Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist: role in restricted conformational mobility and compaction of native state

Understanding the mechanism for sucrose-induced protein stabilization is important in many diverse fields, ranging from biochemistry and environmental physiology to pharmaceutical science. Timasheff and Lee [Lee, J. C. & Timasheff, S. N. (1981) J. Biol. Chem. 256, 7193-7201] have established that thermodynamic stabilization of proteins by sucrose is due to preferential exclusion of the sugar from the protein's surface, which increases protein chemical potential. The current study measures the preferential exclusion of 1 M sucrose from a protein drug, recombinant interleukin 1 receptor antagonist (rhIL-1ra). It is proposed that the degree of preferential exclusion and increase in chemical potential are directly proportional to the protein surface area and that, hence, the system will favor the protein state with the smallest surface area. This mechanism explains the observed sucrose-induced restriction of rhIL-1ra conformational fluctuations, which were studied by hydrogen-deuterium exchange and cysteine reactivity measurements. Furthermore, infrared spectroscopy of rhlL-1ra suggested that a more ordered native conformation is induced by sucrose. Electron paramagnetic resonance spectroscopy demonstrated that in the presence of sucrose, spin-labeled cysteine 116 becomes more buried in the protein's interior and that the hydrodynamic diameter of the protein is reduced. The preferential exclusion of sucrose from the protein and the resulting shift in the equilibrium between protein states toward the most compact conformation account for sucrose-induced effects on rhIL-1ra.     

Effects of Al3+ and Be2+ ions combined with NaF on ciliary process adenylyl cyclase activity and aqueous humor dynamics in the rabbit eye

PURPOSE: The activity of Al3+, Ga3+, and Be2+ ions in the presence of NaF to directly activate G-proteins was investigated by their potentiative effect on forskolin (FSK)-activated adenylyl cyclase in rabbit ciliary process membranes and their effects on aqueous humor dynamics in vivo. METHODS: Adenylyl cyclase (AC) was determined by radiometric conversion of ATP to cAMP by the particulate fraction of rabbit ciliary processes. Intravitreal injections of sterile solutions of analytical grade salts were made into the center of the vitreous in a volume of 20 microliters. Intraocular pressure, aqueous humor flow, and uveoscleral outflow measurements were made by pneumatonometry, fluorophotometry, and fluorescein-dextran method, respectively. Outflow facility was determined by tonography in the intact eyes and by two-level constant pressure perfusion in cannulated eyes. RESULTS: Both Al3+ (EC50, 40 mumol/l) and Be2+ (EC50, 11 mumol/l) in the presence of 0.5-2 mM NaF activated the stimulatory G-protein Gs. Ga3+ was ineffective and did not antagonize the activation by Al3+. Intravitreal injections of Al3+ (1 mumol/eye) or Be2+ (0.5 or 1 mumol/eye) had no significant intraocular pressure (IOP) effect, nor did 1.5 or 3 mumol/eye of NaF, but when either cation was injected together with NaF, IOP decreased by up to 40% for up to 140 hr. At the time of maximum IOP effect (72 hr) aqueous humor flow determined by fluorophotometry was decreased in BeCl2+ NaF-treated eyes by 40% relative to BeCl2-treated eyes; however, tonographic facility of outflow was unaffected. Uveoscleral flow was also decreased by 38% in BeCl2+ NaF treated eyes. CONCLUSIONS: These findings support the hypothesis that Gs activation of ciliary process adenylyl cyclase decreases aqueous humor formation rate in rabbit eyes, and that activation of G-proteins mediates contraction of ciliary muscles causing a decrease of aqueous humor outflow via the uveoscleral route. The results suggest that G-proteins putatively involved in trabecular facility changes are less sensitive to activation by BeF3- than are other parameters of aqueous humor dynamics.     

Combining multiple knowledge bases

Combining knowledge present in multiple knowledge base systems into a single knowledge base is discussed. A knowledge based system can be considered an extension of a deductive database in that it permits function symbols as part of the theory. Alternative knowledge bases that deal with the same subject matter are considered. The authors define the concept of combining knowledge present in a set of knowledge bases and present algorithms to maximally combine them so that the combination is consistent with respect to the integrity constraints associated with the knowledge bases. For this, the authors define the concept of maximality and prove that the algorithms presented combine the knowledge bases to generate a maximal theory. The authors also discuss the relationships between combining multiple knowledge bases and the view update problem     

Determination of precompression and compression force levels to minimize tablet friability using simplex

Pilot simplex experiments for improving the tablet strength of three aspirin tablet formulations based on precompression and compression forces were presented. As each simplex moved towards the direction of the optimum, the friability was being minimized and the crushing strength was concomittantly being maximized. Because it followed a systematic direction, simplex process would locate a local optimum rapidly. The appropriate levels of precompression and compression forces that produced tablets with the desired strength were attained in five trials. By contrast, random search for this force combination required at least ten trials. Simplex technique is a cost and time effective means for determining the precompression and compression forces that will reduce the friability or increase the hardness of a tablet formulation. Results appeared to also indicate that crushing strength might be a more reliable measure of tablet strength than friability.     

Laparoscopic identification of preperitoneal nerve anatomy in the inguinal area

The laparoscopic approach has recently been utilized for inguinal hernia repair. Nerve injuries are now being reported. The femoral branch of the genitofemoral nerve and the lateral cutaneous nerve of the thigh appear most at risk. The purpose of this study was to determine the feasibility of identifying these nerves laparoscopically on either a routine or selective basis. Twenty patients scheduled for laparoscopic inguinal hernia repair were prospectively selected. An attempt was made to identify these nerves so that optimum placement of staples could occur. The femoral branch of the genitofemoral nerve was identified in 19 of 20 patients and the lateral cutaneous nerve of the thigh in 18 of 20 patients. A review of 125 laparoscopic inguinal hernia repairs revealed five nerve injuries (4%). Details are given and recommendations discussed. Knowledge of preperitoneal anatomy and awareness of the location of these nerves should lead to a safer dissection and more accurate application of staples, hopefully decreasing the incidence of nerve injury.     

Beliefs, time and incomplete information in multiple encounter negotiations among autonomous agents

In negotiations among autonomous agents over resource allocation, beliefs about opponents, and about opponents’ beliefs, become particularly important when there is incomplete information. This paper considers interactions among self‐motivated, rational, and autonomous agents, each with its own utility function, and each seeking to maximize its expected utility. The paper expands upon previous work and focuses on incomplete information and multiple encounters among the agents. It presents a strategic model that takes into consideration the passage of time during the negotiation and also includes belief systems. The paper provides strategies for a wide range of situations. The framework satisfies the following criteria: symmetrical distribution, simplicity, instantaneously, efficiency and stability. This revised version was published online in June 2006 with corrections to the Cover Date.