Temperature transitions of protein properties in human red blood cells

Human red blood cells (RBC) undergo a sudden change from blocking to passing through 1.3 +/- 0.2-micrometer micropipettes at a transition temperature (Tc) of 36.4 degrees C. For resealed RBC ghosts this transition occurs at 28.3 degrees C (Tg). These findings are attributed to an elastomeric transition of hemoglobin from being gel-like to a fluid and to an elastomeric transition of membrane proteins such as spectrin. Spectrin shows a uniform distribution along the aspirated RBC tongue above Tg in contrast to the linear gradient below Tg.     

Capillary tube resistive thermal cycling

A system that performs rapid thermal cycling of microliter and smaller liquid volumes inside glass capillary tubes that have an optically transparent thin film of indium-tin oxide (ITO) covering the exterior is described. The ITO film acts as both a heater and a temperature sensor, while cooling is accelerated with forced air. Unlike existing batch-mode thermal cycling systems, this system allows control over each sample's temperature profile. Temperature transition rates of 44 degrees Celsius per second during heating and 15 degrees Celsius per second during cooling have been achieved, allowing successful polymerase chain reaction (PCR) experiments to be performed in 20 min. Capillary external temperature can be regulated typically to within +/- 0.25 degrees Celsius, and peak temperatures more than 800 degrees Celsius have been demonstrated. Capillary internal (sample) temperatures at present are controllable typically to within 2 degrees Celsius. The resistive film can be used as a temperature sensor, and the optical transparency of the thin-film coating could permit fluorescent monitoring of the sample during thermal cycling, making this method well suited for real-time quantitative PCRs.     

Calorimetric determination of inhibition of ice crystal growth by antifreeze protein in hydroxyethyl starch solutions

Differential scanning calorimetry and cryomicroscopy were used to investigate the effects of type I antifreeze protein (AFP) from winter flounder on 58% solutions of hydroxyethyl starch. The glass, devitrification, and melt transitions noted during rewarming were unaffected by 100 micrograms/ml AFP. Isothermal annealing experiments were undertaken to detect the effects of AFP-induced inhibition of ice crystal growth using calorimetry. A premelt endothermic peak was detected during warming after the annealing procedure. Increasing the duration or the temperature of the annealing for the temperature range from -28 and -18 degrees C resulted in a gradual increase in the enthalpy of the premelt endotherm. This transition was unaffected by 100 micrograms/ml AFP. Annealing between -18 and -10 degrees C resulted in a gradual decrease in the premelt peak enthalpy. This process was inhibited by 100 micrograms/ml AFP. Cryomicroscopic examination of the samples revealed that AFP inhibited ice recrystallization during isothermal annealing at -10 degrees C. Annealing at lower temperatures resulted in minimal ice recrystallization and no visible effect of AFP. Thus, the 100 micrograms/ml AFP to have a detectable influence on thermal events in the calorimeter, conditions must be used that result in significant ice growth without AFP and visible inhibition of this process by AFP.     

Influence of Phase Transitions on the Macrokinetics of the Gaseous Reduction of Iron Oxide

The reduction of magnetite pellets is studied by thermogravimetry in a hydrogen flow upon linear heating. A multiple decrease in the reduction rate is observed at the degrees of metallization higher than 70% in a temperature range of 920–950°C. The observed effect of a decrease in the reduction rate by four to five times on heating is related to the α → γ phase transition of iron. Temperature cycling (heating–holding–heating–holding…) in the phase transition range shows that this effect is reversible; i.e., the reduction rate increases on cooling, unlike that on heating. The average temperature of the beginning of the effect in heating–cooling cycles (913°C) turns out to be close to the thermodynamic temperature (911°C) of the α → γ phase transition of iron. Isothermal studies of the reduction rate of pellets in a temperature range of 900–1000°C also confirm this phenomenon. The effect of a decrease in the reduction rate is assumed to appear due to a decrease in the effective coefficient of gas diffusion in γ-iron as compared to α-iron.     

