Thermodynamics of the hydration of non-polar substances

We re-examine the numerical value and physical significance of T(S) the temperature where the entropy of transfer Delta(L)(W)S from the pure hydrocarbon liquid into water is zero. It is shown that the numerical value of T(S) depends on the convention adopted for calculating Delta(L)(W)G from solubility data at 25 degrees C and on the Delta(L)(W)C(P) fitting function. It is concluded that the interpretation of T(S) as the temperature where hydration ceases cannot be sustained. As previously reported [R.L. Baldwin. N. Muller, Proc. Natl. Acad. Sci. USA, 89 (1992) 7110], hydration must vanish at a temperature T' > T(S), where its experimental manifestation, i.e., Delta(L)(W)C(P), is zero. We discuss the concept of water relaxation around a non-polar solute molecule and its relation to the hydration process.     

A note on the nonautonomous delay Beverton-Holt model

It is well known that the periodic cycle {x(n)} of a periodically forced nonlinear difference equation is attenuant (resonant) if av(x(n)) < av(K(n))(av(x(n)) > av(K(n))),where {K ( n )} is the carrying capacity of the environment and av(t(n)) = (1/p)∑(p?1) (i=0) ti (arithmetic mean of the p-periodic cycle {t ( n )}). In this article, we extend the concept of attenuance and resonance of periodic cycles using the geometric mean for the average of a periodic cycle. We study the properties of the periodically forced nonautonomous delay Beverton-Holt model x(n+1) = r(n)x(n)/1 + (r(n?l) ? 1)x(n?k)/K(n?k), n= 0, 1, . . . , where {K ( n )} and {r ( n )} are positive p-periodic sequences; (K ( n )>0, r ( n )>1) as well as k and l are nonnegative integers. We will show that for all positive solutions {x ( n )} of the previous equation lim sup (n→∞) (∏(n?1)(i=0)xi)(1/n) ≤ ((∏(p?1)(i=0)ri)(1/p) ? 1)(∏(p?1)(i=0)(ri ? 1))(?1/p)(∏(p?1)(i=0)Ki)(1/p). In particular, in the case where {x(n)} is a p-periodic solution of the above equation (assuming that such solution exists) and r ( n )=r>1, the periodic cycle is g-attenuant, that is (∏(p?1)(i=0)x(i))(1/p)<(∏(p?1)(i=0)K(i))(p?1) Surprisingly, the obtained results show that the delays k and l do not play any role.     

Predicted permeability parameters of human ovarian tissue cells to various cryoprotectants and water

This study presents a generic numerical model to simulate the coupled solute and solvent transport in human ovarian tissue sections during addition and removal of chemical additives or cryoprotective agents (CPA). The model accounts for the axial and radial diffusion of the solute (CPA) as well as axial convection of the CPA, and a variable vascular surface area (A) during the transport process. In addition, the model also accounts for the radial movement of the solvent (water) into and out of the vascular spaces. Osmotic responses of various cells within an human ovarian tissue section are predicted by the numerical model with three model parameters: permeability of the tissue cell membrane to water (L(p)), permeability of the tissue cell membrane to the solute or CPA (omega) and the diffusion coefficient of the solute or CPA in the vascular space (D). By fitting the model results with published experimental data on solute/water concentrations within an human ovarian tissue section, I was able to determine the permeability parameters of ovarian tissue cells in the presence of 1.5M solutions of each of the following: dimethyl sulphoxide (DMSO), propylene glycol (PROH), ethylene glycol (EG), and glycerol (GLY), at two temperatures (4 degrees C and 27 degrees C). Modeling Approach 1: Assuming a constant value of solute diffusivity (D = 1.0 x 10(-9) m(2)/sec), the best fit values of L(p) ranged from 0.35 x 10(-14) to 1.43 x 10(-14) m(3)/N-sec while omega ranged from 2.57 x 10(-14) to 70.5 x 10(-14) mol/N-sec. Based on these values of L(p) and omega, the solute reflection coefficient, sigma defined as sigma = 1-omega v(CPA)/L(P) ranged from 0.9961 to 0.9996. Modeling Approach 2: The relative values of omega and sigma from our initial modeling suggest that the embedded ovarian tissue cells are relatively impermeable to all the CPAs investigated (or omega approximately 0 and sigma approximately 1.0). Consequently the model was modified and used to predict the values of L(p) and D assuming omega = 0 and sigma = 1.0. The best fit values of L(p) ranged from 0.44 x 10(-14) to 1.2 x 10(-14) m(3)/N-sec while D ranged from 0.85 x 10(-9) to 2.08 x 10(-9) m(2)/sec. Modeling Approach 3: Finally, the best fit values of D from modeling approach 2 were incorporated into model 1 to re-predict the values of L(p) and omega. It is hoped that the ovarian tissue cell parameters reported here will help to optimize chemical loading and unloading procedures for whole ovarian tissue sections and consequently, tissue cryopreservation procedures.     

