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Manipulating the Intracellular Trafficking of Nucleic Acids
Published in Kenneth L. Brigham, Gene Therapy for Diseases of the Lung, 2020
Kathleen E. B Meyer, Lisa S. Uyechi, Francis C. Szoka
An understanding of intracellular macromolecular transport requires a general familiarity of the structure and properties of the cytoplasm, which consists of a filamentous network embedded in an aqueous, gel-like matrix. The matrix is composed of a concentrated mixture of soluble proteins and RNA and contains membrane-bound organelles that are linked to cytoskeletal fibers (14-18). The cytoplasm has the properties of a viscoelastic, contractile gel, making it difficult to model the diffusion of macromolecules. Measurement of the viscosity of the cytoplasm using low-molecular-weight molecules in several mammalian cell types has indicated viscosities similar to that of bulk water (19-21). However, macromolecular crowding is also an important feature of the cytoplasmic environment (Fig. 1). The protein concentration in the cytoplasm may be as high as 200 to 300 mg/ml, which gives it a colloid density similar to that found in solutions of 12.4% Ficoll (22) and 13% dextran (23). In this crowded milieu the translational diffusion of molecules the size of proteins is predicted to be reduced approximately threefold owing to macromolecular collisions (22,24-26).
Physiology of the Fascia
Published in David Lesondak, Angeli Maun Akey, Fascia, Function, and Medical Applications, 2020
In densification-like areas, high-molecular-weight and semi-flexible chains of HA are key factors leading to the high viscosity due to mutual macromolecular crowding.32 It is becoming commonplace to use dynamic ultrasonography methods in order to visualize the gliding between different structures and to collect data regarding the movement of one structure in comparison to others. In elastography images, the densification seems to be visible in the deep fascia. In particular, myofascial pain seems to be associated with not only reduction of gliding between fascial sublayers but also changes in the elasticity of the deep fascia. Elastography imaging seems to be a useful tool to help clinicians in diagnosing myofascial pain owing to its ability to quantify the tissue stiffness (Figure 4.3).
Beyond Enzyme Kinetics
Published in Clive R. Bagshaw, Biomolecular Kinetics, 2017
The conditions of in vitro experiments are controllable (e.g., concentrations, pH, temperature), and indeed, these parameters are usually exploited in the experimental design in order to maximize the amount of information obtained. The results from in vitro experiments can be extrapolated to in vivo conditions, as far as they are known, but there often remains problems such as choosing the appropriate buffer anion to mimic intracellular conditions where macromolecules provide the counter ion. Also, it may be difficult to define the local concentrations of a species within a cell where compartmentation and macromolecular crowding will grossly affect the rate of second-order reactions.
Measuring the effects of macromolecular crowding on antibody function with biolayer interferometry
Published in mAbs, 2019
Dorothy M. Kim, Xiao Yao, Ram P. Vanam, Michael S. Marlow
Macromolecular crowding is ubiquitous in biology. The resulting non-ideal interactions between proteins in crowded solutions are predicted to profoundly affect protein behavior and function.10,39,40 The specific nature of these highly non-linear effects is often difficult to predict, as evidenced by divergent conclusions in several reports.41,42 A limited number of studies using different macromolecular crowding agents have shown considerable consequences for equilibrium constants and reaction rates, often on the order of several logs.10,41,43–45 Together, this highlights the need for techniques capable of readily providing information on the effect of non-ideality in conditions closely replicating physiological environments. Here, we examined how physiological concentrations of albumin-affected mAb function with two complimentary techniques, CG-MALS and BLI. CG-MALS, a powerful and well-established tool that enables measurement of the CVC between two species, was used to obtain an initial understanding of non-specific interactions in the systems. We then utilized a BLI method, with steady-state analysis adapted for non-ideal solution conditions, to first replicate our CG-MALS results, and then extend these observations by performing equilibrium measurements of antigen binding under physiological concentrations of HSA. While orthogonal methods such as AUC with fluorescence detection can measure specific interactions in non-ideal conditions,7,46 BLI is advantageous as a convenient and high-throughput method to assess binding interactions with the inherent flexibility to test many different conditions at high concentrations of crowding agents, and can therefore provide information about binding in various environments in a small set of experiments. This approach is an easy and efficient way to eliminate mAbs or other molecules from consideration during the screening process, early in discovery research.