectin, laminin and keratin; however, mucin and hemoglobin were not susceptible to proteolysis. As metallopeptidases participate in different cellular metabolic pathways in fungal cells, we also tested the influence of 1,10-phenanthroline and EGTA on P. verrucosa development. Contrarily to EGTA, 1,10-phenanthroline inhibited the fungal viability (MIC 0.8 µg/ml), showing fungistatic effect, and induced profound morphological alterations as visualized by transmission electron microscopy. In addition, 1,10-phenanthroline arrested the filamentation process in P. verrucosa. Our results corroborate the supposition that metallopeptidase inhibitors/chelating agents have potential to control crucial biological events in fungal agents of chromoblastomycosis.

Substance P(1-7) antagonizes substance P-induced aversive behaviour in mice.

PubMed

Sakurada, T; Kuwahara, H; Takahashi, K; Sakurada, S; Kisara, K; Terenius, L

1988-12-19

Substance P (SP) and its fragments were administered intrathecally into awake mice. SP and C-terminal fragments caused dose-dependent reciprocal hindlimb scratching responses. SP(5-11) was more potent than SP not only in inducing scratching response but also in inducing aversive behaviour including licking and biting. SP(1-7) induced no behavioural reactions. However, when low doses of SP(1-7) (1.0-4.0 pmol) were injected simultaneously with SP or SP(5-11) (0.1 nmol), aversive behaviours induced by SP or SP(5-11) were significantly reduced. These results indicate that SP(1-7) formed endogenously could modulate the actions of SP or SP(5-11) in the spinal cord.

Decreased collagen types I and IV, laminin, CK-19 and α-SMA expression after bone marrow cell transplantation in rats with liver fibrosis.

PubMed

Carvalho, S N; Lira, D C; Oliveira, G P; Thole, A A; Stumbo, A C; Caetano, C E; Marques, R G; Carvalho, L

2010-11-01

Bone marrow cells have frequently been tested in animal models of liver fibrosis to assess their role in hepatic regeneration. The mononuclear fraction of bone marrow cells is of particular interest, as many studies show that these cells may be beneficial to treat hepatic fibrosis. In this study, we used the bile duct ligation model to induce hepatic fibrosis in an irreversible manner, and rats were treated with bone marrow mononuclear (BMMN) cells after fibrosis was established. Analysis of collagen types I and IV, laminin and α-SMA showed a decreased expression of these proteins in fibrotic livers after 7 days of BMMN cell injection. Moreover, cytokeratin-19 analysis showed a reduction in bile ducts in the BMMN cell-treated group. These results were accompanied by ameliorated levels of hepatic enzymes GPT (Glutamic-pyruvic transaminase), GOT (glutamic-oxaloacetic transaminase) and alkaline phosphatase (AP). Therefore, we showed that BMMN cells decrease hepatic fibrosis by significantly reducing myofibroblast numbers and through reduction of the collagen and laminin-rich extracellular matrix of fibrotic septa and hepatic sinusoids.

The Haemophilus influenzae Hap Autotransporter Binds to Fibronectin, Laminin, and Collagen IV

PubMed Central

Fink, Doran L.; Green, Bruce A.; St. Geme III, Joseph W.

2002-01-01

Nontypeable Haemophilus influenzae (NTHI) initiates infection by colonizing the upper respiratory tract mucosa. NTHI disease frequently occurs in the context of respiratory tract inflammation, where organisms encounter damaged epithelium and exposed basement membrane. In this study, we examined interactions between the H. influenzae Hap adhesin and selected extracellular matrix proteins. Hap is an autotransporter protein that undergoes autoproteolytic cleavage, with release of the adhesive passenger domain, Haps, from the bacterial cell surface. We found that Hap promotes bacterial adherence to purified fibronectin, laminin, and collagen IV and that Hap-mediated adherence is enhanced by inhibition of autoproteolysis. Adherence is inhibited by pretreatment of bacteria with a polyclonal antiserum recognizing Haps. Purified Haps binds with high affinity to fibronectin, laminin, and collagen IV but not to collagen II. Binding of Haps to fibronectin involves interaction with the 45-kDa gelatin-binding domain but not the 30-kDa heparin-binding domain of fibronectin. Taken together, these observations suggest that interactions between Hap and extracellular matrix proteins may play an important role in NTHI colonization of the respiratory tract. PMID:12183535

Formation of a1 Ions Directly from Oxazolone b2 Ions: an Energy-Resolved and Computational Study

NASA Astrophysics Data System (ADS)

Bythell, Benjamin J.; Harrison, Alex G.

2015-05-01

It is well-known that oxazolone b2 ions fragment extensively by elimination of CO to form a2 ions, which often fragment further to form a1 ions. Less well-known is that some oxazolone b2 ions may fragment directly to form a1 ions. The present study uses energy-resolved collision-induced dissociation experiments to explore the occurrence of the direct b2→a1 fragmentation reaction. The experimental results show that the direct b2→a1 reaction is generally observed when Gly is the C-terminal residue of the oxazolone. When the C-terminal residue is more complex, it is able to provide increased stability of the a2 product in the b2→a2 fragmentation pathway. Our computational studies of the relative critical reaction energies for the b2→a2 reaction compared with those for the b2→a1 reaction provide support that the critical reaction energies are similar for the two pathways when the C-terminal residue of the oxazolone is Gly. By contrast, when the nitrogen of the oxazolone ring in the b2 ion does not bear a hydrogen, as in the Ala-Sar and Tyr-Sar (Sar = N-methylglycine) oxazolone b2 ions, a1 ions are not formed but rather neutral imine elimination from the N-terminus of the b2 ion becomes a dominant fragmentation reaction. The M06-2X/6-31+G(d,p) density functional theory calculations are in general agreement with the experimental data for both types of reaction. In contrast, the B3LYP/6-31+G(d,p) model systematically underestimates the barriers of these SN2-like b2→a1 reaction. The difference between the two methods of barrier calculation are highly significant ( P < 0.001) for the b2→a1 reaction, but only marginally significant ( P = 0.05) for the b2→a2 reaction. The computations provide further evidence of the limitations of the B3LYP functional when describing SN2-like reactions.

Modulation of [3H]DAGO binding by substance P (SP) and SP fragments in the mouse brain and spinal cord via MU1 interactions.

PubMed

Krumins, S A; Kim, D C; Seybold, V S; Larson, A A

1989-01-01

Binding of [3H]DAGO to fresh, frozen or beta-funaltrexamine (beta-FNA) pretreated membranes of mouse brain and spinal cord was extensively studied using substance P (SP) or SP fragments as potential competitors and/or modulators. The objective was to determine whether SP exerts its analgesic effect by interacting with mu opioid receptors. The affinity of DAGO was reduced and binding capacity was increased in the presence of SP or the N-terminal SP fragments SP(1-9) and SP(1-4) but not the C-terminal SP fragment SP(5-11). Because sub-nanomolar concentrations of SP or N-terminal SP fragments displaced [3H] DAGO binding to a minor but detectable degree, it is suggested that SP interacts with mu 1 sites through its N-terminus portion. The effect of SP on DAGO binding was less in the spinal cord compared to the rest of the brain. Modulation of DAGO binding by SP was enhanced in the brain after pretreatment of membranes with the narcotic antagonist beta-FNA. These results suggest a novel mechanism for the analgesic action of SP.

Specific Activation of K-RasG12D Allele in the Bladder Urothelium Results in Lung Alveolar and Vascular Defects

PubMed Central

Kanasaki, Megumi; Vong, Sylvia; Rovira, Carlota; Kalluri, Raghu

2014-01-01

K-ras is essential for embryogenesis and its mutations are involved in human developmental syndromes and cancer. To determine the consequences of K-ras activation in urothelium, we used uroplakin-II (UPK II) promoter driven Cre recombinase mice and generated mice with mutated KrasG12D allele in the urothelium (UPK II-Cre;LSL-K-rasG12D). The UPK II-Cre;LSL-K-rasG12D mice died neonatally due to lung morphogenesis defects consisting of simplification with enlargement of terminal air spaces and dysmorphic pulmonary vasculature. A significant alteration in epithelial and vascular basement membranes, together with fragmentation of laminin, points to extracellular matrix degradation as the causative mechanism of alveolar and vascular defects. Our data also suggest that altered protease activity in amniotic fluid might be associated with matrix defects in lung of UPK II-Cre;LSL-K-rasG12. These defects resemble those observed in early stage human neonatal bronchopulmonary dysplasia (BPD), although the relevance of this new mouse model for BPD study needs further investigation. PMID:24760005

Hypocretin-1 Levels Associate with Fragmented Sleep in Patients with Narcolepsy Type 1.

PubMed

Alakuijala, Anniina; Sarkanen, Tomi; Partinen, Markku

2016-05-01

We aimed to analyze nocturnal sleep characteristics of patients with narcolepsy type 1 (narcolepsy with cataplexy) measured by actigraphy in respect to cerebrospinal fluid hypocretin-1 levels of the same patients. Actigraphy recording of 1-2 w and hypocretin-1 concentration analysis were done to thirty-six unmedicated patients, aged 7 to 63 y, 50% female. Twenty-six of them had hypocretin-1 levels under 30 pg/mL and the rest had levels of 31-79 pg/mL. According to actigraphy, patients with very low hypocretin levels had statistically significantly longer sleep latency (P = 0.033) and more fragmented sleep, indicated by both the number of immobile phases of 1 min (P = 0.020) and movement + fragmentation index (P = 0.049). There were no statistically significant differences in the actual sleep time or circadian rhythm parameters measured by actigraphy. Actigraphy gives additional information about the stabilization of sleep in patients with narcolepsy type 1. Very low hypocretin levels associate with more wake intruding into sleep. © 2016 Associated Professional Sleep Societies, LLC.

Oxidized phosphatidylcholines are produced in renal ischemia reperfusion injury

PubMed Central

Solati, Zahra; Edel, Andrea L.; Shang, Yue; O, Karmin

2018-01-01

Background The aim of this study was to determine the individual oxidized phosphatidylcholine (OxPC) molecules generated during renal ischemia/ reperfusion (I/R) injury. Methods Kidney ischemia was induced in male Sprague–Dawley rats by clamping the left renal pedicle for 45 min followed by reperfusion for either 6h or 24h. Kidney tissue was subjected to lipid extraction. Phospholipids and OxPC species were identified and quantitated using liquid chromatography coupled to electrospray ionization tandem mass spectrometry using internal standards. Result We identified fifty-five distinct OxPC in rat kidney following I/R injury. These included a variety of fragmented (aldehyde and carboxylic acid containing species) and non-fragmented products. 1-stearoyl-2-linoleoyl-phosphatidylcholine (SLPC-OH), which is a non-fragmented OxPC and 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PAzPC), which is a fragmented OxPC, were the most abundant OxPC species after 6h and 24 h I/R respectively. Total fragmented aldehyde OxPC were significantly higher in 6h and 24h I/R groups compared to sham operated groups (P = 0.03, 0.001 respectively). Moreover, levels of aldehyde OxPC at 24h I/R were significantly greater than those in 6h I/R (P = 0.007). Fragmented carboxylic acid increased significantly in 24h I/R group compared with sham and 6h I/R groups (P = 0.001, 0.001). Moreover, levels of fragmented OxPC were significantly correlated with creatinine levels (r = 0.885, P = 0.001). Among non-fragmented OxPC, only isoprostanes were elevated significantly in 6h I/R group compared with sham group but not in 24h I/R group (P = 0.01). No significant changes were observed in other non-fragmented OxPC including long chain products and terminal furans. Conclusion We have shown for the first time that bioactive OxPC species are produced in renal I/R and their levels increase with increasing time of reperfusion in a kidney model of I/R and correlate with severity of I/R injury. Given the pathological activity of fragmented OxPCs, therapies focused on their reduction may be a mechanism to attenuate renal I/R injury. PMID:29684044

Third prize: the impact of fluid environment manipulation on shockwave lithotripsy artificial calculi fragmentation rates.

PubMed

Méndez-Probst, Carlos E; Fernadez, Alfonso; Erdeljan, Petar; Vanjecek, Maaike; Cadieux, Peter A; Razvi, Hassan

2011-03-01

Studies have suggested that shockwave lithotripsy (SWL) stone fragmentation rates can be affected by characteristics of the fluid media surrounding the stone, although evidence to implicate the impact of urine specific gravity (SG) is limited and inconclusive. Our aim is to further explore the impact fluid media and SGs have on stone fragmentation using a variable focus lithotripter. Artificial stones were presoaked for 24 hours in urine and then shocked in various fluid media including artificial urine (SG 1.010 control, 1.020, and 1.07), human pooled urine (HPU), degassed HPU, Pentastarch, 100% and 30% contrast, degassed 30% contrast, 100% ethanol, deionized water (dH(2)O), degassed dH(2)O, 5% glucose, Ringer lactate, 0.9% saline, glycerol, whole blood, and lubricating gel. After soaking, SWL using the Modulith SLX-F2 electromagnetic lithotripter was performed. Fragments were dried and sieved using a 4-mm diameter opening grid. Fragments >4 mm were weighed and fragmentation coefficients (FCs) calculated (pre-SWL weight - post-SWL weight)/(pre-SWL weight) × 100. Fifteen stones were shocked for each fluid group. Fluid type, viscosity, and degassing all significantly impacted stone fragmentation. While the solutions' SG, per se, did not appear to affect stone fragmentation, the use of degassed 30% contrast significantly improved stone destruction over the SG 1.010 artificial urine control (95.3% vs 71.4, P < 0.01). Furthermore, degassing improved comminution rates by increasing the number of completely fragmented stones (FC = 100%). Using degassed 30% contrast, 12/15 stones were completely fragmented, compared with only 2/15 in the control group (P = 0.007). Among the whole blood, glycerol, and lubricating gel groups, only 1/15, 0/15, and 1/15 stones reached 100% FC respectively in the narrow focus, possibly because of the detrimental impact of increased viscosity. Different fluid media can significantly affect FC in vitro. Among the various fluids tested, degassed 30% contrast significantly increased the FC and total number of completely fragmented stones.

Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix.

PubMed Central

Sanderson, I R; Ezzell, R M; Kedinger, M; Erlanger, M; Xu, Z X; Pringault, E; Leon-Robine, S; Louvard, D; Walker, W A

1996-01-01

The differentiation of small intestinal epithelial cells may require stimulation by microenvironmental factors in vivo. In this study, the effects of mesenchymal and luminal elements in nonmalignant epithelia] cells isolated from the human fetus were studied in vitro. Enterocytes from the human fetus were cultured and microenvironmental factors were added in stages, each stage more closely approximating the microenvironment in vivo. Four stages were examined: epithelial cells derived on plastic from intestinal culture and grown as a cell clone, the same cells grown on connective tissue support, primary epithelial explants grown on fibroblasts with a laminin base, and primary epithelial explants grown on fibroblasts and laminin with n-butyrate added to the incubation medium. The epithelial cell clone dedifferentiated when grown on plastic; however, the cells expressed cytokeratins and villin as evidence of their epithelial cell origin. Human connective tissue matrix from Engelbreth-Holm-Swarm sarcoma cells (Matrigel) modulated their phenotype: alkaline phosphatase activity increased, microvilli developed on their apical surface, and the profile of insulin-like growth factor binding proteins resembled that secreted by differentiated enterocytes. Epithelial cells taken directly from the human fetus as primary cultures and grown as explants on fibroblasts and laminin expressed greater specific enzyme activities in brush border membrane fractions than the cell clone. These activities were enhanced by the luminal molecule sodium butyrate. Thus the sequential addition of connective tissue and luminal molecules to nonmalignant epithelia] cells in vitro induces a spectrum of changes in the epithelial cell phenotype toward full differentiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8755542

The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells

PubMed Central

Imbeault, Sophie; Gauvin, Lianne G; Toeg, Hadi D; Pettit, Alexandra; Sorbara, Catherine D; Migahed, Lamiaa; DesRoches, Rebecca; Menzies, A Sheila; Nishii, Kiyomasa; Paul, David L; Simon, Alexander M; Bennett, Steffany AL

2009-01-01

Background Gap junction protein and extracellular matrix signalling systems act in concert to influence developmental specification of neural stem and progenitor cells. It is not known how these two signalling systems interact. Here, we examined the role of ECM components in regulating connexin expression and function in postnatal hippocampal progenitor cells. Results We found that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are detected in distinct neural progenitor cell populations cultured in the absence of exogenous ECM. Multipotential Type 1 cells express Cx26, Cx30, and Cx43 protein. Their Type 2a progeny but not Type 2b and 3 neuronally committed progenitor cells additionally express Cx37, Cx40, and Cx45. Cx29 and Cx47 protein is detected in early oligodendrocyte progenitors and mature oligodendrocytes respectively. Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30. These changes are associated with decreased neurogenesis. Further, laminin elicits the appearance of Cx32 protein in early oligodendrocyte progenitors and Cx36 protein in immature neurons. These changes impact upon functional connexin-mediated hemichannel activity but not gap junctional intercellular communication. Conclusion Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells. PMID:19236721

Loss-of-function FERMT1 mutations in kindler syndrome implicate a role for fermitin family homolog-1 in integrin activation.

PubMed

Lai-Cheong, Joey E; Parsons, Maddy; Tanaka, Akio; Ussar, Siegfried; South, Andrew P; Gomathy, Sethuraman; Mee, John B; Barbaroux, Jean-Baptiste; Techanukul, Tanasit; Almaani, Noor; Clements, Suzanne E; Hart, Ian R; McGrath, John A

2009-10-01

Kindler syndrome is an autosomal recessive disorder characterized by skin atrophy and blistering. It results from loss-of-function mutations in the FERMT1 gene encoding the focal adhesion protein, fermitin family homolog-1. How and why deficiency of fermitin family homolog-1 results in skin atrophy and blistering are unclear. In this study, we investigated the epidermal basement membrane and keratinocyte biology abnormalities in Kindler syndrome. We identified altered distribution of several basement membrane proteins, including types IV, VII, and XVII collagens and laminin-332 in Kindler syndrome skin. In addition, reduced immunolabeling intensity of epidermal cell markers such as beta1 and alpha6 integrins and cytokeratin 15 was noted. At the cellular level, there was loss of beta4 integrin immunolocalization and random distribution of laminin-332 in Kindler syndrome keratinocytes. Of note, active beta1 integrin was reduced but overexpression of fermitin family homolog-1 restored integrin activation and partially rescued the Kindler syndrome cellular phenotype. This study provides evidence that fermitin family homolog-1 is implicated in integrin activation and demonstrates that lack of this protein leads to pathological changes beyond focal adhesions, with disruption of several hemidesmosomal components and reduced expression of keratinocyte stem cell markers. These findings collectively provide novel data on the role of fermitin family homolog-1 in skin and further insight into the pathophysiology of Kindler syndrome.

Serum Liver Fibrosis Markers in the Prognosis of Liver Cirrhosis: A Prospective Observational Study

PubMed Central

Qi, Xingshun; Liu, Xu; Zhang, Yongguo; Hou, Yue; Ren, Linan; Wu, Chunyan; Chen, Jiang; Xia, Chunlian; Zhao, Jiajun; Wang, Di; Zhang, Yanlin; Zhang, Xia; Lin, Hao; Wang, Hezhi; Wang, Jinling; Cui, Zhongmin; Li, Xueyan; Deng, Han; Hou, Feifei; Peng, Ying; Wang, Xueying; Shao, Xiaodong; Li, Hongyu; Guo, Xiaozhong

2016-01-01

Background The prognostic role of serum liver fibrosis markers in cirrhotic patients remains unclear. We performed a prospective observational study to evaluate the effect of amino-terminal pro-peptide of type III pro-collagen (PIIINP), collagen IV (CIV), laminin (LN), and hyaluronic acid (HA) on the prognosis of liver cirrhosis. Material/Methods All patients who were diagnosed with liver cirrhosis and admitted to our department were prospectively enrolled. PIIINP, CIV, LN, and HA levels were tested. Results Overall, 108 cirrhotic patients were included. Correlation analysis demonstrated that CIV (coefficient r: 0.658, p<0.001; coefficient r: 0.368, p<0.001), LN (coefficient r: 0.450, p<0.001; coefficient r: 0.343, p<0.001), and HA (coefficient r: 0.325, p=0.001; coefficient r: 0.282, p=0.004) levels, but not PIIINP level (coefficient r: 0.081, p=0.414; coefficient r: 0.090, p=0.363), significantly correlated with Child-Pugh and MELD scores. Logistic regression analysis demonstrated that HA (odds ratio=1.00003, 95% confidence interval [CI]=1.000004–1.000056, p=0.022) was significantly associated with the 6-month mortality. Receiver operating characteristics analysis demonstrated that the area under the curve (AUC) of HA for predicting the 6-month mortality was 0.612 (95%CI=0.508–0.709, p=0.1531). Conclusions CIV, LN, and HA levels were significantly associated with the severity of liver dysfunction, but might be inappropriate for the prognostic assessment of liver cirrhosis. PMID:27480906

Central cardiovascular and behavioral effects of carboxy- and amino-terminal fragments of substance P in conscious rats.

PubMed

Tschöpe, C; Jost, N; Unger, T; Culman, J

1995-08-28

The central cardiovascular and behavioral effects of carboxy- (SP 5-11, SP 6-11, SP 7-11, SP 8-11) and amino- (SP 1-7, SP 1-9) terminal substance P (SP) fragments were compared with those of SP 1-11 in conscious rats. In addition, the ability of these SP-fragments to induce desensitization of the central NK1 receptor was investigated. SP 1-11 (50 pmol) injected i.c.v. induced an increase in mean arterial blood pressure (MAP), heart rate (HR) and a typical behavioral response consisting of face washing (FW), hindquarter grooming (HQG) and wet-dog shakes (WDS). The cardiovascular and behavioral responses to equimolar doses of SP 5-11 and SP 6-11 were similar to those of SP 1-11, however, only SP 5-11 induced exactly the same behavioral pattern as SP 1-11. SP 6-11 was more potent in inducing FW and WDS than SP 1-11 or SP 5-11. The carboxy-terminal SP-fragments, SP 7-11 and SP 8-11, and the amino-terminal SP-fragments, SP 1-7, SP 1-9, did not elicit any significant cardiovascular or behavioral responses. Pretreatment with SP 1-11 reduced the cardiovascular and behavioral responses to subsequent injections of SP 1-11. Of all SP-fragments tested, only SP 5-11 was able to attenuate the cardiovascular and behavioral responses to SP 1-11. Our results demonstrate that SP 6-11 represents the shortest carboxy-terminal amino acid sequence, that after i.c.v. injection, elicits the same cardiovascular response as SP 1-11, but fails to desensitize the NK1 receptor. The carboxy-terminal fragment, SP 5-11, is the shortest amino acid sequence which produces the same pattern of central cardiovascular and behavioral responses as SP 1-11 and also retains the ability to desensitize the NK1 receptor like SP 1-11.

In vitro DNA fragmentation of mitochondrial DNA caused by single-stranded breakage related to macroplasmodial senescence of the true slime mold, Physarum polycephalum.

PubMed

Abe, T; Takano, H; Sasaki, N; Mori, K; Kawano, S

2000-02-01

We found that mitochondrial DNA (mtDNA) isolated from Physarum polycephalum fragmented itself in weak ionic solutions. The mtDNA was dissolved in STE (saline Tris-EDTA: 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA), TE (10 mM Tris-HCl, 1 mM EDTA) and DW, and then electrophoresed in an agarose gel. The intact 86-kbp mtDNA band was seen in STE, but several novel bands appeared in TE and DW. In TE, two discrete bands appeared at 6.7-kbp (alpha-band) and 5.0-kbp (beta-band), whereas at least 17 discrete bands were observed in distilled water (DW). These fragmentation patterns were not stoichiometric, as seen when using restriction endonucleases, but were clearly different from the degradation of DNA caused by a physical shearing force or a contaminating nuclease. In this paper, we characterize this in vitro fragmentation of mtDNA from P. polycephalum. We located 19 fragments, including the alpha and beta fragments, on a mtDNA restriction map, and demonstrated that these cleavage sites were S1 nuclease-sensitive regions, which are single-stranded DNA regions such as nicks and gaps in the mtDNA. The alpha and beta fragments are derived from the region encoding ribosomal RNAs (rRNAs) and the ATP synthase (atpA) gene, while the other 17 fragments are not derived from any specific region, but the cleavage sites are located throughout the mtDNA molecule. In P. polycephalum, it is well known that the growth rate of macroplasmodia decreases with aging. Equal amounts of mtDNA from juvenile and aged macroplasmodia were electrophoresed and the frequency of the beta fragment in each sample was measured. The ratio of the beta band to the total signal including background was estimated to be 3.3-4.0% in juvenile macroplasmodia, whereas it increased to 8.3-28.2% in aged macroplasmodia. This result suggests that the in vitro fragmentation of mtDNA is associated with macroplasmodial senescence. The single-stranded breakage of mtDNA of P. polycephalum may accumulate with age.

Enzymes and chelating agent in cotton pretreatment.

PubMed

Csiszár, E; Losonczi, A; Szakács, G; Rusznák, I; Bezúr, L; Reicher, J

2001-08-23

Desized cotton fabric and cotton seed-coat fragments (impurities) have been treated with commercial cellulase (Celluclast 1.5 L), hemicellulase-pectinase (Viscozyme 120 L) and xylanase (Pulpzyme HC) enzymes. Seed-coat fragments hydrolyzed much faster than the cotton fabric itself. This relative difference in hydrolysis rates makes possible a direct enzymatic removal of seed-coat fragments from desized cotton fabric. Addition of chelating agents such as ethylenediamine-tetra-acetic acid (EDTA) markedly enhanced the directed enzyme action. Pretreatments carried out in acidic solution at pH 5 increased the lightness of seed-coat fragments, contrary to the samples treated in neutral medium at pH 7. Alkaline scouring resulted in darker seed-coat fragments except for the samples pretreated with Pulpzyme HC plus EDTA. This effect is similar to that observed in the biobleaching process in pulp and paper industry.

Site-directed protein recombination as a shortest-path problem.

PubMed

Endelman, Jeffrey B; Silberg, Jonathan J; Wang, Zhen-Gang; Arnold, Frances H

2004-07-01

Protein function can be tuned using laboratory evolution, in which one rapidly searches through a library of proteins for the properties of interest. In site-directed recombination, n crossovers are chosen in an alignment of p parents to define a set of p(n + 1) peptide fragments. These fragments are then assembled combinatorially to create a library of p(n+1) proteins. We have developed a computational algorithm to enrich these libraries in folded proteins while maintaining an appropriate level of diversity for evolution. For a given set of parents, our algorithm selects crossovers that minimize the average energy of the library, subject to constraints on the length of each fragment. This problem is equivalent to finding the shortest path between nodes in a network, for which the global minimum can be found efficiently. Our algorithm has a running time of O(N(3)p(2) + N(2)n) for a protein of length N. Adjusting the constraints on fragment length generates a set of optimized libraries with varying degrees of diversity. By comparing these optima for different sets of parents, we rapidly determine which parents yield the lowest energy libraries.