Analytical ultracentrifugation in a Gibbsian perspective

The analytical ultracentrifuge has come into new intensive use following complete instrumental redesign and the use of advanced computer technologies for the analysis and interpretation of experimental results. Major attention is now devoted to the evaluation of interactions between similar and dissimilar biological macromolecules in dilute and concentrated systems. Electrostatically charged biological solute systems additionally comprise low molecular weight charged and non-charged cosolvents. Solvent/cosolvent interactions, insufficiently considered in most current analytical ultracentrifugation analyses, may quantitatively affect solute/solute interactions. For comprehensive analysis the Svedberg derivation considering a buoyant molar mass (1 - rho0 partial specific volume)M2 and valid at vanishing solute concentration for strictly two component systems only, should be replaced, following classical thermodynamic analysis, by the ratio (delta rho/delta c2)(mu)/d pi/dc2 of the density increment at constant chemical potential of diffusible cosolvents, to the derivative of the osmotic pressure with solute concentration. Disregard of the solvent/cosolvent and solute/cosolvent interactions should be avoided.     

Quantitative structure-selectivity relationship for M2 selectivity between M1 and M2 of piperidinyl piperidine derivatives as muscarinic antagonists

Muscarinic M2 receptor antagonists with high subtype selectivity (M2/M1) will decrease the toxicity in central nervous system in treatment of AD. The exploration of quantitative structure-selectivity relationship (QSSR) to muscarinic M2 receptor antagonists will provide design information for drug with fewer side effects. In this paper, CoMFA models of pK(i)(M1), pK(i)(M2) and p[K(i)(M2)/K(i)(M1)] (pK(i)(M2)-pK(i)(M1)) were used to study the subtype selectivity (M2/M1) of piperidinyl piperidine derivatives as muscarinic M2 subtype receptor antagonists. The parameters of the three models are: 0.633, 0.636 and 0.726 for cross-validated r(2) (r(cv)(2)), 0.109, 0.204 and 0.09 for the Standard error of estimate (SD), respectively. The results show the model of p[K(i)(M2)/K(i)(M1)] is the best one for design of piperidinyl piperidine derivatives as muscarinic antagonists with high subtype selectivity (M2/M1).     

Validity/reliability of sweat analysis by whole-body washdown vs. regional collections

