Thermodynamics of oligo (A) N -2poly(U) from the dependence of the temperature of the helix-coil transition on oligomer concentration

On the basis of elementary two-state, ideal solution thermocynamics, a modified expression for the melting of oligo. polynucleotide helices is derived which is applicable to variations in T_m^N and/or oligomer concentration, C_m with oligomer length, N: ((I)) ΔH_r is the enthalpy per helix residue, i.e., per base-pair or base-triplet, V_r^f is the thermodynamic “available” or “reaction” volume, in liters/mole of helical residues; and n is the number of polynucleotide strands, e.g., n = 2 for oligo (A)_N·2 poly(U)∞. Some earlier treatments have engendered confusion in the interpretation of the “reaction volume,” but with the derivation herein, the entropic origin and physical significance of V_r^f is unequivocal. The following approximation was arrived at for the reduction expected in the configurational entropy, ΔS_r^{conf, ∞}, for (A)∞·2(U)∞, when the poly(A), strand is substituted for by an equivalent strand of contiguous oligo(A)_N,′s: ((II)) This adjustment of ΔS_r^{conf, ∞} represents the source of the coefficient to 1/T_m^∞ in expression (I). The expectation that ΔS_r^{conf, N} < ΔS_r^{conf, ∞} is due to the effect of releasing normal internucleotide configurational restrictions every Nth residue in one-third of the strands of the (A)_N·2(U)∞ helix. Although the reduction in ΔS_r^{conf, ∞} (II) may seem small (i.e., only 5.5% for the tetramer), its effect on the magnitude of V_r^f in expression (I) is exponential. Thus, without these considerations the quantitative applicability of earlier expressions is questionable. By examining the variation in T_m^N with c_m for a single N, all assumptions, required for evaluating V_r^f or the entropic effects of discontinuities in the (A)_N strand are avoided in the determination of a reliable enthalpy. We have therefore examined the system ((III)) and obtained a ΔH_r = 12.58 ± 0.08 kcal per mole (A)·2(U) base-triplets between 5 and 2.5°C. That this value for ΔH_r is in such excellent agreement with all calorimetric values reported for (A)∞·2(U)∞ suggests that the enthalpy for reaction(III) is not significantly affected by disconnections in the backbone of (A)₄·2(U)∞. From (I), V_r^f = 6.0 × 10^?4 1/mole or 1 Å ³per helical residue. ΔH_r°, corrected for residual single-strand stacking in (A)₄, is in excellent agreement with that found earlier for (A)₁·2(U)∞. A residual heat capacity of 90 kcal(±20) per mole (A)·2(U) base-triplets per °C is deduced from the decrease of ΔH_r° with temperature.     

The binding of Mg++ ions to polyadenylate, polyuridylate, and their complexes

The binding of Mg ⁺⁺ to polyadenylate (poly A), Polyuridylate(poly U), and their complexes, poly (A + U) and poly (A + 2U), was studied by means of a technique in which the dye eriochrome black T is used to measure the concentration of free Mg^?. The apparent binding constant K_X = [MgN]/[Mg⁺⁺][N], N = site for Mg⁺⁺ binding (the phosphate group of the nucleotide), was found to decrease rapidly as the extent of binding increased and, at low extents of binding, as the concentration of Na^? increased in poly A, poly (A + U), and poly (A + 2U), and somewhat less so in poly U. K_x is generally in the range 10⁴ > K_X > 10². The cause of these dependences is apparently, primarily, the displacement of Na⁺ by Mg⁺⁺ in poly U and poly (A + U) on the basis of the similarity of extents of displacement measured in this work and those measured potentiometrically. was calculated and was found to approach zero as the concentration of Na⁺ increased. In poly U, poly (A + U), and poly (A + 2U) at low ΔH′ v.H. > 0, about + 2 kcal/“mole.” In poly A, also at low salt, ΔH′ v.H. ≈ ?4 kcal/“mol” for the initial binding of Mg⁺⁺, and increases to +2 kcal/“mol” at saturation. This enthalpic variation probably accounts for the anticooperativity in the binding of Mg⁺⁺ not ascribable to the displacement of Na⁺⁺.     