Hyaluronic acid-laminin hydrogels increase neural stem cell transplant retention and migratory response to SDF-1α.

PubMed

Addington, C P; Dharmawaj, S; Heffernan, J M; Sirianni, R W; Stabenfeldt, S E

2017-07-01

The chemokine SDF-1α plays a critical role in mediating stem cell response to injury and disease and has specifically been shown to mobilize neural progenitor/stem cells (NPSCs) towards sites of neural injury. Current neural transplant paradigms within the brain suffer from low rates of retention and engraftment after injury. Therefore, increasing transplant sensitivity to injury-induced SDF-1α represents a method for increasing neural transplant efficacy. Previously, we have reported on a hyaluronic acid-laminin based hydrogel (HA-Lm gel) that increases NPSC expression of SDF-1α receptor, CXCR4, and subsequently, NPSC chemotactic migration towards a source of SDF-1α in vitro. The study presented here investigates the capacity of the HA-Lm gel to promote NPSC response to exogenous SDF-1α in vivo. We observed the HA-Lm gel to significantly increase NPSC transplant retention and migration in response to SDF-1α in a manner critically dependent on signaling via the SDF-1α-CXCR4 axis. This work lays the foundation for development of a more effective cell therapy for neural injury, but also has broader implications in the fields of tissue engineering and regenerative medicine given the essential roles of SDF-1α across injury and disease states. Copyright © 2016 Elsevier B.V. All rights reserved.

Hyperoxia Causes Mitochondrial Fragmentation in Pulmonary Endothelial Cells by Increasing Expression of Pro-Fission Proteins.

PubMed

Ma, Cui; Beyer, Andreas M; Durand, Matthew; Clough, Anne V; Zhu, Daling; Norwood Toro, Laura; Terashvili, Maia; Ebben, Johnathan D; Hill, R Blake; Audi, Said H; Medhora, Meetha; Jacobs, Elizabeth R

2018-03-01

We explored mechanisms that alter mitochondrial structure and function in pulmonary endothelial cells (PEC) function after hyperoxia. Mitochondrial structures of PECs exposed to hyperoxia or normoxia were visualized and mitochondrial fragmentation quantified. Expression of pro-fission or fusion proteins or autophagy-related proteins were assessed by Western blot. Mitochondrial oxidative state was determined using mito-roGFP. Tetramethylrhodamine methyl ester estimated mitochondrial polarization in treatment groups. The role of mitochondrially derived reactive oxygen species in mt-fragmentation was investigated with mito-TEMPOL and mitochondrial DNA (mtDNA) damage studied by using ENDO III (mt-tat-endonuclease III), a protein that repairs mDNA damage. Drp-1 (dynamin-related protein 1) was overexpressed or silenced to test the role of this protein in cell survival or transwell resistance. Hyperoxia increased fragmentation of PEC mitochondria in a time-dependent manner through 48 hours of exposure. Hyperoxic PECs exhibited increased phosphorylation of Drp-1 (serine 616), decreases in Mfn1 (mitofusion protein 1), but increases in OPA-1 (optic atrophy 1). Pro-autophagy proteins p62 (LC3 adapter-binding protein SQSTM1/p62), PINK-1 (PTEN-induced putative kinase 1), and LC3B (microtubule-associated protein 1A/1B-light chain 3) were increased. Returning cells to normoxia for 24 hours reversed the increased mt-fragmentation and changes in expression of pro-fission proteins. Hyperoxia-induced changes in mitochondrial structure or cell survival were mitigated by antioxidants mito-TEMPOL, Drp-1 silencing, or inhibition or protection by the mitochondrial endonuclease ENDO III. Hyperoxia induced oxidation and mitochondrial depolarization and impaired transwell resistance. Decrease in resistance was mitigated by mito-TEMPOL or ENDO III and reproduced by overexpression of Drp-1. Because hyperoxia evoked mt-fragmentation, cell survival and transwell resistance are prevented by ENDO III and mito-TEMPOL and Drp-1 silencing, and these data link hyperoxia-induced mt-DNA damage, Drp-1 expression, mt-fragmentation, and PEC dysfunction. © 2018 American Heart Association, Inc.

Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

PubMed

Heightman, Tom D; Berdini, Valerio; Braithwaite, Hannah; Buck, Ildiko M; Cassidy, Megan; Castro, Juan; Courtin, Aurélie; Day, James E H; East, Charlotte; Fazal, Lynsey; Graham, Brent; Griffiths-Jones, Charlotte M; Lyons, John F; Martins, Vanessa; Muench, Sandra; Munck, Joanne M; Norton, David; O'Reilly, Marc; Palmer, Nick; Pathuri, Puja; Reader, Michael; Rees, David C; Rich, Sharna J; Richardson, Caroline; Saini, Harpreet; Thompson, Neil T; Wallis, Nicola G; Walton, Hugh; Wilsher, Nicola E; Woolford, Alison J-A; Cooke, Michael; Cousin, David; Onions, Stuart; Shannon, Jonathan; Watts, John; Murray, Christopher W

2018-05-31

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.

Preoperative Planning and Intraoperative Technique for Accurate Translation of a Distal First Metatarsal Osteotomy.

PubMed

Wynes, Jacob; Lamm, Bradley M; Andrade, Bijan J; Malay, D Scot

2016-01-01

We used preoperative radiographic and intraoperative anatomic measurements to predict and achieve, respectively, the precise amount of capital fragment lateral translation required to restore anatomic balance to the first metatarsophalangeal joint. Correlation was used to relate the amount of capital fragment translation and operative reduction of the first intermetatarsal angle (IMA), hallux abductus angle (HAA), tibial sesamoid position (TSP), metatarsus adductus angle, and first metatarsal length. The mean capital fragment lateral translation was 5.54 ± 1.64 mm, and the mean radiographic reductions included a first IMA of 5.04° ± 2.85°, an HAA of 9.39° ± 8.38°, and a TSP of 1.38 ± 0.9. These changes were statistically (p < .001) and clinically (≥32.55%) significant. The mean reduction of the metatarsus adductus angle was 0.66° ± 4.44° and that for the first metatarsal length was 0.33 ± 7.27 mm, and neither of these were statistically (p = .5876 and 0.1247, respectively) or clinically (≤3.5%) significant. Pairwise correlations between the amount of lateral translation of the capital fragment and the first IMA, HAA, and TSP values were moderately positive and statistically significant (r = 0.4412, p = .0166; r = 0.5391, p = .0025; and r = 0.3729, p = .0463; respectively). In contrast, the correlation with metatarsus adductus and the first metatarsal shortening were weak and not statistically significant (r = 0.2296, p = .2308 and r = -0.2394, p = .2109, respectively). The results of our study indicate that predicted preoperative and executed intraoperative lateral translation of the capital fragment correlates with statistically and clinically significant reductions in the first IMA, HAA, and TSP. Copyright © 2016 American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.

The effect of different posts on fracture strength of roots with vertical fracture and re-attached fragments.

PubMed

Ozcopur, B; Akman, S; Eskitascioglu, G; Belli, S

2010-08-01

The aim of this in vitro study was to test the effect of different post systems on fracture strength of roots with re-attached fragments. Root canals of eighty extracted single-rooted human teeth were instrumented (ProFile) and randomly divided into two groups. The roots in the first group were vertically cracked, and the fragments were re-attached using Super Bond C&B (Sun Medical, Tokya, Japan). The roots in the second group were kept sound. Obturation of the roots was performed with MetaSEAL (Sun Medical) and gutta-percha. Post spaces were prepared, and the roots were restored with one of the followings: UniCore (Ultradent), Everstick (Stick Tech), Ribbond (Ribbond), ParaPost (Coltene/Whaledent) (n = 10). Four mm high build-ups were created (Clearfil DC Bond Core; Kuraray, Tokyo, Japan). Compressive loading of the samples was performed after 24 h (1 mm min(-1)). Mean load necessary to fracture each sample was recorded (Newton) and statistically analysed (One-way anova, t-tests). ParaPost showed the highest fracture strength among the roots with re-attached fragments (P < 0.05). UniCore and ParaPost systems showed similar fracture strength in the sound roots (P > 0.05). Re-attached fragments significantly reduced the fracture strength of roots in UniCore group (P = 0.000). Ribbond post showed mostly repairable fractures. Metal post (ParaPost) showed the highest fracture strength in the roots with re-attached fragments; however, fracture pattern was 41% non-repairable. Re-attached fragments significantly reduced the fracture strength of the roots in UniCore group. Prefabricated posts showed similar fracture strength in the sound roots. Customized post systems EverStick and Ribbond showed mostly repairable failure after loading in sound roots or roots with re-attached fragments.

Molecular Dynamics Simulations of a Cyclic DP-240 Amylose Fragment in a Periodic Cell: Glass Transition Temperature and Water Diffusion

USDA-ARS?s Scientific Manuscript database

Molecular dynamics simulations using AMB06C, an in-house carbohydrate force field, (NPT ensembles, 1atm) were carried out on a periodic cell that contained a cyclic-DP-240 amylose fragment and TIP3P water molecules. Molecular conformation and movement of the amylose fragment and water molecules at ...

Over expression of anti-MUC1 single-domain antibody fragments in the yeast Pichia pastoris.

PubMed

Rahbarizadeh, Fatemeh; Rasaee, Mohammad J; Forouzandeh, Mehdi; Allameh, Abdol-Amir

2006-02-01

The methylotrophic yeast Pichia pastoris has become a highly popular expression host system for the recombinant production of a wide variety of proteins, such as antibody fragments. Camelids produce functional antibodies devoid of light chains and constant heavy-chain domain (CH1). The antigen binding fragments of such heavy chain antibodies are therefore comprised in one single domain, the so-called VH of the camelid heavy chain antibody (VHH). To test the feasibility of expressing VHHs in the yeast, which on account of their small size and antigen recognition properties would have a major impact on antibody engineering strategies, we constructed two VHH genes encoding the single-domain antibody fragments with specificity for a cancer associated mucin, MUC1. The recombinant strains of the yeast P. pastoris were developed which secrete single-domain antibody fragment to the culture supernatant as a biologically active protein. Supplementation of medium with sorbitol (in pre-induction phase) and casamino acid or EDTA (in induction phase) provided ideal condition of increasing the yield of VHH production compared to culture condition devoid of above recipe. The secreted protein was purified following a 80% ammonium sulfate precipitation step, followed by a affinity chromatography column. The specific activity in enzyme-linked immunosorbant assay (ELISA) of the purified yeast VHH was higher than that of a bacterial periplasmic counterpart. These results reaffirm that the yeast P. pastoris is a suitable host for high level and correctly folded production of VHH antibody fragments with potential in vivo diagnostic and therapeutic applications. This is the first report of expression of VHH in P. pastoris.

Upregulation of adhesion complex proteins and fibronectin by human keratinocytes treated with an aqueous extract from the leaves of Chromolaena odorata (Eupolin).

PubMed

Phan, T T; Allen, J; Hughes, M A; Cherry, G; Wojnarowska, F

2000-01-01

The fresh leaves and extract of the plant Chromolaena odorata are a traditional herbal treatment in developing countries for burns, soft tissue wounds and skin infections. We have previously shown that the extract had an effect on the growth and proliferation of keratinocytes and fibroblasts in culture. This study has demonstrated that Eupolin extract increased expression of several components of the adhesion complex and fibronectin by human keratinocytes. Using indirect immunofluorescence we found increased expression (dose-dependent) of laminin 5, laminin 1, collagen IV, and fibronectin. The expression of the b1 and b4 integrins was upregulated by the extract at low concentrations (0.1 and 1 microg/ml), but the expression was decreased at higher doses of Eupolin (10 microg-150 microg/ml). A number of clinical studies carried out by Vietnamese and international medical investigators have demonstrated the efficacy of this extract on the wound healing process. In this study we have shown that Eupolin stimulated the expression of many proteins of the adhesion complex and fibronectin by human keratinocytes. The adhesion complex proteins are essential to stabilise epithelium and this effect could contribute to the clinical efficacy of Eupolin in healing.

In vitro fragmentation efficiency of holmium: yttrium-aluminum-garnet (YAG) laser lithotripsy--a comprehensive study encompassing different frequencies, pulse energies, total power levels and laser fibre diameters.

PubMed

Kronenberg, Peter; Traxer, Olivier

2014-08-01

To assess the fragmentation (ablation) efficiency of laser lithotripsy along a wide range of pulse energies, frequencies, power settings and different laser fibres, in particular to compare high- with low-frequency lithotripsy using a dynamic and innovative testing procedure free from any human interaction bias. An automated laser fragmentation testing system was developed. The unmoving laser fibres fired at the surface of an artificial stone while the stone was moved past at a constant velocity, thus creating a fissure. The lithotripter settings were 0.2-1.2 J pulse energies, 5-40 Hz frequencies, 4-20 W power levels, and 200 and 550 μm core laser fibres. Fissure width, depth, and volume were analysed and comparisons between laser settings, fibres and ablation rates were made. Low frequency-high pulse energy (LoFr-HiPE) settings were (up to six times) more ablative than high frequency-low pulse energy (HiFr-LoPE) at the same power levels (P < 0.001), as they produced deeper (P < 0.01) and wider (P < 0.001) fissures. There were linear correlations between pulse energy and fragmentation volume, fissure width, and fissure depth (all P < 0.001). Total power did not correlate with fragmentation measurements. Laser fibre diameter did not affect fragmentation volume (P = 0.81), except at very low pulse energies (0.2 J), where the large fibre was less efficient (P = 0.015). At the same total power level, LoFr-HiPE lithotripsy was most efficient. Pulse energy was the key variable that drove fragmentation efficiency. Attention must be paid to prevent the formation of time-consuming bulky debris and adapt the lithotripter settings to one's needs. As fibre diameter did not affect fragmentation efficiency, small fibres are preferable due to better scope irrigation and manoeuvrability. © 2013 The Authors. BJU International © 2013 BJU International.

Role of extracellular matrix in regulation of staurosporine-induced apoptosis in breast cancer cells.

PubMed

Vasaturo, F; Malacrino, C; Sallusti, E; Coppotelli, G; Birarelli, P; Giuffrida, A; Albonici, L; Simonelli, L; Modesti, A; Modesti, M; Scarpa, S

2005-04-01

Autocrine and paracrine mechanisms modulate the synthesis and secretion of extracellular matrix (ECM); moreover, each component of the ECM is capable of modulating the synthesis and release of other ECM molecules. Therefore, the synthesis of ECM glycoprotein fibronectin and laminin was studied in the human breast cancer cell lines MCF7 and MDA MB 23, plated on different ECM. Our results showed that the cells plated on a fibronectin substrate increased laminin synthesis: this event correlated with an increase in alpha2 and alpha3 integrin subunits. Staurosporine-induced apoptosis was then analyzed in the cell lines plated on different ECM. Staurosporine treatment determined the apoptosis of 35 and 33% respectively of MDA MB 231 and MCF7; these values increased to 60 and 64% in cells plated on laminin, to 48 and 63% in cells plated on fibronectin and to 64 and 69% in cells plated on matrigel. Moreover, staurosporine treatment decreased bcl-2 expression in the cells plated on fibronectin and laminin. Yet, staurosporine treatment determined PARP cleavage and PARP partial disappearance when the cells were plated on matrigel. Finally, a partial loss of function mutant Ras protein that activated only Raf pathway, was expressed in MCF7, in order to identify whether the increase of apoptosis induced by extracellular matrix involved the Raf/MAP kinase pathway. The increase of apoptosis of the cells plated on matrigel suggested that the activation of the Raf pathway is probably involved in the decrease of survival on matrigel. These data demonstrate that the modification of ECM modulates the apoptotic process of breast cancer cells and suggest that it is worthwhile to dissect the role of ECM in the control of apoptotic process.

Detection of microinvasion in vulvar and cervical intraepithelial neoplasia using double immunostaining for cytokeratin and basement membrane components.

PubMed

Rush, Demaretta; Hyjek, Elizabeth; Baergen, Rebecca N; Ellenson, Lora H; Pirog, Edyta C

2005-06-01

Identification of early invasion in vulvar intraepithelial neoplasia 3 (VIN 3) and cervical intraepithelial neoplasia 3 (CIN 3) may be difficult with the use of routine hematoxylin-eosin staining. Presence of obscuring inflammation and tangential tissue sectioning are the most common diagnostic pitfalls. To examine the utility of double immunostaining for cytokeratin-collagen IV or cytokeratin-laminin in the detection of early invasion in VIN 3 and CIN 3. The study group consisted of 10 cases of "VIN 3, suspicious for invasion" and 10 cases of "CIN 3, suspicious for invasion." The negative control group consisted of VIN 3 (n = 15) and CIN 3 (n = 10). The positive control group consisted of cases of invasive vulvar carcinoma (n = 11) and invasive cervical carcinoma (n = 25). All cases were double immunostained for cytokeratin and collagen IV and, in a separate reaction, for cytokeratin and laminin. The continuity of the basement membrane and the presence of stromal invasion were assessed in the stained sections. The staining for collagen IV and laminin yielded identical results. A well-defined, continuous basement membrane was visualized in all cases of VIN 3 and CIN 3. A discontinuous or absent basement membrane was observed around the malignant cells on the invasive tumor front in all cases of vulvar and cervical carcinoma. In 2 of 10 cases of VIN 3, suspicious for invasion and in 4 of 10 cases of CIN 3, suspicious for invasion definitive foci of microinvasion were identified with the use of double immunostaining. A well-defined, continuous basement membrane was present in the remaining cases "suspicious for invasion." Double immunostaining for cytokeratin- collagen IV or cytokeratin-laminin is useful for evaluation of early invasion in equivocal cases of VIN 3 and CIN 3.

Factors affecting calcium oxalate dihydrate fragmented calculi regrowth

PubMed Central

Costa-Bauzá, A; Perelló, J; Isern, B; Sanchis, P; Grases, F

2006-01-01

Background The use of extracorporeal shock wave lithotripsy (ESWL) to treat calcium oxalate dihydrate (COD) renal calculi gives excellent fragmentation results. However, the retention of post-ESWL fragments within the kidney remains an important health problem. This study examined the effect of various urinary conditions and crystallization inhibitors on the regrowth of spontaneously-passed post-ESWL COD calculi fragments. Methods Post-ESWL COD calculi fragments were incubated in chambers containing synthetic urine varying in pH and calcium concentration: pH = 5.5 normocalciuria (3.75 mM), pH = 5.5 hypercalciuria (6.25 mM), pH = 6.5 normocalciuria (3.75 mM) or pH = 6.5 hypercalciuria (6.25 mM). Fragment growth was evaluated by measuring increases in weight. Fragment growth was standardized by calculating the relative mass increase. Results Calcium oxalate monohydrate (COM) crystals formed on COD renal calculi fragments under all conditions. Under pH = 5.5 normocalciuria conditions, only COM crystals formed (growth rate = 0.22 ± 0.04 μg/mg·h). Under pH = 5.5 hypercalciuria and under pH = 6.5 normocalciuria conditions, COM crystals and a small number of new COD crystals formed (growth rate = 0.32 ± 0.03 μg/mg·h and 0.35 ± 0.05 μg/mg·h, respectively). Under pH = 6.5 hypercalciuria conditions, large amounts of COD, COM, hydroxyapatite and brushite crystals formed (growth rate = 3.87 ± 0. 34 μg/mg·h). A study of three crystallization inhibitors demonstrated that phytate completely inhibited fragment growth (2.27 μM at pH = 5.5 and 4.55 μM at pH = 6.5, both under hypercalciuria conditions), while 69.0 μM pyrophosphate caused an 87% reduction in mass under pH = 6.5 hypercalciuria conditions. In contrast, 5.29 mM citrate did not inhibit fragment mass increase under pH = 6.5 hypercalciuria conditions. Conclusion The growth rate of COD calculi fragments under pH = 6.5 hypercalciuria conditions was approximately ten times that observed under the other three conditions. This observation suggests COD calculi residual fragments in the kidneys together with hypercalciuria and high urinary pH values may be a risk factor for stone growth. The study also showed the effectiveness of specific crystallization inhibitors in slowing calculi fragment growth. PMID:16822299

Isolation, purification and characterization of collagenase from hepatopancreas of the land snail Achatina fulica.

PubMed

Indra, D; Ramalingam, K; Babu, Mary

2005-09-01

Collagenase (matrix metalloproteinase-1, EC:3.4.24.7) was isolated from the hepatopancreas of Achatina fulica and characterized for its enzymatic activity and immunological properties. Procollagenase was isolated using ammonium sulphate precipitation and gel filtration, followed by purification by reverse-phase high performance liquid chromatography in the presence of trifluoroacetic acid and by dialysis in neutral buffer. In the presence of SDS and beta-mercaptoethanol, the procollagenase resolved into two subunits with molecular masses of 63 and 28 kDa, respectively. The 63 kDa fragment retained its ability to bind and degrade gelatin, but the 28 kDa was inactive. Analysis by 2D gel electrophoresis revealed that the 63 kDa fragment was basic (pIs 7.6, 7.8 and 8.15), while the 28 kDa fragment was acidic (pI 4.7 and 5.1). Western blot analysis confirmed the identity of collagenase, as only matrix metalloproteinase-1 rabbit antibodies against human matrix metalloproteinase-1 (N-terminal region) recognized both the isolated procollagenase and the 63 kDa fragment.

Effects of Sleep Fragmentation on Glucose Metabolism in Normal Subjects

PubMed Central

Stamatakis, Katherine A.

2010-01-01

Background: Sleep disorders are increasingly associated with insulin resistance, glucose intolerance, and type 2 diabetes mellitus. Whether the metabolic toll imposed by sleep-related disorders is caused by poor-quality sleep or due to other confounding factors is not known. The objective of this study was to examine whether experimental sleep fragmentation across all sleep stages would alter glucose metabolism, adrenocortical function, and sympathovagal balance. Methods: Sleep was experimentally fragmented across all stages in 11 healthy, normal volunteers for two nights using auditory and mechanical stimuli. Primary outcomes included insulin sensitivity (SI), glucose effectiveness (SG), and insulin secretion, as determined by the intravenous glucose tolerance test. Secondary outcomes included measures of sympathovagal balance and serum levels of inflammatory markers, adipokines, and cortisol. Results: Following two nights of sleep fragmentation, SI decreased from 5.02 to 3.76 (mU/L)−1min−1 (P < .0001). SG, which is the ability of glucose to mobilize itself independent of an insulin response, also decreased from 2.73 × 10−2 min−1 to 2.16 × 10−2 min−1 (P < .01). Sleep fragmentation led to an increase in morning cortisol levels and a shift in sympathovagal balance toward an increase in sympathetic nervous system activity. Markers of systemic inflammation and serum adipokines were unchanged with sleep fragmentation. Conclusions: Fragmentation of sleep across all stages is associated with a decrease in SI and SG. Increases in sympathetic nervous system and adrenocortical activity likely mediate the adverse metabolic effects of poor sleep quality. PMID:19542260

A common variant association study in ethnic Saudi Arabs reveals novel susceptibility loci for hypertriglyceridemia.

PubMed

Ram, R; Wakil, S M; Muiya, N P; Andres, E; Mazhar, N; Hagos, S; Alshahid, M; Meyer, B F; Morahan, G; Dzimiri, N

2017-03-01

Hypertriglyceridemia (hTG) is a lipid disorder, resulting from an elevation in triglyceride levels, with a strong genetic component. It constitutes a significant risk factor for coronary artery disease (CAD), a leading cause of death worldwide. In this study, we performed a common variant association study for hTG in ethnic Saudi Arabs. We genotyped 5501 individuals in a two-phase experiment using Affymetrix Axiom ® Genome-Wide CEU 1 Array (Affymetrix, Santa Cruz, CA) that contains a total of 587,352 single nucleotide polymorphisms (SNPs). The lead variant was the rs1558861 [1.99 (1.73-2.30); p = 7.37 × 10 -22 ], residing on chromosome (chr) 11 at the apolipoprotein A-I/A-5 (APOA1/APOA5) locus. The rs780094 [1.34 (1.21-1.49); p = 8.57 × 10 -8 ] on chr 2 at the glucokinase regulatory protein (GCKR) locus was similarly significantly associated, while the rs10911205 [1.29 (1.16-1.44); p = 3.52 × 10 -6 ] on chr1 at the laminin subunit gamma-1 (LAMC1) locus showed suggestive association with disease. Furthermore, the rs17145738 [0.68 (0.60-0.77); p = 6.69 × 10 -9 ] on chr7 at the carbohydrate-responsive element-binding protein-encoding (MLXIPL) gene locus displayed significant protective characteristics, while another variant rs6982502 [0.76 (0.68-0.84); p = 5.31 × 10 -7 ] on chr8 showed similar but weaker properties. These findings were replicated in 317 cases vs 1415 controls from the same ethnic Arab population. Our study identified several variants across the human genome that are associated with hTG in ethnic Arabs. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Electromagnetic Dissociation Cross Sections for High LET Fragments

NASA Technical Reports Server (NTRS)

Norbury, John

2016-01-01

Nuclear interaction cross sections are used in space radiation transport codes to calculate the probability of fragment emission in high energy nucleus-nucleus collisions. Strong interactions usually dominate in these collisions, but electromagnetic (EM) interactions can also sometimes be important. Strong interactions typically occur when the projectile nucleus hits a target nucleus, with a small impact parameter. For impact parameters larger than the sum of the nuclear radii, EM reactions dominate and the process is called electromagnetic dissociation (EMD) if one of the nuclei undergo fragmentation. Previous models of EMD have been used to calculate single proton (p) production, single neutron (n) production or light ion production, where a light ion is defined as an isotope of hydrogen (H) or helium (He), such as a deuteron (2H), a triton (3H), a helion (3He) or an alpha particle (4He). A new model is described which can also account for multiple nucleon production, such as 2p, 2n, 1p1n, 2p1n, 2p2n, etc. in addition to light ion production. Such processes are important to include for the following reasons. Consider, for example, the EMD reaction 56Fe + Al --> 52Cr + X + Al, for a 56Fe projectile impacting Al, which produces the high linear energy transfer (LET) fragment 52Cr. In this reaction, the most probable particles representing X are either 2p2n or 4He. Therefore, production of the high LET fragment 52Cr, must include the multiple nucleon production of 2p2n in addition to the light ion production of 4He. Previous models, such as the NUCFRG3 model, could only account for the 4He production process in this reaction and could not account for 2p2n. The new EMD model presented in this work accounts for both the light ion and multiple nucleon processes, and is therefore able to correctly account for the production of high LET products such as 52Cr. The model will be described and calculations will be presented that show the importance of light ion and multiple nucleon production. The work will also show that EMD reactions contribute most to those fragments with the highest LET.

