THE IONIC ACTIVITY OF GELATIN

2.5 and 1.25 per cent gelatin have been titrated potentiometrically in the absence of salts and in the presence of two concentrations (0.0750 and 0.0375µ) of NaCl, MgCl₂, K₂SO₄, and MgSO₄. The data have been used to calculate values of ± S = v^z – (v – 1)^z, where v^z = v ² – (v ² – v) r_x/18. The maximum and minimum values of S with NaCl were used to calculate the mean distance (r_x) between like charges in gelatin. This is found to be 18 Å.u. or over (between acid or basic groups) which agrees with the probable value and the titration index dispersion. Thus the data with NaCl are shown to be normal and to obey the equation found to hold for simple weak electrolytes; namely, pK'' – pK = Sa See PDF for Equation where S is related to the valence and distance by the above equations. Using the NaCl data as a standard the deviations (ΔS) produced by the other salts are calculated and are found to agree quantitatively with the deviations calculated from equations derived for the simple weak electrolytes. This shows that in gelatin, as in the simple electrolytes, the deviations are related to the "apparent valences" (values which are a function of the true valence and the distance between the groups). The maximum "apparent valences" of gelatin are 2.4 for acid groups (in alkaline solution) and 1.8 for basic groups (in acid solution). These values correspond to the hypothetical condition of zero distance between the groups. They have no physical significance but have a practical utility first as mentioned above, and second in that they may be used in the unmodified Debye-Hückel equation to give the maximum effect of gelatin on the ionic strength. The true effect is probably even lower than these values would indicate. The data indicate that gelatin is a weak polyvalent ampholyte having distant groups and that the molecule has an arborescent structure with interstices permeated by molecules of the solvent and other solutes. The size and shape probably vary with the pH.     

Conceptual Error in Determination of NAD-Malic Enzyme in Extracts Containing NAD-Malic Dehydrogenase

The typical procedure for determining NAD⁺-malic enzyme (EC 1.1.1.39) is to calculate the enzyme rate to be ΔA₃₄₀/Δ time after the endogenous NAD⁺-malic dehydrogenase (EC 1.1.1.37) catalyzed reaction has reached equilibrium. This ignores the equilibrium shift of oxaloacetic acid and NADH during the course of the NAD⁺-malic enzyme reaction and causes an error that varies depending on the reagent [malate], [NAD⁺], pH and final [NADH]. For a ΔA₃₄₀ of 0.02, the error is about 80% and for a ΔA₃₄₀ of 0.30, 20%. We develop this argument, give supportive data and present a simple method to circumvent the error.     

Correlation between the Carbon Isotope Discrimination in Leaf Starch and Sugars of C(3) Plants and the Ratio of Intercellular and Atmospheric Partial Pressures of Carbon Dioxide

Carbon isotope discrimination (Δ) was analyzed in leaf starch and soluble sugars, which represent most of the recently fixed carbon. Plants of three C₃ species (Populus nigra L. × P. deltoides Marsh., Gossypium hirsutum L. and Phaseolus vulgaris L.) were kept in the dark for 24 hours to decrease contents of starch and sugar in leaves. Then gas exchange measurements were made with constant conditions for 8 hours, and subsequently starch and soluble sugars were extracted for analysis of carbon isotope composition. The ratio of intercellular, p_i, and atmospheric, p_a, partial pressures of CO₂, was calculated from gas exchange measurements, integrated over time and weighted by assimilation rate, for comparison with the carbon isotope ratios in soluble sugars and starch. Carbon isotope discrimination in soluble sugars correlated strongly (r = 0.93) with p_i/p_a in all species, as did Δ in leaf starch (r = 0.84). Starch was found to contain significantly more ¹³C than soluble sugar, and possible explanations are discussed. The strong correlation found between Δ and p_i/p_a suggests that carbon isotope analysis in leaf starch and soluble sugars may be used for monitoring, indirectly, the average of p_i/p_a weighted by CO₂ assimilation rate, over a day. Because p_i/p_a has a negative correlation with transpiration efficiency (mol CO₂/mol H₂O) of isolated plants, Δ in starch and sugars may be used to predict differences in this efficiency. This new method may be useful in ecophysiological studies and in selection for improved transpiration efficiency in breeding programs for C₃ species.     