Six subjects (25.3 +/- 3.3 yr, mean +/- SD) exercised for 60 min at 42 +/- 4 [low (L)], 55 +/- 6 [moderate (M)], and 67 +/- 4 %VO2max [high (H)] in a moderate environment. Sweat collected from upper back (UB), lower back (LB), midchest (MC), stomach (S), and thigh (T) areas as well as by whole-body washdown (W) was analyzed for urea nitrogen (N). With the exception of the L where all regional measures were similar, all sites overestimated W (several significantly, P less than 0.05). Regression analysis estimations of W (mg/h) from regional collections were as follows--L: W = 0.727 (S) - 1.366(UB) + 1.181(T) + 65.470 +/- 29.5, R = 0.90; M: W = 0.598(MC) - 0.649(UB) + 0.244(LB) + 43.238 +/- 30.4, R = 0.99; H: W = 0.274(S) - 0.560(T) + 0.223(MC) + 131.104 +/- 4.3, R = 0.99; All Intensities: W = 0.497(MC) - 0.483(T) + 0.112(LB) + 69.554 +/- 31.5, R = 0.96. W recovery of exogenous urea N applied to each subject's body was 98.3 +/- 2.7% (mean +/- SE). Interinvestigator reliability coefficient (r = 0.511) was significant (P less than 0.01) but relatively low and the between investigator urea N recovery (93.3 +/- 3.7 vs. 103.2 +/- 3.5%) was significantly different (P less than 0.05). Repeated W determinations by the same investigator were not different (P greater than 0.05), but intrainvestigator reliability coefficients differed widely (0.385 vs. 0.820). Together, these data indicate that W solute recovery can be high; however, both inter- and intrainvestigator reliability can vary.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attenuation by sphingosine-1-phosphate of rat microvessel acute permeability response to bradykinin is rapidly reversible

To evaluate the hypothesis that sphingosine-1-phosphate (S1P) and cAMP attenuate increased permeability of individually perfused mesenteric microvessels through a common Rac1-dependent pathway, we measured the attenuation of the peak hydraulic conductivity (L(p)) in response to the inflammatory agent bradykinin (BK) by either S1P or cAMP. We varied the extent of exposure to each agent (test) and measured the ratio L(p)(test)/L(p)(BK alone) for each vessel (anesthetized rats). S1P (1 μM) added at the same time as BK (concurrent, no pretreatment) was as effective to attenuate the response to BK (L(p) ratio: 0.14 ± 0.05; n = 5) as concurrent plus pretreatment with S1P for 30 min (L(p) ratio: 0.26 ± 0.06; n = 11). The same pretreatment with S1P, but with no concurrent S1P, caused no inhibition of the BK response (L(p) ratio 1.07 ± 0.11; n = 8). The rapid on and off action of S1P demonstrated by these results was in contrast to cAMP-dependent changes induced by rolipram and forskolin (RF), which developed more slowly, lasted longer, and resulted in partial inhibition when given either as pretreatment or concurrent with BK. In cultured endothelium, there was no Rac activation or peripheral cortactin localization at 1 min with RF, but cortactin localization and Rac activation were maximal at 1 min with S1P. When S1P was removed, Rac activation returned to control within 2 min. Because of such differing time courses, S1P and cAMP are unlikely to act through fully common effector mechanisms.     

Inactivation of cysteine proteases by (acyloxy)methyl ketones using S'-P' interactions

We have synthesized (acyloxy)methyl ketone inactivators of papain, cathepsin B, and interleukin-1beta conversion enzyme (ICE) that interact with both the S and S' subsites. The value of k(inact)/K(i) for these inactivators is strongly dependent on the leaving group. For example, Z-Phe-Gly-CH(2)-X is a poor inactivator of papain when X is OCOCH(3) (k(inact)/K(i) = 2.5 M(-)(1) s(-)(1)) but becomes a potent inactivator when X is OCO-L-Leu-Z (k(inact)/K(i) = 11 000 M(-)(1) s(-)(1)). Since these leaving groups have similar chemical reactivities, the difference in potency must be attributed to interactions with the S' sites. The potency of the leaving group correlates with the P' specificity of papain. Similar results are also observed for the inactivation of cathepsin B by these compounds. A series of inactivators with the general structure Fmoc-L-Asp-CH(2)-X were designed to inactivate ICE. No inhibition was observed when X was OCOCH(3). In contrast, ICE is inactivated when X is OCO-D-Pro-Z (k(inact)/K(i) = 131 M(-)(1) s(-)(1)). These results demonstrate that S'-P' interactions can be utilized to increase the efficacy and selectivity of (acyloxy)methyl ketone inactivators.     

