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1.
BMC Res Notes ; 14(1): 359, 2021 Sep 15.
Article in English | MEDLINE | ID: mdl-34526111

ABSTRACT

OBJECTIVE: Extracellular matrix proteins play important roles in embryonic development and antibodies that specifically detect these proteins are essential to understanding their function. The zebrafish embryo is a popular model for vertebrate development but suffers from a dearth of authenticated antibody reagents for research. Here, we describe a novel antibody designed to detect the minor fibrillar collagen chain Col11a1a in zebrafish (AB strain). RESULTS: The Col11a1a antibody was raised in rabbit against a peptide comprising a unique sequence within the zebrafish Col11a1a gene product. The antibody was affinity-purified and characterized by ELISA. The antibody is effective for immunoblot and immunohistochemistry applications. Protein bands identified by immunoblot were confirmed by mass spectrometry and sensitivity to collagenase. Col11a1a knockout zebrafish were used to confirm specificity of the antibody. The Col11a1a antibody labeled cartilaginous structures within the developing jaw, consistent with previously characterized Col11a1 antibodies in other species. Col11a1a within formalin-fixed paraffin-embedded zebrafish were recognized by the antibody. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research.


Subject(s)
Collagen Type XI , Zebrafish , Animals , Antibodies , Extracellular Matrix Proteins , Peptides , Rabbits
2.
Int J Mol Sci ; 22(12)2021 Jun 09.
Article in English | MEDLINE | ID: mdl-34207917

ABSTRACT

Osteoarthritis is a major concern in the United States and worldwide. Current non-surgical and surgical approaches alleviate pain but show little evidence of cartilage restoration. Cell-based treatments may hold promise for the regeneration of hyaline cartilage-like tissue at the site of injury or wear. Cell-cell and cell-matrix interactions have been shown to drive cell differentiation pathways. Biomaterials for clinically relevant applications can be generated from decellularized porcine auricular cartilage. This material may represent a suitable scaffold on which to seed and grow chondrocytes to create new cartilage. In this study, we used decellularization techniques to create an extracellular matrix scaffold that supports chondrocyte cell attachment and growth in tissue culture conditions. Results presented here evaluate the decellularization process histologically and molecularly. We identified new and novel biomarker profiles that may aid future cartilage decellularization efforts. Additionally, the resulting scaffold was characterized using scanning electron microscopy, fluorescence microscopy, and proteomics. Cellular response to the decellularized scaffold was evaluated by quantitative real-time PCR for gene expression analysis.


Subject(s)
Cell Differentiation , Chondrogenesis , Ear Cartilage/chemistry , Extracellular Matrix/chemistry , Materials Testing , Tissue Scaffolds/chemistry , Animals , Cell Line , Humans , Swine
3.
Int J Mol Sci ; 21(6)2020 Mar 20.
Article in English | MEDLINE | ID: mdl-32244989

ABSTRACT

The Center of Biomedical Research Excellence in Matrix Biology strives to improve our understanding of extracellular matrix at molecular, cellular, tissue, and organismal levels to generate new knowledge about pathophysiology, normal development, and regenerative medicine. The primary goals of the Center are to i) support junior investigators, ii) enhance the productivity of established scientists, iii) facilitate collaboration between both junior and established researchers, and iv) build biomedical research infrastructure that will support research relevant to cell-matrix interactions in disease progression, tissue repair and regeneration, and v) provide access to instrumentation and technical support. A Pilot Project program provides funding to investigators who propose applying their expertise to matrix biology questions. Support from the National Institute of General Medical Sciences at the National Institutes of Health that established the Center of Biomedical Research Excellence in Matrix Biology has significantly enhanced the infrastructure and the capabilities of researchers at Boise State University, leading to new approaches that address disease diagnosis, prevention, and treatment. New multidisciplinary collaborations have been formed with investigators who may not have previously considered how their biomedical research programs addressed fundamental and applied questions involving the extracellular matrix. Collaborations with the broader matrix biology community are encouraged.


