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1.  Optimal 3-D culture of primary articular chondrocytes for use in the Rotating Wall Vessel Bioreactor 
INTRODUCTION
Reliable culturing methods for primary articular chondrocytes are essential to study the effects of loading and unloading on joint tissue at the cellular level. Due to the limited proliferation capacity of primary chondrocytes and their tendency to dedifferentiate in conventional culture conditions, long-term culturing conditions of primary chondrocytes can be challenging. The goal of this study was to develop a suspension culturing technique that not only would retain the cellular morphology but also maintain gene expression characteristics of primary articular chondrocytes.
METHODS
Three-dimensional culturing methods were compared and optimized for primary articular chondrocytes in the rotating wall vessel bioreactor, which changes the mechanical culture conditions to provide a form of suspension culture optimized for low shear and turbulence. We performed gene expression analysis and morphological characterization of cells cultured in alginate beads, Cytopore-2 microcarriers, primary monolayer culture, and passaged monolayer cultures using reverse transcription-PCR and laser scanning confocal microscopy.
RESULTS
Primary chondrocytes grown on Cytopore-2 microcarriers maintained the phenotypical morphology and gene expression pattern observed in primary bovine articular chondrocytes, and retained these characteristics for up to 9 days.
DISCUSSION
Our results provide a novel and alternative culturing technique for primary chondrocytes suitable for studies that require suspension such as those using the rotating wall vessel bioreactor. In addition, we provide an alternative culturing technique for primary chondrocytes that can impact future mechanistic studies of osteoarthritis progression, treatments for cartilage damage and repair, and cartilage tissue engineering.
doi:10.3357/ASEM.3905.2014
PMCID: PMC4207436  PMID: 25199120
rotating wall vessel; chondrocytes; bioreactor; microcarrier; RWV
2.  Immunolocalization of an Amino-Terminal Fragment of Apolipoprotein E in the Pick's Disease Brain 
PLoS ONE  2013;8(12):e80180.
Although the risk factor for apolipoprotein E (apoE) polymorphism in Alzheimer's disease (AD) has been well described, the role that apoE plays in other neurodegenerative diseases, including Pick's disease, is not well established. To examine a possible role of apoE in Pick's disease, an immunohistochemical analysis was performed utilizing a novel site-directed antibody that is specific for an amino-terminal fragment of apoE. Application of this antibody, termed the amino-terminal apoE cleavage fragment (nApoECF) antibody, consistently labeled Pick bodies within area CA1 of the hippocampus in 4 of the 5 cases examined. Co-localization of the nApoECF antibody with PHF-1, a general marker for Pick bodies, as well as with an antibody to caspase-cleaved tau (TauC3) was evident within the hippocampus. While staining of the nApoECF antibody was robust in area CA1, little co-localization with PHF-1 in Pick bodies within the dentate gyrus was observed. A quantitative analysis indicated that approximately 86% of the Pick bodies identified in area CA1 labeled with the nApoECF antibody. The presence of truncated apoE within Pick bodies suggests a broader role of apoE beyond AD and raises the question as to whether this protein contributes to pathogenesis associated with Pick's disease.
doi:10.1371/journal.pone.0080180
PMCID: PMC3846660  PMID: 24312462
3.  Proteomic analysis of Col11a1-associated protein complexes 
Proteomics  2011;11(24):4660-4676.
Cartilage plays an essential role during skeletal development within the growth plate and in articular joint function. Interactions between the collagen fibrils and other extracellular matrix molecules maintain structural integrity of cartilage, orchestrate complex dynamic events during embryonic development, and help to regulate fibrillogenesis. To increase our understanding of these events, affinity chromatography and liquid chromatography/tandem mass spectrometry were used to identify proteins that interact with the collagen fibril surface via the amino terminal domain of collagen alpha 1(XI) a protein domain that is displayed at the surface of heterotypic collagen fibrils of cartilage. Proteins extracted from fetal bovine cartilage using homogenization in high ionic strength buffer were selected based on affinity for the amino terminal noncollagenous domain of collagen alpha 1(XI). Mass spectrometry was used to determine the amino acid sequence of tryptic fragments for protein identification. Extracellular matrix molecules and cellular proteins that were identified as interacting with the amino terminal domain of collagen alpha 1(XI) directly or indirectly, included proteoglycans, collagens, and matricellular molecules, some of which also play a role in fibrillogenesis, while others are known to function in the maintenance of tissue integrity. Characterization of these molecular interactions will provide a more thorough understanding of how the extracellular matrix molecules of cartilage interact and what role collagen XI plays in the process of fibrillogenesis and maintenance of tissue integrity. Such information will aid tissue engineering and cartilage regeneration efforts to treat cartilage tissue damage and degeneration.
doi:10.1002/pmic.201100058
PMCID: PMC3463621  PMID: 22038862
arthritis; cartilage; collagen fibril; extracellular matrix; interactions
4.  Depletion of Beclin-1 due to proteolytic cleavage by caspases in the Alzheimer’s disease brain 
Neurobiology of disease  2010;43(1):68-78.
