scholarly journals Characterization of an Adenovirus Vector Containing a Heterologous Peptide Epitope in the HI Loop of the Fiber Knob

1998 ◽  
Vol 72 (3) ◽  
pp. 1844-1852 ◽  
Author(s):  
Victor Krasnykh ◽  
Igor Dmitriev ◽  
Galina Mikheeva ◽  
C. Ryan Miller ◽  
Natalya Belousova ◽  
...  
Keyword(s):  
Recombinant Adenovirus ◽  
Adenovirus Vector ◽  
Cell Types ◽  
Peptide Sequence ◽  
Cell Surface Receptor ◽  
Cell Binding ◽  
Peptide Epitope ◽  
Present Generation ◽  
Surface Receptor

ABSTRACT The utility of the present generation of recombinant adenovirus vectors for gene therapy applications could potentially be improved by designing targeted vectors capable of gene delivery to selected cell types in vivo. In order to achieve such targeting, we are investigating the possibilities of incorporation of ligands in the adenovirus fiber protein, which mediates primary binding of adenovirus to its cell surface receptor. Based on the proposed structure of the cell-binding domain of the fiber, we hypothesized that the HI loop of the fiber knob can be utilized as a convenient locale for incorporation of heterologous ligands. In this study, we utilized recombinant fiber proteins expressed in baculovirus-infected insect cells to demonstrate that the incorporation of the FLAG octapeptide into the HI loop does not ablate fiber trimerization and does not disturb formation of the cell-binding site localized in the knob. We then generated a recombinant adenovirus containing this modified fiber and showed that the short peptide sequence engineered in the knob is compatible with the biological functions of the fiber. In addition, by using a ligand-specific antibody, we have shown that the peptide incorporated into the knob remains available for binding in the context of mature virions containing modified fibers. These findings suggest that heterologous ligands can be incorporated into the HI loop of the fiber knob and that this locale possesses properties consistent with its employment in adenovirus retargeting strategies.

scholarly journals In vivo paracrine interaction between urokinase and its receptor: effect on tumor cell invasion.

1991 ◽  
Vol 115 (4) ◽  
pp. 1107-1112 ◽  
Author(s):  
L Ossowski ◽  
G Clunie ◽  
M T Masucci ◽  
F Blasi
Keyword(s):  
Chorioallantoic Membrane ◽  
Fold Increase ◽  
Cell Types ◽  
Cell Surface Receptor ◽  
In Vivo Model ◽  
Specific Cell ◽  
Upa Receptor ◽  
Surface Receptor

Numerous studies have linked the production of increased levels of urokinase type plasminogen activator (uPA) with the malignant phenotype. It has also been shown that a specific cell surface receptor can bind uPA through a domain distinct and distant from the proteolytic domain. In an in vivo model of invasion, consisting of experimentally modified chorioallantoic membrane (CAM) of a chick embryo, only cells that concurrently expressed both uPA and a receptor for uPA, and in which the receptor was saturated with uPA, were efficient in invasion. To test whether uPA produced by one cell can, in a paracrine fashion, affect the invasive capacity of a receptor-expressing cell, we transfected LB6 mouse cells with human uPA (LB6[uPA]), or human uPA-receptor cDNA (LB6[uPAR]). LB6(uPA) cells released into the medium 1-2 Ploug units of human uPA per 10(6) cells in 24 h. The LB6(uPAR) cells expressed on their surface approximately 12,000 high affinity (Kd 1.7 x 10(-10) M uPA binding sites per cell. Unlabeled LB6(uPA) and 125-IUdR-labeled LB6(uPAR) cells were coinoculated onto experimentally wounded and resealed CAMs and their invasion was compared to that of homologous mixtures of labeled and unlabeled LB6(uPAR) or LB6(uPA) cells. Concurrent presence of both cell types in the CAMs resulted in a 1.8-fold increase of invasion of the uPA-receptor expressing cells. A four-fold stimulation of invasion was observed when cells were cocultured in vitro, prior to in vivo inoculation. Enhancement of invasion was prevented in both sets of experiments by treatment with specific antihuman uPA antibodies, indicating that uPA was the main mediator of the invasion-enhancing, paracrine effect on the receptor-expressing cells.

scholarly journals A mouse Fas-associated protein with homology to the human Mort1/FADD protein is essential for Fas-induced apoptosis.

