Role of ionization of the phosphate cosubstrate on phosphorolysis by purine nucleoside phosphorylase (PNP) of bacterial (<Emphasis Type="Italic">E. coli</Emphasis>) and mammalian (human) origin

Kinetics of the reactions of purine nucleoside phosphorylases (PNP) from E. coli (PNP-I, the product of the deoD gene) and human erythrocytes with their natural substrates guanosine (Guo), inosine (Ino), a substrate analogue N(7)-methylguanosine (m⁷Guo), and orthophosphate (P_i, natural cosubstrate) and its thiophosphate analogue (SP_i), found to be a weak cosubstrate, have been studied in the pH range 5–8. In this pH range Guo and Ino exist predominantly in the neutral forms (pK_a 9.2 and 8.8); m⁷Guo consists of an equilibrium mixture of the cationic and zwitterionic forms (pK_a 7.0); and P_i and SP_i exhibit equilibria between monoanionic and dianionic forms (pK_a 6.7 and 5.4, respectively). The phosphorolysis of m⁷Guo (at saturated concentration) with both enzymes exhibits Michaelis kinetics with SP_i, independently of pH. With P_i, the human enzyme shows Michaelis kinetics only at pH ∼5. However, in the pH range 5–8 for the bacterial enzyme, and 6–8 for the human enzyme, enzyme kinetics with P_i are best described by a model with high- and low-affinity states of the enzymes, denoted as enzyme-substrate complexes with one or two active sites occupied by P_i, characterized by two sets of enzyme-substrate dissociation constants (apparent Michaelis constants, K _m1 and K _m2) and apparent maximal velocities (V _max1 and V _max2). Their values, obtained from non-linear least-squares fittings of the Adair equation, were typical for negative cooperativity of both substrate binding (K _m1 < K _m2) and enzyme kinetics (V _max1/K _m1 > V _max2/K _m2). Comparison of the pH-dependence of the substrate properties of P_i versus SP_i points to both monoanionic and dianionic forms of P_i as substrates, with a marked preference for the dianionic species in the pH range 5–8, where the population of the P_i dianion varies from 2 to 95%, reflected by enzyme efficiency three orders of magnitude higher at pH 8 than that at pH 5. This is accompanied by an increase in negative cooperativity, characterized by a decrease in the Hill coefficient from n _H ∼1 to n _H ∼0.7 for Guo with the human enzyme, and to n _H ∼0.7 and 0.5 for m⁷Guo with the E. coli and human enzymes, respectively. Possible mechanisms of cooperativity are proposed. Attention is drawn to the substrate properties of SP_i in relation to its structure.     

Long-term changes in abundance of bats as revealed by their frequency in tawny owls’ diet

Percentage of bats in tawny owls’ diet was compared in three periods: I — before 1976, II — 1976–1992, III — 1993–2009, by using the published and unpublished material from Poland (only samples over 100 vertebrate prey items). This species of owl showed an opportunistic predation on bats and took them more frequently in periods of higher abundance. Before the mass use of toxic pesticides in Poland, in the period I bats constituted more than 2% of vertebrates in four out of five diet samples (median 2.4%). The lowest bat abundance occurred in Poland in the 1980s and resulted in the lowest percentage of bats taken by owls in the period II (n = 11, median 0.2%). Due to the recovery of bat populations in the period III, the percentage of bats in tawny owls’ diet increased (n = 23, median 0.7%). In large samples (over 200 vertebrate items, n = 21) collected in central and north-eastern Poland the percentage of bats increased from 1980 to 2009 (the estimated average value at the end of that period slightly exceeded 1%). Samples collected at the same five sites in 1975–1992 and again in 2000–2009, confirmed the increasing trend in percentage of bats captured by tawny owls noted in last years.     