The Beclin-1 protein is essential for the initiation of autophagy and recent studies suggest this function may be compromised in Alzheimer’s disease (AD). In addition, in vitro studies have supported a loss of function of Beclin-1 due to proteolytic modification by caspases. In the present study we examined whether caspase-cleavage of Beclin-1 occurs in the AD brain by designing a site-directed caspase-cleavage antibody based upon a known cleavage site within the protein at position D149. We confirmed that Beclin-1 is an excellent substrate for caspase-3 and demonstrate cleavage led to the formation of a 35 kDa C-terminal fragment labeled by our novel antibody following Western blot analysis. Application of this antibody termed Beclin-1 caspase-cleavage product antibody or BeclinCCP in frontal cortex tissue sections revealed strong immunolabeling within astrocytes that localized with plaque-regions and along blood vessels in all AD cases examined. In addition, weaker, more variable BeclinCCP labeling was also observed within neurofibrillary tangles that co-localized with the early tau conformational marker, MC-1 as well as the late tangle marker, PHF-1. Collectively, these data support a depletion of Beclin-1 in AD following caspase-cleavage.
doi:10.1016/j.nbd.2010.11.003
PMCID: PMC3066274  PMID: 21081164
Beclin-1; Alzheimer’s disease; Astrocytes; Caspase
5.  The expression patterns of minor fibrillar collagens during development in zebrafish 
Gene expression patterns : GEP  2010;10(7-8):315-322.
Minor fibrillar collagens are recognized as the organizers and nucleators during collagen fibrillogenesis but likely serve additional functions. The minor fibrillar collagens include collagens type V and type XI. Mutations of collagen type V and XI can cause Ehlers Danlos, Stickler’s, and Marshall’s syndromes in human. We have characterized the spatiotemporal expression patterns of Col11a1, Col11a2, Col5a1 as well as Col5a3 in zebrafish embryos by in situ hybridization. Col5a1 is expressed in developing somites, neural crest, the head mesenchyme, developing cranial cartilage, pharyngeal arches and vertebrae. Col5a3 is detected in the notochord, mesenchyme cells in the eyes and lens. Both Col11a1 and Col11a2 have similar expression patterns, including notochord, otic vesicle, and developing cranial cartilages. Zebrafish may therefore serve as a valuable vertebrate model system for the study of diseases associated with collagens type V and XI mutations.
doi:10.1016/j.gep.2010.07.002
PMCID: PMC2956583  PMID: 20647059
minor fibrillar collagens; zebrafish; development; in situ hybridization; Danio rerio
6.  Isoform-specific Heparan Sulfate Binding within the Amino-terminal Noncollagenous Domain of Collagen α1(XI)* 
The Journal of biological chemistry  2006;281(51):39507-39516.
Collagen type XI is a constituent of the pericellular matrix of chondrocytes and plays a role in the regulation of fibrillogenesis. The amino-terminal domain of collagen type XI α1 chain is a noncollagenous structure that has been identified on the surface of cartilage collagen fibrils. The biochemical composition of the amino-terminal domain varies due to alternative splicing of the primary transcript. Recombinantly expressed α1(XI) amino-terminal domain isoforms were used in this study to investigate potential interactions. Purified products were analyzed for heparan sulfate binding properties. The results demonstrated that two additional binding sites exist within the α1(XI) amino-terminal domain, one within the amino propeptide and one within the variable region of the amino-terminal domain. Analysis of relative affinities indicated that the site located within the amino propeptide (site 1) was of similar affinity to sites that exist within the major triple helix of collagen type XI. Substitution of amino acid residues 147 to 152 within the amino propeptide by site-directed mutagenesis resulted in altered affinity for heparan sulfate. The binding site located within the variable region (site 2) demonstrated significantly higher affinity than other sites within the molecule. Displacement of collagen type XI within the pericellular matrix was observed in cell culture in the presence of excess heparan sulfate and by treatment with heparinase. These studies suggest two additional binding sites located within the noncollagenous amino-terminal domain that may play a role in the function of collagen type XI. The localization of collagen type XI within the pericellular matrix may be dependent upon interactions with heparan sulfate proteoglycans, and these are likely to take place in an isoform-specfic manner.