The α-Secretase-derived N-terminal Product of Cellular Prion, N1, Displays Neuroprotective Function in Vitro and in Vivo*

PubMed Central

Guillot-Sestier, Marie-Victoire; Sunyach, Claire; Druon, Charlotte; Scarzello, Sabine; Checler, Frédéric

2009-01-01

Cellular prion protein (PrPc) undergoes a disintegrin-mediated physiological cleavage, generating a soluble amino-terminal fragment (N1), the function of which remained unknown. Recombinant N1 inhibits staurosporine-induced caspase-3 activation by modulating p53 transcription and activity, whereas the PrPc-derived pathological fragment (N2) remains biologically inert. Furthermore, N1 protects retinal ganglion cells from hypoxia-induced apoptosis, reduces the number of terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling-positive and p53-immunoreactive neurons in a pressure-induced ischemia model of the rat retina and triggers a partial recovery of b-waves but not a-waves of rat electroretinograms. Our work is the first demonstration that the α-secretase-derived PrPc fragment N1, but not N2, displays in vivo and in vitro neuroprotective function by modulating p53 pathway. It further demonstrates that distinct N-terminal cleavage products of PrPc harbor different biological activities underlying the various phenotypes linking PrPc to cell survival. PMID:19850936

Identification and phylogenetic analysis of novel cytochrome P450 1A genes from ungulate species.

PubMed

Darwish, Wageh Sobhy; Kawai, Yusuke; Ikenaka, Yoshinori; Yamamoto, Hideaki; Muroya, Tarou; Ishizuka, Mayumi

2010-09-01

As part of an ongoing effort to understand the biological response of wild and domestic ungulates to different environmental pollutants such as dioxin-like compounds, cDNAs encoding for CYP1A1 and CYP1A2 were cloned and characterized. Four novel CYP1A cDNA fragments from the livers of four wild ungulates (elephant, hippopotamus, tapir and deer) were identified. Three fragments from hippopotamus, tapir and deer were classified as CYP1A2, and the other fragment from elephant was designated as CYP1A1/2. The deduced amino acid sequences of these fragment CYP1As showed identities ranging from 76 to 97% with other animal CYP1As. The phylogenetic analysis of these fragments showed that both elephant and hippopotamus CYP1As made separate branches, while tapir and deer CYP1As were located beside that of horse and cattle respectively in the phylogenetic tree. Analysis of dN/dS ratio among the identified CYP1As indicated that odd toed ungulate CYP1A2s were exposed to different selection pressure.

Formation of fibroblastic reticular network in the brain after infection with neurovirulent murine coronavirus.

PubMed

Watanabe, Rihito; Kakizaki, Masatoshi; Ikehara, Yuzuru; Togayachi, Akira

2016-12-01

cl-2 virus is an extremely neurovirulent murine coronavirus. However, during the initial phase of infection between 12 and 24 h post-inoculation (hpi), the viral antigens are detected only in the meninges, followed by viral spread into the ventricular wall before invasion into the brain parenchyma, indicating that the viruses employ a passage between the meninges and ventricular wall as an entry route into the brain parenchyma. At 48 hpi, the passage was found to be constructed by ER-TR7 antigen (ERag)-positive fibers (ERfibs) associated with laminin and collagen III between the fourth ventricle and meninges at the cerebellopontine angle. The construct of the fibers mimics the reticular fibers of the fibroblastic reticular network, which comprises a conduit system in the lymphoid organs. In the meninges, ERfibs together with collagen fibers, lining in a striped pattern, made up a pile of thin sheets. In the brain parenchyma, mature ERfibs associated with laminin were found around blood vessels. Besides mature ERfibs, immature Erfibs without associations with other extracellular matrix components like laminin and collagen appeared after infection, suggesting that the CNS creates a unique conduit system for immune communication triggered by viral invasion. © 2016 Japanese Society of Neuropathology.

C-TERMINAL FRAGMENT OF TRANSFORMING GROWTH FACTOR BETA-INDUCED PROTEIN (TGFBIp) IS REQUIRED FOR APOPTOSIS IN HUMAN OSTEOSARCOMA CELLS

PubMed Central

Zamilpa, Rogelio; Rupaimoole, Rajesha; Phelix, Clyde F.; Somaraki-Cormier, Maria; Haskins, William; Asmis, Reto; LeBaron, Richard G.

2009-01-01

Transforming growth factor beta induced protein (TGFBIp), is secreted into the extracellular space. When fragmentation of C-terminal portions is blocked, apoptosis is low, even when the protein is overexpressed. If fragmentation occurs, apoptosis is observed. Whether full-length TGFBIp or integrin-binding fragments released from its C-terminus is necessary for apoptosis remains equivocal. More importantly, the exact portion of the C-terminus that conveys the pro-apoptotic property of TGFBIp is uncertain. It is reportedly within the final 166 amino acids. We sought to determine if this property is dependent upon the final 69 amino acids containing the integrin-binding, EPDIM and RGD, sequences. With MG-63 osteosarcoma cells, transforming growth factor (TGF)-β1 treatment increased expression of TGFBIp over 72 hours (p<0.001). At this time point, apoptosis was significantly increased (p<0.001) and was prevented by an anti-TGFBIp, polyclonal antibody (p<0.05). Overexpression of TGFBIp by transient transfection produced a 2-fold increase in apoptosis (p<0.01). Exogenous purified TGFBIp at concentrations of 37 to 150 nM produced a dose dependent increase in apoptosis (p<0.001). Mass spectrometry analysis of TGFBIp isolated from conditioned medium of cells treated with TGF-β1 revealed truncated forms of TGFBIp that lacked integrin-binding sequences in the C-terminus. Recombinant TGFBIp truncated, similarly, at amino acid 614 failed to induce apoptosis. A recombinant fragment encoding the final 69 amino acids of the TGFBIp C-terminus produced significant apoptosis. This apoptosis level was comparable to that induced by TGF-β1 upregulation of endogenous TGFBIp. Mutation of the integrin-binding sequence EPDIM, but not RGD, blocked apoptosis (p<0.001). These pro-apoptotic actions are dependent on the C-terminus most likely to interact with integrins. PMID:19505574

DNA fingerprinting and analysis of population structure in the chestnut blight fungus, Cryphonectria parasitica.

PubMed

Milgroom, M G; Lipari, S E; Powell, W A

1992-06-01

We analyzed DNA fingerprints in the chestnut blight fungus, Cryphonectria parasitica, for stability, inheritance, linkage and variability in a natural population. DNA fingerprints resulting from hybridization with a dispersed moderately repetitive DNA sequence of C. parasitica in plasmid pMS5.1 hybridized to 6-17 restriction fragments per individual isolate. In a laboratory cross and from progeny from a single perithecium collected from a field population, the presence/absence of 11 fragments in the laboratory cross and 12 fragments in the field progeny set segregated in 1:1 ratios. Two fragments in each progeny set cosegregated; no other linkage was detected among the segregating fragments. Mutations, identified by missing bands, were detected for only one fragment in which 4 of 43 progeny lacked a band present in both parents; no novel fragments were detected in any progeny. All other fragments appeared to be stably inherited. Hybridization patterns did not change during vegetative growth or sporulation. However, fingerprint patterns of single conidial isolates of strains EP155 and EP67 were found to be heterogenous due to mutations that occurred during culturing in the laboratory since these strains were first isolated in 1976-1977. In a population sample of 39 C. parasitica isolates, we found 33 different fingerprint patterns with pMS5.1. Most isolates differed from all other isolates by the presence or absence of several fragments. Six fingerprint patterns each occurred twice. Isolates with identical fingerprints occurred in cankers on the same chestnut stems three times; isolates within the other three pairs were isolated from cankers more than 5 m apart. The null hypothesis of random mating in this population could not be rejected if the six putative clones were removed from the analysis. Thus, a rough estimate of the clonal fraction of this population is 6 in 39 isolates (15.4%).

Lithotripsy of gallstones by means of a quality-switched giant-pulse neodymium:yttrium-aluminum-garnet laser. Basic in vitro studies using a highly flexible fiber system.

PubMed

Hochberger, J; Gruber, E; Wirtz, P; Dürr, U; Kolb, A; Zanger, U; Hahn, E G; Ell, C

1991-11-01

The quality-switched neodymium:yttrium-aluminum-garnet laser represents a new instrument for athermal fragmentation of gallstones by transformation of optical energy into mechanical energy in the form of shock waves via local plasma formation. A highly flexible 300-micron fiber transmission system was used in basic investigations to determine the influence of varying pulse repetition rates (5-30 Hz) and pulse energies (15 and 20 mJ) on shock wave intensity and stone fragmentation in vitro for 105 biliary calculi of known size and chemical composition. After performance of 1200 shock wave pressure measurements using polyvinylidenefluoride hydrophones, stone fragmentation was analyzed by determination of fragment removal rates (volume of fragments removed per fragmentation time), ablation rates (mean volume removed per laser pulse), and median fragment sizes for each laser setting. With the quality-switched neodymium:yttrium-aluminum-garnet laser system, all concrements could be reliably disintegrated into small fragments (median diameter, 0.7-1.7 mm). Compared with pure cholesterol stones, a significantly higher fragment removal rate was achieved in cholesterol stones containing 30% calcium phosphate (P = 0.039), in cholesterol stones containing 20% pigment (P = 0.015), and in pure pigment stones (P = 0.007). Fragment removal rates, local shock wave pressures, and median grain sizes were significantly higher at a pulse energy of 20 mJ than with 15 mJ. Shock wave pressures showed a distinct dependence on pulse repetition rates at 20 mJ, yet not at 15 mJ. Because there is no evident hazard of thermal damage to tissue using the quality-switched neodymium:yttrium-aluminum-garnet laser, it appears to be a promising device for nonsurgical biliary stone therapy.

A comparison of the fragmentation thresholds and inertial cavitation doses of different ultrasound contrast agents

NASA Astrophysics Data System (ADS)

Chen, Wen-Shiang; Matula, Thomas J.; Brayman, Andrew A.; Crum, Lawrence A.

2003-01-01

Contrast bubble destruction is important in several new diagnostic and therapeutic applications. The pressure threshold of destruction is determined by the shell material, while the propensity for of the bubbles to undergo inertial cavitation (IC) depends both on the gas and shell properties of the ultrasound contrast agent (UCA). The ultrasonic fragmentation thresholds of three specific UCAs (Optison, Sonazoid, and biSpheres), each with different shell and gas properties, were determined under various acoustic conditions. The acoustic emissions generated by the agents, or their derivatives, characteristic of IC after fragmentation, was also compared, using cumulated broadband-noise emissions (IC ``dose''). Albumin-shelled Optison and surfactant-shelled Sonazoid had low fragmentation thresholds (mean=0.13 and 0.15 MPa at 1.1 MHz, 0.48 and 0.58 MPa at 3.5 MHz, respectively), while polymer-shelled biSpheres had a significant higher threshold (mean=0.19 and 0.23 MPa at 1.1 MHz, 0.73 and 0.96 MPa for thin- and thick-shell biSpheres at 3.5 MHz, respectively, p<0.01). At comparable initial concentrations, surfactant-shelled Sonazoid produced a much larger IC dose after shell destruction than did either biSpheres or Optison (p<0.01). Thick-shelled biSpheres had the highest fragmentation threshold and produced the lowest IC dose. More than two and five acoustic cycles, respectively, were necessary for the thin- and thick-shell biSpheres to reach a steady-state fragmentation threshold.

Tissue-engineered cartilaginous constructs for the treatment of caprine cartilage defects, including distribution of laminin and type IV collagen.

PubMed

Jeng, Lily; Hsu, Hu-Ping; Spector, Myron

2013-10-01

The purpose of this study was the immunohistochemical evaluation of (1) cartilage tissue-engineered constructs; and (2) the tissue filling cartilage defects in a goat model into which the constructs were implanted, particularly for the presence of the basement membrane molecules, laminin and type IV collagen. Basement membrane molecules are localized to the pericellular matrix in normal adult articular cartilage, but have not been examined in tissue-engineered constructs cultured in vitro or in tissue filling cartilage defects into which the constructs were implanted. Cartilaginous constructs were engineered in vitro using caprine chondrocyte-seeded type II collagen scaffolds. Autologous constructs were implanted into 4-mm-diameter defects created to the tidemark in the trochlear groove in the knee joints of skeletally mature goats. Eight weeks after implantation, the animals were sacrificed. Constructs underwent immunohistochemical and histomorphometric evaluation. Widespread staining for the two basement membrane molecules was observed throughout the extracellular matrix of in vitro and in vivo samples in a distribution unlike that previously reported for cartilage. At sacrifice, 70% of the defect site was filled with reparative tissue, which consisted largely of fibrous tissue and some fibrocartilage, with over 70% of the reparative tissue bonded to the adjacent host tissue. A novel finding of this study was the observation of laminin and type IV collagen in in vitro engineered cartilaginous constructs and in vivo cartilage repair samples from defects into which the constructs were implanted, as well as in normal caprine articular cartilage. Future work is needed to elucidate the role of basement membrane molecules during cartilage repair and regeneration.

Tissue-Engineered Cartilaginous Constructs for the Treatment of Caprine Cartilage Defects, Including Distribution of Laminin and Type IV Collagen

PubMed Central

Jeng, Lily; Hsu, Hu-Ping

2013-01-01

The purpose of this study was the immunohistochemical evaluation of (1) cartilage tissue-engineered constructs; and (2) the tissue filling cartilage defects in a goat model into which the constructs were implanted, particularly for the presence of the basement membrane molecules, laminin and type IV collagen. Basement membrane molecules are localized to the pericellular matrix in normal adult articular cartilage, but have not been examined in tissue-engineered constructs cultured in vitro or in tissue filling cartilage defects into which the constructs were implanted. Cartilaginous constructs were engineered in vitro using caprine chondrocyte-seeded type II collagen scaffolds. Autologous constructs were implanted into 4-mm-diameter defects created to the tidemark in the trochlear groove in the knee joints of skeletally mature goats. Eight weeks after implantation, the animals were sacrificed. Constructs underwent immunohistochemical and histomorphometric evaluation. Widespread staining for the two basement membrane molecules was observed throughout the extracellular matrix of in vitro and in vivo samples in a distribution unlike that previously reported for cartilage. At sacrifice, 70% of the defect site was filled with reparative tissue, which consisted largely of fibrous tissue and some fibrocartilage, with over 70% of the reparative tissue bonded to the adjacent host tissue. A novel finding of this study was the observation of laminin and type IV collagen in in vitro engineered cartilaginous constructs and in vivo cartilage repair samples from defects into which the constructs were implanted, as well as in normal caprine articular cartilage. Future work is needed to elucidate the role of basement membrane molecules during cartilage repair and regeneration. PMID:23672504

Colony-Forming Progenitor Cells in the Postnatal Mouse Liver and Pancreas Give Rise to Morphologically Distinct Insulin-Expressing Colonies in 3D Cultures

PubMed Central

Jin, Liang; Feng, Tao; Chai, Jing; Ghazalli, Nadiah; Gao, Dan; Zerda, Ricardo; Li, Zhuo; Hsu, Jasper; Mahdavi, Alborz; Tirrell, David A.; Riggs, Arthur D.; Ku, Hsun Teresa

2014-01-01

In our previous studies, colony-forming progenitor cells isolated from murine embryonic stem cell-derived cultures were differentiated into morphologically distinct insulin-expressing colonies. These colonies were small and not light-reflective when observed by phase-contrast microscopy (therefore termed “Dark” colonies). A single progenitor cell capable of giving rise to a Dark colony was termed a Dark colony-forming unit (CFU-Dark). The goal of the current study was to test whether endogenous pancreas, and its developmentally related liver, harbored CFU-Dark. Here we show that dissociated single cells from liver and pancreas of one-week-old mice give rise to Dark colonies in methylcellulose-based semisolid culture media containing either Matrigel or laminin hydrogel (an artificial extracellular matrix protein). CFU-Dark comprise approximately 0.1% and 0.03% of the postnatal hepatic and pancreatic cells, respectively. Adult liver also contains CFU-Dark, but at a much lower frequency (~0.003%). Microfluidic qRT-PCR, immunostaining, and electron microscopy analyses of individually handpicked colonies reveal the expression of insulin in many, but not all, Dark colonies. Most pancreatic insulin-positive Dark colonies also express glucagon, whereas liver colonies do not. Liver CFU-Dark require Matrigel, but not laminin hydrogel, to become insulin-positive. In contrast, laminin hydrogel is sufficient to support the development of pancreatic Dark colonies that express insulin. Postnatal liver CFU-Dark display a cell surface marker CD133+CD49flowCD107blow phenotype, while pancreatic CFU-Dark are CD133-. Together, these results demonstrate that specific progenitor cells in the postnatal liver and pancreas are capable of developing into insulin-expressing colonies, but they differ in frequency, marker expression, and matrix protein requirements for growth. PMID:25148366

Enhancing Peripheral Nerve Regeneration with a Novel Drug Delivering Nerve Conduit

DTIC Science & Technology

2014-10-01

tubes of the PLGA nerve conduit. A polyether sulfone (PES) filter membrane, a polydimethyl siloxane (PDMS) plug and silicone sealant (RTV silicone, Dow...harvested from chicken embryos. 1 DRG/well was plated in 24-well plates coated with laminin (1 µg/ml). Finally, different doses of NGF, GDNF and NGF...application in nerve regeneration, Biomedical Microdevices, in preparation, acknowledgement of federal support (yes) Books or other non-periodical, one

[Preparation of monoclonal antibody against 4-amylphenol and homology modeling of its Fv fragment].

PubMed

Cheng, Lei; Wu, Haizhen; Fei, Jing; Zhang, Lujia; Ye, Jiang; Zhang, Huizhan

2017-03-01

Objective To prepare and characterize a monoclonal antibody (mAb) against 4-amylphenol (4-AP), clone its cDNA sequence and make homology modeling for its Fv fragment. Methods A high-affinity anti-4-AP mAb was generated from a hybridoma cell line F10 using electrofusion between splenocytes from APA-BSA-immunized mouse and Sp2/0 myeloma cells. Then we extracted the mRNA of F10 cells and cloned the cDNA of mAb. The homology modeling and molecular docking of its Fv fragment was conducted with biological software. Results Under the optimum conditions, the ic-ELISA equation was y=A 2 +(A 1 -A 2 )/(1+(x/x 0 ) p ) (A 1 =1.28; A 2 =-0.066; x 0 =12560.75; p=0.74) with a correlation coefficient (R 2 ) of 0.997. The lowest detectable limit was 0.65 μg/mL. The heavy and light chains of mAb respectively belonged to IgG1 and Kappa. The homology modeling and molecular docking studies revealed that the binding of 4-Ap and mAb was attributed to the hydrogen bond and hydrophobic interactions. Conclusion The study successfully established a stable 4-AP mAb-secreting hybridoma cell line. The study on spatial structure of Fv fragment using homology modeling provided a reference for the development and design of single chain variable fragments.

A dual role for Integrin α6β4 in modulating hereditary neuropathy with liability to pressure palsies.

PubMed

Poitelon, Yannick; Matafora, Vittoria; Silvestri, Nicholas; Zambroni, Desirée; McGarry, Claire; Serghany, Nora; Rush, Thomas; Vizzuso, Domenica; Court, Felipe A; Bachi, Angela; Wrabetz, Lawrence; Feltri, Maria Laura

2018-05-01

Peripheral myelin protein 22 (PMP22) is a component of compact myelin in the peripheral nervous system. The amount of PMP22 in myelin is tightly regulated, and PMP22 over or under-expression cause Charcot-Marie-Tooth 1A (CMT1A) and Hereditary Neuropathy with Pressure Palsies (HNPP). Despite the importance of PMP22, its function remains largely unknown. It was reported that PMP22 interacts with the β4 subunit of the laminin receptor α6β4 integrin, suggesting that α6β4 integrin and laminins may contribute to the pathogenesis of CMT1A or HNPP. Here we asked if the lack of α6β4 integrin in Schwann cells influences myelin stability in the HNPP mouse model. Our data indicate that PMP22 and β4 integrin may not interact directly in myelinating Schwann cells, however, ablating β4 integrin delays the formation of tomacula, a characteristic feature of HNPP. In contrast, ablation of integrin β4 worsens nerve conduction velocities and non-compact myelin organization in HNPP animals. This study demonstrates that indirect interactions between an extracellular matrix receptor and a myelin protein influence the stability and function of myelinated fibers. © 2018 International Society for Neurochemistry.

Role of mTOR, Bad, and Survivin in RasGAP Fragment N-Mediated Cell Protection

PubMed Central

Yang, Jiang-Yan; Widmann, Christian

2013-01-01

Partial cleavage of p120 RasGAP by caspase-3 in stressed cells generates an N-terminal fragment, called fragment N, which activates an anti-apoptotic Akt-dependent survival response. Akt regulates several effectors but which of these mediate fragment N-dependent cell protection has not been defined yet. Here we have investigated the role of mTORC1, Bad, and survivin in the capacity of fragment N to protect cells from apoptosis. Neither rapamycin, an inhibitor of mTORC1, nor silencing of raptor, a subunit of the mTORC1 complex, altered the ability of fragment N from inhibiting cisplatin- and Fas ligand-induced death. Cells lacking Bad, despite displaying a stronger resistance to apoptosis, were still protected by fragment N against cisplatin-induced death. Fragment N was also able to protect cells from Fas ligand-induced death in conditions where Bad plays no role in apoptosis regulation. Fragment N expression in cells did neither modulate survivin mRNA nor its protein expression. Moreover, the expression of cytoplasmic survivin, known to exert anti-apoptotic actions in cells, still occurred in UV-B-irradiated epidermis of mouse expressing a caspase-3-resistant RasGAP mutant that cannot produce fragment N. Additionally, survivin function in cell cycle progression was not affected by fragment N. These results indicate that, taken individually, mTOR, Bad, or Survivin are not required for fragment N to protect cells from cell death. We conclude that downstream targets of Akt other than mTORC1, Bad, or survivin mediate fragment N-induced protection or that several Akt effectors can compensate for each other to induce the pro-survival fragment N-dependent response. PMID:23826368

PHOTORESTORATION OF FRAGMENTS OF IRRADIATED AMOEBA (in French)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Skreb, Y.; Bevilacqua, L.

1962-11-01

Studies were made on the restcration of fragments of amoeba following 1200 ergs/mm/sup 2/ ultraviolet radiation. An excellent restoration of the total quantity of RNA was found. The numbers corresponding to the nucleated fragments illuminated were even superior to those corresponding to the controls. The ratio between illuminated and irradiated fragments was 1.43 and among the anucleated was 1.25. The results confirmed the fact that photorestoration exerts some actual beneficial effects that manifest themselves with different intensity following the cellular constituent considered. It was concluded that the chromophore exists in the cytoplasm of the amoeba since the consequences of themore » photorestoration manifests itself almost in the same manner in the two types of fragments that are concerned with the evolution of RNA. (P.C.H.)« less

Purification and characterization of substance P endopeptidase activities in the rat spinal cord.

PubMed

Karlsson, K; Eriksson, U; Andrén, P; Nyberg, F

1997-02-01

Two enzymes with substance P degrading activity were purified from the membrane bound fraction of the rat spinal cord. The purified enzymes were characterized with regard to biochemical and kinetic properties. One of the enzymes exhibited close similarity to neutral endopeptidase 24.11 (NEP, EC 3.4.24.11), while the other resembled a substance P converting endopeptidase (SPE), which has previously been identified and purified from human cerebrospinal fluid (CSF). Detergent treated spinal cord homogenates from male Sprague Dawley rats were purified by anion-exchange chromatography (DEAE-sepharose CL-6B), hydrophobic-interaction chromatography (phenyl-sepharose CL-4B) and molecular sieving (Sephadex G-50). Two fractions with enzymes differing in size were recovered and allowed for further purification to apparent homogeneity by ion-exchange chromatography and molecular sieving on a micro-purification system (SMART). The enzyme activities were monitored by following the conversion of synthetic substance P using a radioimmunoassay specific for the heptapeptide product, substance P (1-7). By SDS-polyacrylamide gel electrophoresis of the purified enzymes molecular weights of 43 and 70 kDa were estimated for the SPE-like and NEP-like activity, respectively. A K(m) of 5 microM was determined for the conversion of substance P to its (1-7) fragment by the SPE-like activity. Reversed-phase HPLC together with mass spectrometry permitted identification of all fragments released from substance P by the peptidases. The released fragments were for both enzymes identified as substance P (1-7), substance P (8-11), substance P (1-8), substance P (9-11). The NEP-like enzyme preparation also gave substance P (1-6) as a major product.

PrP(C) homodimerization stimulates the production of PrPC cleaved fragments PrPN1 and PrPC1.

PubMed

Béland, Maxime; Motard, Julie; Barbarin, Alice; Roucou, Xavier

2012-09-19

An endoproteolytic cleavage termed α-cleavage between residues 111/112 is a characteristic feature of the cellular prion protein (PrP(C)). This cleavage generates a soluble N-terminal fragment (PrPN1) and a glycosylphosphatidylinositol-anchored C-terminal fragment (PrPC1). Independent studies demonstrate that modulating PrP(C) α-cleavage represents a potential therapeutic strategy in prion diseases. The regulation of PrP(C) α-cleavage is unclear. The only known domain that is essential for the α-cleavage to occur is a hydrophobic domain (HD). Importantly, the HD is also essential for the formation of PrP(C) homodimers. To explore the role of PrP(C) homodimerization on the α-cleavage, we used a well described inducible dimerization strategy whereby a chimeric PrP(C) composed of a modified FK506-binding protein (Fv) fused with PrP(C) and termed Fv-PrP is incubated in the presence of a dimerizer AP20187 ligand. We show that homodimerization leads to a considerable increase of PrP(C) α-cleavage in cultured cells and release of PrPN1 and PrPC1. Interestingly, enforced homodimerization increased PrP(C) levels at the plasma membrane, and preventing PrP(C) trafficking to the cell surface inhibited dimerization-induced α-cleavage. These observations were confirmed in primary hippocampal neurons from transgenic mice expressing Fv-PrP. The proteases responsible for the α-cleavage are still elusive, and in contrast to initial studies we confirm more recent investigations that neither ADAM10 nor ADAM17 are involved. Importantly, PrPN1 produced after PrP(C) homodimerization protects against toxic amyloid-β (Aβ) oligomers. Thus, our results show that PrP(C) homodimerization is an important regulator of PrP(C) α-cleavage and may represent a potential therapeutic avenue against Aβ toxicity in Alzheimer's disease.

Transverse-momentum dependent modification of dynamic texture in central Au+Au collisions at sqrt(sNN) = 200 GeV

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adams, J.; Aggarwal, M.M.; Ahammed, Z.

