Browsing by Subject "collagen"

Now showing 1 - 8 of 8
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    A Computational Study of the Role of Hydration in the Assembly of Collagen and Other Bio laments
    (2012-10-19) Mayuram Ravikumar, Krishnakumar
    Hydration is known to be crucial in biomolecular interactions including ligand binding and self-assembly. In our earlier studies we have shown the key role of water in stabilizing the specific parts of the collagen triple helix depending on the imino acid content. We further showed that the primary hydration shell around collagen could act as a lubricating layer aiding in collagen assembly. But key details on the structure and dynamics of water near protein surfaces and its role in protein-protein interactions remain unclear. In the current study we have developed a novel method to analyze hydration maps around peptides at 1-A resolution around three self-assembling lament systems with known structures, that respectively have hydrated (collagen), dry non-polar and dry polar (amyloid) interfaces. Using computer simulations, we calculate local hydration maps and hydration forces. We find that the primary hydration shells are formed all over the surface, regardless of the types of the underlying amino acids. The weakly oscillating hydration force arises from coalescence and depletion of hydration shells as two laments approach, whereas local water diffusion, orientation, or hydrogen bonding events have no direct effect. Hydration forces between hydrated, polar, and non-polar interfaces differ in the amplitude and phase of the oscillation relative to the equilibrium surface separation. Therefore, water-mediated interactions between these protein surfaces ranging in character from ?hydrophobic? to ?hydrophilic,? have a common molecular origin based on the robustly formed hydration shells, which is likely applicable to a broad range of biomolecular assemblies whose interfacial geometry is similar in length scale to those of the present study. In a related study through simulations we show that the rate of tissue optical clearing by chemical agents correlated with the preferential formation of hydrogen bond bridges between agent and collagen. Hydrogen bond bridge formation disrupts the collagen hydration layer and facilitates replacement by a chemical agent to destabilize the tertiary structure of collagens thereby reducing light scattering. This study suggests that the clearing ability of an alcohol not only depends on its molecular size, but also on the position of hydroxyl groups on its backbone.
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    Cellular Response to Ordered Collagen Layers on Mica
    (2012-07-16) Leow, Wee Wen
    Extracellular microenvironment, including its components and biophysical parameters such as matrix structure and stiffness, is a crucial determinant of cellular function. There exists interdependency between cellular behaviors and the extracellular matrix (ECM), whereby cells are constantly sensing and modifying their surroundings in response to physical stress or during processes like wound repair, cancer cell invasion, and morphogenesis, to create an environment which supports adaptation. To date, knowledge of the distinct regulatory mechanisms of this complex relationship is little, while the urge is evident as it plays a significant role in understanding tissue remodeling. Cells are observed to align with the parallel arrays of collagen fibrils found in tissues such as bone, tendon, and cornea, suggesting the importance of ordered matrices in defining cell functions. In this study, epitaxial growths of ordered two-dimensional collagen matrices were created, with parallelly aligned fibrils on muscovite mica, and novel triangular pattern matrix on phlogopite mica. Using Fluorescence and Atomic Force Microscopy, we were able to observe cell polarization along with stress fiber formation and matrix deformation at high resolution. Cells were observed to be able to penetrate between collagen fibrils and generate traction anisotropically to polarize. These ordered collagen matrices serve as an excellent model to study cellular remodeling of ECM in vitro, in which this fundamental apprehension of cell-matrix relationship is of crucial importance to manipulate the system and obtain desired cell functions.