doi:10.1074/jbc.M608551200
PMCID: PMC2948787  PMID: 17062562
7.  Characterization of collagenous matrix assembly in a chondrocyte model system 
Collagen is a major component of the newly synthesized pericellular microenvironment of chondrocytes. Collagen types II, IX, and XI are synthesized and assembled into higher ordered complexes by a mechanism in which type XI collagen plays a role in nucleation of new fibrils, and in limiting fibril diameter. This study utilizes a cell line derived from the Swarm rat chondrosarcoma that allows the accumulation and assembly of pericellular matrix. Immunofluorescence and atomic force microscopy were used to assess early intermediates of fibril formation. Results indicate that this cell line synthesizes and secretes chondrocyte-specific pericellular matrix molecules including types II, IX, and XI collagen and is suitable for the study of newly synthesized collagen matrix under the experimental conditions used. AFM data indicate that small fibrils or assemblies of microfibrils are detectable and may represent precursors of the ~20 nm thin fibrils reported in cartilage. Treatment with hyaluronidase indicates that the dimensions of the small fibrils may be dependent upon the presence of hyaluronan within the matrix. This study provides information on the composition and organization of the newly synthesized extracellular matrix that plays a role in establishing the material properties and performance of biological materials such as cartilage.
doi:10.1002/jbm.a.32078
PMCID: PMC2842207  PMID: 18496861
cartilage; collagen; extracellular matrix; immunofluorescence; atomic force microscopy; chondrocyte; microfibril
8.  Expression, Purification, and Refolding of Recombinant Collagen α1(XI) Amino Terminal Domain Splice Variants 
The amino terminal domain of collagen type XI α1 chain is a noncollagenous structure that is essential for the regulation of fibrillogenesis in developing cartilage. The amino terminal domain is alternatively spliced at the mRNA level, resulting in proteins expressed as splice variants. These splice variants, or isoforms, have unique distribution in growing tissues, alluding to distinct roles in development. We report here a rapid and straightforward method for expression, purification and in vitro folding of recombinant collagen XI isoforms α1(XI) NTD[p7] and α1(XI) NTD[p6b+7]. The recombinant isoforms were expressed in Escherichia coli as bacterial inclusion bodies. Unfolded carboxy terminal polyhistidine tagged proteins were purified via nickel affinity chromatography and refolded with specific protocols optimized for each isoform. Purity was assessed by SDS-PAGE and correct secondary structure by a comparison of circular dichroism data with that obtained for Npp. Protein expression and purification of the recombinant collagen XI splice variants will allow further studies to elucidate the structure and molecular interactions with components of the extracellular matrix. This research will clarify the mechanism of collagen XI mediated regulation of collagen fibrillogenesis.
doi:10.1016/j.pep.2006.10.016
PMCID: PMC2713663  PMID: 17166742
9.  A LysR-Type Regulator, CidR, Is Required for Induction of the Staphylococcus aureus cidABC Operon 
Journal of Bacteriology  2005;187(17):5893-5900.
The Staphylococcus aureus cidABC and lrgAB operons have been shown to regulate murein hydrolase activity and affect antibiotic tolerance. The cid operon enhances murein hydrolase activity and antibiotic sensitivity, whereas the lrg operon inhibits these processes. Based on these findings and the structural similarities of the cidA and lrgA gene products to the bacteriophage holin family of proteins, we have proposed that the cid and lrg operons encode holin- and antiholin-like proteins, respectively, that function to control the murein hydrolase activity produced by the bacteria. Analysis of cid operon transcription revealed the presence of two transcripts, one spanning all three cid genes and whose expression is induced by growth in the presence of acetic acid and the other spanning cidB and cidC only that is produced in a sigma B-dependent manner. The cidABC operon lies immediately downstream from the cidR gene, encoding a potential LysR-type transcriptional regulator. In this study, we demonstrate that cidR is involved in the regulation of cidABC expression. Northern blot analyses revealed that the cidR gene product positively regulates cidABC expression by increasing transcription in the presence of acetic acid produced as a result of the metabolism of glucose. As expected for an operon that encodes a positive effector of murein hydrolase activity, the upregulation of cidABC expression resulted in increased murein hydrolase activity produced by these cells. Furthermore, it was demonstrated that antibiotic tolerance and stationary-phase survival of S. aureus are affected by the cidR gene. Taken together, these results demonstrate that the cidR gene product functions as a transcriptional activator of cidABC transcription in response to acetic acid accumulation in the growth medium.
doi:10.1128/JB.187.17.5893-5900.2005
PMCID: PMC1196168  PMID: 16109930

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