THEORY AND MEASUREMENT OF VISUAL MECHANISMS : III. ΔI AS A FUNCTION OF AREA,INTENSITY, AND WAVE-LENGTH,FOR MONOCULAR AND BINOCULAR STIMULATION

Measurements of ΔI as a function of retinal area illuminated have been obtained at various levels of standard intensity I ₁, using "white" light and light of three modal wave-lengths (λ465, 525, 680), for monocular stimulation and for simultaneous excitation of the two eyes ("binocular"), using several methods of varying (rectangular) area and retinal location, with control of exposure time. For data homogeneous with respect to method of presentation, log ΔI_m = -Z log A + C, where ΔI = Ĩ ₂ – I ₁, A is area illuminated, and C is a terminal constant (= log ΔI_m for A = 1 unit) depending on the units in which ΔI and A are expressed, and upon I ₁. The equation is readily deduced on dimensional grounds, without reference to specific theories of the nature of ΔI or of retinal area in terms of its excitable units. Z is independent of the units of I and A. Experimentally it is found to be the same for monocular and binocular excitations, as is to be expected. Also as is expected it is not independent of λ, and it is markedly influenced by the scheme according to which A is varied; it depends directly upon the rate at which potentially excitable elements are added when A is made to increase. For simultaneous excitation of the two eyes (when of very nearly equivalent excitability), ΔĪ_B is less than for stimulation of either eye alone, at all levels of I ₁, A, λ. The mean ratio (ΔĪ_L + ΔĪ_R)/2 to ΔI^B was 1.38. For white light, doubling A on one retina reduces ΔI_m in the ratio 1.21, or a little less than for binocular presentation under the same conditions. These facts are consistent with the view that the properties of ΔI are quantitatively determined by events central to the retina. The measure σ_1ΔI of organic variation in discrimination of intensities and ΔI_m are found to be in simple proportion, independent of I ₁, A, λ (and exposure time). Variability (σ_1ΔI) is not a function of the mode of presentation, save that it may be slightly higher when both retinas are excited, and its magnitude (for a given level of ΔI_m) is independent of the law according to which the adjustable intensity I ₂ is instrumentally controlled.     

Permeability of the Ehrlich Ascites Tumor Cell to Water

The osmotic permeability coefficient for water has been measured for the Ehrlich mouse ascites tumor cell. Measurements were made of the rate of cell shrinkage in hyperosmotic solutions of NaCI, a functionally impermeable solute. During the first 9 months of weekly serial transplantation the mean was 6.4 µ³/µ³/atm. ± 0.8 (S.E.). By the end of the 2nd year the permeability coefficient was much lower and averaged 1.6 ± 0.09. There were no significant differences in the volume of the tumor cells which could explain the discrepancy on the basis of a change in the volume to surface area ratio. Studies of the effect of temperature were done and Eyring's theory of absolute reaction rates was applied to the data. The apparent energy of activation was 9.6 kcal./mol and ΔS‡ was 39.1 entropy units. The thermodynamic data are twice as high as data reported by Wang for self-diffusion and viscous properties of water. Two alternate explanations have been advanced based on the pore hypothesis of membrane permeability. One explains the thermodynamic data from a change in the A'/Δx available for water movement; the other assumes A'/Δx constant and bases the results on the interaction of water dipoles with each other and the membrane.     

The Sodium-Potassium Exchange Pump: Relation of Metabolism to Electrical Properties of the Cell: I. Theory

The Na-K exchange pump is represented as a net stoichiometrically coupled reaction, r, involving ATP, Na⁺, and K⁺, and is located in the active region of the cell membrane. The reaction rate is J_r = L_rr (-ΔF_r), where ΔF_r is the free energy change of the reaction. ΔF_r includes membrane potential ø₂ in the absence of 1:1 coupling between Na⁺ and K⁺, and the reaction rate is potential dependent under these conditions. At the same time the pump will produce a potential H which is the difference between membrane potential and the diffusion potential as calculated with constant field assumptions. In the absence of 1:1 coupling, the pump is electrogenic. The feedback relation between reaction rate and membrane potential makes the membrane resistance in the presence of the pump less than or equal to the resistance in its absence, at the same membrane potential. H depends on stoichiometry, reaction rate, and passive ionic conductances. Experimental verification of the model will depend on the accuracy of permeability determinations. Dissipation and efficiency of transport can be calculated also.     

