Extracellular carbonic anhydrase of skeletal muscle associated with the sarcolemma

We report here 1) the synthesis and properties of a new macromolecular carbonic anhydrase inhibitor, Prontosil-dextran, 2) its application to determine the localization of a previously described extracellular carbonic anhydrase in skeletal muscle, and 3) the application of a recently published histochemical technique using dansylsulfonamide to the same problem. Stable macromolecular inhibitors of molecular weights of 5,000, 100,000 and 1,000,000 were produced by covalently coupling the sulfonamide Prontosil to dextrans. Their inhibition constants towards bovine carbonic anhydrase II are 1-2 X 10(-7) M. The Prontosil-dextrans, PD 5,000, PD 100,000, and PD 1,000,000, were used in studies of the washout of H14CO3-) from the perfused rabbit hindlimb. This washout is slow due to the presence of an extracellular carbonic anhydrase and can be markedly accelerated by PD 5,000 but not by PD 100,000 and PD 1,000,000. Since PD 5,000 is accessible to the entire extracellular space and PD 100,000 and PD 1,000,000 are confined to the intravascular space, we conclude that the extracellular carbonic anhydrase of skeletal muscle is located in the interstitium. The histochemical studies show a strong staining of the sarcolemma of the muscle fibers with high oxidative capacity. It appears likely, therefore, that the extracellular carbonic anhydrase of skeletal muscle is associated with muscle plasma membranes with its active site directed toward the interstitial space.     

Preliminary crystallographic studies on bovine lactoferrin

The purification of bovine lactoferrin, its crystallization at low ionic strength, and preliminary X-ray crystallographic data are reported. The crystals, which grow from a two-phase system, are radiation-stable and suitable for a medium-resolution X-ray analysis. They are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 138.4 A, b = 87.1 A, c = 73.6 A, and one protein molecule in the asymmetric unit.     

Simulations of Flexible Manifolds

Monte Carlo simulations of flexible two-dimensional model membranes embedded in three space dimensions are reported. We explain in detail the techniques how to simulate fluid open membranes and fluid closed membranes (vesicles). It is shown that polymerized open membranes are rough and flat. Accordingly, the two larger components of the inertia tensor are proportional to the number of monomers of the surface, λ₃ ≈ λ₂ ～ N, whereas the smallest λ₁ ～ N ^0.65. Polymerized vesicles are isotropic and their mean square radius of gyration is R ² ～ λ _k ～ N. In contrast, fluid membranes and vesicles exhibit crumpled shapes with λ _k ～ N ^0.8 for k = 1,2,3. A monomer on a fluid surface exhibits a time-dependent mean squared displacement of r ² (t) ～ t ^0.8.     

The Dark Recovery Rate in the Photocycle of the Bacterial Photoreceptor YtvA Is Affected by the Cellular Environment and by Hydration

We report thermal recovery kinetics of the lit state into the parental dark state, measured for the blue light-sensing photoreceptor YtvA inside overexpressing E. coli and B. subtilis bacterial cells, performed for the wild type and several mutated proteins. Recovery was followed as a recovery of the fluorescence, as this property is only found for the parental but not for the photochemically generated lit state. When cells were deposited onto a microscope glass plate, the observed thermal recovery rate in the photocycle was found ca. ten times faster in comparison to purified YtvA in solution. When the E. coli or B. subtilis colonies were soaked in an isotonic buffer, the dark relaxation became again much slower and was very similar to that observed for YtvA in solution. The observed effects show that rate constants can be tuned by the cellular environment through factors such as hydration.     

Process-induced cell cycle oscillations in CHO cultures: Online monitoring and model-based investigation

The influence of process strategies on the dynamics of cell population heterogeneities in mammalian cell culture is still not well understood. We recently found that the progression of cells through the cell cycle causes metabolic regulations with variable productivities in antibody-producing Chimese hamster ovary (CHO) cells. On the other hand, it is so far unknown how bulk cultivation conditions, for example, variable nutrient concentrations depending on process strategies, can influence cell cycle-derived population dynamics. In this study, process-induced cell cycle synchronization was assessed in repeated-batch and fed-batch cultures. An automated flow cytometry set-up was developed to measure the cell cycle distribution online, using antibody-producing CHO DP-12 cells transduced with the cell cycle-specific fluorescent ubiquitination-based cell cycle indicator (FUCCI) system. On the basis of the population-resolved model, feeding-induced partial self-synchronization was predicted and the results were evaluated experimentally. In the repeated-batch culture, stable cell cycle oscillations were confirmed with an oscillating G1 phase distribution between 41% and 72%. Furthermore, oscillations of the cell cycle distribution were simulated and determined in a (bolus) fed-batch process with up to cells/ml. The cell cycle synchronization arose with pulse feeding only and ceased with continuous feeding. Both simulated and observed oscillations occurred at higher frequencies than those observable based on regular (e.g., daily) sample analysis, thus demonstrating the need for high-frequency online cell cycle analysis. In summary, we showed how experimental methods combined with simulations enable the improved assessment of the effects of process strategies on the dynamics of cell cycle-dependent population heterogeneities. This provides a novel approach to understand cell cycle regulations, control cell population dynamics, avoid inadvertently induced oscillations of cell cycle distributions and thus to improve process stability and efficiency.