Mycoplasma hyorhinis infection of pigs selectively bred for high and low immune response

Fourier transform infrared microspectroscopy (FTIR) was used to study glasses of pure carbohydrates and in the cytoplasm of desiccation tolerant plant organs. The position of the OH stretching vibration band (vOH) shifted with temperature. Two linear regression lines were observed in vOH against temperature plots. The temperature at the point of intersection between these two lines coincided with the glass transition temperature (Tg), as determined by other methods. The temperature at the intersection point decreased with increasing water content, which further validates that, indeed, Tg was observed. Tg values that were determined for dry glucose, sucrose, maltose, trehalose and raffinose glasses were 27, 57, 91, 108 and 108 degrees C, respectively. The shift of vOH with temperature, the wavenumber-temperature coefficient (WTC), was higher in sugar glasses having higher Tg. This suggests that glasses are more loosely packed when they have higher Tg. For Typha latifolia pollen and dried Craterostigma plantagineum leaves we obtained similar vOH vs. temperature plots as for carbohydrate glasses, indicating that a glass transition was observed. The Tg in dry pollen was ca. 45 degrees C and in dry plant leaves ca. 65 degrees C, with WTC values comparable to those observed in the carbohydrates. The Tg values in these tissues decreased with increasing water contents. Our data suggest that the carbohydrates that are present in the cytoplasm are primary factors contributing to the glassy state. We conclude that FTIR provides new insights in the structure of glasses in carbohydrates and in biological tissues.     

Differential inhibition of lysophosphatidate signaling by volatile anesthetics

cAMP receptor protein (CRP) is involved in regulation of expression of several genes in Escherichia coli. The protein is a homodimer and each monomer is folded into two distinct structural domains. The mechanism of the biological activity of the protein may involve the interaction between the subunits and domains. In order to determine the interaction between the subunits or domains of CRP, we have studied the reversible denaturation of the protein by guanidine hydrochloride. The unfolding and refolding kinetics of CRP was monitored using stopped-flow fluorescence spectroscopy at 20 degrees C and pH 7.9. The results of CRP denaturation indicate that the transition can be described by a three-state model: (CRP native)2<=> 2 (CRP native)<=>2 (CRP denatured). The faster process, characterized by the relaxation time tau 2 = 80 +/- 3 ms, corresponds to the dissociation of CRP dimer into monomers. The slower process has the relaxation time tau t = 1.9 +/- 0.1 s and corresponds to the cooperative unfolding of CRP monomer. The free energy change in the absence of denaturant upon CRP dissociation is delta G dis degrees = 46.9 +/- 2.5 kJ/mol and for monomer unfolding delta G unf degrees = 30.9 +/- 1.3 kJ/mol. The thermal unfolding of CRP was studied by circular dichroism and fluorescence spectroscopy at various guanidine hydrochloride concentrations. It has been found that the native protein is maximally stable at about 21 +/- 0.3 degrees C and is denatured upon heating and cooling from this temperature. The apparent free energy change for CRP unfolding at 21 degrees C is equal to 30.5 +/- 0.4 kJ/mol and the apparent specific heat change is equal to delta Cp, app = 10.7 +/- 0.7 kJ mol-1 K-1. The predicted values of cold denaturation midpoint is equal to tau G = -18.8 +/- 1.5 degrees C and for high-temperature transition tau G = 63.1 +/- 1.5 degrees C. The predicted midpoint of high-temperature unfolding transition is about the same as determined experimentally.     

Calorimetry of apolipoprotein-A1 binding to phosphatidylcholine-triolein-cholesterol emulsions