Plant dispersal, neighbourhood size and isolation by distance

A theoretical relationship between isolation by distance or spatial genetic structure (SGS) and seed and pollen dispersal is tested using extensive spatial-temporal simulations. Although for animals Wright's neighbourhood size N(e) = 4pisigma(2)(t) has been ascertained also, where sigma(2)(t) is the axial variance of distances between parents and offspring, and it was recently confirmed that N(e) = 4pi(sigma(2)(f) + sigma(2)(m))/2 when dispersal of females and males differ, the situation for plants had not been established. This article shows that for a very wide range of conditions, neighbourhood size defined by Crawford's formula N(e) = 4pi(sigma(2)(s) + sigma(2)(p)/2) fully determines SGS, even when dispersal variances of seed (sigma(2)(s)) and pollen sigma(2)(p)) differ strongly. Further, self-fertilization with rate s acts as zero-distance pollen dispersal, and N(e) = 4pi[sigma(2)(s) + sigma(2)(p)(1 - s)/2] fully determines SGS, for most cases where there are both likely parameter values and substantial SGS. Moreover, for most cases, there is a loglinear relationship, I(1) = 0.587 - 0.117 ln(N(e)), between SGS, as measured by I(1), Moran's coefficient for adjacent individuals, and N(e). However, there are several biologically significant exceptions, namely for very low or large N(e), SGS exceeds the loglinear values. There are also important exceptions to Crawford's formula. First, plants with low seed dispersal, high outcross pollen dispersal and high selfing rate show larger SGS than predicted. Second, in plants with very low (near zero) seed dispersal, selfing decreases SGS, opposite expectations. Finally, in some cases seed dispersal is more critical than pollen dispersal, in a manner inconsistent with Crawford's formula.     

Pressure dependence of the photocycle kinetics of bacteriorhodopsin

The pressure dependence of the photocycle kinetics of bacteriorhodopsin from Halobacterium salinarium was investigated at pressures up to 4 kbar at 25 degrees C and 40 degrees C. The kinetics can be adequately modeled by nine apparent rate constants, which are assigned to irreversible transitions of a single relaxation chain of nine kinetically distinguishable states P(1) to P(9). All states except P(1) and P(9) consist of two or more spectral components. The kinetic states P(2) to P(6) comprise only the two fast equilibrating spectral states L and M. From the pressure dependence, the volume differences DeltaV(o)(LM) between these two spectral states could be determined that range from DeltaV(o)(LM) = -11.4 +/- 0.7 ml/mol (P(2)) to DeltaV(o)(LM) = 14.6 +/- 2.8 mL/mol (P(6)). A model is developed that explains the dependence of DeltaV(o)(LM) on the kinetic state by the electrostriction effect of charges, which are formed and neutralized during the L/M transition.     

The affinity-enhancing roles of flexible linkers in two-domain DNA-binding proteins.

Recently many attempts have been made to design high-affinity DNA-binding proteins by linking two domains. Here a theory for guiding these designs is presented. Flexible linkers may play three types of roles: (a) linking domains which by themselves are unfolded and bind to DNA only as a folded dimer (as in a designed single-chain Arc repressor), (b) connecting domains which can separately bind to DNA (as in the Oct-1 POU domain), and (c) linking a DNA-binding domain with a dimerization domain (as in the lambda repressor). In (a), the linker keeps the protein as a folded dimer so that it is always DNA-binding-competent. In (b), the linker is predicted to enhance DNA-binding affinity over those of the individual domains (with dissociation constants K(A) and K(B)) by p(d(0))/K(B) or p(d(0))/K(A), where p(d(0)) = (3/4pil(p)bL)(3/2) exp(-3d(0)(2)/4l(p)bL)(1 - 5l(p)/4bL +...) is the probability density for the end-to-end vector of the linker with L residues to have a distance d(0). In (c), the linker is predicted to enhance the binding affinity by K(d)(C)/p(d(0)), where K(d)(C) is the dimer dissociation constant for the dimerization domain. The predicted affinity enhancements are found to be actually reached by the Oct-1 POU domain and lambda repressor. However, there is room for improvement in many of the recently designed proteins. The theoretical limits presented should provide a useful guide for current efforts of designing DNA-binding proteins.     