1996 ◽  
Vol 16 (6) ◽  
pp. 2756-2763 ◽  
Author(s):  
J Zhang ◽  
A Winoto
Keyword(s):  
Tumor Necrosis Factor Receptor ◽  
Cell Types ◽  
Cell Surface Receptor ◽  
Mouse Cdna ◽  
Acid Protein ◽  
Surface Receptor ◽  
Induced Apoptosis ◽  
Necrosis Factor ◽  
Amino Acid Protein

The Fas cell surface receptor belongs to the tumor necrosis factor receptor family and can initiate apoptosis in a variety of cell types. Using the Fas cytoplasmic domain as bait in a yeast two-hybrid screening, we isolated a mouse cDNA encoding a 205-amino-acid protein. Its predicted protein sequence shows 68% identity and 80% similarity with the sequence of recently described human Mort/FADD. This protein, most likely the mouse homolog of human FADD, associates with Fas in vivo only upon the induction of cell death. A fraction of this protein is highly phosphorylated at serine/threonine residues, with both phosphorylated and unphosphorylated forms being capable of binding to FAS. Stable expression of a truncated form of the Mort/FADD protein protects cells from Fas-mediated apoptosis by interfering with the wild-type protein-Fas interaction. Thus, mouse Mort/FADD is an essential downstream component that mediates Fas-induced apoptosis.

scholarly journals RGD-containing peptides inhibit the synthesis of myelin-like membrane by cultured oligodendrocytes.

1988 ◽  
Vol 107 (4) ◽  
pp. 1551-1559 ◽  
Author(s):  
M C Cardwell ◽  
L H Rome
Keyword(s):  
Morphological Characteristics ◽  
Cell Surface Receptor ◽  
External Control ◽  
Adhesion Assay ◽  
Cell Binding ◽  
Surface Receptor ◽  
Dependent Cell ◽  
Vitro Development

A synthetic peptide derived from the fibronectin cell-binding domain, GRGDSP, inhibits the adhesion of rat oligodendrocytes to a number of substrates. However, while GRGDSP inhibited the adhesion of cells in a short term adhesion assay, the presence of the peptide did not prevent cells from adhering and thriving in longer term culture. The morphological characteristics of individual cells cultured with 0.1 mg/ml GRGDSP were similar to untreated cultures; small rounded cell bodies radiating numerous fine processes. Peptide-treated cultures were inhibited in their ability to produce myelin specific components. The characteristic developmental peak in sulfolipid synthesis which occurs both in vivo and in vitro was completely inhibited when cells were cultured with GRGDSP. In addition, the synthesis of myelin basic protein was inhibited. Ultrastructurally, cells treated with GRGDSP showed a greatly reduced number of multilamellar myelin-like membrane figures than cells grown without peptide or those grown with GRADSP. Cultured oligodendrocytes did not become sensitive to inhibition of sulfolipid synthesis by GRGDSP until a period immediately preceding the peak in sulfolipid biosynthesis. The effects of pretreatment with peptide for 5 d before this time were completely reversible. Pretreatment which extended into the time of peak myelin synthesis resulted in permanent impairment in the cell's ability to synthesize sulfolipid. The oligodendrocyte's ability to synthesize a myelin-like membrane in culture is, in part, inherent since it occurs in the absence of neurons. The present results indicate that myelin membrane production is also subject to external control since it appears that occupancy of an RGD-dependent cell surface receptor during a critical period of in vitro development is required for the oligodendrocyte to produce myelin-like membrane.

scholarly journals Endocytic Delivery of Vancomycin Mediated by a Synthetic Cell Surface Receptor:  Rescue of Bacterially Infected Mammalian Cells and Tissue Targeting In Vivo

2007 ◽  
Vol 129 (2) ◽  
pp. 268-269 ◽  
Author(s):  
Siwarutt Boonyarattanakalin ◽  
Jianfang Hu ◽  
Sheryl A. Dykstra-Rummel ◽  
Avery August ◽  
Blake R. Peterson
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Cell Surface Receptor ◽  
Synthetic Cell ◽  
Tissue Targeting ◽  
Surface Receptor

Regulation of N-myc gene expression: use of an adenovirus vector to demonstrate posttranscriptional control

1990 ◽  
Vol 10 (12) ◽  
pp. 6700-6708
Author(s):  
L E Babiss ◽  
J M Friedman
Keyword(s):  
Recombinant Adenovirus ◽  
Wilms Tumor ◽  
Fetal Brain ◽  
Adenovirus Vector ◽  
Cell Types ◽  
Adult Brain ◽  
Posttranscriptional Control ◽  
Equivalent Rate ◽  
Myc Gene ◽  
Recombinant Adenovirus Vector