2005-01-10

Correlations in the hadron distributions produced in relativistic Au+Au collisions are studied in the discrete wavelet expansion method. The analysis is performed in the space of pseudorapidity (|{eta}| {le} 1) and azimuth (full 2{pi}) in bins of transverse momentum (p{sub t}) from 0.14 {le} p{sub t} {le} 2.1 GeV/c. In peripheral Au+Au collisions a correlation structure ascribed to minijet fragmentation is observed. It evolves with collision centrality and p{sub t} in a way not seen before which suggests strong dissipation of minijet fragmentation in the longitudinally-expanding medium.

Synchronous detection of ebolavirus conserved RNA sequences and ebolavirus-encoded miRNA-like fragment based on a zwitterionic copper (II) metal-organic framework.

PubMed

Qiu, Gui-Hua; Weng, Zi-Hua; Hu, Pei-Pei; Duan, Wen-Jun; Xie, Bao-Ping; Sun, Bin; Tang, Xiao-Yan; Chen, Jin-Xiang

2018-04-01

From a three-dimensional (3D) metal-organic framework (MOF) of {[Cu(Cmdcp)(phen)(H 2 O)] 2 ·9H 2 O} n (1, H 3 CmdcpBr = N-carboxymethyl-(3,5-dicarboxyl)pyridinium bromide, phen = phenanthroline), a sensitive and selective fluorescence sensor has been developed for the simultaneous detection of ebolavirus conserved RNA sequences and ebolavirus-encoded microRNA-like (miRNA-like) fragment. The results from molecular dynamics simulation confirmed that MOF 1 absorbs carboxyfluorescein (FAM)-tagged and 5(6)-carboxyrhodamine, triethylammonium salt (ROX)-tagged probe ss-DNA (probe DNA, P-DNA) by π … π stacking and hydrogen bonding, as well as additional electrostatic interactions to form a sensing platform of P-DNAs@1 with quenched FAM and ROX fluorescence. In the presence of targeted ebolavirus conserved RNA sequences or ebolavirus-encoded miRNA-like fragment, the fluorophore-labeled P-DNA hybridizes with the analyte to give a P-DNA@RNA duplex and released from MOF 1, triggering a fluorescence recovery. Simultaneous detection of two target RNAs has also been realized by single and synchronous fluorescence analysis. The formed sensing platform shows high sensitivity for ebolavirus conserved RNA sequences and ebolavirus-encoded miRNA-like fragment with detection limits at the picomolar level and high selectivity without cross-reaction between the two probes. MOF 1 thus shows the potential as an effective fluorescent sensing platform for the synchronous detection of two ebolavirus-related sequences, and offer improved diagnostic accuracy of Ebola virus disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate.

PubMed

Chang, J Y

1985-09-02

alpha-Thrombin cleavage of 30 polypeptide hormones and their derivatives were analysed by quantitative amino-terminal analysis. The polypeptides included secretin, vasoactive intestinal polypeptide, cholecystokinin fragment, dynorphin A, somatostatins, gastrin-releasing peptide, calcitonins and human parathyroid hormone fragment. Most of them were selected mainly on the ground that they contain sequence structures homologous to the well known tripeptide substrates of alpha-thrombin. All selected polypeptides have one single major cleavage site and both Arg-Xaa and Lys-Xaa bonds were found to be selectively cleaved by alpha-thrombin. Under fixed conditions (1 nmol polypeptide/0.5 NIH unit alpha-thrombin in 20 microliters of 50 mM ammonium bicarbonate at 25 degrees C), the time required for 50% cleavage ranges from less than 1 min to longer than 24 h. Heparin invariably enhanced thrombin cleavage on all polypeptide analysed. The optimum cleavage site for alpha-thrombin has the structures of (a) P4-P3-Pro-Arg-P1'-P2', where P3 and P4 are hydrophobic amino acid and P1', P2' are nonacidic amino acids and (b) P2-Arg-P1', where P2 or P1' are Gly. The requirement for hydrophobic P3 and P4 was further demonstrated by the drastic decrease of thrombin cleavage rates in both gastrin-releasing peptide and calcitonins after chemical removal of hydrophobic P3 and P4 residues. The requirement for nonacidic P1' and P2' residues was demonstrated by the drastic increase of thrombin cleavage rates in both calcitonin and parathyroid hormone fragments, after specific chemical modification of acidic P1' and P2' residues. These findings confirm the importance of hydrophobic P2-P4 residues for thrombin specificity and provide new evidence to indicate that apolar P1' and P2' residues are also crucial for thrombin specificity. It is concluded that specific cleavage of polypeptides by alpha-thrombin can be reasonably predicted and that chemical modification can be a useful tool in enhancing thrombin cleavage.

The interrelationship of complement-activation fragments and angiogenesis-related factors in early pregnancy and their association with pre-eclampsia

PubMed Central

Lynch, AM; Murphy, JR; Gibbs, RS; Levine, RJ; Giclas, PC; Salmon, JE; Holers, VM

2016-01-01

Objective To determine the interrelationships during early pregnancy of complement-activation fragments Bb, C3a and sC5b-9, and angiogenesis-related factors placental growth factor (PiGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), and their associations with pre-eclampsia. Design Prospective cohort study. Setting Denver complement study (June 2005–June 2008). Population A total of 668 pregnant women with singleton gestations, recruited between 10 and 15 weeks of gestation. Methods Using univariable and multivariable logistic regression analysis, concentrations of complement-activation fragments and angiogenesis-related factors were compared between 10 and 15 weeks of gestation in women who subsequently did or did not develop pre-eclampsia. Interrelationships between these variables were tested using the non-parametric Spearman rank correlation coefficient. Main outcome measure Pre-eclampsia. The association of complement-activation fragments and angiogenesis-related factors with obesity was also examined. Results The mean (±SD) levels of complement Bb in early pregnancy among women who did and did not develop pre-eclampsia were 0.84 (±0.26) µg/ml and 0.69 (±0.2) µg/ml, respectively (P = 0.001). Concentrations of PiGF were significantly (P = 0.01) lower (31 ± 12 pg/ml) in early pregnancy in the pre-eclamptic group of women, as compared with the normotensive group (39 ± 32 pg/ml). The adjusted odds ratio (AOR) of Bb and PiGF were 2.1 (CI = 1.4–3.1, P < 0.0003) and 0.2 (CI = 0.07–0.7, P = 0.01), respectively. There was no significant difference in the levels of C3a, sC5b-9, sFlt-1 and sEng in early pregnancy among women who developed pre-eclampsia, compared with women who remained normotensive during pregnancy. Higher levels of Bb (P = 0.0001) and C3a (P = 0.03), and lower levels of sFlt-1 (P = 0.0002) and sEng (P = 0.0001) were found among women with obesity, compared with non-obese controls. No meaningful relationships were found between the complement-activation fragments and the angiogenesis-related factors. Conclusions In this cohort during early pregnancy, increased concentrations of complement-activation factor Bb and lower concentrations of PiGF were associated with the development of pre-eclampsia later in pregnancy. PMID:20074261

Atypical Chemokine Receptor 1 polymorphism cannot be used as an indicator of liver fibrosis progression in Hepatitis C virus positive patients

PubMed Central

Shao, Lin-Nan; Zhang, Shu-Ting; Zhou, Shi-Hang; Yu, Wei-Jian; Liu, Ming

2017-01-01

Background & Objective: Atypical chemokine receptor 1(ACKR1) represents an atypical chemokine receptor that can bind promiscuously to various chemokines. Chemokines play a crucial role to recruit leukocyte subsets migration through the endothelium and into liver against the virus during the progression of hepatitis C virus (HCV) infection. Most HCV positive patients can lead to liver fibrosis. Hyaluronic acid (HA), laminin (LN), collagen IV(C-IV) and amino-terminal pro-peptide of Type-III pro-collagen (PIII NP) are indices of the extent of liver fibrosis. The aim of this study was to investigate the association between ACKR1 polymorphism and liver fibrosis with these four serum liver markers in HCV positive patients. Methods: From April 2015 to December 2015, a total of 210 patients (109 males and 101 females) with chronic HCV infection at Dalian Infectious Hospital were recruited to participate in this study. ACKR1 genotyping was using TaqMan probes method. HA, LN, C-IV and PIII NP were detected by using diagnostic kits. Results: We compared serum levels of HA, LN, C-IV and PIII NP between FY*A/FY*A and FY*A/FY*B patients and the differences were not significant (P=0.905, P=0.298, P=0.880 and P=0.470, respectively). Conclusions: This study has attempted to elucidate the role of ACKR1 polymorphism in liver fibrosis progression of HCV infection, our results demonstrated that ACKR1 polymorphism is not directly associated with the fibrogenesis in HCV positive patients. PMID:29142552

Identification of a Supramolecular Functional Architecture of Streptococcus mutans Adhesin P1 on the Bacterial Cell Surface*

PubMed Central

Heim, Kyle P.; Sullan, Ruby May A.; Crowley, Paula J.; El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Tang, Wenxing; Besingi, Richard; Dufrene, Yves F.; Brady, L. Jeannine

2015-01-01

P1 (antigen I/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical extraction were significantly diminished in adherence to immobilized salivary agglutinin but remained immunoreactive and were readily aggregated by fluid-phase salivary agglutinin. Bacterial adherence was restored by incubation of postextracted cells with P1 fragments that contain each of the two known adhesive domains. In contrast to untreated cells, glutaraldehyde-treated bacteria gained reactivity with anti-C-terminal monoclonal antibodies (mAbs), whereas epitopes recognized by mAbs against other portions of the molecule were masked. Surface plasmon resonance experiments demonstrated the ability of apical and C-terminal fragments of P1 to interact. Binding of several different anti-P1 mAbs to unfixed cells triggered release of a C-terminal fragment from the bacterial surface, suggesting a novel mechanism of action of certain adherence-inhibiting antibodies. We also used atomic force microscopy-based single molecule force spectroscopy with tips bearing various mAbs to elucidate the spatial organization and orientation of P1 on living bacteria. The similar rupture lengths detected using mAbs against the head and C-terminal regions, which are widely separated in the tertiary structure, suggest a higher order architecture in which these domains are in close proximity on the cell surface. Taken together, our results suggest a supramolecular organization in which additional P1 polypeptides, including the C-terminal segment originally identified as antigen II, associate with covalently attached P1 to form the functional adhesive layer. PMID:25666624

Identification of a supramolecular functional architecture of Streptococcus mutans adhesin P1 on the bacterial cell surface.

PubMed

Heim, Kyle P; Sullan, Ruby May A; Crowley, Paula J; El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Tang, Wenxing; Besingi, Richard; Dufrene, Yves F; Brady, L Jeannine

2015-04-03

P1 (antigen I/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical extraction were significantly diminished in adherence to immobilized salivary agglutinin but remained immunoreactive and were readily aggregated by fluid-phase salivary agglutinin. Bacterial adherence was restored by incubation of postextracted cells with P1 fragments that contain each of the two known adhesive domains. In contrast to untreated cells, glutaraldehyde-treated bacteria gained reactivity with anti-C-terminal monoclonal antibodies (mAbs), whereas epitopes recognized by mAbs against other portions of the molecule were masked. Surface plasmon resonance experiments demonstrated the ability of apical and C-terminal fragments of P1 to interact. Binding of several different anti-P1 mAbs to unfixed cells triggered release of a C-terminal fragment from the bacterial surface, suggesting a novel mechanism of action of certain adherence-inhibiting antibodies. We also used atomic force microscopy-based single molecule force spectroscopy with tips bearing various mAbs to elucidate the spatial organization and orientation of P1 on living bacteria. The similar rupture lengths detected using mAbs against the head and C-terminal regions, which are widely separated in the tertiary structure, suggest a higher order architecture in which these domains are in close proximity on the cell surface. Taken together, our results suggest a supramolecular organization in which additional P1 polypeptides, including the C-terminal segment originally identified as antigen II, associate with covalently attached P1 to form the functional adhesive layer. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Erv1p from Saccharomyces cerevisiae is a FAD-linked sulfhydryl oxidase.

PubMed

Lee, J; Hofhaus, G; Lisowsky, T

2000-07-14

The yeast ERV1 gene encodes a small polypeptide of 189 amino acids that is essential for mitochondrial function and for the viability of the cell. In this study we report the enzymatic activity of this protein as a flavin-linked sulfhydryl oxidase catalyzing the formation of disulfide bridges. Deletion of the amino-terminal part of Erv1p shows that the enzyme activity is located in the 15 kDa carboxy-terminal domain of the protein. This fragment of Erv1p still binds FAD and catalyzes the formation of disulfide bonds but is no longer able to form dimers like the complete protein. The carboxy-terminal fragment contains a conserved CXXC motif that is present in all homologous proteins from yeast to human. Thus Erv1p represents the first FAD-linked sulfhydryl oxidase from yeast and the first of these enzymes that is involved in mitochondrial biogenesis.

Evolution of the P/Shoemaker-Levy 9 'Gang of Four' Region

NASA Technical Reports Server (NTRS)

1994-01-01

This series of eight NASA Hubble Space Telescope 'snapshots' shows the evolution of the P-Q complex, also called the 'gang of four' region, of comet P/Shoemaker-Levy 9.

The eight individual frames chronicle changes in the comet during the 12 months before colliding with Jupiter. The sequence shows that the relative separations of the various cometary fragments, thought to range in size from about 500 meters to almost 4 km (2.5 miles) across, changed dramatically over this period. The apparent separation of Q1 and Q2 was only about 1100 kilometers (680 miles) on 1 July 1993 and increased to 28,000 kilometers (17,400 miles) by 20 July 1994.

The P-Q complex demonstrates that further fragmentation occurred after the breakup of the parent body in July 1992. Fragments Q1 and Q2 were probably together at some point in a single body. However, it is not clear how P1 and P2, and the P and Q objects are related.

Between 24 January and 30 March 1994, the P2 nucleus broke-up into two separate fragments, one of which disappeared by late June. (It might be present in the mid-May image.) The P1 nucleus had a 'streaked' appearance on 24 January 1994 and then became a barely discernible 'puff' through mid-May. It was not detected in subsequent observations.

Throughout the period, most nuclei were within a 4000 kilometer-wide (2500 miles) spherical cloud of dust, called a coma. However, shortly before impact, the coma around each nucleus became highly elongated along the comet's travel path due to 'stretching' by Jupiter's rapidly increasing gravity.

This stretching is dramatic in the image of the Q-complex taken on 20 July 1994, just 10 hours before collision. Despite the coma's changes, HST images show that the core of each nucleus always remained concentrated. This shows that the nuclei were probably not catastrophically fragmenting, at least not up to 10 hours before impact.

The first HST image was taken on 1 July 1993 with the Planetary Camera before the December 1993 HST servicing mission. All other images were taken with the WFPC-2. (The image taken on 17 May 1994 was taken in 'wide-field' mode and has a lower resolution than the other WFPC-2 images). The images were taken in visible light. The different shades of red are a false-color representation of the different intensities of light reflecting off the comet's dust. Each frame covers a region 90,000 by 30,000 kilometers (56,000 by 18,600 miles).

This image and other images and data received from the Hubble Space Telescope are posted on the World Wide Web on the Space Telescope Science Institute home page at URL http://oposite.stsci.edu/pubinfo/

Catalytic domains of the LAR and CD45 protein tyrosine phosphatases from Escherichia coli expression systems: Purification and characterization for specificity and mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cho, Hyeongjin; Ramer, S.E.; Itoh, Michiyasu

1992-01-14

The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates. A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme. A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high k{sub cat}/K{sub M}more » value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of {sup 18}O from {sup 18}O{sub 4} inorganic phosphate into H{sub 2} {sup 16}O at rates of {approximately}1 {times} 10{sup {minus}2} s{sup {minus}1}. The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate. Brief incubation of all three PTPase fragments with a ({sup 32}P)phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of {sup 32}P-labeled enzymes. Pulse/chase studies on the LAR {sup 32}P-enzyme showed turnover of the labeled phosphoryl group.« less

A novel mutation of laminin β2 (LAMB2) in two siblings with renal failure.

PubMed

Falix, Farah A; Bennebroek, Carlien A M; van der Zwaag, Bert; Lapid-Gortzak, Ruth; Florquin, Sandrine; Oosterveld, Michiel J S

2017-04-01

This report describes a novel mutation of LAMB2, the gene associated with Pierson syndrome (microcoria-congenital nephrosis syndrome), in two female siblings. The c.970T>C p.(Cys324Arg) mutation in the LAMB2 gene affects one of the eight highly conserved cysteine residues within the first EGF-like module of the laminin β2 protein. These residues form disulfide bonds in order to achieve a correct 3D structure of the protein. The reported phenotype is considered a relatively mild variant of Pierson syndrome and is associated with later-onset (18 months) therapy-resistant nephrotic syndrome leading to renal failure, and ocular abnormalities consisting of high myopia, microcoria, diverse retinal abnormalities, hence a low level of visual acuity. Importantly, the reported LAMB2 mutation was associated with normal neurological development in both siblings. this report presents the variability of the renal, ocular and neurological phenotypes associated with LAMB2 mutations and underscores the importance of ophthalmologic examination in all children with unexplained renal insufficiency or nephrotic syndrome. What is known • LAMB2 mutations are associated with Pierson syndrome • Pierson syndrome is associated with congenital nephrotic syndrome, microcoria and neurological deficits What is new • A novel mutation in the LAMB2 gene in two female siblings • Genotype and clinical phenotype description of a novel LAMB2 mutation.

[Applylication of new type combined fragments: nrDNA ITS+ nad 1-intron 2 for identification of Dendrobium species of Fengdous].

PubMed

Geng, Li-xia; Zheng, Rui; Ren, Jie; Niu, Zhi-tao; Sun, Yu-long; Xue, Qing-yun; Liu, Wei; Ding, Xiao-yu

2015-08-01

In this study, 17 kinds of Dendrobium species of Fengdous including 39 individuals were collected from 4 provinces. Mitochondrial gene sequences co I, nad 5, nad 1-intron 2 and chloroplast gene sequences rbcL, matK amd psbA-trnH were amplified from these materials, as well as nrDNA ITS. Furthermore, suitable sequences for identification of Dendrobium species of Fengdous were screened by K-2-P and P-distance. The results showed that during the mentioned 7 sequences, nrDNA ITS, nad 1-intron 2 and psbA-trnH which had a high degree of variability could be used to identify Dendrobium species of Fengdous. However, single fragment could not be used to distinguish D. moniliforme and D. huoshanense. Moreover, compared to other combined fragments, new type combined fragments nrDNA ITS+nad 1-intron 2 was more effective in identifying the original plants of Dendrobium species and could be used to identify D. huoshanense and D. moniliforme. Besides, according to the UPGMA tree constructed with nrDNA ITS+nad 1-intron 2, 3 inspected Dendrobium plants were identified as D. huoshanense, D. moniliforme and D. officinale, respectively. This study identified Dendrobium species of Fengdous by combined fragments nrDNA ITS+nad 1-intron 2 for the first time, which provided a more effective basis for identification of Dendrobium species. And this study will be helpful for regulating the market of Fengdous.

Telomere Fragment Induced Amnion Cell Senescence: A Contributor to Parturition?

PubMed Central

Polettini, Jossimara; Behnia, Faranak; Taylor, Brandie D.; Saade, George R.; Taylor, Robert N.; Menon, Ramkumar

2015-01-01

Oxidative stress (OS)-induced senescence of the amniochorion has been associated with parturition at term. We investigated whether telomere fragments shed into the amniotic fluid (AF) correlated with labor status and tested if exogenous telomere fragments (T-oligos) could induce human and murine amnion cell senescence. In a cross-sectional clinical study, AF telomere fragment concentrations quantitated by a validated real-time PCR assay were higher in women in labor at term compared to those not in labor. In vitro treatment of primary human amnion epithelial cells with 40 μM T-oligos ([TTAGGG]2) that mimic telomere fragments, activated p38MAPK, produced senescence-associated (SA) β-gal staining and increased interleukin (IL)-6 and IL-8 production compared to cells treated with complementary DNA sequences (Cont-oligos, [AATCCC]2). T-oligos injected into the uteri of pregnant CD1 mice on day 14 of gestation, led to increased p38MAPK, SA-β-gal (SA β-gal) staining in murine amniotic sacs and higher AF IL-8 levels on day 18, compared to saline treated controls. In summary, term labor AF samples had higher telomere fragments than term not in labor AF. In vitro and in situ telomere fragments increased human and murine amnion p38MAPK, senescence and inflammatory cytokines. We propose that telomere fragments released from senescent fetal cells are indicative of fetal cell aging. Based on our data, these telomere fragments cause oxidative stress associated damages to the term amniotic sac and force them to release other DAMPS, which, in turn, provide a sterile immune response that may be one of the many inflammatory signals required to initiate parturition at term. PMID:26397719

Serum Liver Fibrosis Markers for Predicting the Presence of Gastroesophageal Varices in Liver Cirrhosis: A Retrospective Cross-Sectional Study

PubMed Central

Li, Hongyu; Chen, Jiang; Xia, Chunlian; Peng, Ying; Dai, Junna; Hou, Yue; Deng, Han; Li, Jing; Guo, Xiaozhong

2015-01-01

Background and Aims. A retrospective cross-sectional study was conducted to evaluate the role of hyaluronic acid (HA), laminin (LN), amino-terminal propeptide of type III procollagen (PIIINP), and collagen IV (CIV) in predicting the presence of gastroesophageal varices (GEVs) in patients with liver cirrhosis. Methods. We enrolled 118 patients with liver cirrhosis who underwent the tests for the four serum liver fibrosis markers and upper gastrointestinal endoscopy at the same admissions. The predictive values of the four serum liver fibrosis markers were evaluated by the areas under the receiving operator characteristics curves (AUROCs) with 95% confidence intervals (CIs). Results. The prevalence of GEVs was 88% (104/118). The AUROCs for HA, LN, PIIINP, and CIV levels in predicting the presence of GEVs were 0.553 (95% CI: 0.458 to 0.644, P = 0.5668), 0.490 (95% CI: 0.397 to 0.584, P = 0.9065), 0.622 (95% CI: 0.528 to 0.710, P = 0.1099), and 0.560 (95% CI: 0.466 to 0.652, P = 0.4909). The PIIINP level at a cut-off value of 31.25 had a sensitivity of 73.1% and a specificity of 57.1%. Conclusions. The present study did not recommend HA, LN, PIIINP, and CIV levels to evaluate the presence of GEVs in liver cirrhosis. PMID:26770190

Immunohistochemical and histomorphometric evaluation of vascular distribution in intact canine cranial cruciate ligament.

PubMed

Hayashi, Kei; Bhandal, Jitender; Kim, Sun Young; Rodriguez, Carlos O; Entwistle, Rachel; Naydan, Diane; Kapatkin, Amy; Stover, Susan M

2011-02-01

To (1) describe vascular distribution in the grossly intact canine cranial cruciate ligament (CCL) using immunohistochemical techniques specific to 2 components of blood vessels (factor VIII for endothelial cells, laminin for basement membrane); and (2) compare the vascularity in different areas of interest (craniomedial versus caudolateral bands; core versus epiligamentous regions; and proximal versus middle versus distal portions) in the intact normal canine CCL. In vitro study. Large, mature dogs (n=7) of breeds prone to CCL disease that were euthanatized for nonorthopedic conditions. Intact CCL were collected from fresh canine cadavers free from stifle pathology. CCL tissue was processed for immunohistochemistry and stained for factor VIII and laminin. Vascular density was determined by histomorphometric analysis. Specific vascular staining was sparsely identified throughout the CCL; however, the proximal portion of the CCL appears to have a greater number of vessels than the middle or distal portion of the ligament. The CCL is a hypovascular tissue and its vascular distribution is not homogeneous. © Copyright 2010 by The American College of Veterinary Surgeons.

Tryptic digestion of human GPIIIa. Isolation and biochemical characterization of the 23 kDa N-terminal glycopeptide carrying the antigenic determinant for a monoclonal antibody (P37) which inhibits platelet aggregation.

PubMed Central

Calvete, J J; Rivas, G; Maruri, M; Alvarez, M V; McGregor, J L; Hew, C L; Gonzalez-Rodriguez, J

1988-01-01

Early digestion of pure human platelet glycoprotein IIIa (GPIIIa) leads to a single cleavage of the molecule at 23 kDa far from one of the terminal amino acids. Automated Edman degradation demonstrates that GPIIIa and the smaller (23 kDa) tryptic fragment share the same N-terminal amino acid sequence. A further cleavage occurs in the larger fragment (80 kDa), reducing its apparent molecular mass by 10 kDa. The 23 kDa fragment remains attached to the larger ones in unreduced samples. Stepwise reduction of early digested GPIIIa with dithioerythritol selectively reduces the single disulphide bond joining the smaller (23 kDa) to the larger (80/70 kDa) fragments. Two fractions were obtained by size-exclusion chromatography of early digested GPIIIa after partial or full reduction and alkylation. The larger-size fraction contains the 80/70 kDa fragments, while the 23 kDa fragment is isolated in the smaller. The amino acid compositions of these fractions do not differ very significantly from the composition of GPIIIa; however the 23 kDa fragment contains only 10.2% by weight of sugars and is richer in neuraminic acid. Disulphide bonds are distributed four in the 23 kDa glycopeptide and 20-21 in the 80/70 kDa glycopeptide. The epitope for P37, a monoclonal antibody which inhibits platelet aggregation [Melero & González-Rodríguez (1984) Eur. J. Biochem. 141, 421-427] is situated within the first 17 kDa of the N-terminal region of GPIIIa, which gives a special functional interest to this extracellular region of GPIIIa. On the other hand, the epitopes for GPIIIa-specific monoclonal antibodies, P6, P35, P40 and P97, which do not interfere with platelet aggregation, are located within the larger tryptic fragment (80/70 kDa). Thus, the antigenic areas available in the extracellular surface of GPIIIa for these five monoclonal antibodies are now more precisely delineated. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2455507

The Split Nucleus of Comet Wilson (C/1986 P1 = 1987 VII).