Polynucleotides. XI. Thermodynamics of (A) N -2(U) infinity from the dependence of T m N on oligomer length

The variation in the helix-coil transition temperature, T_m^N, with oligomer length, N, for the system ((I)) has been examined. The results for N = 4-13, measured in 0.2M Na+, have been analyzed in terms of the expression of Blake (1972): ((II)) where c_m is the free oligomer concentration at T_m^N, and V_r^f is the thermodynamic free volume available to a helical base-triplet residue. The correlation coefficient for the fit to expression (II) of data obtained over a 50° temperature range is 0.997 when ΔH_r = ?12.6 kcal/mole of base-triplets (independent of oligomer length (N ? 4) or temperature), the value previously obtained from both calorimetry of (A)_∞·2(U)_∞ and (A)₄ concentration dependence of T_m. It is found that V_r^f = 8.0 × 10^?4 1/mole (± 30%) or 1.33 ų per helical base-triplet, and is constant with temperature. A maximum value for V_r^f of 21.0 × 10^?4 1/M (± 1.3%), equivalent to 3.54 ų per helical basetriplet is obtained by the same treatment of the helix-coil transition data for the three-stranded helix formed by adenosine (N = 1) and 2(U)_∞ obtained by Davies and Davidson (1971).     

Definition and validation of operating equations for poly(vinyl alcohol)‐poly(lactide‐co‐glycolide) microfiltration membrane‐scaffold bioreactors

The aim of this work is to provide operating data for biodegradable hollow fiber membrane bioreactors. The physicochemical cell culture environment can be controlled with the permeate flowrate, so this aim necessitates the provision of operating equations that enable end‐users to set the pressures and feed flowrates to obtain their desired culture environment. In this paper, theoretical expressions for the pure water retentate and permeate flowrates, derived using lubrication theory, are compared against experimental data for a single fiber poly(vinyl alcohol)–poly(lactide‐co‐glycolide) crossflow module to give values for the membrane permeability and slip. Analysis of the width of the boundary layer region where slip effects are important, together with the sensitivity of the retentate and permeate equations to the slip parameter, show that slip is insignificant for these membranes, which have a mean pore diameter of 1.1 µm. The experimental data is used to determine a membrane permeability, of k = 1.86 × 10^?16 m², and to validate the model. It was concluded that the operating equation that relates the permeate to feed ratio, c, lumen inlet flowrate, Q _l,in, lumen outlet pressure, P ₁, and ECS outlet pressure, P ₀, is (1) where A and B are constants that depend on the membrane permeability and geometry (and are given explicitly). Finally, two worked examples are presented to demonstrate how a tissue engineer can use Equation (1) to specify operating conditions for their bioreactor. Biotechnol. Bioeng. 2010;107: 382–392. © 2010 Wiley Periodicals, Inc.     

The mechanism of sex determination in Rumex acetosella

Summary Cytogenetic studies were made with particular emphasis on the sex-determining mechanism in Rumex acetosella (6 x = 42) and its hybrids (F ₁, F ₂, BC ₁ and BC ₂) with R. hastatulus (synthetic 4 x = 16 = 4 A +4 X = and 4 x = 18 = 4 A + 2 (X Y ₁ Y ₂) = ). Rumex acetosella was almost strictly dioecious with 5050 male and female. Breeding tests revealed that the males were heterogametic. The longest chromosomes (S), usually two, are the sex chromosomes of this hexaploid species. The S chromosomes are homomorphic in both male and female. The sex chromosome: autosome ratios, and the strong epistatic male effect of the S ^M chromosome in the polyploid dioecious species and in the hybrids, are evidence of an X/Y Melandrium type sex-determining mechanism controlled by a single pair of homomorphic sex chromosomes. Thus, the sex chromosome formula of the males was S ^F S ^M and that of females was S ^F S ^F. The present approach is a new method for resolving the sex-determining mechanism in a dioecious species.     