The point of maximum cell water volume excursion in case of presence of an impermeable solute

A relativistic permeability model of cell osmotic response (Cryobiology 40:64-83; 41:366-367) is applied to a two-solute system with one impermeable solute. The use of the normalized water volume (w), and the amount of intracellular permeable solute (x), which is the product of the water volume and intracellular osmolality (y), as the main variables allowed us to obtain a homogeneous differential equation dx(Delta)/dw(Delta)=f(x(Delta)/w(Delta)), where w(Delta)=w-w(f), x(Delta)=x-x(f), and f refers to the final (equilibrium) values. The solution of this equation is an explicit function, w(Delta)=g(x(Delta)), which is given in the text. This approach allows us to obtain an analytical (exact) expression of the water volume at the moment of the maximum excursion (water extremum w(m)). Results are compared with numeration of basic osmotic equations and with approximation given in (Cryobiology 40:64-83). Assumption that, dw/dt approximately 0 gives good approximations of the kinetics of water and permeable CPA after the point of maximum volume excursion (the slow phase of osmotic response). Practical aspects of the relativistic permeability approach are also discussed.     

Evidence for protein dielectric relaxations in reaction centers associated with the primary charge separation detected from Rhodospirillum rubrum chromatophores by combined photovoltage and absorption measurements in the 1-15 ns time range

Fast photovoltage measurements in Rhodospirillum rubrum chromatophores in the nanosecond time range, escorted by time-resolved absorption measurements, are described. Under reducing conditions, the photovoltage decayed significantly faster than the spectroscopically detected charge recombination of the radical pair P(+)H(A)(-). This indicates the occurrence of considerable dielectric relaxations. Our data and data from the literature were analyzed by means of a reaction scheme consisting of three states, namely, A, P, and P(+)H(A)(-). A time-dependent DeltaG(t) was introduced by assuming a time-dependent rate constant of the back-reaction, k(-1)(t). With the exception of the latter rate constant, all other parameters of the model are reliably known within narrow limits. This allowed us to distinguish between the three cases assumed for DeltaG degrees (t): (1)DeltaG degrees (t) = constant; (2)DeltaG degrees (t) as published by Peloquin et al. [Peloquin, J. M., Williams, J. C., Lin, X. M., Alden, R. G., Taguchi, A. K. W., Allen, J. P., and Woodbury, N. W. (1994) Biochemistry 33, 8089-8100]; and a (3)DeltaG degrees (t) that fits the present data. The assumption that (1)DeltaG degrees (t) = constant is incompatible with our photovoltage data, and (2)DeltaG degrees (t) is incompatible with the constraint that the ratio of fluorescence yields in the closed and open state is F(m)/F(o) approximately 2. We specify a (3)DeltaG degrees (t) that should be valid for photosynthetic reaction centers in vivo. Furthermore, the overall kinetics of the electric relaxation, e(t), in response to the primary charge separation were determined.     

Bioprocess parameters and oxygen transfer characteristics in beta-lactamase production by Bacillus species

