Toward the Development of Metal‐Free Synthetic Nucleases: Cleavage of a Model Substrates by 1,4‐Diazabicyclo[2.2.2]Octane Derivatives

Artificial ribonucleases of A_nBCL series were synthesized by solid‐phase method. They consist of a hydrophobic alkyl radical A (n = 3–12 carbon atoms), an “RNA‐binding domain” B (bisquaternary salt of 1,4‐diazabicyclo[2.2.2]octane), a “catalytic domain” C (histidine residue) and a “linker” L that joins the domains B and C. The effect of the alkyl radical on the catalytic properties of the chemical catalyst was studied using three activated phosphate ester substrates: p‐nitrophenyl phosphate, bis‐p‐nitrophenyl phosphate, and thymidine‐3′‐p‐nitrophenyl phosphate.     

Direct differential absorbance profiles of denaturing transitions in ribosomal and mRNA.

A direct method for measuring the derivative of thermal transition profiles (ΔA/ΔT), first described by Pavlov and Lyubchenko [Biopolymers 17 , 795–798 (1978)], is applied to the secondary structure of several eukaryotic ribosomal and messenger RNAs. The method consists of generating an effective ΔT between a sample and reference cuvette by altering the T_m (midpoint denaturation temperature) of one of the solutions with respect to the other. This can be done by changing salt concentration, solvent, pH, or ligands. Scanning the two cuvettes by varying the wavelength at different temperatures permits detailed examination of the base composition of differentially melting domains. We report here the ΔA/ΔT profiles generated by monovalent ion concentration differences for a number of high-molecular-weight natural RNAs, as well as the synthetic polynucleotides poly(rA) and “random” poly[r(A,G,U,C)]. The 18S and 28S rRNAs from chick embryos exhibit a reproducible series of peaks in the ΔA/ΔT profiles at low salt with ΔT = 4K. The high-temperature transitions in 28S rRNA appear to contain G·C base pairs exclusively, in contrast to those in 18S rRNA or any natural mRNA. Each mRNA we have examined (bacteriophage MS2, globin mRNA from rabbit reticulocytes, and procollagen mRNA from chick embryos) exhibits a distinctive ΔA/ΔT profile in low salt. The stability of many of the transitions in each of the mRNAs is no greater than that of the secondary structure in random poly(A,G,U,C) in low salt. More than 50% of the base pairing in procollagen mRNA actually “melts” below the mean for the random copolymer, indicating that despite its high G·C content, this mRNA contains a secondary structure that is exceptionally low in stability.     

Evidence for a glucosyl-enzyme intermediate in the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside

The reaction of almond β-glucosidase with $\text{p}$-nitrophenyl-β-$\text{D}$-glucoside has been investigated over the temperature range +25° to ?45° using 50% aqueous dimethyl sulfoxide (DMSO) as solvent. At temperatures below those at which turnover occurs a “burst” of $\text{p}$-nitrophenol proportional to the enzyme concentration is observed. Such a “burst” suggests the existence of a glucosyl-enzyme intermediate whose breakdown is rate-limiting, and provides a method for measuring the active-site normality. At pH 5.9, 25°, the presence of 50% DMSO causes an increase in K_m from 1.7×10^?3M (0%) to 1.7×10^?2M, whereas V_max is unchanged. The DMSO thus apparently acts as a competitive inhibitor with K_i = 0.7M. The Arrhenius plot for turnover is linear over the accessible temperature range with E_a = 23.0 ± 2.0 kcal/mole.     

The aggregative numerical response of polyphagous predators to aphids in cereal fields: attraction to what?

We tested in a field experiment two hypotheses for why polyphagous predators aggregate at concentrations of aphids: 1) because they are attracted directly to aphids as prey, or 2) because they are attracted to alternative prey that aggregate around the honeydew produced by aphids. Small plots were established in the field with two experimental treatments, aphid addition and honey spraying, and a watersprayed control, each replicated 10 times. Arthropods were recorded by pitfall traps and sticky traps in each plot. Diptera were significantly more abundant in the honey plots. Of the predators, Agonum dorsale, “All carabids” and Philonthus sp. were most abundant in the honey plots; Tachyporus spp. and carabid and staphylinid larvae were most abundant in the aphid addition plots. It is suggested that these results reflect differences among the predators in their ability to tolerate and utilise aphids as food.     