The effect of bisulfite modification on the template activity of DNA for DNA polymerase I

Cytosine residues of poly(C) and heat-denatured calf thymus DNA were transformed into 5,6-dihydrouracil-6-sulfonate (U(SO⁻₃)) residues by treatment with bisulfite. The poly(U(SO⁻₃)₂, C₃) and poly(U(SO⁻₃)₉, C₁) prepared did not form inter-base binding with either poly(A) or poly(I) as judged by the absence of hypochromicity in ultraviolet absorbance. U(SO⁻₃) residues in the DNA inactivated it to serve as template for E.coli DNA polymerase I, while the template activity was restored by conversion of the U(SO⁻₃) residues into U.     

Gas exchange strategy in the Nile crocodile: a morphometric study

Summary The respiratory surface area (S_AR) per kilogram body mass (M_B), the harmonic mean thickness of the air-blood barrier (_htR) in the gas exchange tissue, and the anatomical diffusion factor (ADF=S_AR/_htR per M_B) were calculated for four juvenile Nile crocodiles. The ADF of three small specimens (mean M_B=3.59 kg) was 625 cm²·m^–1·kg^–1. The values varied considerably among individuals and were similar to that of a 5.68-kg specimen (593 cm²·m^–1·kg^–1). Only 9% of the ADF is located in the anterior third of the lung, which because of its conical shape makes up only 14 percent of the total lung volume. Particularly in the middle third of the lung, the proximal region near the intrapulmonary bronchus displays a greater ratio of respiratory/non-respiratory surface areas than do more distally located sampling sites. The _htR is also significantly smaller proximally than distally. The cumulative ADF per unit M_B is greater than that previously reported for this species on the basis of overall estimates of S_AR and _htR, but is still less than that of lizards and testudinids. The disposition of ADF between distal air storage region and the intrapulmonary bronchus is consistent with a bidirectional cross-current gas exchange model.Abbreviations ADF anatomical diffusion factor - %AR percent of S_A included in the effective respiratory zone - M _B body mass - NVP non-ventilatory period - %P percent of total lung volume containing parenchyma - S _A total surface area of intrapulmonary septa - S _ANR that portion ofS _A lying out the effective respiratory zone - S _V surface-to-volume ratio in the parenchyma - _htR harmonic mean thickness of the air-blood tissue barrier within the respiratory zone - V _P parenchymal volume - VP ventilatory period     

Macrocyclic zinc(II) complexes for selective recognition of nucleobases in single- and double-stranded polynucleotides

 As an extension of our earlier discoveries that Zn^II-cyclen complex (1) (cyclen=1,4,7,10-tetraazacyclododecane) and Zn^II-acridine-pendant cyclen complex Zn^II-N-(9-acridin)ylmethyl-cyclen (3) are the first compounds to selectively recognize thymidine and uridine nucleosides in aqueous solution at physiological pH, the interaction of these and a relevant complex, bis(Zn^II-cyclen) (7), has been investigated with a series of polynucleotides, single-stranded poly(U) and poly(G), and double-stranded poly(A)·poly(U), poly(dA)·poly(dT) and poly(dG)·poly(dC). These Zn^II-cyclen complexes interact with the imide-containing nucleobases in the single-stranded poly(U), unperturbed by the presence of the anionic phosphodiester backbone. The affinity constant of 1 for each N(3)-deprotonated uracil base in poly(U) is determined to be log K= 5.1 by a kinetic measurement, which is almost the same as log K=5.2 for the interaction of 1 with uridine. Thus, they disrupt the A-U (or A-T) hydrogen bonds to unzip the duplex of poly(A)·poly(U) or poly(dA)·poly(dT), as demonstrated by lowering of the melting temperatures (T _m) of poly(A)·poly(U) and poly(dA)·poly(dT) in 5 mM Tris-HCl buffer (pH 7.6, 10 mM NaCl) with increase in their concentrations. The order of the denaturing efficiency is well correlated with that of the 1 : 1 affinity constants for each complex with uracil or thymine;7>3>1. The comparison of circular dichroism (CD) spectra for poly(A)·poly(U), poly(A), and poly(U) in the presence of 3 has revealed a structural change from poly(A)·poly(U) to two single strands, poly(A) and poly(U), caused by 3 binding exclusively to uracils in poly(U). On the other hand, the acridine-pendant cyclen complex 3, which earlier was found to associate with guanine by the Zn^II coordinating with guanine N(7), in addition to the π-π stacking, interacts with guanine in the double helix of poly(dG)·poly(dC) from outside and stabilized the double-stranded structure, as indicated by higher T _m. Received: 31 December 1997 / Accepted: 23 February 1998     

