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The titer of the scrapie agent was determined by measurements of time intervals from inoculation to onset of illness and from inoculation to death. Both intervals were found to be inversely proportional to the size of the dose injected intracerebrally into random-bred weanling Syrian hamsters. The logarithms of the time intervals minus a time factor were linear functions of the logarithm of the inoculum size. The time factors were determined by regression analysis in order to maximize these linear relationships. An equation relating the titer of the inoculum to the dilution of the sample and the length of the time intervals was developed. This equation facilitates the use of a computerized data base. Validation of these relationships was provided by comparing samples for which the agent was measured both by end-point titration and by time interval assay. Agreement between the two methods was generally within +/-0.5 log10 median infective dose units. No differences between the molecular properties of the agents from hamster and murine sources were observed using primarily the incubation time interval method with the former and end-point titration with the latter. The advantages of this new approach based on time interval measurements are considerable with respect to time and resources.

Using primary and secondary structure information of an RNA molecule, the program RNA2D3D automatically and rapidly produces a first-order approximation of a 3-dimensional conformation consistent with this information. Applicable to structures of arbitrary branching complexity and pseudoknot content, it features efficient interactive graphical editing for the removal of any overlaps introduced by the initial generating procedure and for making conformational changes favorable to targeted features and subsequent refinement. With emphasis on fast exploration of alternative 3D conformations, one may interactively add or delete base-pairs, adjacent stems can be coaxially stacked or unstacked, single strands can be shaped to accommodate special constraints, and arbitrary subsets can be defined and manipulated as rigid bodies. Compaction, whereby base stacking within stems is optimally extended into connecting single strands, is also available as a means of strategically making the structures more compact and revealing folding motifs. Subsequent refinement of the first-order approximation, of modifications, and for the imposing of tertiary constraints is assisted with standard energy refinement techniques. Previously determined coordinates for any part of the molecule are readily incorporated, and any part of the modeled structure can be output as a PDB or XYZ file. Illustrative applications in the areas of ribozymes, viral kissing loops, viral internal ribosome entry sites, and nanobiology are presented.

The scrapie agent causes a progressive degeneration of the central nervous system of animals after a prolonged incubation period. Measurements of incubation period length, defined as the time from inoculation to the onset of clinical signs of neurological dysfunction, were related to the titer of the agent and the dilution of the inoculated sample. Equations defining the relationship provide a new assay for the agent requiring fewer animals than end point titrations. By use of this incubation period assay, the scrapie agent from hamster brain was found to have an s20,w of < 300 S but > 30 S assuming rho p = 1.2 g/cm3. A partially purified fraction P3 was obtained by differential centrifugation and sodium deoxycholate extraction. When P3 was extracted with phenol, virtually no infectivity was found in the aqueous phase even after examining such variables as pH, salt concentration, and predigestion of samples with proteinase K. Nonionic and nondenaturing, anionic detergents did not inactivate the scrapie agent; in contrast, denaturing detergents inactivated the agent. Sodium dodecyl sulfate (NaDodSO4) inactivated greater than 90% of the agent at a NaDodSO4 to protein ratio of 1.8 g/g. Inactivation by NaDodSO4 appears to be a cooperative process. Addition of a nonionic detergent to form mixed micelles with NaDodSO4 prevented inactivation of the agent by NaDodSO4. Weak chaotropic ions do not inactivate the scrapie agent while strong chaotropic ions like SCN- and Cl3CCOO- destroy infectivity at concentrations of 0.2 M. These data provide evidence in support of a protein component within the scrapie agent which is essential for maintenance of infectivity. Thus, it is unlikely that the scrapie agent is composed only of a "naked" nucleic acid as is the case for the plant viroids.