On the difference between gauss-markov and least squares estimates

For the usual full rank univariate least squares regression model y = XB + e, E(e) = 0, E(ee) = A, the equality of the estimates occurs when B-B^* = (XA^?1X)^?1XA^-1y-(XX)^?1Xy = 0. A necessary and sufficient condition for this equality is that A has some N - k + 1 roots equal where N is the rank of A and k is the rank of X.     

Receptor-Agonist Interactions in Service-Theoretic Perspective,Effects of Molecular Timing on the Shape of Dose-Response Curves

Service-theoretic concepts and methods, widely used in other fields (e.g., telecommunication and operations research), are useful also in a biochemical setting because the treatment of biocatalysts (enzymes, receptors) as servers and their ligands as customers, based on the established formal methods of service or queuing theory, may lead to insights and results unobtainable by conventional, mass-action-law-based theories. In this article, we apply the service-theoretic approach to receptor-agonist systems and show how by changing the stochastic time pattern of “operationally relevant” point events (e.g., instants of agonist arrival, instants of postclimax agonist departure) a great variety of dose-response curves may be generated, even in very simple reaction schemes, which, according to mass action kinetics, invariably lead to hyperbolic r(A) curves (r and A stand for response and agonist concentration, respectively). The molecular timing inherent to a hyperbolic response system is not optimal: for instance, at the agonist concentration A₅₀, half of the agonist molecules are rejected (“lost”) because of unfortunate timing of the arrival events. The fraction of lost arrivers can be diminished considerably if the arrivals are better timed: “sub-Poisson” arrivals improve the timing and, thus, convert hyperbolic r(A) curves into “lifted” nonhyperbolic ones. Conversely, “super-Poisson” arrivals make the nonoptimal timing in hyperbolic response systems even worse and, thus, convert hyperbolic r(A) curves into “depressed” nonhyperbolic ones. Furthermore, under special timing conditions, nonhyperbolic r(A) curves can be generated, which are partly lifted, partly depressed relative to the reference hyperbola, and which resemble in shape well-known nonhyperbolic forms of enzyme and receptor kinetics (negatively cooperative, positively cooperative, and sigmoidal kinetics). In addition unusual (undulatory and sawtooth-like) r(A) curves can be generated solely by changing the temporal pattern of arrival and service completion instants. Virtually any shape of dose-response curves may be obtained by allowing for probability distributions whose characteristic shape varies with their mean; we call such distributions “variomorphic” and apply them to the arrival process of agonist molecules.     

Studies on lsoamylase

A method for determining the isoamylase activity is devised. A properly diluted enzyme solution is allowed to act at 20°C on a 1% solution of glutinous-rice starch buffered to pH 6.2. After 24 hours, 0.2 cc of the reaction mixture is mixed with 2 cc of 0.01 N I₂ solution, and diluted to 25 cc. Optical density (E) of the solution at 620 mµ is read in a Pulfrich photometer, using a 1-cm cell. The increment of E at 620 mµ is proportional to enzyme activity within certain ranges. The amount of enzyme is expressed in the “isoamylase unit”, i.e., an increase of E 0.200 being taken as 10 units.

A pH-activity curve is given for yeast isoamylase, which shows a sharp maximum at pH 6.2.     

Microbiological Redox Potential Control to Improve the Efficiency of Chalcopyrite Bioleaching

The effect of controlling the redox potential (E_h) on chalcopyrite bioleaching kinetics was studied as a new aspect of redox control during chalcopyrite bioleaching, and its mechanism was investigated by employing the “normalized” solution redox potential (E_normal) and the reaction kinetics model. Different E_h ranges were established by use of different acidophiles (Sulfobacillus acidophilus YTF1; Sulfobacillus sibiricus N1; Acidimicrobium ferrooxidans ICP; Acidiplasma sp. Fv-AP). Cu dissolution was very susceptible to real-time change in E_h during the reaction. It was found that efficiency of bioleaching of chalcopyrite can be effectively evaluated on the basis of E_normal, since it is normalized for real-time fluctuations of concentrations of major metal solutes during bioleaching. For steady Cu solubilization during bioleaching at a maximum rate, it was important to maintain a redox potential range of 0 ≤ E_normal ≤ 1 (?0.35 mV optimal) at the mineral surface by employing a “weak” ion-oxidizer. This led to a copper recovery of > 75%. At higher E_normal levels (E_normal > 1 by “strong” microbial Fe²⁺ oxidation), Cu solubilization was slowed by diffusion through the product film at the mineral surface (< 50% Cu recovery) caused by low reactivity of the chalcopyrite and by secondary passivation of the chalcopyrite surface, mainly by jarosite.     