Additive genetic variance within populations derived by single-seed descent and pod-bulk descent

Summary Breeders of self-pollinated legumes commonly use single-seed descent (SSD) or pod-bulk descent (PBD) to produce segregating populations of highly inbred individuals. We presented equations for the expected value of the additive genetic variance within populations derived by SSD (E(V _A)_SSD) and PBD (E(V _A)_PBD) in terms of the initial population size (N ₀), the number of seed harvested per pod (M), the probability of survival of an individual (), and the generation at which the population is evaluated (S _t). Differences between (E(V _A)_SSD) and (E(V _A)_PBD) are due to differences in the expected amount of random drift which occurs with the two methods after the S ₀ generation. With both methods, random drift occurs when progeny are sampled from heterozygous parents. An additional component of random drift occurs when sampled progeny fail to survive during SSD, or when sampling occurs amoung families during PBD. For values of N ₀, M, , and S _t that are typical of soybean (Glycine max (L.) Merr.) breeding programs, (E(V _A)_SSD) will be greater than (E(V _A)_PBD). The ratio of (E(V _A)_SSD) to (E(V _A)_PBD) will: (1) increase as M and increase; (2) approach a value of 1.00 as N ₀ increases; and (3) be a curvilinear function of S _t. Plant breeders should compare SSD and PBD based upon values of (E(V _A)_SSD) and (E(V _A)_PBD) and the expected cost of carrying out the two methods.Contribution No. 2910 of the South Carolina Agricultural Experiment Station, Clemson University     

Constrained C-terminal hexapeptide neurotensin analogues containing a 3-oxoindolizidine skeleton

Summary In order to enforce different spatial orientations in the C-terminal hexapeptide of neurotensin (NT_8–13) and to gain information about the importance of the 10–11 peptide bond for binding to NT receptors, the Pro¹⁰-Tyr¹¹ fragment has been replaced with (2R,8S,8aR)-, (2S,8S,8aR)-, (2S,8S,8aS)-, (2S,8R,8aS)- and (2R,8R,8aS)-8-amino-2-benzyl-3-oxoindolizidine-2-carboxylic acid. Molecular dynamics calculations and energy minimization studies have shown that, contrarily to the Pro-Tyr moiety, none of these indolizidines display a tendency to adopt type I and III -turns, but those having (8S,8aR) or (8R,8aS) stereochemistry essentially adopt extended conformations and the (8S,8aS) stereoisomer prefers a nonstandard folding. The four diastereomeric NT_8–13 analogues incorporating (8S,8aR) or (8R,8aS) indolizidines displayed binding affinities for the brain NT receptor similar to that of [Ala¹¹]-NT_8–13 and only five- to ninefold lower than that of the corresponding analogue, [Phe¹¹]NT_8–13. Although this slight decrease could be attributed to differences in conformational behavior between these constrained NT_8–13 analogues and [Phe¹¹]NT_8–13 or NT_8–13, it is not clear whether the -turn around Pro¹⁰-AA¹¹ (AA=Phe, Tyr) is conserved upon receptor binding. An excessive restriction in the motions of the aromatic side chain, imposed by the highly steric constraint of the indolizidine moiety, emerges as an alternative explanation. The findings reported here demonstrate the possibility of replacing the Pro¹⁰-Tyr¹¹ dipeptide in NT_8–13 with a non-peptide residue without affecting considerably the affinity for brain NT receptors.     