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    Identification of a mutation in COL4A5 causative for X-linked Alport syndrome in the domestic dog and analysis of gene expression in the kidneys of affected and nonaffected siblings
    (Texas A&M University, 2004-09-30) Cox, Melissa Luanne
    The domestic dog, Canis lupus familiaris, plays many roles in the lives of humans. Additionally, the dog is recognized for its potential as a model for many human hereditary diseases. Thus, the genetics and genomics of the dog are being studied extensively in order to facilitate its use as a model, as well as to help the dog for its own sake. As part of this research effort, our laboratory has added type I markers (i.e., the acidic and basic keratins, c-kit, type I and IV collagens, and the gene encoding uromodulin) to the emerging map of the canine genome. The mapping of genes, particularly those in large gene families such as the collagens, is valuable because it rapidly increases the density of gene loci on the map and provides insight regarding conservation of synteny between the dog and other mammals. The major focus of work reported here is the genetics of X-linked Alport syndrome (XLAS), a terminal renal disease that affects the human and the dog. The disease results from mutations in COL4A5, a type IV collagen gene. Reported here are the 1) sequencing and mapping of the canine cDNA encoding uromodulin, 2) mapping of the type I and type IV collagen genes, 3) sequencing of the full-length cDNA of canine COL4A5, 4) identification of a 10 bp deletion in COL4A5, causative for XLAS in our colony of mixed breed dogs, 5) development of a genetic test for identification of affected and carrier dogs in the colony and 6) assessment of gene expression in the kidneys of normal and XLAS-dogs. This assessment was performed using a canine-specific oligonucleotide microarray. XLAS dogs demonstrated up-regulation of many genes involved in extracellular matrix reorganization, cell structure, and immune response, as expected in a glomerulopathy with tubulointerstitial nephritis. Trends were verified by quantitative RT-PCR. A review of the current status of canine genetics research, and current understanding of hereditary diseases in the dog, concludes this dissertation.
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    Identification of Scaffold Proteins RACK1 and Hic-5 as Regulators of Endothelial Sprouting Resoponses
    (2014-12-12) Dave, Jui Mayank
    Angiogenesis is defined as growth of new blood vessels from pre-existing ones and occurs during normal physiological development as well as pathological conditions like cancer. During angiogenesis, normally quiescent endothelial cells (ECs) are activated in response to external pro-angiogenic cues and undergo rapid morphogenic changes such as proliferation, migration, invasion, and lumen formation to extend new sprouts into surrounding three-dimensional (3D) matrix. To better understand angiogenic regulation, we utilized an in vitro model wherein sphingosine 1-phosphate (S1P) and pro-angiogenic growth factors (GF) synergize to induce rapid and robust endothelial sprouting in 3D collagen matrices. In a proteomic screen designed to identify molecules relevant to angiogenesis, we found up-regulated levels of receptor for activated C kinase 1 (RACK1) and the intermediate filament protein, vimentin. RACK1 depletion reduced EC invasion. Silencing of vimentin or RACK1 decreased cell adhesion and attenuated with focal adhesion kinase (FAK) activation, which is indispensable for successful angiogenesis. Moreover, pro-angiogenic GFs enhanced RACK1 and vimentin association. RACK1, vimentin, and FAK, formed an intermolecular complex during S1P- and GF- induced invasion. Also, depletion of RACK1 decreased vimentin and FAK association, suggesting a role for RACK1 in stabilizing vimentin-FAK interactions during sprouting. In an independent study, we identified focal adhesion (FA) scaffold protein, hydrogen peroxide inducible clone 5 (Hic-5), as a critical regulator of angiogenesis. Hic-5 depletion interfered with endothelial invasion and lumen formation. S1P induced rapid Hic-5 translocation to FAs and Hic-5 silencing attenuated FAK expression and activation. S1P induced a novel interaction between Hic-5 and membrane type 1 matrix-metalloproteinase (MT1-MMP). In vitro binding experiments revealed that LIM2 domain of Hic-5 was required for MT1-MMP binding. Moreover, Hic-5 and MT1-MMP levels were up-regulated in detergent resistant membrane fractions of invading ECs, indicative of their crosstalk. Hic-5 silencing interfered with S1P- induced MT1-MMP membrane translocation, a critical event for successful angiogenesis. Since MT1-MMP and FAK interaction has been reported to be essential for matrix degradation at FA sites, we further tested if Hic-5 mediated this interaction. Our results indicated that presence of Hic-5 significantly enhanced FAK and MT1-MMP complex formation. In conclusion, we report that scaffold proteins RACK1 and Hic-5 regulate successful endothelial sprouting responses in 3D matrices.