The stability of RNA hairpin loops containing A-U-G: An-U-G-Um

RNA oligomers with the sequence A_n-U-G-U_m, n = 7–9, m = 5–10, have been synthesized and found to form hairpin loops in 21 mM or 101 mM Na⁺. The hairpin loops displayed melting temperatures 13°–29°C greater than that of the hairpin loop A₆-C₈-U₆ in the same solvent. The increased stability of these hairpin loops is attributed to the presence of the trinucleotide A-U-G in the loop. Circular dichroism (CD) spectra were taken of the hairpin loops A₇-U-G-U_6,7,8, A₈-U-G-U_6,7,8, and A₉-U-G-U₆ in 21 mM Na⁺, and compared with circular dichroism spectra of A₆-U₆ in 1 M Na⁺. Difference spectra were calculated between each A_n-U-G-U_m and 11.5 mM (nucleotide) A₆-U₆ at similar temperatures and identical singlestrand fractions to give the “experimental” CD spectrum of the unbase-paired nucleotides in the loop, assuming, five, six, or seven base pairs. CD spectra were calculated for each of the assumed unbase-paired sequences using the measured CD spectra of ApA, A-U-G-, A, and U, and compared with the experimental spectra. The best agreement was found for hairpin-loop models containing five base pairs and five to eight unbase-paired nucleotides in the loop.     

Non-linear arrhenius plots and the analysis of reaction and motional rates in biological membranes

We have systematically derived the rate-temperature relationships for a variety of models of membrane rate processes (particularly enzymic reactions) in order to predict the Arrhenius plot shape(s) appropriate to each model. We have explicitly considered the fact that most thermotropic changes in biological systems extend over finite and sometimes very broad temperature ranges. The rate-temperature relationships for most of the models considered can be expressed in a common, rather simple mathematical form suitable for application in computer data analysis. Only a few models predict Arrhenius plots with the “biphasic linear” form commonly reported in studies of membrane enzymes. However, many of the models yield plots which can be fitted to two intersecting lines within a quite modest experimental error, especially if the change in the slope of the plot around its “break” corresponds to a change in activation enthalpy of less than 15–20 kcal mol^?1. In general, Arrhenius-type plots of motional and reaction rates in membranes are found to be capable of indicating the midpoint but not the endpoints or the overall width of thermotropic transitions in the state of membrane components. Our findings clearly indicate a need for a more rigorous analysis of Arrhenius plot data in terms of graph shapes other than sets of intersecting lines and for more cautious interpretation of Arrhenius plot “breaks” with regard to their physical basis.     

Plant species richness and species-area relations in a shortgrass steppe in Colorado

Abstract. Plant species richness and species-area relations were examined for three landscapes (toposequences), each with a summit or upland, a midslope and a toeslope or lowland, in a shortgrass steppe in Colorado. The number of plant species in the largest plot size (0.16 ha) varied from 38 to 53. Neither the exponential relationship: s = a + b log A, nor the power function: S = cAz fit the data equally well in all situations. The processes acting upon species diversity here seem to operate at two spatial scales. The number of species in plots smaller than 3 m² was independent of the total number of species in the 0.16-ha plots and was constrained by the presence of the dominant bunchgrasses. Beyond 3 m², species number in each plot size was a function of the total number of species in the 0.16-ha plot.     

Multiple source pools for Galápagos plant species richness: a critical analysis of the line of sight connectivity index

An index (C_i*E) combining the number of line‐of‐sight islands (C_i) within a radius i and target island elevation (E) has been proposed as an improved predictive model of plant species richness (S_t) in the Galápagos Archipelago. We examined this index critically and found that several major flaws preclude it from being a useful predictive tool for the archipelago. Although the number of collecting trips to an island was reported over 20 years ago to have substantial predictive value for reported plant species richness in the Galápagos Islands, this relationship was ignored in multiple regression analyses of the index. When we included the number of collecting trips in different multiple regression analyses of the index, C_i*E had less predictive power than collecting trips or ceased to be significant at all. Additionally, the strong significant relationship between elevation and area in the Galápagos Archipelago results in area having a major confounding influence on the C_i*E index. When elevation is removed from the C_i*E index, the predictive power of C_i is far less than area alone. Finally, the data used to construct and correlate the C_i*E index with (S_t) were based only on a subset of the islands and species lists that were incomplete or out of date. Species richness on islands can be related to the interaction of different factors, so development and testing of indices like C_i*E is not inappropriate. However, it is important to examine the interrelationships among the components of these indices thoroughly, and not ignore the effect of factors already known to have high predictive power. We propose several ways in which more meaningful indices of source pool(s) capacity can be constructed.     