The thermotropic properties of triolein-rich, low-cholesterol dipalmitoyl phosphatidylcholine (DPPC) emulsion particles with well-defined chemical compositions (approximately 88% triolein, 1% cholesterol, 11% diacyl phosphatidylcholine) and particle size distributions (mean diameter, approximately 1000-1100 A) were studied in the absence and presence of apolipoprotein-A1 by a combination of differential scanning and titration calorimetry. The results are compared to egg yolk PC emulsions of similar composition and size. Isothermal titration calorimetry at 30 degrees C was used to saturate the emulsion surface with apo-A1 and rapidly quantitate the binding constants (affinity Ka = 11.1 +/- 3.5 x 10(6) M-1 and capacity N = 1.0 +/- 0.09 apo-A1 per 1000 DPPC) and heats of binding (enthalpy H = -940 +/- 35 kcal mol-1 apo-A1 or -0.92 +/- 0.12 kcal mol-1 DPPC). The entropy of association is -3070 cal deg-1 mol-1 protein or -3 cal deg-1 mol-1 DPPC. Without protein on the surface, the differential scanning calorimetry heating curve of the emulsion showed three endothermic transitions at 24.3 degrees C, 33.0 degrees C, and 40.0 degrees C with a combined enthalpy of 1.53 +/- 0.2 kcal mol-1 DPPC. With apo-A1 on the surface, the heating curve showed the three transitions more clearly, in particular, the second transition became more prominent by significant increases in both the calorimetric and Van't Hoff enthalpies. The combined enthalpy was 2.70 +/- 0.12 kcal mol-1 DPPC and remained constant upon repeated heating and cooling. Indicating that the newly formed DPPC emulsion-Apo-A1 complex is thermally reversible during calorimetry. Thus there is an increase in delta H of 1.17 kcal mol-1 DPPC after apo-A1 is bound, which is roughly balanced by the heat released during binding (-0.92 kcal) of apo-A1. The melting entropy increase, +3.8 cal deg-1 mol-1 DPPC of the three transitions after apo-A1 binds, also roughly balances the entropy (-3 cal deg-1 mol-1 DPPC) of association of apo-A1. These changes indicate that apo-A1 increases the amount of ordered gel-like phase on the surface of DPPC emulsions when added at 30 degrees C. From the stoichiometry of the emulsions we calculate that the mean area of DPPC at the triolein/DPPC interface is 54.5 A2 at 41 degrees C and 54.2 A2 at 30 degrees C. The binding of apo-A1 at 30 degrees C to the emulsion reduces the surface area per DPPC molecule from 54.2 A2 to 50.8 A2. At 30 degrees apo-A1 binds with high affinity and low capacity to the surface of DPPC emulsions and increases the packing density of the lipid domain to which it binds. Apo-A1 was also titrated onto DPPC emulsions at 45 degrees C. This temperature is above the gel liquid crystal transition. No heat was released or adsorbed. Furthermore, egg yolk phosphatidylcholine emulsions of nearly identical composition were also titrated at 30 degrees C with apo-A1 and were euthermic. Association constants were previously measured using a classical centrifugation assay and were used to calculate the entropy of apo-A1 binding (+28 cal deg-1 mol-1 apo-A1). This value indicates that apo-A1 binding to a fluid surface like egg yolk phosphatidylcholine or probably DPPC at 45 degrees C is hydrophobic and is consistent with hydrocarbon lipid or protein moities coming together and excluding water. Thus the binding of apo-A1 to partly crystalline surfaces is entropically negative and increases the order of the already partly ordered phases, whereas binding to liquid surfaces is mainly an entropically driven hydrophobic process.     

Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.     

Thermodynamic analysis of human plasma apolipoprotein C-1: high-temperature unfolding and low-temperature oligomer dissociation

Thermal and chemical unfolding of lipid-free apolipoprotein C-1 (apoC-1), a 6-kDa protein component of very low density and high-density lipoproteins, was analyzed by far-UV CD. In neutral 1 mM Na2HPO4 solutions containing 6-7 micrograms/mL protein, the apoC-1 monomer is approximately 30% alpha-helical at 0-22 degrees C and unfolds reversibly from about 22-80 degrees C with Tm = 51 +/- 3 degrees C and van't Hoff enthalpy delta Hv(Tm) = 19 +/- 3 kcal/mol. The apparent free energy of the monomer stabilization determined from the chemical unfolding at 0 degree C, delta G(0 degree C) = 2.8 +/- 0.8 kcal/mol, decreases by about 1 kcal/mol upon heating to 25 degrees C. A small apparent heat capacity increment suggests the absence of a substantial hydrophobic core for the apoC-1 molecule. At pH 7, increasing apoC-1 concentration above 10 micrograms/mL leads to self-association and formation of additional alpha-helices that unfold upon both heating and cooling from room temperature. The CD data indicate that the high-temperature transition reflects a complete monomer unfolding and the low-temperature transition reflects oligomer dissociation into stable monomers. This suggests the importance of hydrophobic interactions for apoC-1 self-association. Close proximity between the high- and low-temperature transitions and the absence of a plateau in the chemical unfolding curves recorded from oligomeric apoC-1 indicate marginal oligomer stability and suggest that in vivo apoC-1 transfer is mediated via the complexes with other apolipoproteins and/or lipids.     