After screening potential beta-lactamase producers in a medium containing penicillin G, an inducible (Bacillus subtilis NRS 1125) and a constitutive (Bacillus licheniformis 749/C ATCC 25972) beta-lactamase producer were selected. As the highest enzyme activity was obtained with B. licheniformis 749/C, the effects of the concentration of carbon sources, i.e., glucose, fructose, sucrose, citric acid, and glycerol, and nitrogen sources, i.e., (NH(4))(2)HPO(4), NH(4)Cl, yeast extract, casamino acids and peptone, pH, and temperature on beta-lactamase production were investigated with B. licheniformis 749/C in laboratory scale bioreactors. Among the investigated media, the highest volumetric activity was obtained as 270 U cm(-)(3) in the medium containing 10.0 kg m(-)(3) glucose, 1.18 kg m(-)(3) (NH(4))(2)HPO(4), 8.0 kg m(-)(3) yeast extract, and the salt solution at 32 degrees C and pH(0) = 6.0. By using the designed medium, fermentation and oxygen transfer characteristics of the bioprocess were investigated at V = 3.0 dm(3) bioreactor systems with a V(R) = 1.65 dm(3) working volume at Q(O)/V(R) = 0.5 vvm and N = 500 min(-1). At the beginning of the process the Damk?hler number was <1, indicating that the process was at biochemical reaction limited condition; at t = 2-5 h both mass-transfer and biochemical reaction resistances were effective; and at t = 6-10 h (Da >1) the bioprocess was at mass transfer limited condition. Overall oxygen transfer coefficients (K(L)a) varied between 0.01 and 0.03 s(-)(1), enhancement factor (K(L)a/K(L)a(O)) varied between 1.2 and 2.3, and volumetric oxygen uptake rate varied between 0.001 and 0.003 mol m(-)(3) s(-)(1) throughout the bioprocess. The specific oxygen uptake and the specific substrate consumption rates were the highest at t = 2 h and then decreased with the cultivation. The maximum yield of cells on substrate and the maximum yield of cells on oxygen values were obtained, respectively, as Y(X/S) = 0.34 and Y(X/O) = 1.40, at t = 5 h, whereas the highest yield of substrate on oxygen was obtained as Y(S/O) = 6.94 at t = 3.5 h. The rate of oxygen consumption for maintenance and the rate of substrate consumption for maintenance values were found, respectively, as m(O) = 0.13 kg kg(-)(1) h(-)(1) and m(S) = 3.02 kg kg(-)(1) h(-)(1).     

Role of a glycocalyx on coronary arteriole permeability to proteins: evidence from enzyme treatments

Whereas the glycocalyx of endothelial cells has been shown to influence solute flux from capillary microvessels, little is known about its contribution to the movement of macromolecules across the walls of other microvessels. We evaluated the hypothesis that a glycocalyx contributes resistance to protein flux measured in coronary arterioles. Apparent solute permeability (P(s)) to two proteins of different size and similar charge, alpha-lactalbumin (alpha-lactalb) and porcine serum albumin (PSA), was determined in arterioles isolated from the hearts of 43 female Yucatan miniature swine. P(s) was assessed in arterioles with an "intact" glycocalyx under control conditions and again after suffusion with adenosine (Ado, 10(-5) M, n = 42 arterioles, N = 29 pigs). In a second set of experiments (n = 21 arterioles, N = 21 pigs) arteriolar P(s) was determined before and after perfusion with enzyme (pronase or heparinase), which was used to digest the glycocalyx. P(s) was assessed a third time on those microvessels after exposure to Ado. Consistent with the hypothesis, P(s) for PSA (P(PSA)(s)) and P(s) for alpha-lactalb (P(alpha-lactalb)(s)) increased from basal levels following enzyme treatment. Subsequent suffusion with Ado, a significant metabolite known to alter coronary vascular smooth muscle tone and permeability, resulted in a significant reduction of basal P(alpha-lactalb)(s) in both untreated and enzyme-treated arterioles. Furthermore, in untreated arterioles, P(PSA)(s) was unchanged by Ado suffusion, whereas Ado induced a pronounced reduction in P(PSA)(s) of enzyme-treated vessels. These data demonstrate that in intact coronary arterioles an enzyme-sensitive layer, most likely at the endothelial cell surface, contributes significantly to net barrier resistance to solute flux.     