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    Relationships between Beef Postharvest Biochemical Factors and Warner-Bratzler Shear Force
    (2013-04-01) Orozco Hernandez, Pilar
    Biochemical changes in muscle postmortem have been associated with initial beef tenderness early postmortem, and with improvements in tenderness during postmortem storage, defined as meat aging. Differences in the initial contractile state of the sarcomere, the ionic environment of the sarcoplasm including pH, the activity of neutral proteolytic enzymes, and collagen content and solubility have been associated with beef tenderness. In Phase I, steaks from four genetic lines of steers and heifers were used to understand the biochemical differences between tough and tender steaks. The most tender (< 30 N Warner Bratzler shear force (WBS)) and toughest Longissimus steaks (< 30 N WBS) from Angus, Braford, Brangus, and Simbrah heifers and steers were used. For Phase II, samples were obtained from a subset of Santa Cruz yearling heifers selected based of genotypes for tenderness (tough and tender) using a commercial genetic marker. Within genotype for tenderness, each animal was randomly assigned to one of four growth enhancement treatments. The most tender (< 30 N WBS) and toughest Longissimus steaks (< 30 N WBS) were selected for use in this study. In Phase I, tough steaks after 3, 10, and 17d postmortem had higher (P < 0.0005) WBS values than tender steaks. Tender steaks came from carcass with slightly higher (P = 0.008) marbling score and (P = 0.01) Quality grade. Sarcomere length, total and soluble collagen, potassium concentration, and m and ?calpain did not differ (P > 0.05) between tough and tender steaks. Sodium concentration at 10 d was higher (P = 0.03) in tough steaks, but only account for 0.05% of the variation in WBS at 3d. Tender steaks had less (P = 0.04) intact desmin at 24h, but intact desmin was not correlated (P > 0.05) with WBS. In Phase II, tough steaks after 3, 10, and 17d postmortem had higher (P < 0.0001) WBS values than tender steaks. Tender steaks came from carcass with slightly higher (P < 0.03) marbling score and (P = 0.02) Quality grade. Tender teaks were slightly lighter (P = 0.02), with more red (P = 0.02) and yellow (P = 0.007) color, and had slightly lower (P = 0.02) pH, compared with tough steaks. Sarcomere length, total and soluble collagen, sodium and potassium concentration, and m and ?calpain did not differ (P > 0.05) between tough and tender steaks. Tender steaks had less (P < 0.0001) intact desmin at 17d postmortem than tough steaks. Intact desmin at 17d was responsible for 4%, 47%, and 30% of WBS variation after 3, 10, and 17d postmortem, respectively. The slight difference in marbling and quality grade did not account for a significant amount of variation in WBS. However, meat color and pH accounted for variation in shear WBS. Calcium flux may have influenced meat tenderness by activation of calpains and may have altered protein to protein interactions. Results suggested that marbling, ? calpain activity, and desmin degradation, and to a lesser extent pH and meat color contributed to meat tenderness.
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    Study of Endothelial Morphogenesis in Three-Dimensional Collagen Matrices
    (2011-08-08) Su, Shih-Chi
    Sprouting angiogenesis is a multi-step process consisting of basement membrane degradation, endothelial cell (EC) activation, proliferation, invasion, lumen formation, and stabilization. Such complexity reveals that the orchestration of individual genes and multiple signaling pathways are required. To better understand the mechanisms that direct the transformation of adherent ECs on the surface of collagen matrices to multicellular invading sprouts, we analyzed differential gene expression with time using an in vitro model of EC invasion driven by the combination of sphingosine-1-phosphate (S1P) and angiogenic growth factors. Gene expression changes were confirmed by real-time PCR and Western blot analyses. In addition, we have undertaken a proteomic screen to dissect downstream targets of the S1P receptors that possibly regulate EC invasion. Gene silencing or overexpression were used to examine the involvement and role of downstream targets of S1P in EC invasion into three-dimensional collagen matrices. We demonstrated that various cell adhesion molecule genes involved in adherens junction and cell-extracellular matrix (ECM) interactions were upregulated; whereas a set of genes associated with tight junctions were downregulated. Numerous genes encoding ECM proteins and proteases were induced, indicating that biosynthesis and remodeling of ECM is indispensable for sprouting angiogenesis. Knockdown of a highly upregulated gene, A Disintegrin and Metalloproteinase with Thrombospondin-type repeats-1 (ADAMTS1), decreased invasion responses, confirming a role for ADAMTS1 in mediating EC invasion. Furthermore, differential expression of multiple members of the Wnt (wingless) and Notch pathways were observed. Functional experiments indicated that inhibition and activation of the Notch signaling pathway stimulated and inhibited EC invasion responses, respectively. In addition, we identified annexin 2 as a regulator of endothelial morphogenesis. We observed that S1P triggered annexin 2 translocation from cytosol to plasma membrane and its association with vascular endothelial (VE)-cadherin. Moreover, annexin 2 depletion attenuated Akt activation, which was associated with increased phosphorylation of VE-cadherin and endothelial barrier leakage. Disrupting homotypic VE-cadherin interactions resulted in decreased Akt (but not Erk1/2) activation. Furthermore, expression of constitutively active Akt restored reduced EC invasion observed with annexin 2 and VE-cadherin knockdown. Collectively, we report that annexin 2 regulates endothelial morphogenesis through an adherens junction-mediated pathway upstream of Akt.
