The complex folding behavior of HIV-1-protease monomer revealed by optical-tweezer single-molecule experiments and molecular dynamics simulations.

We have used optical tweezers and molecular dynamics simulations to investigate the unfolding and refolding process of a stable monomeric form of HIV-1-protease (PR). We have characterized the behavior under tension of the native state (N), and that of the ensemble of partially folded (PF) conformations the protein visits en route to N, which collectively act as a long-lived state controlling the slow kinetic phase of the folding process. Our results reveal a rich network of unfolding events, where the native state unfolds either in a two-state manner or by populating an intermediate state I, while the PF state unravels through a multitude of pathways, underscoring its structural heterogeneity. Refolding of mechanically denatured HIV-1-PR monomers is also a multiple-pathway process. Molecular dynamics simulations allowed us to gain insight into possible conformations the protein adopts along the unfolding pathways, and provide information regarding possible structural features of the PF state.

Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme.

Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 +/- 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol.

Axotomy affects density but not properties of potassium leak channels, in the leech AP neurons.

Leech AP neurons react to axotomy by increasing excitability and resting potential of the cell body membrane. In a previous report we described single potassium channels contributing to the leak conductance in the soma membrane of AP cells. Here we compare both properties and density of single potassium leak channels in cell-free patches from normal and axotomized AP neurons. We show that properties such as single channel conductance, outward rectification, time constants of open and shut interval distributions and absence of inactivation do not significantly differ between normal and axotomized cells. On the other hand, we find that the number of channels per patch progressively increases with time after axotomy. We conclude that changes in density rather than alterations in properties of single channels can account for the increase in the resting potential, observed after axotomy.

Model instructional program for mainstreaming handicapped children.

The purpose of this study was to implement and test the effectiveness of an instructional program designed to teach normal children about cerebral palsy. Presentation of the program to 17 first-grade children resulted in a significant increase in the students' knowledge of the disability, compared to that of a control group. Implications of these results for physical and occupational therapists involved in facilitating placement of handicapped children into the least restrictive environment were discussed.

Concentration of fibronectin in plasma of tumor-bearing mice and synthesis by Ehrlich ascites tumor cells.

In the present paper we have studied: (a) the concentration of fibronectin (FN) in plasma and in ascitic fluid of mice at different times after inoculation of Ehrlich ascites tumor cells; (b) the ability of Ehrlich ascites cells to synthesize and release FN; and (c) the localization of FN in Ehrlich ascites cells by immunofluorescence microscopy. It was found that (a) 4 to 5 days after inoculation of the tumor, the plasma concentration of FN was significantly higher [1.7 +/- 0.07% (S.E.) of total plasma protein] than that in the normal control mice (0.8 +/- 0.035); (b) FN is present in the ascitic fluid in all phases of tumor growth; (c) Ehrlich ascites cells cultured in vitro synthesize and release large amounts of FN in the culture medium; and (d) only about 1 to 2% of the tumor cells show a very small amount of FN, and this is mostly in the area of cell-cell contact.

Influence of reading level difficulty on the disfluencies of normal children.

This study assessed the effect of increasing the difficulty of reading material on the frequency and type of disfluency in the oral reading of normally fluent elementary school children. Eighty children participated in the study. Ten boys and 10 girls each, from Grades 3, 4, 5, and 6, read five 200-word passages, corresponding to one grade below their school placement, the grade of their school placement, and one, two, and three grades above their school placement. Results indicated a significant increase in total moments of disfluency and four specific types of disfluency as the difficulty of reading material increased. The data revealed a tendency for "stuttering" disfluencies to increase more in frequency than "normal" nonfluencies. Results are discussed with respect to theoretical and experimental implications.