NASA Astrophysics Data System (ADS)

Meech, Karen J.; Knopp, Graham P.; Farnham, Tony L.; Green, Daniel

1995-07-01

We present CCD observations of Comet Wilson (C/1986 P1 = 1987 VII) from 26 nights during the time period 1986 October to 1991 February, which brackets perihelion, During the observing run of 1988 February, the comet was observed to have split into two fragments. Our broadband CCD photometry, along with photometry from the International Cometary Quarterly, shows a steady decline in brightness of Comet Wilson post-perihelion, with an outburst between heliocentric distances r = 2.8 and 3.3 AU during 1987 October and November. By r ≈ 7 AU, the fragment had faded with respect to the parent and was no longer centrally condensed. A brightness limit of mR ≈ 25, when the comet was at r = 12.65 AU, constrains the primary nucleus to have a maximum radius between 5 and 7 km, assuming an albedo of 0.04. The accuracy of direct orbital solutions for the parent body and fragment to determine the time of splitting was limited by the presence of significant nongravitational forces and the limited fragment orbital coverage. We used the relative position of the fragment with respect to the parent to calculate a time of splitting which was consistent with the time of the observed outburst. We discuss the possible causes of the splitting. The coma of Comet Wilson was observed to have a surface-brightness profile which fell off as p-1 (characteristic of a canonical steady-state coma under the influence of radiation pressure) for all of the data with the exception of the data taken during 1987 November when the gradient was p-1.3 . This steeper slope was probably caused by the injection of new material into the coma during the outburst. During 1986 October, there was a break in the surface-brightness profile slope which may be interpreted as the distance at which grains are swept into the tail. The profiles suggested grain velocities of a few x 10 2 to 10 m sec -1 for grains between 1 and a few hundred micrometers. Finson-Probstein dust modeling showed that ejection of grains began preperihelion at r gt; 6 AU, peaking near perihelion with a very rapid decline thereafter, with an outburst occurring roughly 170 days post-perihelion. We found no evidence for the existence of discrete features in the coma. Both the primary and fragment nuclei grew redder with time, with the fragment being redder than the parent, consistent with the picture of smaller grains leaving the field of view of the aperture more quickly than larger grains.

Potential Energy Surfaces and Dynamics for Energetic Ionic Liquids

DTIC Science & Technology

2012-04-09

advantage of such architectures12. Very recently, we have implemented the FMO method on the BG/ P system at Argonne National Laboratory, demonstrating that...Molecular Orbital Method”, J. Comp. Theoret. Chem., 6, 1 (2010). 4. T. Nagata, D . Fedorov, K. Kitaura, and M.S. Gordon, “A Combined Effective Fragment...Chem., 3, 177 (2007). 6. T. Nagata, D . Fedorov, K. Kitaura, and M.S. Gordon, “A Combined Effective Fragment Potential - Fragment Molecular Orbital

Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV.

PubMed

Vieira, Monica L; de Morais, Zenaide M; Gonçales, Amane P; Romero, Eliete C; Vasconcellos, Silvio A; Nascimento, Ana L T O

2010-01-01

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease that affects populations worldwide. We have identified in proteomic studies a protein that is encoded by the gene LIC10314 and expressed in virulent strain of L. interrogans serovar Pomona. This protein was predicted to be surface exposed by PSORT program and contains a p83/100 domain identified by BLAST analysis that is conserved in protein antigens of several strains of Borrelia and Treponema spp. The proteins containing this domain have been claimed antigen candidates for serodiagnosis of Lyme borreliosis. Thus, we have cloned the LIC10314 and expressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. This protein is conserved among several species of pathogenic Leptospira and absent in the saprophytic strain L. biflexa. We confirm by liquid-phase immunofluorescence assays with living organisms that this protein is most likely a new surface leptospiral protein. The ability of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC10314, named Lsa63 (Leptospiral surface adhesin of 63kDa), binds strongly to laminin and collagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed during infection since it was recognized by antibodies of serum samples of confirmed-leptospirosis patients in convalescent phase of the disease. Altogether, the data suggests that this novel identified surface protein may be involved in leptospiral pathogenesis. 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor and effectively alleviates acute lung injury induced by H9N2 swine influenza virus.

PubMed

Xu, Ming-Ju; Liu, Bao-Jian; Wang, Cun-Lian; Wang, Guo-Hua; Tian, Yong; Wang, Shao-Hua; Li, Jun; Li, Pei-Yao; Zhang, Rui-Hua; Wei, Dong; Tian, Shu-Fei; Xu, Tong

2017-11-01

Epigallocatechin-3-gallate (EGCG) was found to inhibit the Toll-like receptor 4 (TLR4) pathway involved in influenza virus pathogenesis. Here, the effect of EGCG on TLR4 in an H9N2 virus-induced acute lung injury mouse model was investigated. BALB/c mice were inoculated intranasally with A/Swine/Hebei/108/2002 (H9N2) virus or noninfectious allantoic fluid, and treated with EGCG and E5564 or normal saline orally for 5 consecutive days. PMVECs were treated with EGCG or anti-67kDa laminin receptor (LR). Lung physiopathology, inflammation, oxidative stress, viral replication, and TLR4/NF-κB/Toll-interacting protein (Tollip) pathway in lung tissue and/or PMVECs were investigated. EGCG attenuated lung histological lesions, decreased lung W/D ratio, cytokines levels, and inhibited MPO activity and prolonged mouse survival. EGCG treatment also markedly downregulated TLR4 and NF-κB protein levels but Tollip expression was upregulated compared with that in untreated H9N2-infected mice (P<0.05). In PMVECs, anti-67LR antibody treatment significantly downregulated Tollip levels; however, the TLR4 and NF-κB protein levels dramatically increased compared with that in the EGCG-treated group (P<0.05). EGCG remarkably downregulated TLR4 protein levels through 67LR/Tollip, decreased MPO activity and inflammatory cytokine levels, supporting EGCG as a potential therapeutic agent for managing acute lung injury induced by H9N2 SIV. Copyright © 2017 Elsevier B.V. All rights reserved.

Single Protein Structural Analysis with a Solid-state Nanopore Sensor

NASA Astrophysics Data System (ADS)

Li, Jiali; Golovchenko, Jene; McNabb, David

2005-03-01

We report on the use of solid-state nanopore sensors to detect single polypeptides. These solid-state nanopores are fabricated in thin membranes of silicon nitride by ion beam sculpting...[1]. When an electrically biased nanopore is exposed to denatured proteins in ionic solution, discrete transient electronic signals: current blockages are observed. We demonstrate examples of such transient electronic signals for Bovine Serum Albumin (BSA) and human placental laminin M proteins in Guanidine hydrochloride solution, which suggest that these polypeptides are individually translocating through the nanopore during the detecting process. The amplitude of the current blockages is proportional to the bias voltage. No transient current blockages are observed when proteins are not present in the solution. To probe protein-folding state, pH and temperature dependence experiments are performed. The results demonstrate a solid-state nanopore sensor can be used to detect and analyze single polypeptide chains. Similarities and differences with signals obtained from double stranded DNA in a solid-state nanopore and single stranded DNA in a biological nanopore are discussed. [.1] Li, J., D. Stein, C. McMullan, D. Branton, M.J. Aziz, and J.A. Golovchenko, Ion-beam sculpting at nanometre length scales. Nature, 2001. 412(12 July): p. 166-169.

Kinetic Energy Release of the Singly and Doubly Charged Methylene Chloride Molecule: The Role of Fast Dissociation.

PubMed

Alcantara, K F; Rocha, A B; Gomes, A H A; Wolff, W; Sigaud, L; Santos, A C F

2016-09-01

The center of mass kinetic energy release distribution (KERD) spectra of selected ionic fragments, formed through dissociative single and double photoionization of CH2Cl2 at photon energies around the Cl 2p edge, were extracted from the shape and width of the experimentally obtained time-of-flight (TOF) distributions. The KERD spectra exhibit either smooth profiles or structures, depending on the moiety and photon energy. In general, the heavier the ionic fragments, the lower their average KERDs are. In contrast, the light H(+) fragments are observed with kinetic energies centered around 4.5-5.5 eV, depending on the photon energy. It was observed that the change in the photon energy involves a change in the KERDs, indicating different processes or transitions taking place in the breakup process. In the particular case of double ionization with the ejection of two charged fragments, the KERDs present have characteristics compatible with the Coulombic fragmentation model. Intending to interpret the experimental data, singlet and triplet states at Cl 2p edge of the CH2Cl2 molecule, corresponding to the Cl (2p → 10a1*) and Cl (2p → 4b1*) transitions, were calculated at multiconfigurational self-consistent field (MCSCF) level and multireference configuration interaction (MRCI). These states were selected to form the spin-orbit coupling matrix elements, which after diagonalization result in a spin-orbit manifold. Minimum energy pathways for dissociation of the molecule were additionally calculated aiming to give support to the presence of the ultrafast dissociation mechanism in the molecular breakup.

Construction of a food-grade cloning vector for Lactobacillus plantarum and its utilization in a food model.

PubMed

Rattanachaikunsopon, Pongsak; Phumkhachorn, Parichat

2012-01-01

The development of Lactobacillus plantarum to be used in starter cultures in the food industry has been limited because of the lack of a food-grade cloning vector for the bacterium. In this study, the plasmid pFLP1 was constructed by joining 2 DNA fragments derived from food-approved organisms. The 5.2-kb BamHI/KpnI DNA fragment of pRV566 containing the theta-type replicon of Lactobacillus sakei was ligated to the BamHI/KpnI DNA fragment of a 2.9-kb lactococcal cadmium resistance determinant amplified from pND918. The 8.1-kb newly constructed plasmid could transform L. plantarum N014, a bacteriocin-producing bacteria originally isolated from nham, a traditional Thai fermented sausage. The resulting transformant, L. plantarum N014-FLP, and its parent strain were shown to be very similar in growth rate and bacteriocin activity. In addition, the plasmid was very stable in its host bacteria under nonselective pressure for 100 generations in MRS medium and for 5 days in a nham model. These results suggest that pFLP1 is a potential food-grade cloning vector for L. plantarum.

Targeting human prostate cancer with 111In-labeled D2B IgG, F(ab')2 and Fab fragments in nude mice with PSMA-expressing xenografts.

PubMed

Lütje, Susanne; van Rij, Catharina M; Franssen, Gerben M; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J; Colombatti, Marco; Boerman, Otto C

2015-01-01

D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing subcutaneous prostate cancer xenografts. The optimal time point for imaging was determined in biodistribution and microSPECT imaging studies with (111)In-D2B IgG, (111)In-capromab pendetide, (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments in mice with PSMA-expressing LNCaP and PSMA-negative PC3 tumors at several time points after injection. All (111)In-labeled antibody formats specifically accumulated in the LNCaP tumors, with highest uptake of (111)In-D2B IgG and (111)In-capromab pendetide at 168 h p.i. (94.8 ± 19.2% injected dose per gram (ID/g) and 16.7 ± 2.2% ID/g, respectively), whereas uptake of (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments peaked at 24 h p.i. (12.1 ± 3.0% ID/g and 15.1 ± 2.9% ID/g, respectively). Maximum LNCaP tumor-to-blood ratios were 13.0 ± 2.3 (168 h p.i.), 6.2 ± 0.7 (24 h p.i.), 23.0 ± 4.0 (24 h p.i.) and 4.5 ± 0.6 (168 h p.i.) for (111)In-D2B IgG, (111)In-F(ab')2, (111)In-Fab and (111)In-capromab pendetide, respectively. LNCaP tumors were clearly visualized with microSPECT with all antibody formats. This study demonstrates the feasibility of D2B IgG, F(ab')2 and Fab fragments for targeting PSMA-expressing prostate cancer xenografts. Copyright © 2014 John Wiley & Sons, Ltd.

Effect of fragmentary displacement and morphology in the treatment of comminuted femoral shaft fractures with an intramedullary nail.

PubMed

Lin, Shih-Jie; Chen, Chi-Lung; Peng, Kuo-Ti; Hsu, Wei-Hsiu

2014-04-01

Our study aimed to determine whether the displacement and morphology of a fragment in femur fracture with Arbeitsgemeinschaft für Osteosynthesefragen/Orthopaedic Trauma Association/32-B/32-C (AO/OTA/32-B/32-C) classification affect the outcomes following closed reduction and internal fixation with an interlocking nail. This was a retrospective study. The study was conducted at a Level III trauma centre. A total of 50 consecutive patients presenting femoral shaft fracture with AO/OTA-type 32-B/32-C were included in the present study. Patients were divided into two groups according to the displacement of the fragments. In the large displacement group, patients were further subgrouped according to whether a reversed morphology of the fragment was present. The radiographic union score of femur (RUSF), the mean union time and the re-operation rate were assessed. The union rate of small- and large-gap groups at 12 months postoperatively was 75.9% and 21.1%, respectively (p=0.000). The mean union time of those union cases in these two groups was 7.8 and 13.0 months, respectively (p=0.000). The union rate of the non-reversed and reversed groups at 12 months postoperatively was 30% and 11.1%, respectively (p=0.179). The mean RUSF at 12 months in the non-reversed and reversed groups was 8.8 and 8.3, respectively (p=0.590). However, we found that patients presenting a reversed fragment had an increased risk of more than one re-operation (p=0.030). A fragmentary displacement of >1cm in AO/OTA-type 32-B/32-C femoral shaft fracture after nailing affected bone healing. Among the large-gap group patients, an unreduced reverse fragment presented a negative prognostic factor for re-operation. Prognostic level III. Copyright © 2013 Elsevier Ltd. All rights reserved.

Fluid viscosity affects the fragmentation and inertial cavitation threshold of lipid encapsulated microbubbles

PubMed Central

Helfield, Brandon; Black, John J.; Qin, Bin; Pacella, John; Chen, Xucai; Villanueva, Flordeliza S.

2015-01-01

Ultrasound and microbubble optimization studies for therapeutic applications are often conducted in water/saline, with a fluid viscosity of 1 cP. In an in vivo context, microbubbles are situated in blood, a more viscous fluid (~4 cP). In this study, ultra-high speed microscopy and passive cavitation approaches were employed to investigate the effect of fluid viscosity on microbubble behavior at 1 MHz subject to high pressures (0.25 – 2 MPa). The propensity for individual microbubble (n=220) fragmentation was shown to significantly decrease in 4 cP fluid as compared to 1 cP fluid, despite achieving similar maximum radial excursions. Microbubble populations diluted in 4 cP fluid exhibited decreased wideband emissions (up to 10.2 times), and increasingly distinct harmonic emission peaks (e.g. ultraharmonic) with increasing pressure as compared to 1 cP fluid. These results suggest that in vitro studies should consider an evaluation using physiologic viscosity perfusate before transitioning to in vivo evaluations. PMID:26674676

Slc15a1 is involved in the transport of synthetic F5-peptide into the seminiferous epithelium in adult rat testes

PubMed Central

Su, Linlin; Zhang, Yufei; Cheng, Yan C.; Lee, Will M.; Ye, Keping; Hu, Dahai

2015-01-01

Spermiation and BTB restructuring, two critical cellular events that occur across seminiferous epithelium in mammalian testis during spermatogenesis, are tightly coordinated by biologically active peptides released from laminin chains. Our earlier study reported that F5-peptide, synthesized based on a stretch of 50 amino acids within laminin-γ3 domain IV, could reversibly induce the impairment of spermatogenesis, disruption of BTB integrity, and germ cell loss, and thus is a promising male contraceptive. However, how F5-peptide when administered intratesticularly enters seminiferous tubules and exerts effects beyond BTB is currently unknown. Here we demonstrated that Slc15a1, a peptide transporter also known as Pept1, was predominantly present in peritubular myoid cells, interstitial Leydig cells, vascular endothelial cells and germ cells, while absent in Sertoli cells or BTB site. The steady-state protein level of Slc15a1 in adult rat testis was not affected by F5-peptide treatment. Knockdown of Slc15a1 by in vivo RNAi in rat testis was shown to prevent F5-peptide induced disruptive effects on spermatogenesis. This study suggests that Slc15a1 is involved in the transport of synthetic F5-peptide into seminiferous epithelium, and thus Slc15a1 is a novel target in testis that could be genetically modified to improve the bioavailability of F5-peptide as a prospective male contraceptive. PMID:26537751

Slc15a1 is involved in the transport of synthetic F5-peptide into the seminiferous epithelium in adult rat testes.

PubMed

Su, Linlin; Zhang, Yufei; Cheng, Yan C; Lee, Will M; Ye, Keping; Hu, Dahai

2015-11-05

Spermiation and BTB restructuring, two critical cellular events that occur across seminiferous epithelium in mammalian testis during spermatogenesis, are tightly coordinated by biologically active peptides released from laminin chains. Our earlier study reported that F5-peptide, synthesized based on a stretch of 50 amino acids within laminin-γ3 domain IV, could reversibly induce the impairment of spermatogenesis, disruption of BTB integrity, and germ cell loss, and thus is a promising male contraceptive. However, how F5-peptide when administered intratesticularly enters seminiferous tubules and exerts effects beyond BTB is currently unknown. Here we demonstrated that Slc15a1, a peptide transporter also known as Pept1, was predominantly present in peritubular myoid cells, interstitial Leydig cells, vascular endothelial cells and germ cells, while absent in Sertoli cells or BTB site. The steady-state protein level of Slc15a1 in adult rat testis was not affected by F5-peptide treatment. Knockdown of Slc15a1 by in vivo RNAi in rat testis was shown to prevent F5-peptide induced disruptive effects on spermatogenesis. This study suggests that Slc15a1 is involved in the transport of synthetic F5-peptide into seminiferous epithelium, and thus Slc15a1 is a novel target in testis that could be genetically modified to improve the bioavailability of F5-peptide as a prospective male contraceptive.

Sleep fragmentation and sleep-disordered breathing in individuals living close to main roads: results from a population-based study.

PubMed

Gerbase, M W; Dratva, J; Germond, M; Tschopp, J M; Pépin, J L; Carballo, D; Künzli, N; Probst-Hensch, N M; Adam, M; Zemp Stutz, E; Roche, F; Rochat, T

2014-03-01

Nighttime traffic noise is associated with sleep disturbances, but sleep fragmentation and sleep-disordered breathing (SDB) have not been demonstrated in individuals living near busy roads. We asked 1383 participants to answer a health questionnaire and to undergo 24-h electrocardiogram (ECG). Nocturnal ECG records were used to calculate the very low frequency index (VLFI) interval, a surrogate marker of sleep fragmentation. Distances of participants' addresses to roadways were calculated using the VECTOR25© Swisstopo roads classification, a traffic noise proxy. Distances of homes within 100 or 50 m of major roads defined proximity to busy roads. Adjusted multivariate logistic regressions analyzed associations between the distance of home to main roads and VLFI or self-reported SDB. Distance of participants' homes to main roads was significantly associated with the VLFI in women (odds ratio [OR], 1.58 [confidence interval {CI}, 1.03-2.42]; P = .038) but not in men (OR, 1.35 [CI, 0.77-2.35]; P = .295). Women under hormonal replacement therapy (HRT) were at higher risk for increased VLFI when living close to main roads (OR, 2.10 [CI, 1.20-3.68]; P = .01) than untreated women (P = .584). Associations with self-reported SDB were not statistically relevant. In our large population, women living close to main roads were at significantly higher risk for sleep fragmentation than men. The 2-fold higher risk for menopausal women under HRT underscores the vulnerability of this group. Copyright © 2014 Elsevier B.V. All rights reserved.

Immunoscintigraphy of human pancreatic carcinoma in nude mice with I-131-F(ab')/sub 2/-fragments of monoclonal antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Senekowitsch, R.; Maul, F.D.; Wenisch, H.J.C.

1985-05-01

In the present study radioiodinated F(ab')/sub 2/-fragments of CA19-9 and antibody that reacts specifically with human gastrointestinal cancer were examined for their ability to detect human pancreatic carcinoma hosted in nude mice. Tumor-bearing mice received 80..mu..Ci of I-131-F(ab')/sub 2/ with a specific activity of 1.8..mu..Ci/..mu..g. All mice were imaged after the injection and every 24hr up to 6 days. The retained radioactivity was also registered with a whole-body counter immediately after imaging. As a control F(ab's)/sub 2/ of a nonspecific antibody were administered in parallel to another group of animals bearing the same tumor. Three animals of each group weremore » killed at 1,2,4 and 8 days for determination of the distribution of both labeled antibody-fragments. On scintigraphic images obtained with the CA19-9-F(ab')/sub 2/ the tumors could be visualized 24hr after injection, the best dilineation however was achieved 96hr p.i.. The biodistribution data exhibited a more rapid blood clearance for the specific fragments compared to that for the unspecific ones. Tumors showed an increase in uptake up to 48hr reaching 1.7% of the injected dose per gram, declining to values of 0.08%/g at day 6 p.i.. The highest tumor-to-blood ratios were found after 96h. They were 7 for the CA19-9-fragments compared to 1.5 for the unspecific fragments. The whole body counting revealed a more rapid excretion for the fragments of the specific monoclonal antibodies than for the unspecific ones. In summary the authors were able to show that CA19-9-F(ab')/sub 2/-fragments can be used for immunodetection of human pancreatic carcinoma hosted in nude mice.« less

Analysis of ligand-receptor cross-linked fragments by mass spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Son, C.D.; Sargsyan, H.; Hurst, Gregory

G-protein coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7-TM) configuration. The a-factor receptor (Ste2p) from Saccharomyces cerevisiae is a GPCR that, upon binding of a peptide ligand, transduces a signal to initiate a cascade of events leading to the mating of haploid yeast cells. This study summarizes the application of affinity purification and of matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) experiments using biotinylated photoactivatable a-factor analogs. Affinity purification and enrichment of biotinylated peptides by monomeric avidin beads resulted in mass spectrometric detection of specific signals corresponding to crosslinked fragments ofmore » Ste2p. Data obtained from cyanogen bromide (CNBr) fragments of receptor cross-linked to an a-factor analog with the photoaffinity group p-benzoyl-L-phenylalanine on position 1 were in agreement with the previous results reported by our laboratory suggesting the cross-linking between position 1 of a-factor and a region of Ste2p covering residues 251 294.« less

Food composition and feeding habits of some fresh water fishes in various water systems at Abbassa, Egypt, with special reference to snails transmitting diseases.

PubMed

El Gamal, Abd El-Rahman A; Ismail, Nahed M M

2005-08-01

Study of feeding habits of freshwater fishes collected from ponds at World Fish Center (ICLARM) showed that the African catfish, Clarias gariepinus and Forskal catfish, Bagras bayad had the highest proportion of full stomachs (31-58% & 44-45% respectively). In cichlid fishes, the rate of full stomachs was much lower, being 0.0-12.5% and showed higher incidence of empty stomachs that varied from 37.5% for Oreochromis niloticus to 78.3% for Sarotherodon galilaeus. Food items were analyzed by the percentage of point assessment (P%), abundance (N%) and frequency of occurrence (F%). Results of the three methods of analyses (Index of relative importance, I.R.I) emphasized the importance of plants (1214.7) as a major food resource in the stomach of Nile tilapia, O. niloticus followed by shell fragments (628.5), whereas, snail soft bodies were the main food category in the diet of hybrid tilapia O. niloticus x O. aureus (2539.3). Shell fragments (652) and snail soft bodies (296.9) were the 1st in relative importance as foods of O. aurea. In case of S. galillae, shell fragments (338) came 2nd in I.R.I. after plants (559). Present investigation shows that shell fragments were represented by 11.1% and 15.1% in the diet of African catfish, C. gariepinus by (N%) and (P%) methods, however, they came as the second food item in its diet by I.R.I (1237.3). According to F% method, both shell fragments and Crustacea were present in the diet of C. gariepinus considerable proportions each of 47.4%. Shell fragments were represented by low proportions in the diet of B. bayad 3.9, 2.1 and 22.2 by N%, P% and F% respectively.

Cytoplasmic transfer of heritable elements other than mtDNA from SAMP1 mice into mouse tumor cells suppresses their ability to form tumors in C57BL6 mice.

PubMed

Shimizu, Akinori; Tani, Haruna; Takibuchi, Gaku; Ishikawa, Kaori; Sakurazawa, Ryota; Inoue, Takafumi; Hashimoto, Tetsuo; Nakada, Kazuto; Takenaga, Keizo; Hayashi, Jun-Ichi

2017-11-04

In a previous study, we generated transmitochondrial P29mtSAMP1 cybrids, which had nuclear DNA from the C57BL6 (referred to as B6) mouse strain-derived P29 tumor cells and mitochondrial DNA (mtDNA) exogenously-transferred from the allogeneic strain SAMP1. Because P29mtSAMP1 cybrids did not form tumors in syngeneic B6 mice, we proposed that allogeneic SAMP1 mtDNA suppressed tumor formation of P29mtSAMP1 cybrids. To test this hypothesis, current study generated P29mt(sp)B6 cybrids carrying all genomes (nuclear DNA and mtDNA) from syngeneic B6 mice by eliminating SAMP1 mtDNA from P29mtSAMP1 cybrids and reintroducing B6 mtDNA. However, the P29mt(sp)B6 cybrids did not form tumors in B6 mice, even though they had no SAMP1 mtDNA, suggesting that SAMP1 mtDNA is not involved in tumor suppression. Then, we examined another possibility of whether SAMP1 mtDNA fragments potentially integrated into the nuclear DNA of P29mtSAMP1 cybrids are responsible for tumor suppression. We generated P29 H (sp)B6 cybrids by eliminating nuclear DNA from P29mt(sp)B6 cybrids and reintroducing nuclear DNA with no integrated SAMP1 mtDNA fragment from mtDNA-less P29 cells resistant to hygromycin in selection medium containing hygromycin. However, the P29 H (sp)B6 cybrids did not form tumors in B6 mice, even though they carried neither SAMP1 mtDNA nor nuclear DNA with integrated SAMP1 mtDNA fragments. Moreover, overproduction of reactive oxygen species (ROS) and bacterial infection were not involved in tumor suppression. These observations suggest that tumor suppression was caused not by mtDNA with polymorphic mutations or infection of cytozoic bacteria but by hypothetical heritable cytoplasmic elements other than mtDNA from SAMP1 mice. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The Guanine Nucleotide Exchange Factor Tiam1 Affects Neuronal Morphology; Opposing Roles for the Small GTPases Rac and Rho

PubMed Central

van Leeuwen, Frank N.; Kain, Hendrie E.T.; van der Kammen, Rob A.; Michiels, Frits; Kranenburg, Onno W.; Collard, John G.

1997-01-01

The invasion-inducing T-lymphoma invasion and metastasis 1 (Tiam1) protein functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Here we show that overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the α6β1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid– induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process. PMID:9348295

A local autocrine axis in the testes that regulates spermatogenesis

PubMed Central

Cheng, C. Yan; Mruk, Dolores D.

2014-01-01

Spermiation—the release of mature spermatozoa from Sertoli cells into the seminiferous tubule lumen—occurs by the disruption of an anchoring device known as the apical ectoplasmic specialization (apical ES). At the same time, the blood–testis barrier (BTB) undergoes extensive restructuring to facilitate the transit of preleptotene spermatocytes. While these two cellular events take place at opposite ends of the Sertoli cell epithelium, the events are in fact tightly coordinated, as any disruption in either process will lead to infertility. A local regulatory axis exists between the apical ES and the BTB in which biologically active laminin fragments produced at the apical ES by the action of matrix metalloproteinase 2 can regulate BTB restructuring directly or indirectly via the hemidesmosome. Equally important, polarity proteins play a crucial part in coordinating cellular events within this apical ES–BTB–hemidesmosome axis. Additionally, testosterone and cytokines work in concert to facilitate BTB restructuring, which enables the transit of spermatocytes while maintaining immunological barrier function. Herein, we will discuss this important autocrine-based cellular axis that parallels the hormonal-based hypothalamic–pituitary–testicular axis that regulates spermatogenesis. This local regulatory axis is the emerging target for male contraception. PMID:20571538

Development of oral CTL vaccine using a CTP-integrated Sabin 1 poliovirus-based vector system.