Mg<Superscript>2+ </Superscript>Decreases Arrhenius Energies of Activation for High Temperature Catalysis of Phosphatases in <Emphasis Type="Italic">Thermoactinomyces vulgaris</Emphasis>

The nonspecific acid and alkaline phosphatases of Thermoactinomyces vulgaris were found to be optimally active at 65°C and 70°C, respectively, indicating the thermophilic nature of these enzymes in this obligate thermophile. Mg²⁺, when added in the assay mixture (in the form of MgCl₂), increased the specific activities of these enzymes without affecting their respective temperature optima. This divalent cation decreased the Arrhenius energies of activation (E _A ) of both acid and alkaline phosphatases, as substantiated by Mg²⁺-dependent decrease in the slopes of their Arrhenius plots, which were found to be linear. Thus, Mg²⁺-dependent stimulation of high temperature catalysis of T. vulgaris phosphatases appeared to be accomplished by the decrease in their E _A values by this divalent cation, and such unique feature of these enzymes might be associated with their evolutionary adaptation in this thermophilic actinomycete to support its growth at elevated temperatures. The catalytic role of Mg²⁺ in enhancing the phosphatase activities was specified by the fact that this metal ion was able to recover the enzyme activities inhibited by dialysis and EDTA.     

Kinetics of microbial death by combined treatment with heat and antimicrobial agents

Studies were made of the death kinetics of Escherichia coli cells heated at 46 to 56°C in 0.05M phosphate buffer (pH 7.0) containing either an amphoteric surfactant (Tego 15DL, 1–10 μg/ml) or sorbic acid (0.5 to 3%). A linear relationship was obtained on the Arrhenius plot for the death of cells heated with each antimicrobial agent. The kinetics of the action of the surfactant, however, differed from that of sorbic acid. With the amphoteric surfactant, the activation enthalpy of the death reaction decreased from 108 to 51 kcal/mol as the concentration of surfactant was increased in the range tested although the death rate remained high; whereas with sorbic acid the activation enthalpy remained fairly constant (104 ± 9 kcal/mol) independent of its concentration and the death rate was similarly high. Further, in the action of the amphoteric surfactant, a thermodynamic compensation effect was observed, the compensation temperature being 334°K (61°C), i.e., relatively close to the observed temperatures. For sorbic acid, however, this temperature seemed to be too high to observe when determined from the Arrhenius plot. The data of the dependency of the death-rate constant upon the concentration of antimicrobial agent indicated a similar difference in the action of the two agents. Based on our results and on data obtained by other workers, we propose that antimicrobial agents that enhance cellular death induced by heating can be characterized into two types.     

The convenience of the use of allosteric "probes" for the study of lipid--protein interactions in biological membranes: thermodynamic considerations.

One of the most commonly used methods to study enzyme (protein)-structure interactions at a more specific level is the use of the Arrhenius plots to detect the influence of the “environment” on the enzyme. We want to point out here that the use of a suitable “allosteric enzyme” would be a more sensitive method to detect influences of the environment than the study of Arrhenius plots. According to simple thermodynamic considerations, a change of more than 2.8 kcal/mol in the interaction between enzyme and membrane would be needed to give a noticeable change in the position of the break (T_i) of the corresponding Arrhenius plot. On the other hand, feeble changes in the membrane-enzyme interaction, of the order of 0·7–0·8 kcal/mol, would be enough to give a significant change in the values of n (Hill coefficient).     

Perfect temperature for protein structure prediction and folding

We have investigated the influence of the “noise” of inevitable errors in energetic parameters on-protein structure prediction. Because of this noise, only a part of all the interactions operating in a protein chain can be taken into account, and therefore a search for the energy minimum becomes inadequate for protein structure prediction. One can rather rely on statistical mechanics: a calculation carried out at a temperature T_* somewhat below that of protein melting gives the best possible, though always approximate prediction. The early stages of protein folding also “take into account” only a part of all the interactions; consequently, the same temperature T_* is favorable for the self-organization of native-like intermediates in protein folding. © 1995 Wiley-Liss, Inc.     