Brachyspira (Serpulina) hyodysenteriae gyrB Mutants and Interstrain Transfer of Coumermycin A1 Resistance

To further develop genetic techniques for the enteropathogen Brachyspira hyodysenteriae, the gyrB gene of this spirochete was isolated from a λZAPII library of strain B204 genomic DNA and sequenced. The putative protein encoded by this gene exhibited up to 55% amino acid sequence identity with GyrB proteins of various bacterial species, including other spirochetes. B. hyodysenteriae coumermycin A₁-resistant (Cn^r) mutant strains, both spontaneous and UV induced, were isolated by plating B204 cells onto Trypticase soy blood agar plates containing 0.5 μg of coumermycin A₁/ml. The coumermycin A₁ MICs were 25 to 100 μg/ml for the resistant strains and 0.1 to 0.25 μg/ml for strain B204. Four Cn^r strains had single nucleotide changes in their gyrB genes, corresponding to GyrB amino acid changes of Gly₇₈ to Ser (two strains), Gly₇₈ to Cys, and Thr₁₆₆ to Ala. When Cn^r strain 435A (Gly₇₈ to Ser) and Cm^r Km^r strain SH (ΔflaA1::cat Δnox::kan) were cultured together in brain heart infusion broth containing 10% (vol/vol) heat-treated (56°C, 30 min) calf serum, cells resistant to chloramphenicol, coumermycin A₁, and kanamycin could be isolated from the cocultures after overnight incubation, but such cells could not be isolated from monocultures of either strain. Seven Cn^r Km^r Cm^r strains were tested and were determined to have resistance genotypes of both strain 435A and strain SH. Cn^r Km^r Cm^r cells could not be isolated when antiserum to the bacteriophage-like agent VSH-1 was added to cocultures, and the numbers of resistant cells increased fivefold when mitomycin C, an inducer of VSH-1 production, was added. These results indicate that coumermycin resistance associated with a gyrB mutation is a useful selection marker for monitoring gene exchange between B. hyodysenteriae cells. Gene transfer readily occurs between B. hyodysenteriae cells in broth culture, a finding with practical importance. VSH-1 is the likely mechanism for gene transfer.     

Genetic Evaluation of Physiological Functions of Thiolase Isozymes in the n-Alkane-Assimilating Yeast Candida tropicalis

The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-T1B, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption. The homozygous ct-t1aΔ/t1bΔ null mutation abolished the activity of acetoacetyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3aΔ/t3bΔ null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on n-alkanes (C₁₀ to C₁₃). All thiolase activities in this yeast disappeared with the ct-t1aΔ/t1bΔ and ct-t3aΔ/t3bΔ null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-T1A locus in the ct-t1bΔ null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal β-oxidation. In addition to its essential contribution to the β-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the β-oxidation of a C₄ substrate butyrate.     

Dissecting the Dynamic Pathways of Stereoselective DNA Threading Intercalation

DNA intercalators that have high affinity and slow kinetics are developed for potential DNA-targeted therapeutics. Although many natural intercalators contain multiple chiral subunits, only intercalators with a single chiral unit have been quantitatively probed. Dumbbell-shaped DNA threading intercalators represent the next order of structural complexity relative to simple intercalators, and can provide significant insights into the stereoselectivity of DNA-ligand intercalation. We investigated DNA threading intercalation by binuclear ruthenium complex [μ-dppzip(phen)₄Ru₂]⁴⁺ (Piz). Four Piz stereoisomers are defined by the chirality of the intercalating subunit (Ru(phen)₂dppz) and the distal subunit (Ru(phen)₂ip), respectively, each of which can be either right-handed (Δ) or left-handed (Λ). We used optical tweezers to measure single DNA molecule elongation due to threading intercalation, revealing force-dependent DNA intercalation rates and equilibrium dissociation constants. The force spectroscopy analysis provided the zero-force DNA binding affinity, the equilibrium DNA-ligand elongation Δx_eq, and the dynamic DNA structural deformations during ligand association x_on and dissociation x_off. We found that Piz stereoisomers exhibit over 20-fold differences in DNA binding affinity, from a K_d of 27 ± 3 nM for (Δ,Λ)-Piz to a K_d of 622 ± 55 nM for (Λ,Δ)-Piz. The striking affinity decrease is correlated with increasing Δx_eq from 0.30 ± 0.02 to 0.48 ± 0.02 nm and x_on from 0.25 ± 0.01 to 0.46 ± 0.02 nm, but limited x_off changes. Notably, the affinity and threading kinetics is 10-fold enhanced for right-handed intercalating subunits, and 2- to 5-fold enhanced for left-handed distal subunits. These findings demonstrate sterically dispersed transition pathways and robust DNA structural recognition of chiral intercalators, which are critical for optimizing DNA binding affinity and kinetics.     