Binding of dioxygen to non-metal sites in proteins: exploration of the importance of binding site size versus hydrophobicity in the copper amine oxidase from Hansenula polymorpha

Copper amine oxidases (CAOs) contain 2,4,5-trihydroxyphenylalanyl quinone (TPQ) and a copper ion in their active sites, catalyzing amine oxidation to aldehyde and ammonia concomitant with the reduction of molecular oxygen to hydrogen peroxide. Kinetic studies on the CAO from bovine serum (BSAO) [Su and Klinman (1999) Biochemistry 37, 12513-12525] and the recent reports on the cobalt substituted form of the enzyme from Hansenula polymorpha (HPAO) [Mills and Klinman (2000) J. Am. Chem. Soc. 122, 9897-9904, and Mills et al. (2002) Biochemistry, 41, 10577-10584] support pre-binding of molecular oxygen prior to a rate-limiting electron transfer from the reduced form of TPQ (p-aminohydroquinone form) to dioxygen. Although there is significant sequence homology between BSAO and HPAO, k(cat)/K(m)(O2) for BSAO under the optimal condition is one order of magnitude lower than that for HPAO. From a comparison of amino acid sequences for BSAO and HPAO, together with the X-ray crystal structure of HPAO, a plausible dioxygen pre-binding site has been identified that involves Y407, L425, and M634 in HPAO; the latter two residues are altered in BSAO to A490 and T695. To determine which of these residues plays a greater role in dioxygen chemistry, k(cat)/K(m)(O2) was determined in HPAO for the M634 --> T and L425 --> A mutants. The L425 --> A mutation does not alter k(cat)/K(m)(O2) to a large extent, whereas the M634 --> T decreased k(cat)/K(m)(O2) by one order of a magnitude, creating a catalyst that is similar to BSAO. A series of mutants at M634 (to F, L, and Q) were, therefore, prepared in HPAO and characterized with regard to k(cat)/K(m)(O2) as a function of pH. Structure reactivity correlations show a linear relationship of rate with side chain volume, rather than hydrophobicity, indicating that dioxygen reactivity increases with the bulk of the residue at position 634. This site also shows specificity for O2, in relation to the co-gas N2, since substitution of the inert gas N2 by either Ar or He has no effect on measured rates. In particular, He gas is expected to have little affinity for protein at 1 atmospheric pressure, implying little or no binding by N2 as well.     

Application of the local-bulk partitioning and competitive binding models to interpret preferential interactions of glycine betaine and urea with protein surface

Two thermodynamic models have been developed to interpret the preferential accumulation or exclusion of solutes in the vicinity of biopolymer surface and the effects of these solutes on protein processes. The local-bulk partitioning model treats solute (and water) as partitioning between the region at/or near the protein surface (the local domain) and the bulk solution. The solvent exchange model analyzes a 1:1 competition between water and solute molecules for independent surface sites. Here we apply each of these models to interpret thermodynamic data for the interactions of urea and the osmoprotectant glycine betaine (N,N,N-trimethylglycine; GB) with the surface exposed in unfolding the marginally stable lacI HTH DNA binding domain. The partition coefficient K(P) quantifying accumulation of urea at this protein surface (K(P) approximately equal 1.1) is only weakly dependent on urea concentration up to 6 M urea. However, K(P) quantifying exclusion of GB from the vicinity of this protein surface increases from 0.83 (extrapolated to 0 M GB) to 1.0 (indicating that local and bulk GB concentrations are equal) at 4 M GB (activity > 40 M). We interpret the significant concentration dependence of K(P) for GB, predicted to be general for excluded, nonideal solutes such as GB, as a modest (8%) attenuation of the GB concentration dependence of solute nonideality in the local domain relative to that in the bulk solution. Above 4 M, K(P) for the interaction of GB with the surface exposed in protein unfolding is predicted to exceed unity, which explains the maximum in thermal stability observed for RNase and lysozyme at 4 M GB (Santoro, M. M., Liu, Y. F., Khan, S. M. A., Hou, L. X., and Bolen, D. W. (1992) Biochemistry 31, 5278-5283). Both thermodynamic models provide good two-parameter fits to GB and urea data for lacI HTH unfolding over a wide concentration range. The solute partitioning model allows for a full spectrum of attenuation effects in the local domain, encompasses the cases treated by the competitive binding model, and provides a somewhat better two-parameter fit of effects of high GB concentration on lacI HTH stability. Parameters of this fit should be applicable to isothermal and thermal unfolding data for all proteins with similar compositions of surface exposed in unfolding.     