Subject(s)
Biomedical Research , Cooperative Behavior , Extracellular Matrix/metabolism , Research Personnel , Advisory Committees , Career Choice , Humans , Students
4.
Ann Biomed Eng ; 46(11): 1882-1895, 2018 Nov.
Article in English | MEDLINE | ID: mdl-29873012

ABSTRACT

Ligament wound healing involves the proliferation of a dense and disorganized fibrous matrix that slowly remodels into scar tissue at the injury site. This remodeling process does not fully restore the highly aligned collagen network that exists in native tissue, and consequently repaired ligament has decreased strength and durability. In order to identify treatments that stimulate collagen alignment and strengthen ligament repair, there is a need to develop in vitro models to study fibroblast activation during ligament wound healing. The objective of this study was to measure gene expression and matrix protein accumulation in fibroblast-collagen gels that were subjected to different static stress conditions (stress-free, biaxial stress, and uniaxial stress) for three time points (1, 2 or 3 weeks). By comparing our in vitro results to prior in vivo studies, we found that stress-free gels had time-dependent changes in gene expression (col3a1, TnC) corresponding to early scar formation, and biaxial stress gels had protein levels (collagen type III, decorin) corresponding to early scar formation. This is the first study to conduct a targeted evaluation of ligament healing biomarkers in fibroblast-collagen gels, and the results suggest that biomimetic in-vitro models of early scar formation should be initially cultured under biaxial stress conditions.


Subject(s)
Collagen/metabolism , Extracellular Matrix/metabolism , Fibroblasts/metabolism , Ligaments , Models, Biological , Wound Healing , Animals , Extracellular Matrix/pathology , Fibroblasts/pathology , Gels , Ligaments/injuries , Ligaments/metabolism , Ligaments/pathology , Mice , NIH 3T3 Cells
5.
J Neurosci ; 30(4): 1523-38, 2010 Jan 27.
Article in English | MEDLINE | ID: mdl-20107080

ABSTRACT

Ret signaling is critical for formation of the enteric nervous system (ENS) because Ret activation promotes ENS precursor survival, proliferation, and migration and provides trophic support for mature enteric neurons. Although these roles are well established, we now provide evidence that increasing levels of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) in mice causes alterations in ENS structure and function that are critically dependent on the time and location of increased GDNF availability. This is demonstrated using two different strains of transgenic mice and by injecting newborn mice with GDNF. Furthermore, because different subclasses of ENS precursors withdraw from the cell cycle at different times during development, increases in GDNF at specific times alter the ratio of neuronal subclasses in the mature ENS. In addition, we confirm that esophageal neurons are GDNF responsive and demonstrate that the location of GDNF production influences neuronal process projection for NADPH diaphorase-expressing, but not acetylcholinesterase-, choline acetyltransferase-, or tryptophan hydroxylase-expressing, small bowel myenteric neurons. We further demonstrate that changes in GDNF availability influence intestinal function in vitro and in vivo. Thus, changes in GDNF expression can create a wide variety of alterations in ENS structure and function and may in part contribute to human motility disorders.


Subject(s)
Enteric Nervous System/embryology , Enteric Nervous System/metabolism , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Neurons/metabolism , Stem Cells/metabolism , Animals , Cell Cycle/genetics , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Proliferation , Enteric Nervous System/cytology , Esophagus/embryology , Esophagus/innervation , Gastrointestinal Motility/genetics , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/genetics , Glial Cell Line-Derived Neurotrophic Factor/genetics , Glial Cell Line-Derived Neurotrophic Factor/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Myenteric Plexus/cytology , Myenteric Plexus/embryology , Myenteric Plexus/metabolism , NADPH Dehydrogenase/metabolism , Neurites/metabolism , Neurites/ultrastructure , Neurogenesis/drug effects , Neurogenesis/genetics , Neurons/cytology , Proto-Oncogene Proteins c-ret/genetics , Proto-Oncogene Proteins c-ret/metabolism , Stem Cells/cytology , Stem Cells/drug effects
6.
Cancer Res ; 65(19): 8896-904, 2005 Oct 01.
Article in English | MEDLINE | ID: mdl-16204061