Visual vs Fully Automatic Histogram-Based Assessment of Idiopathic Pulmonary Fibrosis (IPF) Progression Using Sequential Multidetector Computed Tomography (MDCT)

Objectives

To describe changes over time in extent of idiopathic pulmonary fibrosis (IPF) at multidetector computed tomography (MDCT) assessed by semi-quantitative visual scores (VSs) and fully automatic histogram-based quantitative evaluation and to test the relationship between these two methods of quantification.

Methods

Forty IPF patients (median age: 70 y, interquartile: 62-75 years; M:F, 33:7) that underwent 2 MDCT at different time points with a median interval of 13 months (interquartile: 10-17 months) were retrospectively evaluated. In-house software YACTA quantified automatically lung density histogram (10^th-90^th percentile in 5^th percentile steps). Longitudinal changes in VSs and in the percentiles of attenuation histogram were obtained in 20 untreated patients and 20 patients treated with pirfenidone. Pearson correlation analysis was used to test the relationship between VSs and selected percentiles.

Results

In follow-up MDCT, visual overall extent of parenchymal abnormalities (OE) increased in median by 5 %/year (interquartile: 0 %/y; +11 %/y). Substantial difference was found between treated and untreated patients in HU changes of the 40th and of the 80th percentiles of density histogram. Correlation analysis between VSs and selected percentiles showed higher correlation between the changes (Δ) in OE and Δ 40^th percentile (r=0.69; p<0.001) as compared to Δ 80^th percentile (r=0.58; p<0.001); closer correlation was found between Δ ground-glass extent and Δ 40^th percentile (r=0.66, p<0.001) as compared to Δ 80^th percentile (r=0.47, p=0.002), while the Δ reticulations correlated better with the Δ 80^th percentile (r=0.56, p<0.001) in comparison to Δ 40^th percentile (r=0.43, p=0.003).

Conclusions

There is a relevant and fully automatically measurable difference at MDCT in VSs and in histogram analysis at one year follow-up of IPF patients, whether treated or untreated: Δ 40^th percentile might reflect the change in overall extent of lung abnormalities, notably of ground-glass pattern; furthermore Δ 80^th percentile might reveal the course of reticular opacities.     

Heat capacity changes in RNA folding: application of perturbation theory to hammerhead ribozyme cold denaturation

In proteins, empirical correlations have shown that changes in heat capacity (ΔC_P) scale linearly with the hydrophobic surface area buried upon folding. The influence of ΔC_P on RNA folding has been widely overlooked and is poorly understood. In addition to considerations of solvent reorganization, electrostatic effects might contribute to ΔC_Ps of folding in polyanionic species such as RNAs. Here, we employ a perturbation method based on electrostatic theory to probe the hot and cold denaturation behavior of the hammerhead ribozyme. This treatment avoids much of the error associated with imposing two-state folding models on non-two-state systems. Ribozyme stability is perturbed across a matrix of solvent conditions by varying the concentration of NaCl and methanol co-solvent. Temperature-dependent unfolding is then monitored by circular dichroism spectroscopy. The resulting array of unfolding transitions can be used to calculate a ΔC_P of folding that accurately predicts the observed cold denaturation temperature. We confirm the accuracy of the calculated ΔC_P by using isothermal titration calorimetry, and also demonstrate a methanol-dependence of the ΔC_P. We weigh the strengths and limitations of this method for determining ΔC_P values. Finally, we discuss the data in light of the physical origins of the ΔC_Ps for RNA folding and consider their impact on biological function.     