Study of salt effects on adenosine–polyuridylic acid interaction

Complex formation between poly(U) and adenosine in solutions of salts that stabilize (Na₂SO₄), destabilize (NaClO₄), or have little effect on the water structure (NaCl), as well as the poly(U)·poly(A) interaction in NaClO₄, was studied by equilibrium dialysis and uv spectroscopy. At 3°C and neutral pH, Ado·2 poly(U) is formed in 1M NaCl and 0.33M Na₂SO₄. In NaClO₄ solutions under the same conditions, an Ado·poly(U) was found over the whole range of salt concentration investigated (10 mM?1M), which has not been previously observed under any conditions. The Ado-poly(U) was also found in a NaCl/NaClO₄ mixture, the transition from the triple- to the double-helical complex occurring within a narrow range of concentration of added NaClO₄. In the presence of 1M NaCl this transition is observed on adding as little as 10 mM NaClO₄, i.e., at a [ClO]/[Cl^?] ratio of about 1:100. However, when NaClO₄ is added to a 1M solution of the stabilizing salt Na₂SO₄, no transition occurs even at a [ClO]/[SO] ratio of 1:4. Investigation of melting curves and uv spectra has shown that in an equimolar mixture of the polynucleotides, only a double-helical poly(U)·poly(A) exists in 1M NaClO₄ at low temperatures; this also holds for 1M NaCl. This changes to a triple-helical 2 poly(U)·poly(A) and then dissociates as the temperature increases. At low temperatures and the poly(U)/poly(A) concentration ratio of 2:1, a mixture of 2 poly(U)·poly(A) and poly(U)·poly(A) was observed in 1M NaClO₄, in contrast to the case of 1M NaCl. Thus, sodium perchlorate, a strong destabilizer of water structure, promotes formation of double-helical complexes both in the polynucleotide–monomer and the polynucleotide–polynucleotide systems. Beginning with a sufficiently high ionic strength (μ ? 0.9), a further increase in the salt molarity results in an increase of the poly(U)·adenosine melting temperature in both stabilizing and neutral salts and a decrease in the destabilizing salt. In Na₂SO₄ concentrations higher than 1.2M Ado·2 poly(U) precipitates at room temperature. Analysis of the binding isotherms and melting profiles of the complexes between poly(U) and adenosine according to Hill's model shows that the cooperativity of binding, due to adenosine stacking on poly(U), increases in the order NaClO₄ < NaCl < Na₂SO₄. The free energy of adenosine stacking on the template is similar to that of hydrogen bonding between adenosine and poly(U) and ranges from ?1 to ?2 kcal/mol. The values of ΔH_t [the effective enthalpy of adenosine binding to poly(U) next to an occupied site, obtained from the relationship between complex melting temperature and free monomer concentration at the midpoint of the transition] are ?14.2, ?18.3, and ?16.8 kcal/mol for 1M solutions of NaClO₄, NaCl, and Na₂SO₄, respectively. The results indicate that the effects of anions of the salts studied are related to water structure alterations rather than to their direct interaction with the complexes between poly(U) and adenosine.     

Functional similarities of AeEα Ia molecules as determined by analysis with T-cell clones

Recognition of A_eE Ia antigens at the functional level was investigated using T-cell clones. The reactivities of an alloreactive and an antigen-reactive clone, both of which recognized A_eE Ia molecules, were compared on a panel of stimulator/antigen-presenting cells of various genotypes. The two clones recognized all tested A _e ^b E ^x Ia molecules, where x is a haplotype capable of expressing an Ia.7-bearing E polypeptide. Ia antigen recognition by either clone could be inhibited by the monoclonal antibody Y-17, which recognizes a combinatorial serologic determinant on certain A_eE molecules. There were no differences in the recognition of Ia by the alloreactive versus the antigen-reactive clone, suggesting that Ia antigens are recognized by the two clones in a fundamentally similar way. The recognition of these various Ia molecules by the two cloned T-cell lines provides evidence that the E polypeptides from H-2 haplotypes k, d, r, and u are functionally indistinguishable.Abreviations MHC major histocompatibility complex - Mb myoglobin - MLR mixed leukocyte reaction - PBS phosphate buffered saline - APC antigen presenting cell - KLH keyhole limpet hemocyanin - GAT poly (glut⁶⁰ alai³⁰ tyr¹⁰)n - (TG)-A—L poly (L-tyr, L-glu)-poly (D, L-ala)—poly L-lys - GLPhe poly (glu⁵⁵ lys³⁶ phe⁹)n     

The amphiphilic peptide adenoregulin enhances agonist binding to A1-adenosine receptors and [35S]GTPγS to brain membranes