Analysis of binding curves in multivalent antigen-heterogeneous antibody systems

The binding of antibodies to N-valent antigens can be utilized to gain information on the antibody affinity distribution, under the assumption, that the formation of each bond occurs as an independent event. The analysis of the most widely used plots of binding data (double reciprocal, Scatchard, Sips and the so-called “avidity” plot) leads to expressions which correlate asymptotical features of the binding curves to the antibody site concentration to the antigen valence and to the affinity moments <K^?1>, <K> and <K²>. Only in the “avidity” plot is the shape of the curve independent of the valence of the antigen, depending merely on the antibody affinity distribution.     

Sequence-based association analysis of HLA class I and II alleles in Japanese supports conservation of common haplotypes

 Alleles of HLA-A, B, C, DRB1, DQB1, and DPB1 loci were fully determined in 117 healthy Japanese. A ^* 2402, A ^* 3303, A ^* 1101, A ^* 0201, B ^* 4403, B ^* 5201, Cw ^* 0102, Cw ^* 1403, Cw ^* 0304, Cw ^* 0702, Cw ^* 0801, and Cw ^* 1202 showed frequencies of over 10%. Multi-locus haplotype frequencies were estimated by the maximum likelihood method. Strength of association between C and B loci was comparable with that between DRB1 and DQB1 loci. Alleles unidentified by a serological method and having very similar nucleotide sequences (A2: A ^* 0201, A ^* 0206, A ^* 0207, B61: B ^* 4002, B ^* 4006) were carried by different haplotypes. Several frequent five-locus haplotypes were identified including A ^* 3303-Cw ^* 1403-B ^* 4403-DRB1 ^* 1302-DQB1 ^* 0604, and A ^* 2402-Cw ^* 1202-B ^* 5201-DRB1 ^* 1502-DQB1 ^* 0601. These sequence-based haplotypes corresponded to serology-based common haplotypes which have already been described in Japanese. These findings indicate that common HLA haplotypes consist of particular sets of HLA alleles and that these haplotypes have been conserved through recent human evolution. Received: 25 November 1996 / Revised: 20 January 1997     

Species constancy depends on plot size – a problem for vegetation classification and how it can be solved

Question: While it is well known that species richness depends on plot size, it is not generally recognised that the same must be true for constancy. Accordingly, many authors use varying plot sizes when classifying vegetation based on the comparison of constancies between groups of plots. We ask whether the constancy‐area relationship follows a general rule, how strong the effect of plot sizes is on constancies, and if it is possible to correct constancies for area. Location: For empirical evaluation, we use data from plant communities in the Czech Republic, Sweden and Russia. Methods: To assess the potential influence of differences in plot size on constancies, we develop a mathematical model. Then, we use series of nested plot species richness data from a wide range of community types (herbaceous and forest) to determine the parameters of the derived function and to test how much the shape of the constancy‐area relationship depends on taxa or vegetation types. Results: Generally, the constancy‐area relationship can be described by C (A)=1?(1?C₀)^{(A/A0)^d}, with C being constancy, A area, C₀ known constancy on a specific area A₀, and d a damping parameter accounting for spatial autocorrelation. As predicted by this function, constancies in plant communities always varied from values near 0% to near 100% if plot sizes were changed sufficiently. For the studied vegetation types, a two‐ to fourfold increase in plot size resulted in a change of conventional constancy classes, i.e. an increase of constancy by 20% or more. Conclusions: Vegetation classification, which largely relies on constancy values, irrespective of whether traditional or modern fidelity definitions are used, is strongly prone to distorting scale effects when relevés of different plot sizes are combined in studies. The constancy‐area functions presented allow an approximate transformation of constancies to other plot sizes but are flawed by idiosyncrasies in taxa and vegetation types. Thus, we conclude that the best solution for future surveys is to apply uniform plot sizes within a few a priori delimited formations and to determine diagnostic species only within these formations. Finally, we suggest that more detailed analyses of constancy‐area relationships can contribute to a better understanding of species‐area relationships because the latter are the summation of the first for all species.