Prevalence of domestic violence in the city of Durango]

PURPOSE: Thermal tissue damage (TTD) is customarily associated with some lasers. The thermal potential of rotational atherectomy (RA) devices is unknown. We investigated the temperature profile and potential TTD as well as the value of fluid flushing of an RA device. METHODS: We used a high-resolution infrared imaging system that can detect changes as small as 0.1 degree C to measure the temperature changes at the tip of a fast RA device with and without fluid flushing. To assess TTD, segments of porcine aorta were subjected to the rotating tip under controlled conditions, stained by a special histochemical stain (picrisirius red) and examined under normal and polarized light microscopy. RESULTS: There was significant heating of the rotating cam. The mean "peak" temperature rise was 52.8 +/- 16.9 degrees C. This was related to rotational speed; thus the "peak" temperature rise was 88.3 +/- 12.6 degrees C at 80,000 rpm and 17.3 +/- 3.8 degrees C at 20,000 rpm (p < 0.001, t-test). Fluid flushing at 18 ml/min reduced, but did not abolish, heating of the device (11.8 +/- 2.9 degrees C). A crater was observed in all segments exposed to the rotating tip. The following features were most notable: (i) A zone of "thermal" tissue damage extended radially from the crater reaching adventitia in some sections, especially at high speeds. This zone showed markedly reduced or absent birefringence. (ii) Fluid flushing of the catheter reduced the above changes but increased the incidence and extent of dissections in the media, especially when combined with high atherectomy speeds. (iii) These changes were observed in five of six specimens exposed to RA without flushing, but in only one of six with flushing (p < 0.05). (iv) None of the above changes was seen in control segments. CONCLUSION: RA is capable of generating significant heat and potential TTD. Fluid flushing reduced heating and TTD. These findings warrant further studies in vivo, and may influence the design of atherectomy devices.     

Enthalpy relaxation in binary amorphous mixtures containing sucrose

PURPOSE: To compare the enthalpy relaxation of amorphous sucrose and co-lyophilized sucrose-additive mixtures near the calorimetric glass transition temperature, so as to measure the effects of additives on the molecular mobility of sucrose. METHODS: Amorphous sucrose and sucrose-additive mixtures, containing poly(vinylpyrrolidone) (PVP), poly(vinylpyrrolidone-co-vinyl-acetate) (PVPNA) dextran or trehalose, were prepared by lyophilization. Differential scanning calorimetry (DSC) was used to determine the area of the enthalpy recovery endotherm following aging times of up to 750 hours for the various systems. This technique was also used to compare the enthalpy relaxation of a physical mixture of amorphous sucrose and PVP. RESULTS: Relative to sucrose alone, the enthalpy relaxation of co-lyophilized sucrose-additive mixtures was reduced when aged for the same length of time at a comparable degree of undercooling in the order: dextran approximately PVP > PVPNA > trehalose. Calculated estimates of the total enthalpy change required for sucrose and the mixtures to relax to an equilibrium supercooled liquid state (deltaHinfinity) were essentially the same and were in agreement with enthalpy changes measured at longer aging times (750 hours). CONCLUSIONS: The observed decrease in the enthalpy relaxation of the mixtures relative to sucrose alone indicates that the mobility of sucrose is reduced by the presence of additives having a Tg that is greater than that of sucrose. Comparison with a physically mixed amorphous system revealed no such effects on sucrose. The formation of a molecular dispersion of sucrose with a second component, present at a level as low as 10%, thus reduces the mobility of sucrose below Tg, most likely due to the coupling of the molecular motions of sucrose to those of the additive through molecular interactions.     