Summary 1. Adenoregulin is an amphilic peptide isolated from skin mucus of the tree frog,Phyllomedusa bicolor. Synthetic adenoregulin enhanced the binding of agonists to several G-protein-coupled receptors in rat brain membranes.2. The maximal enhancement of agonist binding, and in parentheses, the concentration of adenoregulin affording maximal enhancement were as follows: 60% (20 µM) for A₁-adenosine receptors, 30% (100 µM) for A_2a-adenosine receptors, 20% (2 µM) for ₂-adrenergic receptors, and 30% (100 µM) for 5HT_1A receptors. High affinity agonist binding for A_1-, _2-, and 5HT_1A-receptors was virtually abolished by GTPS in the presence of adenoregulin, but was only partially abolished in its absence. Magnesium ions increased the binding of agonists to receptors and reduced the enhancement elicited by adenoregulin.3. The effect of adenoregulin on binding of N⁶-cyclohexyladenosine ([³H]CHA) to A₁-receptors was relatively slow and was irreversible. Adenoregulin increased the B_max value for [³H]CHA binding sites, and the proportion of high affinity states, and slowed the rate of [³H]CHA dissociation. Binding of the A₁-selective antagonist, [³H]DPCPX, was maximally enhanced by only 13% at 2 µM adenoregulin. Basal and A₁-adenosine receptor-stimulated binding of [³⁵S]GTPS were maximally enhanced 45% and 23%, respectively, by 50 µM adenoregulin. In CHAPS-solubilized membranes from rat cortex, the binding of both [³H]CHA and [³H]DPCPX were enhanced by adenoregulin. Binding of [³H]CHA to membranes from DDT₁ MF-2 cells was maximally enhanced 17% at 20 µM adenoregulin. In intact DDT₁ MF-2 cells, 20 µM adenoregulin did not potentiate the inhibition of cyclic AMP accumulation mediatedvia the adenosine A₁ receptor.4. It is proposed that adenoregulin enhances agonist binding through a mechanism involving enhancement of guanyl nucleotide exchange at G-proteins, resulting in a conversion of receptors into a high affinity state complexed with guanyl nucleotide-free G-protein.     

Inhibition-threshold substrate concentrations

A proposed substrate inhibition model (M. C. Tseng and M. Wayman, Can. J. Microbiol., 21 , 994 (1975)), ((1)) ((2)) derived from yeast growth rates has been applied to data for bacterial growth: Pseudomonas methanica grown on methanol and Arthrobacter AK19 grown on n-butanol. The model represents the experimental data very well.     

Thermal denaturation of Na- and Li-DNA in salt-free solutions

Thermal denaturation of Na- and Li-DNA from chicken erythrocytes was studied by means of scanning microcalorimetry in salt-free solutions at DNA concentrations (C_p) from 4.5 · 10^?2 to 1 · 10^?3 moles of nucleotides/liter (M). Linear dependencies of DNA melting temperature (T_m) vs lgC_p were obtained: ((1)) ((2)) for Na- and Li-DNA, respectively. Microcalorimetry data were compared with the results of spectrophotometric studies at 260 nm of DNA thermal denaturation in Me-DNA + MeCl solutions at C_p ? (6–8) · 10^?5 M and C_s = 0–40 mM (Me is Na or Li, C_s is salt concentration). It was found that Eqs. (1) and (2) are valid in DNA salt-free solutions over the C_p range 6 · 10^?5?4.5 · 10^?2M. Protonation of DNA bases due to the absorption of CO₂ from air in Na-DNA + NaCl solutions affects DNA melting parameters at C_s < 4 mM. Linear dependence of T_m on lga₊ is found in Na-DNA + NaCl at C_s > 0.4 mMin the absence of contact of solutions with CO₂ from air (a₊ is cation activity). A dependence of [dT_m/dlga₊] on Li⁺ activity was observed in Li-DNA + LiCl solutions at C_s < 10 mM: [dT_m/dlga₊] increases from 17°–18° at C_s > 10 mM to 28°–30° at C_s ? 0.2–0.4 mM. Spectrophotometric measurements at 282 nm show that this effect was caused by protonation of bases in fragments of denatured DNA in neutral solutions. The Poisson–Boltzmann (PB) equation was solved for salt-free DNA at the melting point. The linear dependence of T_m vs lgC_p was interpreted in terms of Manning's condensation theory. PB and Manning's theories fit the experimental data if charge density parameter (ξ) of denatured DNA is in the range 1.8–2.1 (assuming for native DNA ξ = 4.2). Specificity of Li ions in interactions with DNA is discussed. © 1994 John Wiley & Sons, Inc.     