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    The Impact of Extracellular Matrix Stiffness on Angiogensis
    (2012-12-06) Lee, Po-Feng 1976-
    Sprouting endothelial cells (ECs) use soluble and insoluble cues to guide migration and expand the existing vascular network to meet changing trophic needs of the tissue during angiogenesis. A noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique was used to optically image endothelial sprouting morphogenesis in three dimensional (3D) collagen matrices with simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Sprout advancement and lumen expansion companying with ECM alteration were the synergistic results of membrane-associated matrix metalloproteinase and cell traction evidenced by proteinase inhibition and Rho-associated kinase (p160ROCK) inhibition experiments. These physical EC-ECM interactions suggest that ECM mechanical properties may influence angiogenic responses. In a 3D angiogenesis model, we measure angiogenic responses as a function of collagen matrix stiffness by inducing collagen cross-linking with microbial transglutaminase (mTG). Collagen matrices stiffen with both mTG treatment and incubation time as evidenced with biaxial mechanical test results and collagen TPF intensity increases with mTG treatment and that the ratio of TPF/SHG correlates with biaxial tested mechanical stiffness. SHG and optical coherence microscopy (OCM) are further used to show that other physical properties of the matrix do not change with mTG treatment, thus providing the same density but different stiffness with which to measure angiogenic responses. Stiffer matrices promote angiogenesis with more invading sprouts that invade deeper. No differences in lumen size were observed between control and mTG stiffened 3D cultures, but there was evidence of greater matrix remodeling in stiffer gels using NLOM. Results of this study show angiogenic responses are enhanced with increasing stiffness and suggest that these properties may be used in tissue engineering and regenerative medicine applications to engineer angiogenesis.
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    Thermal alteration of collagenous tissue subjected to biaxial isometric constraints
    (Texas A&M University, 2005-08-29) Wells, Paul B.
    Clinical thermal therapies are widespread and gaining in appeal due to improved technology of heating devices and promising results. Outcomes of thermal treatment are often unpredictable and suboptimal, however, due in part to a lack of appreciation of the underlying biothermomechanics. There is a pressing need, therefore, to understand better the role of clinically-controllable parameters on the thermal damage processes of tissue. Heretofore, researchers have primarily sought to understand this process through various uniaxial experiments on tissues containing collagen as their primary constituent. Most biological tissues experience multiaxial loading, however, with complex boundary constraints inclusive of both isotonic and isometric conditions. The primary focus of this work is on the isothermal denaturation of fibrillar collagen subjected to a biaxial isometric constraint. Results from our tests reveal a complicated process, the kinetics of which are not easily measured. Evolving isometric contraction forces during heating do not correlate with resultant mechanical behaviors, as thermal shrinkage does in biaxial isotonic tests. Furthermore, resultant mechanical behaviors at variousdurations of heating reveal a two phase process with a rate dependent on the amount of isometric stretch. For tissues heated at 75oC for 15 minutes, at which point the first phase of mechanical alteration dominates for all constraints herein, resultant mechanical behaviors correlate well with the amount of isometric stretch. The correlation is similar to that between isotonic loads and resultant mechanical behaviors from previous studies. In light of the need for a better measure of thermal damage in isometric tests, we performed a histological analysis of tissues heated under varying constraints. Results show a good correlation between the level of isometric constraint and thermally-induced histological aberrations. Finally, we demonstrate that our seemingly limited and qualitative knowledge can be applied well to a specific clinical application: namely, the use of glycerol as a clearing agent for laser therapies. Our results suggest that glycerol is safe to use for such therapies because it increases the thermal stability of fibrillar collagen, and its hyperosmotic effects on mechanical behavior are fully reversed upon rehydration.