Photorespiration in Green Plants During Photosynthesis Estimated by Use of Isotopic CO(2)

Photosynthetic re-absorption of photorespired CO₂ causes underestimation in measured photorespiration and turnover rate of the substrate for photorespiration. Actual values of photorespiration exceed the measured by a factor greater than 1 + R′_w/r_p + [C_L]_x/(r_p·L_x). (R′_w and r_p are the partial resistances to CO₂ uptake between atmosphere, mesophyll evaporating surface, and photosynthetic sink, respectively; L_x is the measured flux of photorespired CO₂ and [C_L]_x is the ambient conc of photorespired CO₂). In 8 species, 1 + R′_w/r_p alone amounted to a correction ranging between 148% and 233%.     

Carbon Isotope Discrimination in Coffee Genotypes Grown under Limited Water Supply

Photosynthetic gas exchange, plant-water relations characteristics, and stable carbon isotope discrimination (Δ) were evaluated for five Coffea arabica L. genotypes growing under two soil moisture regimes in the field. The Δ of leaf tissue was strongly correlated (r = −0.95) with inherent water use efficiency (ratio of assimilation to stomatal conductance; A/g). The variation in inherent water use efficiency (WUE) among genotypes was 30% for plants irrigated weekly. The higher WUE exhibited by some of these plants resulted from reduced g rather than increased photosynthetic capacity at a given g. Withholding irrigation for 1 month caused Δ to decline substantially in expanding leaf tissue of all genotypes. A strong correlation (r = 0.92) was found between Δ and plant hydraulic efficiency estimated as the ratio of g to the diurnal range in leaf water potential (Ψ_l). The Δ values for plants irrigated weekly adequately predicted drought-induced changes in Δ (r = 0.99) and midday Ψ_l (r = 0.95). The results indicated that Δ might be used to evaluate several aspects of plant performance and response to specific environmental conditions, once suitable background physiological data have been gathered.     

Increased Circulating Endothelial Apoptotic Microparticle to Endothelial Progenitor Cell Ratio Is Associated with Subsequent Decline in Glomerular Filtration Rate in Hypertensive Patients

Background

Recent research indicates hypertensive patients with microalbuminuria have decreased endothelial progenitor cells (EPCs) and increased levels of endothelial apoptotic microparticles (EMP). However, whether these changes are related to a subsequent decline in glomerular filtration rate (GFR) remains unclear.

Methods and Results

We enrolled totally 100 hypertensive out-patients with eGFR ≥30 mL/min/1.73 m². The mean annual rate of GFR decline (△GFR/y) was −1.49±3.26 mL/min/1.73 m² per year during the follow-up period (34±6 months). Flow cytometry was used to assess circulating EPC (CD34⁺/KDR⁺) and EMP levels (CD31⁺/annexin V⁺) in peripheral blood. The △GFR/y was correlated with the EMP to EPC ratio (r = −0.465, p<0.001), microalbuminuria (r = −0.329, p = 0.001), and the Framingham risk score (r = −0.245, p = 0.013). When we divided the patients into 4 groups according to the EMP to EPC ratio, there was an association between the EMP to EPC ratio and the ΔGFR/y (mean ΔGFR/y: 0.08±3.04 vs. −0.50±2.84 vs. −1.25±2.49 vs. −4.42±2.82, p<0.001). Multivariate analysis indicated that increased EMP to EPC ratio is an independent predictor of ΔeGFR/y.

Conclusions

An increased circulating EMP to EPC ratio is associated with subsequent decline in GFR in hypertensive patients, which suggests endothelial damage with reduced vascular repair capacity may contribute to further deterioration of renal function in patients with hypertension.     

The measurement of the intrinsic alkaline Bohr effect of various human haemoglobins by isoelectric focusing