Anaerobic digestion of palm oil mill effluent and condensation water waste: an overall kinetic model for methane production and substrate utilization

The process of anaerobic digestion is viewed as a series of reactions which can be described kinetically both in terms of substrate utilization and methane production. It is considered that the rate limiting factor in the digestion of complex wastewaters is hydrolysis and this cannot be adequately described using a Monod equation. In contrast readily assimilable wastewaters conform well to this approach. A generalized equation has thus been derived, based on both the Monod and Contois equations, which serves extreme cases. The model was verified experimentally using continuous feed anaerobic digesters treating palm oil mill effluent (POME) and condensation water from a thermal concentration process. POME represents a complex substrate comprising of unhydrolyzed materials whereas the condensation water is predominantly short chain volatile fatty acids. Substrate removal and methane production in both cases could be predicted accurately using the generalized equation presented.List of Symbols A (=K_skY/K_h) Kinetic parameter - B Specific methane yield, 1 of CH₄/g of substrate added B₀ Maximum specific methane yield, 1 of CH₄/g of substrate added at infinity - C Empirical constant in Contois equation - F Volumetric substrate removal rate, g/l day - k Hydrolysed substrate transport rate coefficient, 1/days - K (=YC) Kinetic parameter in Chen-Hashimoto equation - K _h Substrate hydrolysis rate coefficient, 1/days - K _s Half-saturation constant for hydrolysed substrate, g/l - M _v Volumetric methane production rate, 1 of CH₄/l day - MS Mineral solids, g/l - MSS Mineral suspended soilds, g/l - POME Palm oil mill effluent - R (=S_r/S_T0) Refractory coefficient - S _h Concentration of hydrolysed substrate, g/l - S _u Intracellular concentration of hydrolysed substrate, g/l - S ₀ Input biodegradable substrate concentration, g/l - S Biodegradable substrate concentration in the effluent or in the digester, g/l - S _r Refractory feed substrate concentration, g/l - S _T0 (=S₀+S_r) Total feed substrate concentration, g/l - S _T (S+S_r) Total substrate concentration in the effluent, g/l - TS Total solids, g/l - TSS Total suspended solids, g/l - VFA Total volatile fatty acids, g/l - VS Volatile solids, g/l - VSS Volatile suspended solids, g/l - X Biomass concentration, g/l - Y Biomass yield coefficient, biomass/substrate mass - Hydraulic retention time, days. - Specific growth rate of microorganisms, l/days - _m Maximum specific growth rate of microorganisms, l/days The authors wish to express their gratitude to the Departamento de Postgrado y Especialización del CSIC and to the Consejería de Educación y Ciencia de la Junta de Andalucia for their financial support of this work.     

Effects of β-Amyloid-(25-35) Peptides on Radioligand Binding to Excitatory Amino Acid Receptors and Voltage-Dependent Calcium Channels: Evidence for a Selective Affinity for the Glutamate and Glycine Recognition Sites of the NMDA Receptor