PubMed

Han, Seung-Soo; Lee, Jinjoo; Jung, Yideul; Kang, Myeong-Ho; Hong, Jung-Hyub; Cha, Min-Suk; Park, Yu-Jin; Lee, Ezra; Yoon, Cheol-Hee; Bae, Yong-Soo

2015-09-11

We developed a CTL vaccine vector by modification of the RPS-Vax system, a mucosal vaccine vector derived from a poliovirus Sabin 1 strain, and generated an oral CTL vaccine against HIV-1. A DNA fragment encoding a cytoplasmic transduction peptide (CTP) was integrated into the RPS-Vax system to generate RPS-CTP, a CTL vaccine vector. An HIV-1 p24 cDNA fragment was introduced into the RPS-CTP vector system and a recombinant poliovirus (rec-PV) named vRPS-CTP/p24 was produced. vRPS-CTP/p24 was genetically stable and efficiently induced Th1 immunity and p24-specific CTLs in immunized poliovirus receptor-transgenic (PVR-Tg) mice. In challenge experiments, PVR-Tg mice that were pre-immunized orally with vRPS-CTP/p24 were resistant to challenge with a lethal dose of p24-expressing recombinant vaccinia virus (rMVA-p24). These results suggested that the RPS-CTP vector system had potential for developing oral CTL vaccines against infectious diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

The pPSU Plasmids for Generating DNA Molecular Weight Markers.

PubMed

Henrici, Ryan C; Pecen, Turner J; Johnston, James L; Tan, Song

2017-05-26

Visualizing nucleic acids by gel electrophoresis is one of the most common techniques in molecular biology, and reference molecular weight markers or ladders are commonly used for size estimation. We have created the pPSU1 & pPSU2 pair of molecular weight marker plasmids which produce both 100 bp and 1 kb DNA ladders when digested with two common restriction enzymes. The 100 bp ladder fragments have been optimized to migrate appropriately on both agarose and native polyacrylamide, unlike many currently available DNA ladders. Sufficient plasmid DNA can be isolated from 100 ml E. coli cultures for the two plasmids to produce 100 bp or 1 kb ladders for 1000 gels. As such, the pPSU1 and pPSU2 plasmids provide reference fragments from 50 to 10000 bp at a fraction of the cost of commercial DNA ladders. The pPSU1 and pPSU2 plasmids are available without licensing restrictions to nonprofit academic users, affording freely available high-quality, low-cost molecular weight standards for molecular biology applications.

A High Proliferation Rate is Critical for Reproducible and Standardized Embryoid Body Formation from Laminin-521-Based Human Pluripotent Stem Cell Cultures.

PubMed

Dziedzicka, Dominika; Markouli, Christina; Barbé, Lise; Spits, Claudia; Sermon, Karen; Geens, Mieke

2016-12-01

When aiming for homogenous embryoid body (EB) differentiation, the use of equal-sized EBs is required to avoid a size-induced differentiation bias. In this study we developed an efficient and standardized EB formation protocol for human pluripotent stem cells (hPSC) cultured in a laminin-521-based xeno-free system. As the cell proliferation rate of the cells growing on laminin-521 strongly affected the efficiency of aggregate formation, we found that recently passaged cells, as well as the addition of ROCK inhibitor, were essential for reproducible EB formation from hPSC single-cell suspensions. EBs could be obtained in a variety of differentiation media, in 96-well round-bottom plates and in hanging drops. Gene expression studies on differentially sized EBs from three individual human embryonic stem cell lines demonstrated that the medium used for differentiation influenced the differentiation outcome to a much greater extent than the number of cells used for the initial EB formation. Our findings give a new insight into factors that influence the EB formation and differentiation process. This optimized method allows us to easily manipulate EB formation and provide an excellent starting point for downstream EB-based differentiation protocols.

pLS101 plasmid vector

DOEpatents

Lacks, S.A.; Balganesh, T.S.

1985-02-19

Disclosed is recombinant plasmid pLS101, consisting essentially of a 2.0 Kb ma1M gene fragment ligated to a 4.4 Kb Tcr DNA fragment, which is particularly useful for transforming Gram-positive bacteria. This plasmid contains at least four restriction sites suitable for inserting exogeneous gene sequences. Also disclosed is a method for plasmid isolation by penicillin selection, as well as processes for enrichment of recombinant plasmids in Gram-positive bacterial systems. 5 figs., 2 tabs.

Distribution of Oxytetracycline Resistance Plasmids between Aeromonads in Hospital and Aquaculture Environments: Implication of Tn1721 in Dissemination of the Tetracycline Resistance Determinant Tet A

PubMed Central

Rhodes, Glenn; Huys, Geert; Swings, Jean; Mcgann, Patrick; Hiney, Maura; Smith, Peter; Pickup, Roger W.

2000-01-01

Oxytetracycline-resistant (OTr) mesophilic aeromonads were recovered from untreated hospital effluent (72 isolates) and from fish farm hatchery tanks (91 isolates) at sites within the English Lake District, Cumbria, England. The transfer of OTr plasmids from these isolates was investigated. Using Escherichia coli J53-1 as a recipient, 11 isolates from the hospital site and 6 isolates from the fish farm site transferred OTr plasmids (designated pFBAOT1 to 17). Original isolates were identified using fatty acid methyl ester and fluorescent amplified fragment length polymorphism comparisons as either Aeromonas hydrophila HG3 (eight isolates), A. veronii b.v. sobria HG8 (six isolates), and A. caviae HGB5 (one isolate). One isolate remained unidentified, and one could not be assigned a taxonomic designation beyond the genus level. Plasmids pFBAOT1 to -17 were screened for the presence of the tetracycline resistance determinants Tet A to E and Tet G. Only determinant Tet A (10 plasmids) was detected in these plasmids, with 7 tet gene determinants remaining unclassified. In all cases, Tet A was located on a 5.5-kb EcoRI restriction fragment. Hybridization with inc-rep probes N, P, Q, W, and U showed pFBAOT3, -4, -5, -6, -7, -9, and -11, from the hospital environment, to be IncU plasmids. Further, restriction fragment length polymorphism (RFLP) analyses and DNA probing demonstrated that pFBAOT plasmids were closely related to IncU OTr plasmids pASOT, pASOT2, pASOT3, pRAS1 (originally isolated from A. salmonicida strains from fish farms in Scotland and Norway, respectively), and pIE420 (isolated from a German hospital E. coli strain). In addition, DNA analyses demonstrated that plasmids pRAS1 and pIE420 had identical RFLP profiles and that all fragments hybridized to each other. The presence of tetracycline resistance transposon Tn1721 in its entirety or in a truncated form in these plasmids was demonstrated. These results provided direct evidence that related tetracycline resistance-encoding plasmids have disseminated between different Aeromonas species and E. coli and between the human and aquaculture environments in distinct geographical locations. Collectively, these findings provide evidence to support the hypothesis that the aquaculture and human compartments of the environment behave as a single interactive compartment. PMID:10966404

Creating Novel Activated Factor XI Inhibitors through Fragment Based Lead Generation and Structure Aided Drug Design

PubMed Central

Fjellström, Ola; Akkaya, Sibel; Beisel, Hans-Georg; Eriksson, Per-Olof; Erixon, Karl; Gustafsson, David; Jurva, Ulrik; Kang, Daiwu; Karis, David; Knecht, Wolfgang; Nerme, Viveca; Nilsson, Ingemar; Olsson, Thomas; Redzic, Alma; Roth, Robert; Sandmark, Jenny; Tigerström, Anna; Öster, Linda

2015-01-01

Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrinolytic effects with a low bleeding risk. This motivated a structure aided fragment based lead generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on docking experiments, was performed to generate a FXIa targeted fragment library for an NMR screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket. The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine structures are novel FXIa P1 fragments. The expansion of these fragments towards the FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa inhibitors that we found to bind in the S1-S1’-S2’ FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1’-S2’ binding reference compounds enabled structure guided linking and expansion work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC50 of 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1’-S2’ binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds. PMID:25629509

Transcription Factor Stat5, A Novel Therapeutic Protein, Inhibits Metastatic Potential and Invasive Characteristics of Human Breast Cancer Cells

DTIC Science & Technology

2004-10-01

digestion and cloned into pLoxpNeo upstream of the PGK-neomycin cassette. A 1.3 kb fragment with the first coding exon was amplified by Pfx polymerase ...introducing a XhoI site on the 5’-end of the amplification product. The PCR fragment was cloned blunt into the HinDIII(blunt) site 5’ of the single...functionality of each of the loxP sites was tested in AM-1 cells (Invitrogen) that express Cre recombinase . Step 2: Gene targeting in embryonic stem

A study of the fragmentation of quarks in et- p collisions at HERA

NASA Astrophysics Data System (ADS)

Aid, S.; Andreev, V.; Andrieu, B.; Appuhn, R.-D.; Arpagaus, M.; Babaev, A.; Baehr, J.; Bán, J.; Ban, Y.; Baranov, P.; Barrelet, E.; Barschke, R.; Bartel, W.; Barth, M.; Bassler, U.; Beck, H. P.; Behrend, H.-J.; Belousov, A.; Berger, Ch.; Bernardi, G.; Bernet, R.; Bertrand-Coremans, G.; Besançon, M.; Beyer, R.; Biddulph, P.; Bispham, P.; Bizot, J. C.; Blobel, V.; Borras, K.; Botterweck, F.; Boudry, V.; Braemer, A.; Brasse, F.; Braunschweig, W.; Brisson, V.; Bruncko, D.; Brune, C.; Buchholz, R.; Büngener, L.; Bürger, J.; Büsser, F. W.; Buniatian, A.; Bürke, S.; Burton, M.; Buschhorn, G.; Campbell, A. J.; Carli, T.; Charles, F.; Charlet, M.; Clarke, D.; Clegg, A. B.; Clerbaux, B.; Colombo, M.; Contreras, J. G.; Cormack, C.; Coughlan, J. A.; Courau, A.; Coutures, Ch.; Cozzika, G.; Criegee, L.; Cussans, D. G.; Cvach, J.; Dagoret, S.; Dainton, J. B.; Dau, W. D.; Daum, K.; David, M.; Delcourt, B.; Del Buono, L.; De Roeck, A.; De Wolf, E. A.; Di Nezza, P.; Dollfus, C.; Dowell, J. D.; Dreis, H. B.; Droutskoi, A.; Duboc, J.; Düllmann, D.; Dünger, O.; Duhm, H.; Ebert, J.; Ebert, T. R.; Eckerlin, G.; Efremenko, V.; Egli, S.; Ehrlichmann, H.; Eichenberger, S.; Eichler, R.; Eisele, F.; Eisenhandler, E.; Ellison, R. J.; Elsen, E.; Erdmann, M.; Erdmann, W.; Evrard, E.; Favart, L.; Fedotov, A.; Feeken, D.; Felst, R.; Feltesse, J.; Ferencei, J.; Ferrarotto, F.; Flamm, K.; Fleischer, M.; Flieser, M.; Flügge, G.; Fomenko, A.; Fominykh, B.; Forbush, M.; Formánek, J.; Foster, J. M.; Franke, G.; Fretwurst, E.; Gabathuler, E.; Gabathuler, K.; Gamerdinger, K.; Garvey, J.; Gayler, J.; Gebauer, M.; Gellrich, A.; Genzel, H.; Gerhards, R.; Goerlach, U.; Goerlach, L.; Gogitidze, N.; Goldberg, M.; Goldner, D.; Gonzalez-Pineiro, B.; Gorelov, I.; Goritchev, P.; Grab, C.; Grässler, H.; Grässler, R.; Greenshaw, T.; Grindhammer, G.; Gruber, A.; Gruber, C.; Haack, J.; Haidt, D.; Hajduk, L.; Hamon, O.; Hampel, M.; Hanlon, E. M.; Hapke, M.; Haynes, W. J.; Heatherington, J.; Heinzelmann, G.; Henderson, R. C. W.; Henschel, H.; Herynek, I.; Hess, M. F.; Hildesheim, W.; Hill, P.; Hiller, K. H.; Hilton, C. D.; Hladký, J.; Hoeger, K. C.; Höppner, M.; Horisberger, R.; Hudgson, V. L.; Huet, Ph.; Hütte, M.; Hufnagel, H.; Ibbotson, M.; Itterbeck, H.; Jabiol, M.-A.; Jacholkowska, A.; Jacobsson, C.; Jaffre, M.; Janoth, J.; Jansen, T.; Jönson, L.; Johnson, D. P.; Johnson, L.; Jung, H.; Kalmus, P. I. P.; Kant, D.; Kaschowitz, R.; Kasselmann, P.; Kathage, U.; Katzy, J.; Kaufmann, H. H.; Kazarian, S.; Kenyon, I. R.; Kermiche, S.; Keuker, C.; Kiesling, C.; Klein, M.; Kleinwort, C.; Knies, G.; Ko, W.; Köhler, T.; Köhne, J. H.; Kolanoski, H.; Kole, F.; Kolya, S. D.; Korbel, V.; Korn, M.; Kostka, P.; Kotelnikov, S. K.; Krdmerkämper, T.; Krasny, M. W.; Krehbiel, H.; Krücker, D.; Krüger, U.; Krüner-Marquis, U.; Kubenka, J. P.; Küster, H.; Kuhlen, M.; Kurča, T.; Kurzhöfer, J.; Kuznik, B.; Lacour, D.; Lamarche, F.; Lander, R.; Landon, M. P. J.; Lange, W.; Lanius, P.; Laporte, J.-F.; Lebedev, A.; Leverenz, C.; Levonian, S.; Ley, Ch.; Lindner, A.; Lindström, G.; Link, J.; Linsel, F.; Lipinski, J.; List, B.; Lobo, G.; Loch, P.; Lohmander, H.; Lomas, J.; Lopez, G. C.; Lubimov, V.; Luke, D.; Magnussen, N.; Malinovski, E.; Mani, S.; Maraček, R.; Marage, P.; Marks, J.; Marshall, R.; Martens, J.; Martin, R.; Martyn, H.-U.; Martyniak, J.; Masson, S.; Mavroidis, T.; Maxfield, S. J.; McMahon, S. J.; Mehta, A.; Meier, K.; Mercer, D.; Merz, T.; Meyer, C. A.; Meyer, H.; Meyer, J.; Migliori, A.; Mikocki, S.; Milstead, D.; Moreau, F.; Morris, J. V.; Mroczko, E.; Müller, G.; Müller, K.; Murín, P.; Nagovizin, V.; Nahnhauer, R.; Naroska, B.; Naumann, Th.; Newman, P. R.; Newton, D.; Neyret, D.; Nguyen, H. K.; Nicholls, T. C.; Niebergall, F.; Niebuhr, C.; Niedzballa, Ch.; Nisius, R.; Nowak, G.; Noyes, G. W.; Nyberg-Werther, M.; Oakden, M.; Oberlack, H.; Obrock, U.; Olsson, J. E.; Ozerov, D.; Panaro, E.; Panitch, A.; Pascaud, C.; Patel, G. D.; Peppel, E.; Perez, E.; Phillips, J. P.; Pichler, Ch.; Pieuchot, A.; Pitzl, D.; Pope, G.; Prell, S.; Prosi, R.; Rabbertz, K.; Radel, G.; Raupach, F.; Reimer, P.; Reinshagen, S.; Ribarics, P.; Rick, H.; Riech, V.; Riedlberger, J.; Riess, S.; Rietz, M.; Rizvi, E.; Robertson, S. M.; Robmann, P.; Roloff, H. E.; Roosen, R.; Rosenbauer, K.; Rostovtsev, A.; Rouse, F.; Royon, C.; Rütter, K.; Rusakov, S.; Rybicki, K.; Rylko, R.; Sahlmann, N.; Sanchez, E.; Sankey, D. P. C.; Schacht, P.; Schiek, S.; Schleper, P.; von Schlippe, W.; Schmidt, C.; Schmidt, D.; Schmidt, G.; Schöning, A.; Schröder, V.; Schuhmann, E.; Schwab, B.; Schwind, A.; Sefkow, F.; Seidel, M.; Sell, R.; Semenov, A.; Shekelyan, V.; Sheviakov, I.; Shooshtari, H.; Shtarkov, L. N.; Siegmon, G.; Siewert, U.; Sirois, Y.; Skillicorn, I. O.; Smirnov, P.; Smith, J. R.; Solochenko, V.; Soloviev, Y.; Spiekermann, J.; Spielman, S.; Spitzerx, H.; Starosta, R.; Steenbock, M.; Steffen, P.; Steinberg, R.; Stella, B.; Stephens, K.; Stier, J.; Stiewe, J.; Stosslein, U.; Stolze, K.; Strachota, J.; Straumann, U.; Struczinski, W.; Sutton, J. P.; Tapprogge, S.; Tchernyshov, V.; Thiebaux, C.; Thompson, G.; Truöl, P.; Turnau, J.; Tutas, J.; Uelkes, P.; Usik, A.; Valkár, S.; Valkárová, A.; Vallée, C.; Van Esch, P.; Van Mechelen, P.; Vartapetian, A.; Vazdik, Y.; Verrecchia, P.; Villet, G.; Wacker, K.; Wagener, A.; Wagener, M.; Walker, I. W.; Walther, A.; Weber, G.; Weber, M.; Wegener, D.; Wegner, A.; Wellisch, H. P.; West, L. R.; Willard, S.; Winde, M.; Winter, G.-G.; Wittek, C.; Wright, A. E.; Wünsch, E.; Wulff, N.; Yiou, T. P.; Žáček, J.; Zarbock, D.; Zhang, Z.; Zhokin, A.; Zimmer, M.; Zimmermann, W.; Zomer, F.; Zuber, K.; H1 Collaboration

1995-02-01

Deep inelastic scattering (DIS) events, selected from 1993 data taken by the H1 experiment at HERA, are studied in the Breit frame of reference. The fragmentation function of the quark is compared with those of e+e- data. It is shown that certain aspects of the quarks emerging from within the proton in e-p interactions are essentially the same as those of quarks pair-created from the vacuum in e+e- annihilation. The measured area, peak position and width of the fragmentation function show that the kinematic evolution variable, equivalent to the e+e- squared centre of mass energy, is in the Breit frame the invariant square of the four-momentum transfer. We comment on the extent to which we have evidence for coherence effects in pArton showers.

Dissecting fragment-based lead discovery at the von Hippel-Lindau protein:hypoxia inducible factor 1α protein-protein interface.

PubMed

Van Molle, Inge; Thomann, Andreas; Buckley, Dennis L; So, Ernest C; Lang, Steffen; Crews, Craig M; Ciulli, Alessio

2012-10-26

Fragment screening is widely used to identify attractive starting points for drug design. However, its potential and limitations to assess the tractability of often challenging protein:protein interfaces have been underexplored. Here, we address this question by means of a systematic deconstruction of lead-like inhibitors of the pVHL:HIF-1α interaction into their component fragments. Using biophysical techniques commonly employed for screening, we could only detect binding of fragments that violate the Rule of Three, are more complex than those typically screened against classical druggable targets, and occupy two adjacent binding subsites at the interface rather than just one. Analyses based on ligand and group lipophilicity efficiency of anchored fragments were applied to dissect the individual subsites and probe for binding hot spots. The implications of our findings for targeting protein interfaces by fragment-based approaches are discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

Fragmented esophageal smooth muscle contraction segments on high resolution manometry: a marker of esophageal hypomotility.

PubMed

Porter, R F; Kumar, N; Drapekin, J E; Gyawali, C P

2012-08-01

Esophageal peristalsis consists of a chain of contracting striated and smooth muscle segments on high resolution manometry (HRM). We compared smooth muscle contraction segments in symptomatic subjects with reflux disease to healthy controls. High resolution manometry Clouse plots were analyzed in 110 subjects with reflux disease (50 ± 1.4 years, 51.5% women) and 15 controls (27 ± 2.1 years, 60.0% women). Using the 30 mmHg isobaric contour tool, sequences were designated fragmented if either smooth muscle contraction segment was absent or if the two smooth muscle segments were separated by a pressure trough, and failed if both smooth muscle contraction segments were absent. The discriminative value of contraction segment analysis was assessed. A total of 1115 swallows were analyzed (reflux group: 965, controls: 150). Reflux subjects had lower peak and averaged contraction amplitudes compared with controls (P < 0.0001 for all comparisons). Fragmented sequences followed 18.4% wet swallows in the reflux group, compared with 7.5% in controls (P < 0.0001), and were seen more frequently than failed sequences (7.9% and 2.5%, respectively). Using a threshold of 30% in individual subjects, a composite of failed and/or fragmented sequences was effective in segregating reflux subjects from control subjects (P = 0.04). Evaluation of smooth muscle contraction segments adds value to HRM analysis. Specifically, fragmented smooth muscle contraction segments may be a marker of esophageal hypomotility. © 2012 Blackwell Publishing Ltd.

Observation of medium induced modifications of jet fragmentation in PbPb collisions using isolated-photon-tagged jets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sirunyan, Albert M; et al.

Measurements of fragmentation functions for jets associated with an isolated photon are presented for the first time in pp and PbPb collisions. The analysis uses data collected with the CMS detector at the CERN LHC at a nucleon-nucleon center-of-mass energy of 5.02 TeV. Fragmentation functions are obtained for jets with pmore » $$_\\mathrm{T}^\\text{jet} >$$ 30 GeV in events containing an isolated photon with p$$_\\mathrm{T}^\\gamma>$$ 60 GeV, using charged tracks with transverse momentum p$$_\\mathrm{T}^\\text{trk} >$$ 1 GeV in a cone around the jet axis. The association with an isolated photon constrains the initial p$$_\\mathrm{T}$$ and azimuthal angle of the parton whose shower produced the jet. For central PbPb collisions, modifications of the jet fragmentation functions are observed when compared to those measured in pp collisions, while no significant differences are found in the 50% most peripheral collisions. Jets in central PbPb events show an excess (depletion) of low (high) p$$_\\mathrm{T}$$ particles, with a transition around 3 GeV.« less

ACE-SWICS In Situ Plasma Composition of Fragmented Comet 73P/Schwassmann-Wachmann 3

NASA Astrophysics Data System (ADS)

Gilbert, J. A.; Lepri, S. T.; Rubin, M.; Zurbuchen, T.

2013-12-01

The interiors of comets contain some of the most pristine material in the solar system. Comet 73P/Schwassmann-Wachmann 3, discovered in 1930 with a double nucleus, is a Jupiter-family comet with a 5.34-year period. This comet split into 5 fragments in 1995 and disintegrated into nearly 70 pieces in 2006. In May and June of 2006, recently ionized cometary particles originating from some of these fragments were collected with the ACE-SWICS sensor. Due to a combination of the close proximity of the fragments passing between ACE-SWICS and the Sun, and the instrument characteristics, unique measurements regarding the charge state composition and the elemental abundances of both cometary and heliospheric plasma were made during this time. The cometary material released from some of these fragments can be identified by the concentrations of water-group pick-up ions having a mass-per-charge of 16-18 amu/e. With a focus on Helium, Carbon, and water-group ions, we present an analysis of the cometary plasma. Charge state ratios of C+/O+ fall below 0.1 during detection of comet fragment plasma, and there is a clear increase in He+ during fragment crossings. The C/O ratio and He charge states are used to provide constraints on the activity of the cometary fragments and also the spatial distribution of the extended and ionized cometary tail.

Influence of residual composition on the structure and properties of extracellular matrix derived hydrogels.

PubMed

Claudio-Rizo, Jesús A; Rangel-Argote, Magdalena; Castellano, Laura E; Delgado, Jorge; Mata-Mata, José L; Mendoza-Novelo, Birzabith

2017-10-01

In this work, hydrolysates of extracellular matrix (hECM) were obtained from rat tail tendon (TR), bovine Achilles tendon (TAB), porcine small intestinal submucosa (SIS) and bovine pericardium (PB), and they were polymerized to generate ECM hydrogels. The composition of hECM was evaluated by quantifying the content of sulphated glycosaminoglycans (sGAG), fibronectin and laminin. The polymerization process, structure, physicochemical properties, in vitro degradation and biocompatibility were studied and related to their composition. The results indicated that the hECM derived from SIS and PB were significantly richer in sGAG, fibronectin and laminin, than those derived from TAB and TR. These differences in hECM composition influenced the polymerization and the structural characteristics of the fibrillar gel network. Consequently, the swelling, mechanics and degradation of the hydrogels showed a direct relationship with the remaining composition. Moreover, the cytocompatibility and the secretion of transforming growth factor beta-1 (TGF-β1) by macrophages were enhanced in hydrogels with the highest residual content of ECM biomolecules. The results of this work evidenced the role of the ECM molecules remaining after both decellularization and hydrolysis steps to produce tissue derived hydrogels with structure and properties tailored to enhance their performance in tissue engineering and regenerative medicine applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Cloning of a newly identified heart-specific troponin I isoform, which lacks the troponin T binding portion, using the yeast hybrid system.

PubMed

Suzuki, Hideaki; Arakawa, Yasuhiro; Ito, Masaki; Yamada, Hisashi; Horiguchi-Yamada, Junko

2006-01-01

To elucidate the molecular pathogenesis behind increased levels of laminin in cardiac muscle cells in cardiomyopathy by using a yeast hybrid screen. The present study reports the cloning of a newly identified heart-specific troponin I isoform, which is putatively linked to laminin. Future studies will explore the functional significance of this connection. Yeast two-hybrid screen analysis was performed using MLF1-interacting protein (amino acids 1 to 318) as bait. The human heart complementary DNA library was screened by using the yeast-mating method for overnight culture. Two final positive clones from the heart library were isolated. These two clones encoded the same protein, a short isoform of human cardiac troponin I (TnI) that lacked TnI exons 5 and 6. The TnI isoform has a heart-specific expression pattern and it shares several sequence features with human cardiac TnI; however, it lacks the troponin T binding portion. The heart-specific segment of the human cardiac TnI isoform shares several sequence features with human cardiac TnI, but it lacks the troponin T binding portion. These results suggest that the heart-specific TnI isoform may be involved in cardiac development and disease.

