Vivo 1908 Software Update

[Rafael Nowning]

Spidersilk is an interesting biomaterial for medical applications. Recently, a method for production of recombinant spider silk protein (4RepCT) that forms macroscopic fibres in physiological solution was developed. Herein, 4RepCT and MersilkTM (control) fibres were implanted subcutaneously in rats for seven days, without any negative systemic or local reactions. The tissue response, characterised by infiltration of macrophages and multinucleated cells, was similar with both fibres, while only the 4RepCT-fibres supported ingrowth of fibroblasts and newly formed capillaries. This in vivo study indicates that 4RepCT-fibres are well tolerated and could be used for medical applications, e.g., tissue engineering.

Mitochondria play multiple roles in the cell, including maintaining energy homeostasis in response to environmental and cellular stress. Therefore, a key aspect of normal cellular function is the ability of the cell to respond to stress by stimulating mitochondrial biogenesis and turnover. Impairment of this ability can lead to mitochondrial dysfunction and disruption of cell energetics. Our research focuses on understanding the regulation of mitochondrial metabolism and how this changes with age. We have three related and overlapping research interests. They are to understand

We believe that in order to understand how the body regulates mitochondrial function in response to stress it is necessary to integrate research on molecular, biochemical, and organismal levels. However, due to the lack of necessary tools to measure mitochondrial function in vivo, approaches have typically focused on in vitro measurements, particularly of the electron transport chain (ETC). However, mitochondria are sensitive to many systemic and cellular factors making it difficult to extrapolate results from isolated mitochondria to function in the intact organism. To bridge this gap we have developed state of the art molecular imaging/spectroscopy tools to study oxidative phosphorylation in skeletal muscle of intact organisms. We use optical and magnetic resonance spectroscopies to provide independent measures of O2 and ATP fluxes in vivo. By independently measuring these fluxes we determine several parameters of mitochondrial energetics in intact skeletal muscle, including the coupling of oxidative phosphorylation (P/O), phosphorylation capacity, and the sensitivity of respiration to oxygen content. Our research program combines these in vivo tools with transgenic models and molecular analysis to investigate the cellular and molecular mechanisms responsible for changes in the regulation of energy metabolism with age.

Our work has led to significant insights into mitochondrial function in both normal and aged skeletal muscle. First, we determined that oxygen does not significantly regulate mitochondrial function in mouse skeletal muscle under normal physiological conditions (1). This finding is significant because it allowed us to separate the effects of mitochondrial dysfunction and oxygen limitation in aging and disease. A second paper demonstrated that our measurements of P/O in intact mouse hindlimb agree well with the theoretical values (2). This finding demonstrated the validity of the method, but more importantly established the baseline from which to analyze how mitochondrial function changes in response to environmental and disease stressors. Most recently we found significant mitochondrial uncoupling in resting skeletal muscle of aged mice (3). Our findings indicating significant mitochondrial uncoupling in aged skeletal muscle were recently extended to human muscle in collaboration with colleagues in the Metabolic Spectroscopy Laboratory (4). This uncoupling was associated with greater physiological stress (reduced ATP concentration) at rest and may be a key factor sensitizing the cell to apoptosis.

The focus of our current research is to understand how changes in the ability of the cell to stimulate mitochondrial biogenesis in response to cellular stress affect in vivo mitochondrial function. My research addresses these issues at multiple levels of biological organization. Our strategy pairs in vivo experiments with studies of isolated muscles using pharmacological and transgenic manipulations that affect oxidative metabolism. We can then assess the change in mitochondrial function both in vivo and in vitro. This functional analysis is paired with a molecular analysis of the signaling pathways regulating the response. The long-term goal is to apply a gene expression analysis to identify and gene therapy approach to manipulate key steps signaling nodes responsible for the activation and inhibition of mitochondrial biogenesis. In this way we address how the regulation of mitochondrial function and biogenesis at the tissue, cellular, and molecular levels varies with environment and age.