The neurotoxic fragment corresponding to residues 25-35 of the -amyloid (A) peptide [A-(25-35)] has been shown to exert effects on (+)-[³H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate ([³H]MK-801) binding to the cation channel of the N-methyl-D-aspartate (NMDA) receptor. In the present study, we investigated whether the amidated and carboxylic acid C-terminated forms of A-(25-35) [A-(25-35-NH₂) and A-(25-35-COOH), respectively] exert effects on other excitatory amino acid receptor and cation channel types in rat cortical membranes. Both A-(25-35-NH₂) and A-(25-35-COOH) gave statistically significant dose-dependent inhibitions of [³H]glutamate and [³H]glycine binding to the agonist recognition sites of the NMDA receptor. Ten M A-(25-35-NH₂) and A-(25-35-COOH) gave 25% and 20% inhibitions of [³H]glutamate binding and 75% and 70% inhibitions of [³H]glycine binding, respectively. A-(25-35-NH₂), but not A-(25-35-COOH), gave a small (ca. 17% at 10 M) statistically significant increase of [³H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([³H]AMPA) binding. [³H]kainate binding was not significantly affected by either peptide. Similarly, neither peptide affected either the maximal level or EC₅₀ value for calcium stimulation of [³H]nitrendipine binding. It is concluded that A-(25-35) shows slight affinity for the agonist recognition sites of the NMDA receptor, but not for other excitatory amino acid receptor types or for L-type voltage-dependent calcium channels.     

Simulation of a multicolumn recirculated packed bed reactor (MRPBR) for penicillin acylase

Enzyme reactors for the industrial hydrolysis of penicillin are analyzed in terms of biocatalyst stability to pH. A multicolumn system with packed beds placed in parallel and operating under recirculating conditions is proposed as an adequate reactor for this process. The system is studied both experimentally and with the aid of a simulation program.List of Symbols A transversal area (cm²) - C _A ammonia concentration in the reaction mixture (M) - C ₁ concentration of KH₂PO₄ in buffer (M) - C ₂ concentration of K₂HPO₄ in buffer (M) - d _p biocatalyst diameter (cm) - E enzyme or biocatalyst concentration (gcat l^–1) - K _APA APA non competitive inhibition constant (M) - K _IS excess substrate inhibition constant (M) - Km constant Michaelis-Menten (M) - K _PAA PAA competitive inhibition constant (M) - Q recirculation flow rate (cm³ min^–1) - Q _T recirculation flow rate per column (cm³ min^–1) - Re Reynolds number - S _E substrate concentration entering the neutralization tank (M) - S ₀ initial substrate concentration (M) - S _T substrate concentration in neutralization tank (M) - t time (min) - v _i initial reactor rate (mol min^–1 gcat^–1) - V _s superficial velocity (cm seg^–1) - V _T volume of neutralization tank (cm³) - X _E substrate conversion entering tank - X _T substrate conversion in neutralization tank - X conversion - Z reactor length (cm) - z axial position in reactor (cm) - z ^* non-dimensional axial position in reactor - biocatalyst's density (gcat cm^–3) - p pressure drop in the packed-bed reactor     

New insights into the photochemistry of carotenoid spheroidenone in light-harvesting complex 2 from the purple bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Light-harvesting complex 2 (LH2) from the semi-aerobically grown purple phototrophic bacterium Rhodobacter sphaeroides was studied using optical (static and time-resolved) and resonance Raman spectroscopies. This antenna complex comprises bacteriochlorophyll (BChl) a and the carotenoid spheroidenone, a ketolated derivative of spheroidene. The results indicate that the spheroidenone-LH2 complex contains two spectral forms of the carotenoid: (1) a minor, “blue” form with an S₂ (1¹B _u ⁺ ) spectral origin band at 522 nm, shifted from the position in organic media simply by the high polarizability of the binding site, and (2) the major, “red” form with the origin band at 562 nm that is associated with a pool of pigments that more strongly interact with protein residues, most likely via hydrogen bonding. Application of targeted modeling of excited-state decay pathways after carotenoid excitation suggests that the high (92%) carotenoid-to-BChl energy transfer efficiency in this LH2 system, relative to LH2 complexes binding carotenoids with comparable double-bond conjugation lengths, derives mainly from resonance energy transfer from spheroidenone S₂ (1¹B _u ⁺ ) state to BChl a via the Q_x state of the latter, accounting for 60% of the total transfer. The elevated S₂ (1¹B _u ⁺ ) → Q_x transfer efficiency is apparently associated with substantially decreased energy gap (increased spectral overlap) between the virtual S₂ (1¹B _u ⁺ ) → S₀ (1¹A _g ^? ) carotenoid emission and Q_x absorption of BChl a. This reduced energetic gap is the ultimate consequence of strong carotenoid–protein interactions, including the inferred hydrogen bonding.