The molecular mobility of supercooled amorphous indomethacin as a function of temperature and relative humidity

PURPOSE: To determine the relaxation times of supercooled indomethacin as a function of temperature and relative humidity above Tg, and to analyze the results in the context of being able to predict such behavior at various storage conditions. METHODS: Dielectric relaxation times were measured in the frequency domain (12 to 10(5) Hz) for amorphous indomethacin equilibrated at 0, 56, and 83% relative humidity. The heating rate dependence of Tg for dry supercooled indomethacin was measured with differential scanning calorimetry and used to determine relaxation times. The results were compared with previously published shear relaxation times and enthalpy recovery data. RESULTS: Very good agreement was observed between dielectric and shear relaxation times, and those obtained from the heating rate dependence of the Tg, for dry indomethacin as a function of temperature above Tg. The introduction of water lowered the dielectric relaxation times of supercooled indomethacin without significantly affecting its fragility. The relaxation times below Tg, found to be lower than those predicted by extrapolation of the data obtained above Tg, were analyzed in the context of the Adam-Gibbs-Vogel equation. CONCLUSIONS: The relaxation times of amorphous indomethacin obtained from the heating rate dependence of Tg were in good agreement with those obtained from shear and dielectric measurements, thus validating a relatively simple approach of assessing molecular mobility. The significant molecular mobility of amorphous indomethacin observed below Tg, and the significant plasticizing effects of sorbed water, help to explain why amorphous indomethacin crystallizes well below Tg over relatively short time scales.     

Mechanical properties of some polymer materials used for tooth positioners

The chemical composition, thermal behaviour and mechanical properties of three tooth positioner materials, Urethane P1 (P1), White Rubber (WR) and Elastocryl (EL) were investigated. Infra-red spectrophotometry indicated the P1 polyurethane material to be of the polyether type, and EL to be a blend of poly(ethyl methacrylate) and poly(methyl methacrylate) while WR appeared to be filled cis-poly (isoprene) (natural rubber). The glass transition temperature (Tg) for EL was determined as approximately 10 degrees C, and for both P1 and WR the Tg was less than -50 degrees C. The stress relaxation behaviour was assessed in compression by measuring the stress variation with time. The results for all three materials conformed to the superelastic theory of rubber elasticity. EL exhibited both a more rapid rate and higher degree of stress relaxation than did P1 and WR. Recovery from deformation was assessed by compressing cylinders for given periods of time and then measuring the level of reduced residual strain of the material with time. All three materials exhibited significant residual strain (epsilon(t)) over 'clinically relevant' time periods, and the reduced residual strain (epsilon(t)/epsilon(O)) following deformation was greater for EL than P1 or WR. There was some indication that the three materials have some permanent set following deformation. It was concluded that, in considering desirable mechanical properties of tooth positioner materials, EL is the least suitable of the three examined, with none of the materials being ideal.     

Slow β relaxation in La-based metallic glasses based on mechanical spectroscopy measurements

Dynamic mechanical relaxations of La-based metallic glasses were investigated by mechanical spectroscopy.In the framework of the mixing enthalpy of constituent atoms,it was found thatβrelaxation was less evident by the addition of Cu to replace Ni in the LaCuNiAl glassy alloy.By introducing Cu into the LaNiAl metallic glass,the mixing enthalpy was less negative,which led to weakerβrelaxation of the metallic glasses.Theαrelaxation of the La-based metallic glasses could be described by a Kohlrausch-Williams-Watts(KWW)function with a Kohlrausch exponentβKWW around 0.5.It should be noted that physical aging above the glass transition temperature Tginduced a decrease ofβrelaxation intensity in the La-based metallic glass.     

Calorimetric and freeze fracture analysis of lipid phase transitions and lateral translational motion of intramembrane particles in mitochondrial membranes

Differential scanning calorimetry combined with freeze fracture electron microscopy reveals that thermotropic lipid phase transitions and lateral translational motion of intramembrane particles occur in both membranes of whole, intact rat liver mitochondria and in isolated inner and outer membranes. The onset temperature of the liquid crystalline to gel state lipid phase transition in whole mitochondria and in the isolated outer membrane fraction is biphasic with an initial transition exotherm occurring at 9 degrees C. The onset temperature of the transition exotherm of the isolated inner membrane occurs at -4 degrees C. The onset temperature of the lipid transition endotherm is -15 degrees C for whole mitochondria, the inner membrane, ane the outer membrane fractions. These calorimetric analyses reveal that the bilayer lipid in the inner, energy transducing membrane is more fluid than in the outer membrane. Mitochondrial membranes cooled to temperatures in the region of their transition exotherms and then frozen reveal striking lateral separations between smooth, intramembrane particle-free regions (rich in gel state lipid) and particle-dense regions (rich in integral proteins) in their hydrophobic fracture faces. Such thermotropic lipid-protein lateral separations are completely reversible. These freeze fracture observations suggest that both mitochondrial membranes are naturally fluid to the extent that the integrat membrane proteins can diffuse laterally in the bilayer lipid.     

