The Lys 280 → Gln mutation mimicking disease‐linked acetylation of Lys 280 in tau extends the structural core of fibrils and modulates their catalytic properties

Amyloid fibrillar aggregates isolated from the brains of patients with neurodegenerative diseases invariably have post‐translational modifications (PTMs). The roles that PTMs play in modulating the structures and polymorphism of amyloid aggregates, and hence their ability to catalyze the conversion of monomeric protein to their fibrillar structure is, however, poorly understood. This is particularly true in the case of tau aggregates, where specific folds of fibrillar tau have been implicated in specific tauopathies. Several PTMs, including acetylation at Lys 280, increase aggregation of tau in the brain, and increase neurodegeneration. In this study, tau‐K18 K280Q, in which the Lys 280 → Gln mutation is used to mimic acetylation at Lys 280, is shown, using HX‐MS measurements, to form fibrils with a structural core that is longer than that of tau‐K18 fibrils. Measurements of critical concentrations show that the binding affinity of monomeric tau‐K18 for its fibrillar counterpart is only marginally more than that of monomeric tau‐K18 K280Q for its fibrillar counterpart. Quantitative analysis of the kinetics of seeded aggregation, using a simple Michaelis–Menten‐like model, in which the monomer first binds and then undergoes conformational conversion to β‐strand, shows that the fibrils of tau‐K18 K280Q convert monomeric protein more slowly than do fibrils of tau‐K18. In contrast, monomeric tau‐K18 K280Q is converted faster to fibrils than is monomeric tau‐K18. Thus, the effect of Lys 280 acetylation on tau aggregate propagation in brain cells is expected to depend on the amount of acetylated tau present, and on whether the propagating seed is acetylated at Lys 280 or not.     

Feedback optimization of fed-batch fermentation

The problem of feedback optimization of the feed rate for fed-batch fermentation processes is formulated in the framework of singular control theory and switching hypersurfaces. Using four differential balance equations that describe a general class of fedbatch processes and a general objective function to be minimized, it is shown that under certain restrictions the feedback optimization of the feed rate can be realized as a nonlinear function of the state variables, such as the concentrations of cell mass, substrate and product, and the fermentor volume. The restrictions on the initial conditions, the fermentation kinetics and the objective function, that are needed for realization of the feedback optimization, are provided. Fed-batch fermentation models of lysine and alcohol are used to construct switching curves and to illustrate the feedback optimization of the feed flow rates.     

Simple nonsingular control approach to fed-batch fermentation optimization

Determination of the optimal feed rate for fed-batch fermentation is normally a problem in singular control with a state inequality constraint and as such is, in general, difficult to solve, especially for those described by a large number of dynamic mass balance equations. In this article we use a new set of state variables and the culture volume as the control variable. In this way the problem is converted to one of nonsingular control with the magnitude and rate constraints on the manipulated variable and can be numerically solved by a gradient-based technique, thus avoiding the difficulty associated with singular control problems. Examples are given to illustrate the method.     

Pyridoxal 5'-phosphate mediated inactivation of Escherichia coli DNA polymerase I: identification of lysine-635 as an essential residue for the processive mode of DNA synthesis

Inactivation of Escherichia coli DNA polymerase I by pyridoxal 5'-phosphate treatment results from its reactivity at multiple lysine residues. One of these residues, lysine-758, has been shown to be located at the substrate binding site in DNA polymerase I [Basu, A., & Modak, M. J. (1987) Biochemistry 26, 1704-1709]. We now demonstrate that lysine-635 is another important target of pyridoxylation; modification of this site results in decreased rates of DNA synthesis. Addition of template-primer with or without substrate deoxynucleoside triphosphate protects lysine-635 from pyridoxylation. Analysis of the initiation versus elongation phase of DNA synthesis by lysine-635-modified enzyme revealed that elongation of the DNA chain is severely affected by the lysine-635 modification. We therefore conclude that this lysine residue plays an important role in the processive mode of DNA synthesis by E. coli DNA polymerase I.     

A procedure for easy removal of acrylamide gel rods from the casting glass tubes

A new method specific for the determination of subpicomole quantities of tryptophan has been developed by elaboration of the Pictet-Spengler reaction. It permitted reproducible quantitation of tryptophan in less than 1 μl of plasma ultrafiltrate or 1 mg of brain tissue. Samples deproteinized by trichloroacetic acid were boiled for 15 min with formaldehyde and potassium ferricyanide at controlled acidity, where tryptophan was converted to a single new product identified as 9-hydroxymethyl-β-carboline. It was quantitated by either direct spectrofluorometry or a reversed-phase HPLC system developed for β-carbolines. Under our conditions, peptides containing N-terminal tryptophan such as Trp-Leu and delta sleep-inducing peptide gave N-(9-hydroxymethyl-β-carboline-3-carbonyl) peptides which retained all amino acid residues except tryptophan.     

A molecular model of the complete three-dimensional structure of the Klenow fragment of Escherichia coli DNA polymerase I: binding of the dNTP substrate and template-primer.

A complete three-dimensional structure of the Klenow fragment of Escherichia coli DNA polymerase I (pol I) has been proposed on the basis of molecular modeling and molecular mechanics studies using available C alpha coordinates. The structure seems quite reliable because the overall surface of electrostatic potentials calculated for the molecularly modeled enzyme closely resembles that reported for the X-ray structure. The modeled structure is then used in developing a ternary complex of dTTP and (dA)25-(dT)14 poised in its active site. The orientation of both substrates in the ternary complex was primarily guided by the amino acid residues which had been known to interact with dNTP and DNA substrates from earlier studies. The proposed model (a) explains the geometrical and physicochemical relationship of the two substrates with the various critical amino acid residues involved in the binding process and (b) suggests possible roles for additional residues in the binding and/or polymerization reaction. Furthermore, the ternary complex appears to satisfy many biochemical and genetic data concerning catalytic requirements known to exist for the polymerization reaction.     

Making sense of the combined effect of interleukin-2 and interleukin-4 on lymphocytes using a mathematical model

The cytokines are the information superhighway of the immune system. They are an important component of the integrated behavior of the system. In order to be able to have a good understanding of the immune system, we must be able to model the effect of cytokines and their combined effect. This work is a step in that direction. We study the combined effect of two cytokines: interleukin-2 (IL-2) and interleukin-4 (IL-4) on some cells of the immune system. Interleukin-2 and interleukin-4 are important growth and differentiation factors for B and T cells. Interleukin-4 antagonizes the effect of interleukin-2 on B cells and some T cells while it synergizes with interleukin-2 on other T cells. We build a mathematical model of the interaction of both cytokines on T and B cells as a building block toward a model of the Th1/Th2 cross-regulation. The response of a given cell to the combination of interleukin-2 and interleukin-4 is shown to involve competing dynamical effects which can lead to either antagnostic or synergistic combined effect. Author to whom correspondence should be addressed at Department of Engineering and Public Policy. Work supported by NIH grant nv: Ai31427.     

Intracellular aminopeptidases inStreptomyces lividans 66

Summary We have investigated the aminopeptidase activities present inStreptomyces lividans strains. The majority of these activities proved to be intracellular with multiple active species. Two aminopeptidase P genes were identified to be responsible for the ability to hydrolyze amino terminal peptide bonds adjacent to proline residues. Two other broad spectrum aminopeptidases were found to display homology at both the DNA and protein levels. One showed significant homology to PepN proteins, particularly around the putative zinc-binding residues which are important for catalysis. The second broad spectrum activity was not analyzed in detail but showed a different spectrum of substrate specificity to that of PepN.