A Single Equation to Quantify the Hierarchy in Plant Size Induced by Competition Within Monocultures

The following empirical model: Ra(i) = r(1+ln(w(i)/wm)Kn)(1–(w(i)/W))(1–(y/Y)) which is based on the logistic growth equation, is developedto describe the growth of differently sized individuals withinplant communities. The model is tested against extensive setsof carrot (Daucus carota L.) and red beet (Beta vulgaris L.)data and is shown to fit well. The model was used to predictindividual plant weights in independent data. The agreementsbetween observed and predicted weights were often close butsome systematic deviations did occur. Thus, a single equationdescribed most of the complex interactions that occurred withinmonocultures of annual crop plants. Carrot, Daucus carota L., red beet, Beta vulgaris L., model, growth, variation     

Rapid and quantitative activation of Chlamydia trachomatis ribonucleotide reductase by hydrogen peroxide

We recently showed that the class Ic ribonucleotide reductase (RNR) from the human pathogen Chlamydia trachomatis ( Ct) uses a Mn (IV)/Fe (III) cofactor in its R2 subunit to initiate catalysis [Jiang, W., Yun, D., Saleh, L., Barr, E. W., Xing, G., Hoffart, L. M., Maslak, M.-A., Krebs, C., and Bollinger, J. M., Jr. (2007) Science 316, 1188-1191]. The Mn (IV) site of the novel cofactor functionally replaces the tyrosyl radical used by conventional class I RNRs to initiate substrate radical production. As a first step in evaluating the hypothesis that the use of the alternative cofactor could make the RNR more robust to reactive oxygen and nitrogen species [RO(N)S] produced by the host's immune system [H?gbom, M., Stenmark, P., Voevodskaya, N., McClarty, G., Gr?slund, A., and Nordlund, P. (2004) Science 305, 245-248], we have examined the reactivities of three stable redox states of the Mn/Fe cluster (Mn (II)/Fe (II), Mn (III)/Fe (III), and Mn (IV)/Fe (III)) toward hydrogen peroxide. Not only is the activity of the Mn (IV)/Fe (III)-R2 intermediate stable to prolonged (>1 h) incubations with as much as 5 mM H 2O 2, but both the fully reduced (Mn (II)/Fe (II)) and one-electron-reduced (Mn (III)/Fe (III)) forms of the protein are also efficiently activated by H 2O 2. The Mn (III)/Fe (III)-R2 species reacts with a second-order rate constant of 8 +/- 1 M (-1) s (-1) to yield the Mn (IV)/Fe (IV)-R2 intermediate previously observed in the reaction of Mn (II)/Fe (II)-R2 with O 2 [Jiang, W., Hoffart, L. M., Krebs, C., and Bollinger, J. M., Jr. (2007) Biochemistry 46, 8709-8716]. As previously observed, the intermediate decays by reduction of the Fe site to the active Mn (IV)/Fe (III)-R2 complex. The reaction of the Mn (II)/Fe (II)-R2 species with H 2O 2 proceeds in three resolved steps: sequential oxidation to Mn (III)/Fe (III)-R2 ( k = 1.7 +/- 0.3 mM (-1) s (-1)) and Mn (IV)/Fe (IV)-R2, followed by decay of the intermediate to the active Mn (IV)/Fe (III)-R2 product. The efficient reaction of both reduced forms with H 2O 2 contrasts with previous observations on the conventional class I RNR from Escherichia coli, which is efficiently converted from the fully reduced (Fe 2 (II/II)) to the "met" (Fe 2 (III/III)) form [Gerez, C., and Fontecave, M. (1992) Biochemistry 31, 780-786] but is then only very inefficiently converted from the met to the active (Fe 2 (III/III)-Y (*)) form [Sahlin, M., Sj?berg, B.-M., Backes, G., Loehr, T., and Sanders-Loehr, J. (1990) Biochem. Biophys. Res. Commun. 167, 813-818].