We present evidence that differences in the levels of N-myc mRNA among different cell types are the result of posttranscriptional control. First, we noted that while steady-state mouse N-myc mRNA could be detected only in fetal mouse brain, it was transcribed at an equivalent rate in adult brain, liver, spleen, and placenta and in fetal brain. Similarly, the human N-myc gene was transcribed at an equivalent rate in HeLa cells, which do not accumulate this RNA in the cytoplasm, and cell lines G401 (a Wilms tumor-derived cell line) and SKNMc (established from a primitive neuroepithelioma), which do express N-myc RNA. As expected, the N-myc promoter functioned at equivalent rates, as demonstrated by the level of a reporter gene, when introduced into these cell types by using a recombinant adenovirus vector. The suggestion that posttranscriptional mechanisms control the level of this RNA was supported by the observation that sequences in the N-myc third exon specifically decreased the level of E1A mRNA when these sequences were placed downstream of the E1A promoter in a recombinant adenovirus. Finally, we further localized these sequences to a 600-bp fragment of the third exon by introducing various subclones of this sequence downstream of the E1A promoter in both viral and plasmid vectors.

scholarly journals Porcine Breast Extracellular Matrix Hydrogel for Spatial Tissue Culture

2018 ◽  
Vol 19 (10) ◽  
pp. 2912 ◽  
Author(s):  
Girdhari Rijal ◽  
Jing Wang ◽  
Ilhan Yu ◽  
David Gang ◽  
Roland Chen ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Human Mammary Epithelial Cell ◽  
Tumor Formation ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Breast Diseases ◽  
Porous Scaffolds ◽  
Ecm Proteins ◽  
Surface Receptor

Porcine mammary fatty tissues represent an abundant source of natural biomaterial for generation of breast-specific extracellular matrix (ECM). Here we report the extraction of total ECM proteins from pig breast fatty tissues, the fabrication of hydrogel and porous scaffolds from the extracted ECM proteins, the structural properties of the scaffolds (tissue matrix scaffold, TMS), and the applications of the hydrogel in human mammary epithelial cell spatial cultures for cell surface receptor expression, metabolomics characterization, acini formation, proliferation, migration between different scaffolding compartments, and in vivo tumor formation. This model system provides an additional option for studying human breast diseases such as breast cancer.

scholarly journals Recombinant adenovirus causes prolonged mobilization of macrophages in the anterior chamber of mice

2021 ◽  
Author(s):  
Kacie J Meyer ◽  
Danielle Pellack ◽  
Adam J Hedberg-Buenz ◽  
Nicholas Pomernackas ◽  
Dana Soukup ◽  
...  
Keyword(s):  
Recombinant Adenovirus ◽  
Cell Types ◽  
Slit Lamp ◽  
Correct Treatment ◽  
Intraocular Injection ◽  
Lack Of Information ◽  
Corneal Opacities ◽  
Corneal Damage ◽  
Sequential Images

Purpose: Ocular tissues of mice have been studied in many ways using replication deficient species C type 5 adenoviruses (Ad5) as tools for manipulating gene expression. While refinements to injection protocols and tropism have led to several advances in targeting cells of interest, there remains a relative lack of information concerning how Ad5 may influence other ocular cell types capable of confounding experimental interpretation. Here, a slit-lamp is used to thoroughly photodocument the sequalae of intraocular Ad5 injections over time in mice, with attention to potentially confounding indices of inflammation. Methods: A cohort of C57BL/6J mice were randomly split into 3 groups (Virus, receiving unilateral intraocular injection with 5x107 pfu of a cargo-less Ad5 construct; Saline, receiving unilateral balanced salt solution injection; and Naïve, receiving no injections). A total of 52 eyes from 26 mice were photodocumented via slit-lamp at 4 timepoints (baseline, 1, 3, and 10 weeks following initiation of the experiment) by an observer masked to treatments and other parameters of the experimental design. Following the last in vivo exam, tissues were collected. Based on the slit-lamp data, tissues were studied via immunostaining with the macrophage marker F4/80. Results: The masked investigator was able to use the sequential images from each mouse to assign each mouse into its correct treatment group with near perfect fidelity. Virus injected eyes were characterized by corneal damage indicative of intraocular injection and a prolonged mobilization of clump cells on the surface of the iris. Saline injected eyes had only transient corneal opacities indicative of intraocular injections, and Naïve eyes remained normal. Immunostaining with F4/80 was consistent with ascribing the clump cells visualized via slit-lamp imaging as a type of macrophage.

scholarly journals Extracellular interactions and ligand degradation shape the nodal morphogen gradient