ABSTRACT

Tumor-associated and tumor-infiltrating neutrophils (TAN) and macrophages (TAM) can account for as much as 50% of the total tumor mass in invasive breast carcinomas. It is thought that tumors secrete factors that elicit a wound-repair response from TAMs and TANs and that this response inadvertently stimulates tumor progression. Oncostatin M is a pleiotropic cytokine belonging to the interleukin-6 family that is expressed by several cell types including activated human T lymphocytes, macrophages, and neutrophils. Whereas oncostatin M can inhibit the proliferation of breast cancer cells in vitro, recent studies suggest that oncostatin M may promote tumor progression by enhancing angiogenesis and metastasis. In addition, neutrophils can be stimulated to synthesize and rapidly release large quantities of oncostatin M. In this article, we show that human neutrophils secrete oncostatin M when cocultured with MDA-MB-231 and T47D human breast cancer cells. Neutrophils isolated from whole blood or breast cancer cells alone express little oncostatin M by immunocytochemistry and ELISA, but neutrophils express and release high levels of oncostatin M when they are cocultured with breast cancer cells. In addition, we show that granulocyte-macrophage colony-stimulating factor produced by breast cancer cells and cell-cell contact are both necessary for the release of oncostatin M from neutrophils. Importantly, neutrophil-derived oncostatin M induces vascular endothelial growth factor from breast cancer cells in coculture and increases breast cancer cell detachment and invasive capacity, suggesting that neutrophils and oncostatin M may promote tumor progression in vivo.


Subject(s)
Breast Neoplasms/pathology , Cell Communication/physiology , Neutrophils/cytology , Neutrophils/metabolism , Peptides/metabolism , Antibodies/immunology , Antibodies/pharmacology , Breast Neoplasms/metabolism , Cell Adhesion/physiology , Cell Line, Tumor , Coculture Techniques , Culture Media, Conditioned , Disease Progression , Enzyme-Linked Immunosorbent Assay , Granulocyte-Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/physiology , Humans , Neoplasm Invasiveness , Oncostatin M , Vascular Endothelial Growth Factor A/biosynthesis
7.
Mech Dev ; 122(1): 27-41, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15582775

ABSTRACT

Glial cell-line derived neurotrophic factor (GDNF) is a potent survival factor for motor neurons. Previous studies have shown that some motor neurons depend upon GDNF during development but this GDNF-dependent motor neuron subpopulation has not been characterized. We examined GDNF expression patterns in muscle and the impact of altered GDNF expression on the development of subtypes of motor neurons. In GDNF hemizygous mice, motor neuron innervation to muscle spindle stretch receptors (fusimotor neuron innervation) was decreased, whereas in transgenic mice that overexpress GDNF in muscle, fusimotor innervation to muscle spindles was increased. Facial motor neurons, which do not contain fusimotor neurons, were not changed in number when GDNF was over expressed by facial muscles during their development. Taken together, these data indicate that fusimotor neurons depend upon GDNF for survival during development. Since the fraction of cervical and lumbar motor neurons lost in GDNF-deficient mice at birth closely approximates the size of the fusimotor neuron pool, these data suggest that motor neuron loss in GDNF-deficient mice may be primarily of fusimotor neuron origin.


Subject(s)
Motor Neurons, Gamma/metabolism , Nerve Growth Factors/metabolism , Animals , Axons/metabolism , Axons/ultrastructure , Cell Survival , Facial Muscles/innervation , Facial Muscles/metabolism , Glial Cell Line-Derived Neurotrophic Factor , Mice , Mice, Transgenic , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/ultrastructure , Muscle Spindles/metabolism , Muscle Spindles/ultrastructure , Muscle, Skeletal/innervation , Muscle, Skeletal/metabolism , Muscle, Skeletal/ultrastructure
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