Cloning of a newly identified heart-specific troponin I isoform, which lacks the troponin T binding portion, using the yeast hybrid system

PubMed Central

Suzuki, Hideaki; Arakawa, Yasuhiro; Ito, Masaki; Yamada, Hisashi; Horiguchi-Yamada, Junko

2006-01-01

OBJECTIVE To elucidate the molecular pathogenesis behind increased levels of laminin in cardiac muscle cells in cardiomyopathy by using a yeast hybrid screen. The present study reports the cloning of a newly identified heart-specific troponin I isoform, which is putatively linked to laminin. Future studies will explore the functional significance of this connection. METHODS Yeast two-hybrid screen analysis was performed using MLF1-interacting protein (amino acids 1 to 318) as bait. The human heart complementary DNA library was screened by using the yeast-mating method for overnight culture. RESULTS Two final positive clones from the heart library were isolated. These two clones encoded the same protein, a short isoform of human cardiac troponin I (TnI) that lacked TnI exons 5 and 6. The TnI isoform has a heart-specific expression pattern and it shares several sequence features with human cardiac TnI; however, it lacks the troponin T binding portion. CONCLUSION The heart-specific segment of the human cardiac TnI isoform shares several sequence features with human cardiac TnI, but it lacks the troponin T binding portion. These results suggest that the heart-specific TnI isoform may be involved in cardiac development and disease. PMID:18651010

Endothelin-1 Mediated Induction of Extracellular Matrix Genes in Strial Marginal Cells Underlies Strial Pathology in Alport Mice

PubMed Central

Meehan, Daniel T.; Delimont, Duane; Dufek, Brianna; Zallocchi, Marisa; Phillips, Grady; Gratton, Michael Anne; Cosgrove, Dominic

2016-01-01

Alport syndrome, a type IV collagen disorder, manifests as glomerular disease associated with hearing loss with thickening of the glomerular and strial capillary basement membranes (SCBMs). We have identified a role for endothelin-1 (ET-1) activation of endothelin A receptors (ETARs) in glomerular pathogenesis. Here we explore whether ET-1 plays a role in strial pathology. Wild type (WT) and Alport mice were treated with the ETAR antagonist, sitaxentan. The stria vascularis was analyzed for SCBM thickness and for extracellular matrix (ECM) proteins. Additional WT and Alport mice were exposed to noise or hypoxia and the stria analyzed for hypoxia-related and ECM genes. A strial marginal cell line cultured under hypoxic conditions, or stimulated with ET-1 was analyzed for expression of hypoxia-related and ECM transcripts. Noise exposure resulted in significantly elevated ABR thresholds in Alport mice relative to wild type littermates. Alport stria showed elevated expression of collagen α1(IV), laminin α2, and laminin α5 proteins relative to WT. SCBM thickening and elevated ECM protein expression was ameliorated by ETAR blockade. Stria from normoxic Alport mice and hypoxic WT mice showed upregulation of hypoxia-related, ECM, and ET-1 transcripts. Both ET-1 stimulation and hypoxia up-regulated ECM transcripts in cultured marginal cells. We conclude that ET-1 mediated activation of ETARs on strial marginal cells results in elevated expression of ECM genes and thickening of the SCBMs in Alport mice. SCBM thickening results in hypoxic stress further elevating ECM and ET-1 gene expression, exacerbating strial pathology. PMID:27553900

Treatment with the non-ionic surfactant poloxamer P188 reduces DNA fragmentation in cells from bovine chondral explants exposed to injurious unconfined compression.

PubMed

Baars, D C; Rundell, S A; Haut, R C

2006-06-01

Excessive mechanical loading to a joint has been linked with the development of post-traumatic osteoarthritis (OA). Among the suspected links between impact trauma to a joint and associated degeneration of articular cartilage is an acute reduction in chondrocyte viability. Recently, the non-ionic surfactant poloxamer 188 (P188) has been shown to reduce by approximately 50% the percentage of non-viable chondrocytes 24 h post-injury in chondral explants exposed to 25 MPa of unconfined compression. There is a question whether these acutely 'saved' chondrocytes will continue to degrade over time, as P188 is only thought to act by acute repair of damaged cell membranes. In order to investigate the degradation of traumatized chondrocytes in the longer term, the current study utilized TUNEL staining to document the percentage of cells suffering DNA fragmentation with and without an immediate 24 h period of exposure of the explants to P188 surfactant. In the current study, as in the previous study by this laboratory, chondral explants were excised from bovine metacarpophalangeal joints and subjected to 25 MPa of unconfined compression. TUNEL staining was performed at 1 h, 4 days, and 7 days post-impact. The current study found that P188 was effective in reducing the percentage of cells with DNA fragmentation in impacted explants by approximately 45% at 4 and 7 days post-impact. These data suggest that early P188 intervention was effective in preventing DNA fragmentation of injured chondrocytes. The current hypothesis is that this process was mitigated by the acute repair of damaged plasma membranes by the non-ionic surfactant P188, and that most repaired cells did not continue to degrade as measured by the fragmentation of their DNA.

p65 fragments, homologous to the C2 region of protein kinase C, bind to the intracellular receptors for protein kinase C.

PubMed

Mochly-Rosen, D; Miller, K G; Scheller, R H; Khaner, H; Lopez, J; Smith, B L

1992-09-08

Receptors for activated protein kinase C (RACKs) have been isolated from the particulate cell fraction of heart and brain. We previously demonstrated that binding of protein kinase C (PKC) to RACKs requires PKC activators and is via a site on PKC that is distinct from the substrate binding site. Here, we examine the possibility that the C2 region in the regulatory domain of PKC is involved in binding of PKC to RACKs. The synaptic vesicle-specific p65 protein contains two regions homologous to the C2 region of PKC. We found that three p65 fragments, containing either one or two of these PKC C2 homologous regions, bound to highly purified RACKs. Binding of the p65 fragments and PKC to RACKs was mutually exclusive; preincubation of RACKs with the p65 fragments inhibited PKC binding, and preincubation of RACKs with PKC inhibited binding of the p65 fragments. Preincubation of the p65 fragments with a peptide resembling the PKC binding site on RACKs also inhibited p65 binding to RACKs, suggesting that PKC and p65 bind to the same or nearby regions on RACKs. Since the only homologous region between PKC and the p65 fragments is the C2 region, these results suggest that the C2 region on PKC contains at least part of the RACK binding site.

Role for a region of helically unstable DNA within the Epstein-Barr virus latent cycle origin of DNA replication oriP in origin function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Polonskaya, Zhanna; Benham, Craig J.; Hearing, Janet

The minimal replicator of the Epstein-Barr virus (EBV) latent cycle origin of DNA replication oriP is composed of two binding sites for the Epstein-Barr virus nuclear antigen-1 (EBNA-1) and flanking inverted repeats that bind the telomere repeat binding factor TRF2. Although not required for minimal replicator activity, additional binding sites for EBNA-1 and TRF2 and one or more auxiliary elements located to the right of the EBNA-1/TRF2 sites are required for the efficient replication of oriP plasmids. Another region of oriP that is predicted to be destabilized by DNA supercoiling is shown here to be an important functional component ofmore » oriP. The ability of DNA fragments of unrelated sequence and possessing supercoiled-induced DNA duplex destabilized (SIDD) structures, but not fragments characterized by helically stable DNA, to substitute for this component of oriP demonstrates a role for the SIDD region in the initiation of oriP-plasmid DNA replication.« less

Sequence Analysis of the Cryptic Plasmid pMG101 from Rhodopseudomonas palustris and Construction of Stable Cloning Vectors

PubMed Central

Inui, Masayuki; Roh, Jung Hyeob; Zahn, Kenneth; Yukawa, Hideaki

2000-01-01

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of α-proteobacteria. PMID:10618203

Cloning and sequencing of the pheP gene, which encodes the phenylalanine-specific transport system of Escherichia coli.

PubMed Central

Pi, J; Wookey, P J; Pittard, A J

1991-01-01

The phenylalanine-specific permease gene (pheP) of Escherichia coli has been cloned and sequenced. The gene was isolated on a 6-kb Sau3AI fragment from a chromosomal library, and its presence was verified by complementation of a mutant lacking the functional phenylalanine-specific permease. Subcloning from this fragment localized the pheP gene on a 2.7-kb HindIII-HindII fragment. The nucleotide sequence of this 2.7-kb region was determined. An open reading frame was identified which extends from a putative start point of translation (GTG at position 636) to a termination signal (TAA at position 2010). The assignment of the GTG as the initiation codon was verified by site-directed mutagenesis of the initiation codon and by introducing a chain termination mutation into the pheP-lacZ fusion construct. A single initiation site of transcription 30 bp upstream of the start point of translation was identified by the primer extension analysis. The pheP structural gene consists of 1,374 nucleotides specifying a protein of 458 amino acid residues. The PheP protein is very hydrophobic (71% nonpolar residues). A topological model predicted from the sequence analysis defines 12 transmembrane segments. This protein is highly homologous with the AroP (general aromatic transport) system of E. coli (59.6% identity) and to a lesser extent with the yeast permeases CAN1 (arginine), PUT4 (proline), and HIP1 (histidine) of Saccharomyces cerevisiae. Images PMID:1711024

Knockout of toll-like receptor-2 attenuates both the proinflammatory state of diabetes and incipient diabetic nephropathy.

PubMed

Devaraj, Sridevi; Tobias, Peter; Kasinath, Balakuntalam S; Ramsamooj, Rajendra; Afify, Alaa; Jialal, Ishwarlal

2011-08-01

Type 1 diabetes (T1DM) is a proinflammatory state and confers an increased risk for vascular complications. Toll-like receptors (TLR) could participate in diabetic vasculopathies. Whether TLR activation contributes to the proinflammatory state of T1DM and the pathogenesis of diabetic nephropathy remains unknown. We induced T1DM in TLR2 knockout mice (TLR2-/-) and wild-type littermates (C57BL/6J-WT) using streptozotocin (STZ). Fasting blood, peritoneal macrophages, and kidneys were obtained for flow cytometry, Western blot, microscopy, and cytokine assays at 6 and 14 weeks after induction of diabetes. Macrophage TLR2 expression and MyD88-dependent signaling were increased in diabetic mice (WT+STZ) compared with nondiabetic WT mice. These biomarkers were attenuated in diabetic TLR2-/- macrophages. WT+STZ mice showed increased kidney:body weight ratio due to cell hypertrophy, increased albuminuria, decreased kidney nephrin, podocin, and podocyte number and increased transforming growth factor-β and laminin compared with WT mice. Nephrin, podocin, and podocyte number and effacement were restored, and transforming growth factor-β and laminin levels were decreased in TLR2-/-+ STZ mice kidneys versus WT+STZ. Peritoneal and kidney macrophages were predominantly M1 phenotype in WT+STZ mice; this was attenuated in TLR2-/-+STZ mice. These data support a role for TLR2 in promoting inflammation and early changes of incipient diabetic nephropathy, in addition to albuminuria and podocyte loss.

Expression of laminin-5 and integrins in actinic cheilitis and superficially invasive squamous cell carcinomas of the lip.

PubMed

Peixoto da-Silva, Janaína; Lourenço, Silvia; Nico, Marcello; Silva, Filomena H; Martins, Marília Trierveiler; Costa-Neves, Adriana

2012-10-15

The progression of carcinogenesis entails the detachment of cells, invasion and migration of neoplastic cells. Alterations in epithelial adhesion and basement membrane proteins might mediate the early stages of carcinogenesis. This study investigated the expression of adhesion molecules and the basement membrane protein laminin-5 in actinic cheilitis (AC) and incipient squamous cell carcinoma of the lower lip to understand early photocarcinogenesis. Ln-5γ2 chain as well as α3, β1 subunits of α3β1 heterodimer and β4 subunit of integrin α6β4 were evaluated by immunohistochemistry in 16 cases of AC and 16 cases of superficially invasive squamous cell carcinoma (SISCC). Most AC cases showed reduced expression of β1, β4 and α3 integrins, and SISCCs lacked β1, β4 and α3 integrins in the invasive front. AC cases were negative for the Ln-5γ2 chain. Five cases of SISCC (31%) showed heterogeneous Ln-5γ2 chain expression in the invasive front of the tumor. Integrin β1, β4 and α3 expression is lost during the early stages of lip carcinogenesis. Expression of Ln-5γ2 in the invasive front in cases and its correlation with tumor progression suggest that it mediates the acquisition of the migrating and invading epithelial cell phenotype. Copyright © 2012 Elsevier GmbH. All rights reserved.

In Situ Plasma Measurements of Fragmented Comet 73P Schwassmann-Wachmann 3

NASA Astrophysics Data System (ADS)

Gilbert, J. A.; Lepri, S. T.; Rubin, M.; Combi, M.; Zurbuchen, T. H.

2015-12-01

The interiors of comets contain some of the most pristine material in the solar system. Comet 73P/Schwassmann-Wachmann 3, discovered in 1930, is a Jupiter-family comet with a 5.34-year period. This comet split into 5 fragments in 1995 and disintegrated into nearly 70 major pieces in 2006. In 2006 May and June, recently ionized cometary particles originating from fragments including and surrounding some of these major objects were collected with the ACE/SWICS and Wind/STICS sensors. Due to a combination of the instrument characteristics and the close proximity of the fragments passing between those spacecraft and the Sun, unique measurements regarding the charge state composition and the elemental abundances of both cometary and heliospheric plasma were made during that time. The cometary material released from some of these fragments can be identified by the concentrations of water-group pickup ions having a mass-per-charge ratio of 16-18 amu e-1, indicating that while these fragments are small, they are still actively sublimating. We present an analysis of cometary composition, spatial distribution, and heliospheric interactions, with a focus on helium, C+/O+, and water-group ions.

IN SITU PLASMA MEASUREMENTS OF FRAGMENTED COMET 73P SCHWASSMANN–WACHMANN 3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gilbert, J. A.; Lepri, S. T.; Combi, M.

2015-12-10

The interiors of comets contain some of the most pristine material in the solar system. Comet 73P/Schwassmann–Wachmann 3, discovered in 1930, is a Jupiter-family comet with a 5.34-year period. This comet split into 5 fragments in 1995 and disintegrated into nearly 70 major pieces in 2006. In 2006 May and June, recently ionized cometary particles originating from fragments including and surrounding some of these major objects were collected with the ACE/SWICS and Wind/STICS sensors. Due to a combination of the instrument characteristics and the close proximity of the fragments passing between those spacecraft and the Sun, unique measurements regarding themore » charge state composition and the elemental abundances of both cometary and heliospheric plasma were made during that time. The cometary material released from some of these fragments can be identified by the concentrations of water-group pickup ions having a mass-per-charge ratio of 16–18 amu e{sup −1}, indicating that while these fragments are small, they are still actively sublimating. We present an analysis of cometary composition, spatial distribution, and heliospheric interactions, with a focus on helium, C{sup +}/O{sup +}, and water-group ions.« less

Exogenous Thyropin from p41 Invariant Chain Diminishes Cysteine Protease Activity and Affects IL-12 Secretion during Maturation of Human Dendritic Cells

PubMed Central

Zavašnik-Bergant, Tina; Bergant Marušič, Martina

2016-01-01

Dendritic cells (DC) play a pivotal role as antigen presenting cells (APC) and their maturation is crucial for effectively eliciting an antigen-specific immune response. The p41 splice variant of MHC class II-associated chaperone, called invariant chain p41 Ii, contains an amino acid sequence, the p41 fragment, which is a thyropin-type inhibitor of proteolytic enzymes. The effects of exogenous p41 fragment and related thyropin inhibitors acting on human immune cells have not been reported yet. In this study we demonstrate that exogenous p41 fragment can enter the endocytic pathway of targeted human immature DC. Internalized p41 fragment has contributed to the total amount of the immunogold labelled p41 Ii-specific epitope, as quantified by transmission electron microscopy, in particular in late endocytic compartments with multivesicular morphology where antigen processing and binding to MHC II take place. In cell lysates of treated immature DC, diminished enzymatic activity of cysteine proteases has been confirmed. Internalized exogenous p41 fragment did not affect the perinuclear clustering of acidic cathepsin S-positive vesicles typical of mature DC. p41 fragment is shown to interfere with the nuclear translocation of NF-κB p65 subunit in LPS-stimulated DC. p41 fragment is also shown to reduce the secretion of interleukin-12 (IL-12/p70) during the subsequent maturation of treated DC. The inhibition of proteolytic activity of lysosomal cysteine proteases in immature DC and the diminished capability of DC to produce IL-12 upon their subsequent maturation support the immunomodulatory potential of the examined thyropin from p41 Ii. PMID:26960148

P40 and P90 from Mpn142 are Targets of Multiple Processing Events on the Surface of Mycoplasma pneumoniae

PubMed Central

Widjaja, Michael; Berry, Iain J.; Pont, Elsa J.; Padula, Matthew P.; Djordjevic, Steven P.

2015-01-01

Mycoplasma pneumoniae is a significant cause of community acquired pneumonia globally. Despite having a genome less than 1 Mb in size, M. pneumoniae presents a structurally sophisticated attachment organelle that (i) provides cell polarity, (ii) directs adherence to receptors presented on respiratory epithelium, and (iii) plays a major role in cell motility. The major adhesins, P1 (Mpn141) and P30 (Mpn453), are localised to the tip of the attachment organelle by the surface accessible cleavage fragments P90 and P40 derived from Mpn142. Two events play a defining role in the formation of P90 and P40; removal of a leader peptide at position 26 (23SLA↓NTY28) during secretion to the cell surface and cleavage at amino acid 455 (452GPL↓RAG457) generating P40 and P90. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) analysis of tryptic peptides generated by digesting size-fractionated cell lysates of M. pneumoniae identified 15 cleavage fragments of Mpn142 ranging in mass from 9–84 kDa. Further evidence for the existence of cleavage fragments of Mpn142 was generated by mapping tryptic peptides to proteins recovered from size fractionated eluents from affinity columns loaded with heparin, fibronectin, fetuin, actin, plasminogen and A549 surface proteins as bait. To define the sites of cleavage in Mpn142, neo-N-termini in cell lysates of M. pneumoniae were dimethyl-labelled and characterised by LC-MS/MS. Our data suggests that Mpn142 is cleaved to generate adhesins that are auxiliary to P1 and P30. PMID:28248283

The Research of Acellular pancreatic bioscaffoldas a natural 3D platform In Vitro

NASA Astrophysics Data System (ADS)

Wang, Xin; Li, Zhao

2018-03-01

AIM: To investigate the biochemical and functional properties of a rat acellular pancreatic bioscaffold (APB). METHODS: Fresh pancreata were soaked and perfused. The histological structure, the extracellular matrix (ECM) composition, and the DNA content of the APBs were evaluated. After biocompatibility studies, the proliferation, apoptosis and differentiation of AR42J pancreatic acinar cells cultured on APBs were assessed. RESULTS: The pancreatic tissues became translucent after decellularization. The native macroscopic 3D architecture and the ECM ultrastructure were preserved, with large ductal structures and vascular tissue branching from the greater pancreatic artery, but there were no visible vascular endothelial cells, cellular components or cracked cellular debris. The ECM components, including collagen I, collagen IV, fibronectin, laminin and sGAG, were not decreased after decellularization of the APB (P>0.05) however, the DNA content was decreased significantly (P<0.05). The subcutaneous implantation sites showed low immunological response and low cytotoxicity around the APB. The proliferation rate was higher and the apoptosis rate was lower when AR42J cells were cultured on APB than when they were cultured in media alone, on artificial scaffold or ECM (P<0.05). The gene expression of pancreatic duodenal homeodomain containing transcription factor (PDX-1) and pancreatic exocrine transcription factor (PTF-1) and the protein expression of α-Amy, cytokeratin 7 (CK7) and fetal liver kinase-1 (Flk-1) were higher for the APB group than for the other groups (P<0.001). CONCLUSION: Our findings support the biological utility of whole pancreas APBs as biomaterial scaffolds, which provides an improved approach for regenerative medicine.

Hallucinations and sleep disorders in PD

PubMed Central

Goetz, Christopher G.; Ouyang, Bichun; Negron, Alice; Stebbins, Glenn T.

2010-01-01

Objective: To assess prospectively progression and relationship of hallucinations and sleep disorders over a 10-year longitudinal study of patients with Parkinson disease (PD). Methods: Eighty-nine patients with PD were recruited to fill cells of normal sleep without hallucinations (n = 20), sleep fragmentation only (n = 20), vivid dreams/nightmares (n = 20), hallucinations with insight (n = 20), and hallucinations without insight (n = 9). At baseline, 0.5, 1.5, 4, 6, and 10 years, sleep disorders and hallucinations were assessed by standardized scales with the longitudinal data analyzed by generalized estimating equations with assumptions of linearity in time. Results: At 10 years, we could account for all subjects (27 interviewed, 61 deceased, and 1 too ill for interview). Hallucination prevalence and severity increased over time (p < 0.0001, p = 0.0001). Acting out dreams also increased over time (p = 0.001). In contrast, presence of vivid dreams/nightmares or sleep fragmentation did not increase over time. For all visits, the prevalence of sleep fragmentation did not differ between subjects with vs without hallucinations (odds ratio [OR] = 1.50, p = 0.09). However, severe sleep fragmentation was associated with concurrent hallucinations (OR 2.01, p = 0.006). The presence of hallucinations was also highly associated with concurrent vivid dreams/nightmares (OR = 2.60, p < 0.0001) and with concurrent acting out dreams (OR = 2.38, p = 0.0004). Among the baseline nonhallucinators, no sleep abnormalities at study entry predicted future development of hallucinations. Conclusions: Hallucinations and sleep abnormalities follow very different patterns of progression in PD over 10 years. Whereas patients with hallucinations often have concurrent sleep aberrations, no sleep problem is predictive of future hallucinations. GLOSSARY CI = confidence interval; GEE = generalized estimating equation; MMSE = Mini-Mental State Examination; OR = odds ratio; PD = Parkinson disease; PSQI = Pittsburgh Sleep Quality Index; UPDRS = Unified Parkinson's Disease Rating Scale; UPDRSm = motor section of the Unified Parkinson's Disease Rating Scale. PMID:20962287

The antiproliferative effect of Moringa oleifera crude aqueous leaf extract on cancerous human alveolar epithelial cells

PubMed Central

2013-01-01

Background The incidence of lung cancer is expected to increase due to increases in exposure to airborne pollutants and cigarette smoke. Moringa oleifera (MO), a medicinal plant found mainly in Asia and South Africa is used in the traditional treatment of various ailments including cancer. This study investigated the antiproliferative effect of MO leaf extract (MOE) in cancerous A549 lung cells. Methods A crude aqueous leaf extract was prepared and the cells were treated with 166.7 μg/ml MOE (IC50) for 24 h and assayed for oxidative stress (TBARS and Glutathione assays), DNA fragmentation (comet assay) and caspase (3/7 and 9) activity. In addition, the expression of Nrf2, p53, Smac/DIABLO and PARP-1 was determined by Western blotting. The mRNA expression of Nrf2 and p53 was assessed using qPCR. Results A significant increase in reactive oxygen species with a concomitant decrease in intracellular glutathione levels (p < 0.001) in MOE treated A549 cells was observed. MOE showed a significant reduction in Nrf2 protein expression (1.89-fold, p < 0.05) and mRNA expression (1.44-fold). A higher level of DNA fragmentation (p < 0.0001) was seen in the MOE treated cells. MOE’s pro-apoptotic action was confirmed by the significant increase in p53 protein expression (1.02-fold, p < 0.05), p53 mRNA expression (1.59-fold), caspase-9 (1.28-fold, p < 0.05), caspase-3/7 (1.52-fold) activities and an enhanced expression of Smac/DIABLO. MOE also caused the cleavage and activation of PARP-1 into 89 KDa and 24 KDa fragments (p < 0.0001). Conclusion MOE exerts antiproliferative effects in A549 lung cells by increasing oxidative stress, DNA fragmentation and inducing apoptosis. PMID:24041017

Scaling invariance of spherical projectile fragmentation upon high-velocity impact on a thin continuous shield

DOE Office of Scientific and Technical Information (OSTI.GOV)

Myagkov, N. N., E-mail: nn-myagkov@mail.ru

The problem of aluminum projectile fragmentation upon high-velocity impact on a thin aluminum shield is considered. A distinctive feature of this description is that the fragmentation has been numerically simulated using the complete system of equations of deformed solid mechanics by a method of smoothed particle hydrodynamics in three-dimensional setting. The transition from damage to fragmentation is analyzed and scaling relations are derived in terms of the impact velocity (V), ratio of shield thickness to projectile diameter (h/D), and ultimate strength (σ{sub p}) in the criterion of projectile and shield fracture. Analysis shows that the critical impact velocity V{sub c}more » (separating the damage and fragmentation regions) is a power function of σ{sub p} and h/D. In the supercritical region (V > V{sub c}), the weight-average fragment mass asymptotically tends to a power function of the impact velocity with exponent independent of h/D and σ{sub p}. Mean cumulative fragment mass distributions at the critical point are scale-invariant with respect to parameters h/D and σ{sub p}. Average masses of the largest fragments are also scale-invariant at V > V{sub c}, but only with respect to variable parameter σ{sub p}.« less

Measurement of inclusive jet charged-particle fragmentation functions in Pb+Pb collisions at √ sNN = 2.76 TeV with the ATLAS detector

DOE PAGES

Aad, G.; Abbott, B.; Abdallah, J.; ...

2014-11-04

In this study, measurements of charged-particle fragmentation functions of jets produced in ultra-relativistic nuclear collisions can provide insight into the modification of parton showers in the hot, dense medium created in the collisions. ATLAS has measured jets in √s NN = 2.76 TeV Pb+Pb collisions at the LHC using a data set recorded in 2011 with an integrated luminosity of 0.14 nb –1. Jets were reconstructed using the anti-kt algorithm with distance parameter values R = 0.2, 0.3, and 0.4. Distributions of charged-particle transverse momentum and longitudinal momentum fraction are reported for seven bins in collision centrality for R=0.4 jetsmore » with p T jet. Commensurate minimum p T values are used for the other radii. The ratios of fragment distributions in each centrality bin to those measured in the most peripheral bin are presented. These ratios show a reduction of fragment yield in central collisions relative to peripheral collisions at intermediate z values, 0.04≲z≲0.2, and an enhancement in fragment yield for z≲0.04. A smaller, less significant enhancement is observed at large z and large p T in central collisions.« less

Extraction of jet properties from two particle azimuthal correlations in proton-proton and gold+gold collisions at center of mass energy = 200 GeV

NASA Astrophysics Data System (ADS)

Constantin, Paul

Two particle azimuthal correlation functions measured in pp and AuAu collisions at sNN = 200 GeV at midrapidity with the central arms of the PHENIX detector are analyzed in order to extract the properties of hadronic jets produced in QCD vacuum and highly excited QCD media, like the jet shape parameters jT and kT, the jet conditional yields (number of associated hadrons per high pT trigger hadron) and the jet fragmentation function. The analysis of pp data starts with the measurement of the vacuum fragmentation function from which the pT dependence of the mean fragmentation momentum fraction is extracted. We obtain a constant value of = 0.74 +/- 0.02 for pT above 3 GeV/c. Soft (non-perturbative) parton fragmentation becomes significant below 3 GeV/c and the slope of the fragmentation function is pT dependent in this region. Gaussian fits to jet induced azimuthal correlations are employed to measure the mean jet fragmentation transverse momentum <| jTy|> = 359 +/- 11 (stat) +/- 6 (syst) MeV/c and the mean partonic transverse momentum <|k Ty|> = 964 +/- 49 (stat) +/- 16 (syst) MeV/c. The analysis of AuAu data is based on azimuthal correlation functions between charged hadrons in the 1.5--3 GeV/c and 3--5 GeV/c pT regions in five classes of collision centrality. The mean jet fragmentation transverse momentum <|jTy|> is centrality independent and consistent with the value in pp data mentioned above. A strong broadening with the centrality of the extracted mean partonic transverse momentum <|kTy|> (multiplied by ) is observed. This is an important finding because hadronic jet quenching through energy loss is expected to be accompanied by a broadening effect. Another important finding is that the jet conditional yields in both the near (Deltaφ ˜ 0) and away (Deltaφ ˜ pi) regions exhibit a slightly raising trend with centrality. This was also expected to accompany the jet quenching found at higher pT based on simple energy conservation considerations.