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Yin Wang ◽  
Xi Wang ◽  
Thorsten Wohland ◽  
Karuna Sampath
Keyword(s):  
Correlation Spectroscopy ◽  
Cell Surface Receptor ◽  
Morphogen Gradient ◽  
Axis Formation ◽  
Cellular Interactions ◽  
Fluorescence Correlation ◽  
Selective Ligand ◽  
Surface Receptor ◽  
First Time

The correct distribution and activity of secreted signaling proteins called morphogens is required for many developmental processes. Nodal morphogens play critical roles in embryonic axis formation in many organisms. Models proposed to generate the Nodal gradient include diffusivity, ligand processing, and a temporal activation window. But how the Nodal morphogen gradient forms in vivo remains unclear. Here, we have measured in vivo for the first time, the binding affinity of Nodal ligands to their major cell surface receptor, Acvr2b, and to the Nodal inhibitor, Lefty, by fluorescence cross-correlation spectroscopy. We examined the diffusion coefficient of Nodal ligands and Lefty inhibitors in live zebrafish embryos by fluorescence correlation spectroscopy. We also investigated the contribution of ligand degradation to the Nodal gradient. We show that ligand clearance via degradation shapes the Nodal gradient and correlates with its signaling range. By computational simulations of gradient formation, we demonstrate that diffusivity, extra-cellular interactions, and selective ligand destruction collectively shape the Nodal morphogen gradient.

scholarly journals Characterization of a type IV collagen major cell binding site with affinity to the alpha 1 beta 1 and the alpha 2 beta 1 integrins.

1991 ◽  
Vol 113 (6) ◽  
pp. 1475-1483 ◽  
Author(s):  
P Vandenberg ◽  
A Kern ◽  
A Ries ◽  
L Luckenbill-Edds ◽  
K Mann ◽  
...  
Keyword(s):  
Binding Site ◽  
Type Iv Collagen ◽  
Cell Attachment ◽  
Cell Surface Receptor ◽  
Helical Conformation ◽  
Amino Acid Residues ◽  
Cell Binding ◽  
Type Iv ◽  
Depth Study ◽  
Surface Receptor

The aim of this investigation was to identify the domains of type IV collagen participating in cell binding and the cell surface receptor involved. A major cell binding site was found in the trimeric cyanogen bromide-derived fragment CB3, located 100 nm away from the NH2 terminus of the molecule, in which the triple-helical conformation is stabilized by interchain disulfide bridges. Cell attachment assays with type IV collagen and CB3 revealed comparable cell binding activities. Antibodies against CB3 inhibited attachment on fragment CB3 completely and on type IV collagen to 80%. The ability to bind cells was strictly conformation dependent. Four trypsin derived fragments of CB3 allowed a closer investigation of the binding site. The smallest, fully active triple-helical fragment was (150)3-amino acid residues long. It contained segments of 27 and 37 residues, respectively, at the NH2 and COOH terminus, which proved to be essential for cell binding. By affinity chromatography on Sepharose-immobilized CB3, two receptor molecules of the integrin family, alpha 1 beta 1 and alpha 2 beta 1, were isolated. Their subunits were identified by sequencing the NH2 termini or by immunoblotting. The availability of fragment CB3 will allow for a more in-depth study of the molecular interaction of a short, well defined triple-helical ligand with collagen receptors alpha 1 beta 1 and alpha 2 beta 1.

scholarly journals A ligand-based system for receptor-specific delivery of proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mariano Maffei ◽  
Chiara Morelli ◽  
Ellie Graham ◽  
Stefano Patriarca ◽  
Laura Donzelli ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Protein Delivery ◽  
Genetic Diseases ◽  
Cell Surface Receptor ◽  
Viral Gene ◽  
Promising Alternative ◽  
Large Proteins ◽  
Reporter Mice ◽  
Surface Receptor

AbstractGene delivery using vector or viral-based methods is often limited by technical and safety barriers. A promising alternative that circumvents these shortcomings is the direct delivery of proteins into cells. Here we introduce a non-viral, ligand-mediated protein delivery system capable of selectively targeting primary skin cells in-vivo. Using orthologous self-labelling tags and chemical cross-linkers, we conjugate large proteins to ligands that bind their natural receptors on the surface of keratinocytes. Targeted CRE-mediated recombination was achieved by delivery of ligand cross-linked CRE protein to the skin of transgenic reporter mice, but was absent in mice lacking the ligand’s cell surface receptor. We further show that ligands mediate the intracellular delivery of Cas9 allowing for CRISPR-mediated gene editing in the skin more efficiently than adeno-associated viral gene delivery. Thus, a ligand-based system enables the effective and receptor-specific delivery of large proteins and may be applied to the treatment of skin-related genetic diseases.

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