The intrinsic chemical reaction of adenosine triphosphate (ATP) hydrolysis catalyzed by myosin is modeled by using a combined quantum mechanics and molecular mechanics (QM/MM) methodology that achieves a near ab initio representation of the entire model. Starting with coordinates derived from the heavy atoms of the crystal structure (Protein Data Bank ID code 1VOM) in which myosin is bound to the ATP analog ADP⅐VO 4 ؊ , a minimum-energy path is found for the transformation ATP ؉ H 2O 3 ADP ؉ Pi that is characterized by two distinct events: (i) a low activation-energy cleavage of the P ␥OO␤␥ bond and separation of the ␥-phosphate from ADP and (ii) the formation of the inorganic phosphate as a consequence of proton transfers mediated by two water molecules and assisted by the Glu-459 -Arg-238 salt bridge of the protein. The minimum-energy model of the enzyme-substrate complex features a stable hydrogen-bonding network in which the lytic water is positioned favorably for a nucleophilic attack of the ATP ␥-phosphate and for the transfer of a proton to stably bound second water. In addition, the P␥OO␤␥ bond has become significantly longer than in the unbound state of the ATP and thus is predisposed to cleavage. The modeled transformation is viewed as the part of the overall hydrolysis reaction occurring in the closed enzyme pocket after ATP is bound tightly to myosin and before conformational changes preceding release of inorganic phosphate.ATP hydrolysis ͉ enzymatic catalysis ͉ energy profile ͉ quantum mechanics and molecular mechanics simulations T he mechanism of hydrolysis of adenosine triphosphate (ATP) by myosin, leading to adenosine diphosphate (ADP) and inorganic phosphate (P i ), which constitutes one of the most important enzymatic reactions responsible for energy transduction into the directed movements of adjoining actin filaments, continues to remain a subject of active debates (1-16), a significant part of which relates to what constitutes the acceptor of the proton that must be released by the ''lytic'' water in its nucleophilic attack on the ATP ␥-phosphate.In terms of the generally accepted kinetic scheme (1-3), the relevant ATP-myosin transformations may be described by the equationin which M* and M** indicate conformers of myosin. As reported (1-3), reaction (Eq. 1) occurs with a near unit equilibrium constant K Ͻ 10 and the estimated rate constants k ϩ Ն 160 s Ϫ1 and k Ϫ Ն 18 s Ϫ1 . The rate constant k ϩ ϭ 160 s Ϫ1 can be converted to the free-energy activation barrier ⌬G # Ϸ 14.6 kcal/mol at room temperature, T ϭ 300 K, by applying a simple transition-state theory formula (17) k Ϸ 6⅐10 12 exp͓Ϫ⌬G # ͞RT͔. [2]However, noting that the experimental rate constants of reaction (Eq. 1) incorporate contributions from conformational changes in the protein from M* to M** leads us to expect that the activation energy of the intrinsic chemical reactionwhich excludes conformational rearrangements, should be considerably Ͻ14.6 kcal/mol. However, previous attempts (13, 15, 16) to simulate the mechanism of react...

Accumulation of the GABA inhibitory neurotransmitter for rapid delivery into synapses can be accomplished by GAD65 dependent and independent membrane-targeting of GAD67.

The accuracy of regional myocardial blood flow measurements made with microspheres of different sizes is uncertain. Therefore, we simultaneously injected radioactive microspheres of different sizes into the left atria of dogs and sheep; the microsphere diameters were 25µ, 15µ, 9µ, 1-l0µ, and filtered 1-10µ (most > 7µ). Antipyrine was sometimes simultaneously infused for 15-60 seconds. Myocardial blood flow was altered by hemorrhage, tachycardia, supravalvar aortic constriction, or infusion of methoxamine or adenosine triphosphate; left coronary artery branches were occluded on two occasions. Sometimes the percent of untrapped microspheres was estimated. All sizes of microspheres measured similar total myocardial blood flows when the percent of untrapped microspheres was known. All indicators were distributed identically to the right and left ventricular free walls and septum; ischemic areas had a 1% excess of antipyrine. With any pair of microspheres, the larger had a subendocardial excess except for 25µ microspheres, which were in excess compared with 15µ microspheres in the higher flow layer whether it was subendocardial or subepicardial. The greatest difference for any pair of microspheres was 10.53% of flow in a layer. Antipyrine did not define which size of microspheres measured true regional blood flow, since many previously unemphasized limitations were discovered. Nevertheless, we believe that microspheres 9µ in diameter are probably the best for measuring regional myocardial blood flow.