Circadian rhythms have no effect on cycling performance

The aim of this research was to determine if circadian rhythms have an effect on time trial cycling performance of 15 min duration. Seven males (Mean+/-SD): age, 22.3+/-4.9 yr; height 179.0+/-7.9 cm, body mass 74.5+/-15.5 kg; VO2max 68.0+/-5.7 ml x kg(-1) x min(-1) who were all competitive cyclists or triathletes with previous experience in laboratory testing procedures volunteered to participate in this study. Each of the seven subjects underwent a series of four tests; one VO2 max test, and three 15 min maximal performance tests, at varying times during a 24 hr period. Testing times were at 08.00-10.00; 14.00-16.00 and 20.00-22.00 hours. Heart rate was recorded during the last 10-15 seconds of each minute and blood lactate levels were taken at 5 and 10 min during exercise and again immediately post-exercise. O2 consumption was measured continuously using open circuit spirometry. RPE was measured using the Borg scale at 5 and 10 min during, and again immediately following the completion of testing. Resting oral temperature was the only variable to show a significant time of day effect (p<0.05). Oral temperature during the afternoon was higher than both morning and evening results by 0.76 degrees C and 0.09 degrees C respectively. Total work (kJ) and average power output (W) were recorded at their highest during the morning session and reached a trough during the afternoon session, but these differences were not significant (p = 0.9997 and 0.9972 respectively). The results obtained in this study indicate that while certain biological rhythms are present, they appear to have no effect on this type of cycling performance. Although athletic performance may be enhanced by training programs that are compatible with an individuals body clock, the ability to perform and train at various times has an adaptive response which appears to over-ride these naturally inherent rhythms.     

An evaluation of the sensitivity of subjects with peanut allergy to very low doses of peanut protein: a randomized, double-blind, placebo-controlled food challenge study

PURPOSE: To find out if the physical instability of a lyophilized dosage form is related to molecular mobility below the glass transition temperature. Further, to explore if the stability data generated at temperatures below the glass transition temperature can be used to predict the stability of a lyophilized solid under recommended storage conditions. METHODS: The temperature dependence of relaxation time constant, tau, was obtained for sucrose and trehalose formulations of the monoclonal antibody (5 mg protein/vial) from enthalpy relaxation studies using differential scanning calorimetry. The non-exponentiality parameter, beta, in the relaxation behavior was also obtained using dielectric relaxation spectroscopy. RESULTS: For both sucrose and trehalose formulations, the variation in tau with temperature could be fitted Vogel-Tammann-Fulcher (VTF) equation. The two formulations exhibited difference sensitivities to temperature. Sucrose formulation was more fragile and exhibited a stronger non-Arrhenius behavior compared to trehalose formulation below glass transition. Both formulations exhibited < 2% aggregation at t/tau values < 10, where t is the time of storage. CONCLUSIONS: Since the relaxation times for sucrose and trehalose formulations at 5 degrees C are on the order of 10(8) and 10(6) hrs, it is likely that both formulations would undergo very little (< 2%) aggregation in a practical time scale under refrigerated conditions.     

Effect of metal ion substitutions in concanavalin A on the binding of carbohydrates and on thermal stability

Isothermal titration calorimetry (ITC) measurements were performed on the binding of alpha methyl-D-mannopyranoside, D-mannopyranose, alpha methyl-D-glucopyranoside, and D-glucopyranose (Glu) to cobalt, nickel, and cadmium substituted concanavalin A (Con A) derivatives at pH = 6.9 and at 25 degrees C. The metal substituted Con A derivatives consisted of Co2+, Ni2+, and Cd2+ substituted for the Mn2+ ion in the S1 site of Con A which is about 12.8 A away from the center of the carbohydrate binding site of Con A. The thermodynamic quantities determined from the ITC measurements were the same for most of the binding reactions indicating that the structure of the binding site in solution is the same for all the Con A derivatives in solution and that the presence of different 2+ metal ions in the S1 site has little effect on the binding reactions. Differential scanning calorimetry scans of solutions of the metal ion derivatives of Con A show that the thermodynamics of the unfolding transition for the cobalt and nickel substituted derivatives are the same as for Con A: they dissociate from tetramers into monomers as they unfold around 85 degrees C. The cadmium substituted Con A derivative, however, exhibits an additional transition around 93 degrees C which also appears following the addition of Cd2+ to the Con A solutions. This transition results from the unfolding of a species of Con A with Cd2+ substituted into a third binding site at the monomeric interface of the Con A tetramer. The higher stability of this species is not only exemplified by the higher thermal transition temperature but also by the lack of dissociation as it unfolds. Cd2+ is released from the S3 site upon decreasing the pH from 6.9 to 6.4. ITC measurements on the binding reaction of Cd2+ to Con A show that the binding enthalpy is 40.2 +/- 0.4 kJ mol-1 at 23.4 +/- 0.2 degrees C and the binding reaction exhibits a large heat capacity change of 1.43 +/- 0.41 kJ mol-1 K-1.     