We have used isoelectric focusing to measure the differences between the pI values of various normal and mutant human haemoglobins when completely deoxygenated and when fully liganded with CO. It was assumed that the ΔpI(deox.–ox.) values might correspond quantitatively to the intrinsic alkaline Bohr effect, as most of the anionic cofactors of the haemoglobin molecule are `stripped' off during the electrophoretic process. In haemoglobins known to exhibit a normal Bohr coefficient (ΔlogP₅₀/ΔpH) in solutions, the ΔpI(deox.–ox.) values are lower the higher their respective pI(ox.) values. This indicates that for any particular haemoglobin the ΔpI(deox.–ox.) value accounts for the difference in surface charges at the pH of its pI value. This was confirmed by measuring, by the direct-titration technique, the difference in pH of deoxy and fully liganded haemoglobin A₀ (α₂β₂) solutions in conditions approximating those of the isoelectric focusing, i.e. at 5°C and very low concentration of KCl. The variation of the ΔpH(deox.–ox.) curve as a function of pH (ox.) was similar to the isoelectric-focusing curve relating the variation of ΔpI(deox.–ox.) versus pI(ox.) in various haemoglobins with Bohr factor identical with that of haemoglobin A₀. In haemoglobin A₀ the ΔpI(deox.–ox.) value is 0.17 pH unit, which corresponds to a difference of 1.20 positive charges between the oxy and deoxy states of the tetrameric haemoglobin. This value compares favourably with the values of the intrinsic Bohr effect estimated in back-titration experiments. The ΔpI(deox.–ox.) values of mutant or chemically modified haemoglobins carrying an abnormality at the N- or C-terminus of the α-chains are decreased by 30% compared with the ΔpI value measured in haemoglobin A₀. When the C-terminus of the β-chains is altered, as in Hb Nancy (α₂β^{Tyr-145→Asp}₂), we observed a 70% decrease in the ΔpI value compared with that measured in haemoglobin A₀. These values are in close agreement with the estimated respective roles of the two major Bohr groups, Val-1α and His-146β, at the origin of the intrinsic alkaline Bohr effect [Kilmartin, Fogg, Luzzana & Rossi-Bernardi (1973) J. Biol. Chem. 248, 7039–7043; Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema (1980) J. Mol. Biol. 138, 649–670]. In other mutant haemoglobins it is demonstrated also that the ΔpI(deox.–ox.) value may be decreased or even suppressed when the substitution affects residues involved in the stability of the tetramer. These results support the interpretation proposed by Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema [(1980), J. Mol. Biol. 138, 649–670] for the mechanism of the alkaline Bohr effect, and also indicate that the transition between the two quaternary configurations is a prerequisite for the full expression of the alkaline Bohr effect.     

The Role of Allosteric Coupling on Thermal Activation of Thermo-TRP Channels

Thermo-transient receptor potential channels display outstanding temperature sensitivity and can be directly gated by low or high temperature, giving rise to cold- and heat-activated currents. These constitute the molecular basis for the detection of changes in ambient temperature by sensory neurons in animals. The mechanism that underlies the temperature sensitivity in thermo-transient receptor potential channels remains unknown, but has been associated with large changes in standard-state enthalpy (ΔH^o) and entropy (ΔS^o) upon channel gating. The magnitude, sign, and temperature dependence of ΔH^o and ΔS^o, the last given by an associated change in heat capacity (ΔC_p), can determine a channel’s temperature sensitivity and whether it is activated by cooling, heating, or both, if ΔC_p makes an important contribution. We show that in the presence of allosteric gating, other parameters, besides ΔH^o and ΔS^o, including the gating equilibrium constant, the strength- and temperature dependence of the coupling between gating and the temperature-sensitive transitions, as well as the ΔH^o/ΔS^o ratio associated with them, can also determine a channel’s temperature-dependent activity, and even give rise to channels that respond to both cooling and heating in a ΔC_p-independent manner.     

Energetics of Sodium Transport in Frog Skin : II. The effects of electrical potential on oxygen consumption

Studies were made of the dependence of the rate of oxygen consumption, J_r, on the electrical potential difference, Δψ, across the frog skin. After the abolition of sodium transport by ouabain the basal oxygen consumption was independent of Δψ. In fresh skins J_r was a linear function of Δψ over a range of at least ±70 mv. Treatment with aldosterone stimulated the short-circuit current, I_o, and the associated rate of oxygen consumption, J_ro, and increased their stability; linearity was then demonstrable over a range of ±160 mv. Brief perturbations of Δψ (±30–200 mv) did not alter subsequent values of I_o. Perturbations for 10 min or more produced a "memory" effect both with and without aldosterone: accelerating sodium transport by negative clamping lowered the subsequent value of I_o; positive clamping induced the opposite effect. Changes in J_ro were more readily detectable in the presence of aldosterone; these were in the same direction as the changes in I_o. The linearity of J_r in Δψ indicates the validity of analysis in terms of linear nonequilibrium thermodynamics—brief perturbations of Δψ appear to produce no significant effect on either the phenomenological coefficients or the free energy of the metabolic driving reaction. Hence it is possible to evaluate this free energy.     