Structure-activity studies of bufokinin, substance P and their C-terminal fragments at bufokinin receptors in the small intestine of the cane toad, Bufo marinus.

PubMed

Liu, Lu; Murray, Michael; Burcher, Elizabeth

2002-01-15

Bufokinin is a substance P-related tachykinin peptide with potent spasmogenic actions, isolated from the intestine of the cane toad, Bufo marinus. Bufokinin acts via a tachykinin receptor with similarities to the mammalian NK(1) receptor. In this structure-activity study of bufokinin, substance P (SP) and their C-terminal fragments, we have used isolated segments and homogenates of toad small intestine to compare the contractile potencies and abilities to compete for the binding of [125I]-Bolton-Hunter bufokinin. In general, potency was very similar in both studies (r=0.956) and was primarily related to peptide length, with the natural undecapeptide tachykinins bufokinin - ranakinin>SP- cod SP -trout SP being most potent. The weakest peptides were [Pro(9)]SP, BUF(7-11) and SP(7-11). Bufokinin fragments (BUF) were approximately equipotent to the corresponding SP fragments, with only BUF(5-11) showing unexpectedly low binding affinity. Data obtained with SP, bufokinin and fragments were subjected to quantitative structure--activity (QSAR) analysis which demonstrated that molecular connectivity and shape descriptors yielded significant regression equations (r approximately 0.90). The predictive capacity of the equations was confirmed using ranakinin, trout SP and cod SP, but not using the synthetic analogs [Pro(9)]SP and [Sar(9)]SP. The study suggests that the full undecapeptide sequence of bufokinin is required for optimal activity, with high potency conferred by Lys(1), Pro(2), Gly(9) and probably Tyr(8). The finding that receptor-ligand interactions were correlated with the shape descriptor 2kappa(alpha) and favored by basic and rigid residues at position 1-3 is consistent with an important role of conformation at the N-terminus of bufokinin.

Comparison of gene expression profiles between dental pulp and periodontal ligament tissues in humans

PubMed Central

Gong, Ai-Xiu; Zhang, Jing-Han; Li, Jing; Wu, Jun; Wang, Lin; Miao, Deng-Shun

2017-01-01

There are anatomical and functional differences between human dental pulp (DP) and periodontal ligament (PDL). However, the molecular biological differences and function of these tissues are poorly understood. In the present study, we employed a cDNA microarray array to screen for differentially expressed genes (DEGs) between human DP and PDL tissues, and used the online software WebGestalt to perform the functional analysis of the DEGs. In addition, the STRING database and KEGG pathway analysis were applied for interaction network and pathway analysis of the DEGs. DP and PDL samples were obtained from permanent premolars (n=16) extracted for orthodontic purposes. The results of the microarray assay were confirmed by RT-qPCR. The DEGs were found to be significantly associated with the extracellular matrix and focal adhesion. A total of 10 genes were selected to confirm the results. The mRNA levels of integrin alpha 4 (ITGA4), integrin alpha 8 (ITGA8), neurexin 1 (NRXN1) and contactin 1 (CNTN1) were significantly higher in the DP than in the PDL tissues. However, the levels of collagen type XI alpha 1 (COL11A1), aggrecan (ACAN), collagen type VI alpha 1 (COL6A1), chondroadherin (CHAD), laminin gamma 2 (LAMC2) and laminin alpha 3 (LAMA3) were higher in the PDL than in the DP samples. The gene expression profiles provide novel insight into the characterization of DP and PDL tissues, and contribute to our understanding of the potential molecular mechanisms of dental tissue mineralization and regeneration. PMID:28713908

Cloning of the active thymidine kinase gene of herpes simplex virus type 1 in Escherichia coli K-12.

PubMed

Colbere-Garapin, F; Chousterman, S; Horodniceanu, F; Kourilsky, P; Garapin, A C

1979-08-01

A herpes simplex virus DNA fragment that is produced by digestion with BamHI endonuclease and carries the thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene has been cloned in Escherichia coli. A recombinat plasmid, pFG5, has been analyzed extensively and a detailed restriction map is presented. pFG5 DNA efficiently transforms TK- mouse L cells. The TK coding sequence in the cloned fragment has been localized and a smaller recombinant plasmid, pAG0, also carrying an active TK gene, has been constructed to serve as a more convenient vector for transfer, into TK- cells, of genes previously cloned in E. coli.

The Splitting of Double-component Active Asteroid P/2016 J1 (PANSTARRS)

NASA Astrophysics Data System (ADS)

Moreno, F.; Pozuelos, F. J.; Novaković, B.; Licandro, J.; Cabrera-Lavers, A.; Bolin, Bryce; Jedicke, Robert; Gladman, Brett J.; Bannister, Michele T.; Gwyn, Stephen D. J.; Vereš, Peter; Chambers, Kenneth; Chastel, Serge; Denneau, Larry; Flewelling, Heather; Huber, Mark; Schunová-Lilly, Eva; Magnier, Eugene; Wainscoat, Richard; Waters, Christopher; Weryk, Robert; Farnocchia, Davide; Micheli, Marco

2017-03-01

We present deep imaging observations, orbital dynamics, and dust-tail model analyses of the double-component asteroid P/2016 J1 (J1-A and J1-B). The observations were acquired at the Gran Telescopio Canarias (GTC) and the Canada-France-Hawaii Telescope (CFHT) from mid-March to late July of 2016. A statistical analysis of backward-in-time integrations of the orbits of a large sample of clone objects of P/2016 J1-A and J1-B shows that the minimum separation between them occurred most likely ˜2300 days prior to the current perihelion passage, I.e., during the previous orbit near perihelion. This closest approach was probably linked to a fragmentation event of their parent body. Monte Carlo dust-tail models show that those two components became active simultaneously ˜250 days before the current perihelion, with comparable maximum loss rates of ˜0.7 and ˜0.5 kg s-1, and total ejected masses of 8 × 106 and 6 × 106 kg for fragments J1-A and J1-B, respectively. Consequently, the fragmentation event and the present dust activity are unrelated. The simultaneous activation times of the two components and the fact that the activity lasted 6-9 months or longer, strongly indicate ice sublimation as the most likely mechanism involved in the dust emission process.

Deletion of PRKAA triggers mitochondrial fission by inhibiting the autophagy-dependent degradation of DNM1L.

PubMed

Wang, Qilong; Wu, Shengnan; Zhu, Huaiping; Ding, Ye; Dai, Xiaoyan; Ouyang, Changhan; Han, Young-Min; Xie, Zhonglin; Zou, Ming-Hui

2017-02-01

PRKAA (protein kinase, AMP-activated, α catalytic subunit) regulates mitochondrial biogenesis, function, and turnover. However, the molecular mechanisms by which PRKAA regulates mitochondrial dynamics remain poorly characterized. Here, we report that PRKAA regulated mitochondrial fission via the autophagy-dependent degradation of DNM1L (dynamin 1-like). Deletion of Prkaa1/AMPKα1 or Prkaa2/AMPKα2 resulted in defective autophagy, DNM1L accumulation, and aberrant mitochondrial fragmentation in the mouse aortic endothelium. Furthermore, autophagy inhibition by chloroquine treatment or ATG7 small interfering RNA (siRNA) transfection, upregulated DNM1L expression and triggered DNM1L-mediated mitochondrial fragmentation. In contrast, autophagy activation by overexpression of ATG7 or chronic administration of rapamycin, the MTOR inhibitor, promoted DNM1L degradation and attenuated mitochondrial fragmentation in Prkaa2-deficient (prkaa2 -/- ) mice, suggesting that defective autophagy contributes to enhanced DNM1L expression and mitochondrial fragmentation. Additionally, the autophagic receptor protein SQSTM1/p62, which bound to DNM1L and led to its translocation into the autophagosome, was involved in DNM1L degradation by the autophagy-lysosome pathway. Gene silencing of SQSTM1 markedly reduced the association between SQSTM1 and DNM1L, impaired the degradation of DNM1L, and enhanced mitochondrial fragmentation in PRKAA-deficient endothelial cells. Finally, the genetic (DNM1L siRNA) or pharmacological (mdivi-1) inhibition of DNMA1L ablated mitochondrial fragmentation in the mouse aortic endothelium and prevented the acetylcholine-induced relaxation of isolated mouse aortas. This suggests that aberrant DNM1L is responsible for enhanced mitochondrial fragmentation and endothelial dysfunction in prkaa knockout mice. Overall, our results show that PRKAA deletion promoted mitochondrial fragmentation in vascular endothelial cells by inhibiting the autophagy-dependent degradation of DNM1L.

Traces of pFc' in IVIG interact with human IgG Fc domains and counteract aggregation.

PubMed

Rispens, Theo; Himly, Martin; Ooievaar-De Heer, Pleuni; den Bleker, Tamara H; Aalberse, Rob C

2010-04-16

To prevent multimer formation, intravenous immunoglobulin (IVIG) is often treated with traces of pepsin. So far, the mechanism behind this treatment has been unclear. Recently, we reported that human IgG4 binds other IgG molecules via Fc-Fc interactions. Here we show that IVIG treated with traces of pepsin (Nanogam) inhibits these interactions. We found that--besides IgG4--peptides corresponding to IgG1 and IgG2 pFc' (products of limited pepsin digestion) are responsible for the inhibitory action. Using radiolabeled pFc', it was found that pFc' binds directly to IgG1. Furthermore, recombinant CH3 fragments were found to also possess binding activity, and potencies of inhibition varied over 3 orders of magnitude amongst the subclasses, IgG4 being most potent. We propose that pFc' formation explains how limited pepsin digestion diminishes adverse effects of IVIG. In particular, the presence of this fragment can enhance the stability of IgG products including IVIG and therapeutical monoclonal antibodies. Indeed, using a model system it was found that acid-induced aggregation of IgG is reduced in the presence of pFc', suggesting a 'chaperone-like' activity of this fragment. Thus, pFc' can modulate Fc interactions and may therefore reduce adverse effects of IVIG, in particular by preventing oligomerization. 2010 Elsevier B.V. All rights reserved.

Production of a Recombinant E. coli Expressed Malarial Vaccine from the C-Terminal Fragment of Plasmodium Falciparum 3D7 Merozoite Surface Protein-1.

DTIC Science & Technology

2000-04-01

Reportable Outcomes 1. Manuscript in preparation: Evelina Angov, Barbara Aufiero, Michel Van Handenhove, Christian Ockenhouse, Kent Kester, Doug Walsh, Jana...Burnouf, T., Ouattara, D., Attanath, P., Bouharoun-Tayoun, H., Chantavanich, P., Foucault , C., Chongsuphajaisiddhi, T., and Druilhe, P. (1991

Unique Footprint in the scl1.3 Locus Affects Adhesion and Biofilm Formation of the Invasive M3-Type Group A Streptococcus.

PubMed

Bachert, Beth A; Choi, Soo J; LaSala, Paul R; Harper, Tiffany I; McNitt, Dudley H; Boehm, Dylan T; Caswell, Clayton C; Ciborowski, Pawel; Keene, Douglas R; Flores, Anthony R; Musser, James M; Squeglia, Flavia; Marasco, Daniela; Berisio, Rita; Lukomski, Slawomir

2016-01-01

The streptococcal collagen-like proteins 1 and 2 (Scl1 and Scl2) are major surface adhesins that are ubiquitous among group A Streptococcus (GAS). Invasive M3-type strains, however, have evolved two unique conserved features in the scl1 locus: (i) an IS1548 element insertion in the scl1 promoter region and (ii) a nonsense mutation within the scl1 coding sequence. The scl1 transcript is drastically reduced in M3-type GAS, contrasting with a high transcription level of scl1 allele in invasive M1-type GAS. This leads to a lack of Scl1 expression in M3 strains. In contrast, while scl2 transcription and Scl2 production are elevated in M3 strains, M1 GAS lack Scl2 surface expression. M3-type strains were shown to have reduced biofilm formation on inanimate surfaces coated with cellular fibronectin and laminin, and in human skin equivalents. Repair of the nonsense mutation and restoration of Scl1 expression on M3-GAS cells, restores biofilm formation on cellular fibronectin and laminin coatings. Inactivation of scl1 in biofilm-capable M28 and M41 strains results in larger skin lesions in a mouse model, indicating that lack of Scl1 adhesin promotes bacterial spread over localized infection. These studies suggest the uniquely evolved scl1 locus in the M3-type strains, which prevents surface expression of the major Scl1 adhesin, contributed to the emergence of the invasive M3-type strains. Furthermore these studies provide insight into the molecular mechanisms mediating colonization, biofilm formation, and pathogenesis of group A streptococci.

Fluid Viscosity Affects the Fragmentation and Inertial Cavitation Threshold of Lipid-Encapsulated Microbubbles.

PubMed

Helfield, Brandon; Black, John J; Qin, Bin; Pacella, John; Chen, Xucai; Villanueva, Flordeliza S

2016-03-01

Ultrasound and microbubble optimization studies for therapeutic applications are often conducted in water/saline, with a fluid viscosity of 1 cP. In an in vivo context, microbubbles are situated in blood, a more viscous fluid (∼4 cP). In this study, ultrahigh-speed microscopy and passive cavitation approaches were employed to investigate the effect of fluid viscosity on microbubble behavior at 1 MHz subject to high pressures (0.25-2 MPa). The propensity for individual microbubble (n = 220) fragmentation was found to significantly decrease in 4-cP fluid compared with 1-cP fluid, despite achieving similar maximum radial excursions. Microbubble populations diluted in 4-cP fluid exhibited decreased wideband emissions (up to 10.2 times), and increasingly distinct harmonic emission peaks (e.g., ultraharmonic) with increasing pressure, compared with those in 1-cP fluid. These results suggest that in vitro studies should consider an evaluation using physiologic viscosity perfusate before transitioning to in vivo evaluations. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Sarcospan: a small protein with large potential for Duchenne muscular dystrophy

PubMed Central

2013-01-01

Purification of the proteins associated with dystrophin, the gene product responsible for Duchenne muscular dystrophy, led to the discovery of the dystrophin-glycoprotein complex. Sarcospan, a 25-kDa transmembrane protein, was the last component to be identified and its function in skeletal muscle has been elusive. This review will focus on progress over the last decade revealing that sarcospan is an important regulator of muscle cell adhesion, strength, and regeneration. Investigations using several transgenic mouse models demonstrate that overexpression of sarcospan in the mouse model for Duchenne muscular dystrophy ameliorates pathology and restores muscle cell binding to laminin. Sarcospan improves cell surface expression of the dystrophin- and utrophin-glycoprotein complexes as well as α7β1 integrin, which are the three major laminin-binding complexes in muscle. Utrophin and α7β1 integrin compensate for the loss of dystrophin and the finding that sarcospan increases their abundance at the extra-synaptic sarcolemma supports the use of sarcospan as a therapeutic target. Newly discovered phenotypes in sarcospan-deficient mice, including a reduction in specific force output and increased drop in force in the diaphragm muscle, result from decreased utrophin and dystrophin expression and further reveal sarcospan’s role in determining abundance of these complexes. Dystrophin protein levels and the specific force output of the diaphragm muscle are further reduced upon genetic removal of α7 integrin (Itga7) in SSPN-deficient mice, demonstrating that interactions between integrin and sarcospan are critical for maintenance of the dystrophin-glycoprotein complex and force production of the diaphragm muscle. Sarcospan is a major regulator of Akt signaling pathways and sarcospan-deficiency significantly impairs muscle regeneration, a process that is dependent on Akt activation. Intriguingly, sarcospan regulates glycosylation of a specific subpopulation of α-dystroglycan, the laminin-binding receptor associated with dystrophin and utrophin, localized to the neuromuscular junction. Understanding the basic mechanisms responsible for assembly and trafficking of the dystrophin- and utrophin-glycoprotein complexes to the cell surface is lacking and recent studies suggest that sarcospan plays a role in these essential processes. PMID:23282144

Evolution of post-ESWL residual lithiasis depending on the type of calculus and urine composition.

PubMed

Grases, Felix; Costa-Bauzá, Antonia; Isern, Bernat; Sanchis, Pilar; Perelló, Joan; Hierro, Fernando; Conte Visus, Antonio

2009-07-01

Extracorporeal shock wave lithotripsy (ESWL) is one of the most commonly used procedures for removal of renal calculi from the upper urinary tract, but complete expulsion of the fragments generated is not always achieved. This can lead to new lithiasic episodes, and it is considered that 10-26% of fragmented calculi can undergo regrowth. This in vitro study investigated the influence of fragment and urinary composition on post-ESWL growth of fragments, with the aims of establishing the effect and importance of these parameters, and identifying effective prophylactic measures. Fragments collected from patients immediately following expulsion after ESWL treatment were selected for regrowth experiments. The particles included 24 calcium oxalate monohydrate (COM) fragments, 48 calcium oxalate dihydrate (COD), 24 hydroxyapatite (HAP), and 16 uric acid. In all treatments, calculi fragments showed a considerable capacity to induce growth of calcium oxalate and calcium phosphate. Under normocalciuria conditions, new COM crystals formed; both COM and COD crystals developed under hypercalciuria conditions at a urinary pH < 6.0; and in hypercalciuric conditions and urinary pH > 6.0 both HAP and brushite (BRU) crystals were formed. The highest growth rates were observed for COD calculi fragments under hypercalciuria conditions and at a urinary pH of 6.5, followed by growth on COM and HAP fragments under the same conditions; growth rates under other conditions tested were similar but 10-fold lower. With regard to the role of crystallization inhibitors, phytate exhibited inhibitory effects under all assay conditions. However, citrate had little effect, even at the highest concentration tested (1,000 mg/L). This study demonstrates the importance of avoiding heterogeneous nucleant retention (pre-existing solid microparticles) in renal cavities, as these can act as very efficient inducers of the formation of new calculi, the composition of which is mainly dependant on the urine composition.

Molecular Mechanism of Active Zone Organization at Vertebrate Neuromuscular Junctions

PubMed Central

Nishimune, Hiroshi

2013-01-01

Organization of presynaptic active zones is essential for development, plasticity, and pathology of the nervous system. Recent studies indicate a trans-synaptic molecular mechanism that organizes the active zones by connecting the pre- and the postsynaptic specialization. The presynaptic component of this trans-synaptic mechanism is comprised of cytosolic active zone proteins bound to the cytosolic domains of voltage-dependent calcium channels (P/Q-, N-, and L-type) on the presynaptic membrane. The postsynaptic component of this mechanism is the synapse organizer (laminin β2) that is expressed by the postsynaptic cell and accumulates specifically on top of the postsynaptic specialization. The pre- and the postsynaptic components interact directly between the extracellular domains of calcium channels and laminin β2 to anchor the presynaptic protein complex in front of the postsynaptic specialization. Hence, the presynaptic calcium channel functions as a scaffolding protein for active zone organization and as an ion-conducting channel for synaptic transmission. In contrast to the requirement of calcium influx for synaptic transmission, the formation of the active zone does not require the calcium influx through the calcium channels. Importantly, the active zones of adult synapses are not stable structures and require maintenance for their integrity. Furthermore, aging or diseases of the central and peripheral nervous system impair the active zones. This review will focus on the molecular mechanisms that organize the presynaptic active zones and summarize recent findings at the neuromuscular junctions and other synapses. PMID:22135013

TCDD disrupts posterior palatogenesis and causes cleft palate.

PubMed

Yamada, Tomohiro; Hirata, Azumi; Sasabe, Eri; Yoshimura, Tomohide; Ohno, Seiji; Kitamura, Naoya; Yamamoto, Tetsuya

2014-01-01

Dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) cause cleft palate at a high rate. A post-fusional split may contribute to the pathogenesis, and tissue fragility may be a concern. The objective of this study was to investigate the effects of TCDD on the palatal epithelium, bone and muscle, which contribute to tissue integrity. ICR mice (10-12 weeks old) were used. TCDD was administered on E12.5 at 40 mg/kg. Immunohistochemical staining for AhR, ER-α, laminin, collagen IV, osteopontin, Runx2, MyoD, and desmin were performed. Furthermore, western blot analysis for osteopontin, Runx2, MyoD, and desmin were performed to evaluate protein expression in the palatal tissue. Immunohistologically, there was little difference in the collagen IV and laminin localization in the palatal epithelium between control versus TCDD-treated mice. Runx2 and osteopontin immunoreactivity decreased in the TCDD-treated palatal bone, and MyoD and desmin decreased in the TCDD-treated palatal muscle. AhR and ER-α immunoreactivity were localized to the normal palatal bone, but ER-α was diminished in the TCDD-treated palate. On western blot analysis, Runx2, MyoD, and desmin were all downregulated in the TCDD-treated palate. TCDD may suppress palatal osteogenesis and myogenesis via AhR, and cause cleft palates via a post-fusional split mechanism, in addition to a failure of palatal fusion. Copyright © 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Biofunctionalized peptide-based hydrogels provide permissive scaffolds to attract neurite outgrowth from spiral ganglion neurons.

PubMed

Frick, Claudia; Müller, Marcus; Wank, Ute; Tropitzsch, Anke; Kramer, Benedikt; Senn, Pascal; Rask-Andersen, Helge; Wiesmüller, Karl-Heinz; Löwenheim, Hubert

2017-01-01

Cochlear implants (CI) allow for hearing rehabilitation in patients with sensorineural hearing loss or deafness. Restricted CI performance results from the spatial gap between spiral ganglion neurons and the CI, causing current spread that limits spatially restricted stimulation and impairs frequency resolution. This may be substantially improved by guiding peripheral processes of spiral ganglion neurons towards and onto the CI electrode contacts. An injectable, peptide-based hydrogel was developed which may provide a permissive scaffold to facilitate neurite growth towards the CI. To test hydrogel capacity to attract spiral ganglion neurites, neurite outgrowth was quantified in an in vitro model using a custom-designed hydrogel scaffold and PuraMatrix ® . Neurite attachment to native hydrogels is poor, but significantly improved by incorporation of brain-derived neurotrophic factor (BDNF), covalent coupling of the bioactive laminin epitope IKVAV and the incorporation a full length laminin to hydrogel scaffolds. Incorporation of full length laminin protein into a novel custom-designed biofunctionalized hydrogel (IKVAV-GGG-SIINFEKL) allows for neurite outgrowth into the hydrogel scaffold. The study demonstrates that peptide-based hydrogels can be specifically biofunctionalized to provide a permissive scaffold to attract neurite outgrowth from spiral ganglion neurons. Such biomaterials appear suitable to bridge the spatial gap between neurons and the CI. Copyright © 2016 Elsevier B.V. All rights reserved.

Three-layer microfibrous peripheral nerve guide conduit composed of elastin-laminin mimetic artificial protein and poly(L-lactic acid)

NASA Astrophysics Data System (ADS)

Kakinoki, Sachiro; Nakayama, Midori; Moritan, Toshiyuki; Yamaoka, Tetsuji

2014-07-01

We developed a microfibrous poly(L-lactic acid) (PLLA) nerve conduit with a three-layered structure to simultaneously enhance nerve regeneration and prevent adhesion of surrounding tissue. The inner layer was composed of PLLA microfiber containing 25% elastin-laminin mimetic protein (AG73-(VPGIG)30) that promotes neurite outgrowth. The thickest middle layer was constructed of pure PLLA microfibers that impart the large mechanical stremgth to the conduit. A 10% poly(ethylene glycol) was added to the outer layer to prevent the adhesion with the surrounding tissue. The AG73-(VPGIG)30 composisting of an elastin-like repetitive sequence (VPGIG)30 and a laminin-derived sequence (RKRLQVQLSIRT: AG73) was biosynthesized using Escherichia coli. The PLLA microfibrous conduits were fabricated using an electrospinning procedure. AG73-(VPGIG)30 was successfully mixed in the PLLA microfibers, and the PLLA/AG73-(VPGIG)30 microfibers were stable under physiological conditions. The PLLA/AG73-(VPGIG)30 microfibers enhanced adhesion and neurite outgrowth of PC12 cells. The electrospun microfibrous conduit with a three-layered structure was implanted for bridging a 2.0-cm gap in the tibial nerve of a rabbit. Two months after implantation, no adhesion of surrounding tissue was observed, and the action potential was slightly improved in the nerve conduit with the PLLA/AG73-(VPGIG)30 inner layer.

Exocellular extract of Fusarium oxysporum, fungus free, is able to permeate and act selectively in skin.

PubMed

Sibin Melo, Katia C; Correia, Marcelo H; Svidzinski, Terezinha I E; Hernandes, Luzmarina

2018-05-01

The skin is an important gateway for Fusarium infection in humans. Our hypothesis is that metabolites produced by Fusarium oxysporum should change the barrier structure to permeate the skin. Male Wistar rats received a topical application of a solution (0.05 mg/mL) of Fusarium metabolites. The animals were euthanized 3, 6, 12, 24 h after and the skin was processed for immunostaining by laminin and E-cadherin to investigate whether the Fusarium metabolites can break the barrier of healthy skin. Other techniques were employed: H&E to study the morphology; metalloproteinase-9 (MMP-9), TUNEL, and PCNA immunostaining to evaluate the inflammation, cell death, and proliferation, respectively. There was an inflammatory response mainly centered in the dermis. Qualitatively, the skin of the experimental group showed reduced E-cadherin and laminin immunostaining at 3, 12, and 24 h. Higher intensity staining by TUNEL at 3 h, and PCNA at 6, 12, and 24 h. There was intense MMP-9 activity at 6, 12, and 24 h. None of analyses revealed any changes in the epidermis. It was concluded that the fraction was able to permeate the skin and act selectively in dermis, inducing inflammatory response, increasing MMP-9 immunostaining, inducing apoptosis, and reducing E-cadherin and laminin immunostaining. © 2018 APMIS. Published by John Wiley & Sons Ltd.