The physical state of nafcillin sodium in frozen aqueous solutions and freeze-dried powders

The purpose of this study was to develop a better understanding of the physical chemistry of freeze drying of lyotropic liquid crystals using nafcillin sodium as a model solute. Solutions and freeze-dried powders of nafcillin sodium were studied by polarized light microscopy, differential scanning calorimetry, x-ray powder diffraction, and water vapor adsorption. Differential scanning calorimetry thermograms of nafcillin sodium solutions contain a melting endotherm at approximately -5.5 degrees C and, depending on the concentration and heating rate, a crystallization exotherm immediately after this endotherm followed by the melting endotherm of ice. When the sample is annealed at -4 degrees C, both the endotherm and exotherm are eliminated, and a new endotherm appears at approximately -1 degree C on the shoulder of the ice-melting endotherm. The data are interpreted as melting of a liquid crystalline phase, followed by crystallization. X-ray powder diffractograms of unannealed freeze-dried nafcillin sodium are consistent with a lamellar liquid crystal. Diffractograms of annealed freeze-dried nafcillin sodium indicate crystalline material which is a different crystal form than the monohydrate starting material. Moisture adsorption isotherms of the freeze-dried annealed (crystalline) and unannealed (liquid crystalline) nafcillin sodium show different affinities for moisture compared to the crystalline starting material. Solid-state stability data demonstrate that the freeze-dried liquid crystalline form of nafcillin sodium is much less stable than the freeze-dried crystal-line material. The literature recognizes two types of solute behavior on freezing, where the solute either crystallizes from the freeze concentrate or remains amorphous. Lyotropic liquid crystal formation during freezing represents a separate category of freezing behavior, the physical chemistry of which is worthy of further investigation.     

Influence of various types of species-specific acoustic signals on the heart rate of mammals

INTRODUCTION: Successful radiofrequency ablation of an accessory pathway has been demonstrated to be associated with an electrode-tissue interface temperature of approximately 60 degrees C or an impedance change of -5 to -10 omega. However, the temperature and impedance changes associated with ablation of AV nodal reentrant tachycardia (AVNRT) using the slow pathway approach have not been reported. Therefore, the purpose of this study was to define the temperature and impedance changes achieved during ablation of AVNRT: METHODS AND RESULTS: The study included 35 consecutive patients with AVNRT undergoing radiofrequency ablation of the slow pathway with a fixed power output of 32 W, and using a catheter with a thermistor bead embedded in the distal 4-mm electrode. The procedure was successful in each patient. The steady-state electrode-tissue interface temperature during successful applications of energy was 48.5 +/- 3.3 degrees C (range 42 degrees to 56 degrees C), and the steady-state temperature during ineffective applications was 46.8 degrees +/- 5.5 degrees C (P = 0.03). The mean impedance change during all applications of energy was -1.4 +/- 2.8 omega, and did not differ significantly during effective and ineffective applications. Coagulum formation resulted during five applications (2.7%) in two patients (5.7%). There were no recurrences during 114 +/- 21 days of follow-up. CONCLUSIONS: Successful ablation of AVNRT using fixed power output is achieved at an electrode-tissue interface temperature of approximately 48 degrees C and is associated with a drop in impedance of 1 to 2 omega. These findings suggest that slow pathway ablation requires less heating at the electrode-tissue interface than does accessory pathway or AV junction ablation.