Determination of molecular size of O-(2-hydroxyethyl)cellulose (HEC) and its relationship to the mechanism of enzymatic hydrolysis by cellulases

Z-average root mean square end-to-end distance 〈r_o²〉_z^1/2 and radius of gyration 〈s_o²〉_z^1/2 for 13 samples of O-(2-hydroxyethyl)cellulose (HEC) of different molecular weights were derived from Gel Permeation Chromatography and intrinsic viscosity measurements with water as a solvent. At 40 °C and pH 4.5, contraction of chain dimensions is observed, compared with the sizes observed under neutral conditions at room temperature. The effect is lower for samples with higher molecular weights. Values of 〈r_o²〉₄₀/DP_z also indicate that chain flexibility increases at higher temperature and acidic conditions. From the analysis of molecular weight dependence of 〈s_o²〉 _z^1/2, Flory exponent υ was derived at 40 °C and pH 4.5. A value of υ = 0.70 ± 0.02 was recorded, which indicates that a relatively stiff chain is present under these conditions. Finally, different equations to calculate persistence length L_p were evaluated. Values in the range of 260-400 Å were derived for persistence length. Implications of chain conformation in the enzymatic action of cellulases are also discussed.     

Stepwise Photoinhibition of Photosystem II : Studies with Synechocystis Species PCC 6803 Mutants with a Modified D-E Loop of the Reaction Center Polypeptide D1

Several mutant strains of Synechocystis sp. PCC 6803 with large deletions in the D-E loop of the photosystem II (PSII) reaction center polypeptide D1 were subjected to high light to investigate the role of this hydrophilic loop in the photoinhibition cascade of PSII. The tolerance of PSII to photoinhibition in the autotrophic mutant ΔR225-F239 (PD), when oxygen evolution was monitored with 2,6-dichloro-p-benzoquinone and the equal susceptibility compared with control when monitored with bicarbonate, suggested an inactivation of the Q_B-binding niche as the first event in the photoinhibition cascade in vivo. This step in PD was largely reversible at low light without the need for protein synthesis. Only the next event, inactivation of Q_A reduction, was irreversible and gave a signal for D1 polypeptide degradation. The heterotrophic deletion mutants ΔG240-V249 and ΔR225-V249 had severely modified Q_B pockets, yet exhibited high rates of 2,6-dichloro-p-benzoquinone-mediated oxygen evolution and less tolerance to photoinhibition than PD. Moreover, the protein-synthesis-dependent recovery of PSII from photoinhibition was impaired in the ΔG240-V249 and ΔR225-V249 mutants because of the effects of the mutations on the expression of the psbA-2 gene. No specific sequences in the D-E loop were found to be essential for high rates of D1 polypeptide degradation.     

Photosynthesis Decrease and Stomatal Control of Gas Exchange in Abies alba Mill. in Response to Vapor Pressure Difference

The responses of steady state CO₂ assimilation rate (A), transpiration rate (E), and stomatal conductance (g_s) to changes in leaf-to-air vapor pressure difference (ΔW) were examined on different dates in shoots from Abies alba trees growing outside. In Ecouves, a provenance representative of wet oceanic conditions in Northern France, both A and g_s decreased when ΔW was increased from 4.6 to 14.5 Pa KPa⁻¹. In Nebias, which represented the dry end of the natural range of A. alba in southern France, A and g_s decreased only after reaching peak levels at 9.0 and 7.0 Pa KPa⁻¹, respectively. The representation of the data in assimilation rate (A) versus intercellular CO₂ partial pressure (C_i) graphs allowed us to determine how stomata and mesophyll photosynthesis interacted when ΔW was increased. Changes in A were primarily due to alterations in mesophyll photosynthesis. At high ΔW, and especially in Ecouves when soil water deficit prevailed, A declined, while C_i remained approximately constant, which may be interpreted as an adjustment of g_s to changes in mesophyll photosynthesis. Such a stomatal control of gas exchange appeared as an alternative to the classical feedforward interpretation of E versus ΔW responses with a peak rate of E. The gas exchange response to ΔW was also characterized by considerable deviations from the optimization theory of IR Cowan and GD Farquhar (1977 Symp Soc Exp Biol 31: 471-505).