Topological Constraints and Their Conformational Entropic Penalties on RNA Folds

Functional RNAs can fold into intricate structures using a number of different secondary and tertiary structural motifs. Many factors contribute to the overall free energy of the target fold. This study aims at quantifying the entropic costs coming from the loss of conformational freedom when the sugar-phosphate backbone is subjected to constraints imposed by secondary and tertiary contacts. Motivated by insights from topology theory, we design a diagrammatic scheme to represent different types of RNA structures so that constraints associated with a folded structure may be segregated into mutually independent subsets, enabling the total conformational entropy loss to be easily calculated as a sum of independent terms. We used high-throughput Monte Carlo simulations to simulate large ensembles of single-stranded RNA sequences in solution to validate the assumptions behind our diagrammatic scheme, examining the entropic costs for hairpin initiation and formation of many multiway junctions. Our diagrammatic scheme aids in the factorization of secondary/tertiary constraints into distinct topological classes and facilitates the discovery of interrelationships among multiple constraints on RNA folds. This perspective, which to our knowledge is novel, leads to useful insights into the inner workings of some functional RNA sequences, demonstrating how they might operate by transforming their structures among different topological classes.     

Physical and biological aspects of renal vitrification

Cryopreservation would potentially very much facilitate the inventory control and distribution of laboratory-produced organs and tissues. Although simple freezing methods are effective for many simple tissues, bioartificial organs and complex tissue constructs may be unacceptably altered by ice formation and dissolution. Vitrification, in which the liquids in a living system are converted into the glassy state at low temperatures, provides a potential alternative to freezing that can in principle avoid ice formation altogether. The present report provides a brief overview of the problem of renal vitrification. We report here the detailed case history of a rabbit kidney that survived vitrification and subsequent transplantation, a case that demonstrates both the fundamental feasibility of complex system vitrification and the obstacles that must still be overcome, of which the chief one in the case of the kidney is adequate distribution of cryoprotectant to the renal medulla. Medullary equilibration can be monitored by monitoring urine concentrations of cryoprotectant, and urine flow rate correlates with vitrification solution viscosity and the speed of equilibration. By taking these factors into account and by using higher perfusion pressures as per the case of the kidney that survived vitrification, it is becoming possible to design protocols for equilibrating kidneys that protect against both devitrification and excessive cryoprotectant toxicity.     

Overexpression, Purification, and Generation of a Thermostable Variant ofZymomonas mobilisFructokinase

The gene encoding fructokinase (EC 2.7.1.4) fromZymomonas mobilishas been expressed at high level inEscherichia coliby modifying the ribosome binding site using the polymerase chain reaction. A simple two-step purification from extracts of the recombinant cells results in highly purified enzyme suitable for use in fructose determination. Using the polymerase chain reaction in mutagenic conditions, a variant of fructokinase was isolated which was more thermostable than the wild type, taking the 30 min half-life from 70.1 to 72.4°C. The purified thermostable variant had the same specific activity as the wild type. Sequencing of the variant indicated that only one amino acid was changed, with Ser 69 becoming Ala. Searches of the mutant libraries for variants that were (a) active with glucose or (b) had reduced inhibition by glucose were unsuccessful.     

Binding of plasma fibronectin to the surfaces of BHK cells in suspension at 4 °C

Plasma fibronectin is shown by several different criteria to bind to suspended BHK cells if the binding incubations are carried out at 4 °C. In indirect immunofluorescence experiments, fibronectin bound to suspended BHK cells at 4 °C in a punctate distribution over the entire cell surfaces. Little binding, however, was detected on cells incubated with fibronectin at 37 °C. The fibronectin bound to the cells at 4 °C was functionally active, since these cells subsequently were able to spread on tissue culture dishes in protein-free medium, unlike cells preincubated with fibronectin at 37 °C or in the absence of fibronectin. Also, the cell surface receptors for soluble fibronectin and fibronectin-coated beads appeared to be similar, since cells preincubated with fibronectin at 4 °C subsequently bound fewer fibronectin-coated beads than control cells. In biochemical studies with radiolabeled fibronectin, binding of fibronectin to the cells was shown to increase with incubation time up to 4 h. In competition experiments with unlabeled fibronectin, 30% of the binding of radiolabeled fibronectin could be inhibited.     

Deposition of fibronectin on material surfaces exposed to plasma: quantitative and biological studies

Experiments were carried out to characterize plasma fibronectin deposition onto material surfaces exposed to plasma solutions. Under nonclotting conditions, the amount of fibronectin adsorption on the surfaces, determined by an indirect radioactive antibody assay, was maximal at low plasma concentrations (0.1%). At higher concentrations of plasma, other plasma proteins appeared to compete with and inhibit adsorption of fibronectin. Biological activity (fibronectin-promoted cell spreading) was also greatest at low plasma concentrations and decreased as the plasma concentration was raised. When surfaces were exposed to plasma under clotting conditions (i.e., addition of Ca2+ and thrombin), fibronectin deposition on the surfaces and biological activity remained constant or increased as the plasma concentration was raised. Based on indirect immunofluorescent antibody assays, the fibronectin deposited from clotting plasma appeared to be in a punctate distribution over the entire material surface and occasionally was associated with discrete fibrillar structures. The increased deposition of fibronectin from clotting plasma compared to nonclotting plasma (approximately a 10-fold difference with 10% plasma) was partially a result of covalent crosslinking of fibronectin to fibrin based upon studies with putrescine added to inhibit crosslinking during clotting. On the other hand, the increase in biological activity that occurred if the surfaces were exposed to clotting plasma was completely inhibited by putrescine, indicating that fibronectin had to be crosslinked to fibrin to have biological activity under these conditions. Finally, fibronectin deposition also occurred on surfaces exposed to whole blood and was markedly enhanced when clotting occurred.