To define critical parameters concerning interferon (IFN) effects upon natural killer (NK) cells in vivo, we gave cancer patients serial weekly intramuscular injections of purified lymphoblastoid IFN in six doses ranging from 10(5) to 3 X 10(7) U. Dose sequences were determined by randomly allocating patients to one of six levels in a latin square ordering scheme. NK cell stimulation, a threefold peak increase above preinjection levels of cytolysis (P = 0.022), occurred in peripheral mononuclear cells (PMC) sampled 24 h postinjection, of 3 X 10(6) U, but was not detectable at any dose in PMC sampled 7 d postinjection. No blunting occurred in NK cell responsiveness to repeated injection of IFN dosages a second time at or several weeks after study completion. At IFN doses of 3 X 10(6), 10(7), and 3 X 10(7) U, a negative correlation existed between the amount of IFN injected and the average extent of NK cell activation (r = -0.423, P less than 0.05). This contrasted with the progressively increasing response of NK cells to in vitro incubation with increasing concentration of up to 3,000 U/ml of IFN. Overnight culturing of PMC sampled before IFN injections resulted in a mean 1.9-fold increase in cytolytic activity (P = 0.0005) and a mean 53% decrease in variance (P = 0.024) between serial preinjection NK cell activity determinations. Cell separation procedures may, therefore, have resulted in NK cell inactivation, from which overnight culturing permitted recovery. We found that maximal NK cell activation at a low IFN dose, decreasing NK cell responsiveness at higher doses, and the need to culture PMC to efficiently detect NK cell boosting may account for disparities in reported effects of IFN on NK cell function.

Mesenchymal stem cells (MSCs) are multipotent cells which can give rise to mesenchymal and nonmesenchymal tissues in vitro and in vivo [1]. The distribution of resident MSCs throughout the post-natal organism is mainly related to their existence in perivascular niches [2]. They can differentiate into osteogenic, adipogenic, chondrogenic, myocardial, or neural lineages when exposed to specific stimuli, making them attractive for tissue regeneration [3, 4]. Emerging evidence has shown that MSC transplantation offers a means to stimulate tissue repair either by direct (exogenous) or indirect (endogenous) cell replacement or angiogenesis [5, 6]. In fact, exogenous MSCs have shown the ability to support a paracrine activation of endogenous stem cells for tissue repair by secreting chemokines, as stromal derived factor-1 alpha (SDF-1α), and/or growth factors, as vascular endothelial growth factor. Despite the rapid research advancement, possible tissue repair by adult stem cell therapy is currently hampered in vivo by poor cell viability and delivery efficiency, uncertain differentiating fate, and therefore the use of this approach has raised a number of bioethical questions [7]. Hence, the strong need for more effective therapeutic approaches emphasizing the physiological plasticity of postnatal organs following an injury [8, 9], and more accurate imaging methods to allow a long-term in vivo monitoring of tissue regeneration [10]. Indeed, one of the most important accomplishments of modern physiology is the development of imaging techniques able to explore biochemical/molecular processes in the intact organism, i.e. in the absence of confounding effects inevitably caused by invasive procedures or ex vivo experimental prepar

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

Fibronectin (FN) is a large, modular glycoprotein that generates a polymeric fibrillar network in the extracellular matrix (ECM), and forms soluble, disulfide-linked dimeric protomers in plasma and other body fluids (1, 2). Fibronectin is a ligand for many molecules, including fibrin, heparin, chondroitin sulfate, collagen/gelatin, and integrins. It is involved in multiple cellular processes such as cell adhesion/migration, blood clotting, morphogenesis, tissue repair, and cell signaling. Fibronectin functions are mediated by the insoluble polymeric fibrillar network. Conversion of soluble Fibronectin to Fibronectin fibrils in the ECM is initiated by binding to cell surface integrins, resulting in exposure of cryptic epitopes necessary for polymerization (1). Fibronectin is made up of three types of homologous structural motifs termed FN type I, type II, and type III repeats (3-5). Alternative splicing generates multiple isoforms of Fibronectin which may have insertions of extra type III domains (EDA and EDB) or alteration of the type III connecting segment (IIICS) (5). Differential splicing within the IIICS domain determines the presence of CS1 and CS2 sequences, and its sensitivity to proteases (6, 7). The tilt angle between type III domains #9 and #10 (which contains an RGD motif) determines integrin binding affinity, suggesting how structural differences between fibrillar and soluble Fibronectin may influence their function (8). From the N-terminus to the furin cleavage site at amino acid 1908, human Fibronectin shares 92% amino acid sequence identity with mouse and rat Fibronectin.

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