Role of LTD4 in the Regulatory Volume Decrease Response in Ehrlich Ascites Tumor Cells

Stimulation with leukotriene D₄ (LTD₄) (3–100 nm) induces a transient increase in the free intracellular Ca²⁺ concentration ([Ca²⁺] _i ) in Ehrlich ascites tumor cells. The LTD₄-induced increase in [Ca²⁺] _i is, however, significantly reduced in Ca²⁺-free medium (2 mm EGTA), and under these conditions stimulation with a low LTD₄ concentration (3 nm) does not result in any detectable increase in [Ca²⁺] _i . Addition of LTD₄ (3–100 nm) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD₄-induced (100 nm) acceleration of the RVD response is also seen in Ca²⁺-free medium and also at 3 nm LTD₄, indicating that LTD₄ can open K⁺- and Cl⁻-channels without any detectable increase in [Ca²⁺] _i . Buffering cellular Ca²⁺ with BAPTA almost completely blocks the LTD₄-induced (100 nm) acceleration of the RVD response. Thus, the reduced [Ca²⁺] _i level after BAPTA-loading or buffering of [Ca²⁺] _i seems to inhibit the LTD₄-induced stimulation of the RVD response even though the LTD₄-induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca²⁺] _i . The LTD₄ receptor antagonist L649,923 (1 μm) completely blocks the LTD₄-induced increase in [Ca²⁺] _i and inhibits the RVD response as well as the LTD₄-induced acceleration of the RVD response. When the LTD₄ receptor is desensitized by preincubation with 100 nm LTD₄, a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD₄ plays a role in the activation of the RVD response. LTD₄ seems to activate K⁺ and Cl⁻ channels via stimulation of a LTD₄ receptor with no need for a detectable increase in [Ca²⁺] _i . Received: 25 September 1995/Revised: 25 January 1996     

Heat and mass exchange processes between the surface of the human body and ambient air at various altitudes

 The rates of convection and evaporation at the interface between the human body and the surrounding air are expressed by the parameters convective heat transfer coefficient h _c, in W m^–2°C^–1 and evaporative heat transfer coefficient h _e, W m^–2 hPa^–1. These parameters are determined by heat transfer equations, which also depend on the velocity of the airstream around the body, that is still air (free convection) and moving air (forced convection). The altitude dependence of the parameters is represented as an exponential function of the atmospheric pressure p: h _c∼p ⁿ and h _e∼p ^1–n, where n is the exponent in the heat transfer equation. The numerical values of n are related to airspeed: n=0.5 for free convection, n=0.618 when airspeed is below 2.0 ms^–1 and n=0.805 when airspeed is above 2.0 ms^–1. This study considers the coefficients h _c and h _e with respect to the similarity of the two processes, convection and evaporation. A framework to explain the basis of established relationships is proposed. It is shown that the thickness of the boundary layer over the body surface increases with altitude. As a medium of the transfer processes, the boundary layer is assumed to be a layer of still air with fixed insulation which causes a reduction in the intensity of heat and mass flux propagating from the human body surface to its surroundings. The degree of reduction is more significant at a higher altitude because of the greater thickness of the boundary layer there. The rate of convective and evaporative heat losses from the human body surface at various altitudes in otherwise identical conditions depends on the following factors: (1) during convection – the thickness of the boundary layer, plus the decrease in air density, (2) during evaporation (mass transfer) – the thickness of the boundary layer, plus the increase with altitude in the diffusion coefficient of water vapour in the air. The warming rate of the air volume due to convection and evaporation is also considered. Expressions for the calculation of altitude dependences h _c (p) and h _e (p) are suggested. Received: 23 June 1998 / Accepted: 10 February 1999     

Chloride Conductance and P i Transport are Separate Functions Induced by the Expression of NaPi-1 in Xenopus Oocytes

Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl⁻ conductance (G_Cl), organic anion transport and Na⁺-dependent P _i -uptake. In the present study we investigated the relation between the NaPi-1 induced G_Cl and P _i -induced currents and transport. NaPi-1 expression induced P _i -transport, which was not different at 1–20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1–2 days after cRNA injection. In contrast, G_Cl was augmented at increased amounts of cRNA injection (1–20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. P _i -induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of P _i (≥ 1 mm P _i ). The amplitudes of Ip correlated well with G_Cl. Ip was blocked by the Cl⁻ channel blocker NPPB, partially Na⁺-dependent and completely abolished in Cl⁻ free solution. In contrast, P _i -transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na⁺ and weakly affected by Cl⁻ substitution. Endogenous P _i -uptake in water-injected oocytes amounted in all experiments to 30–50% of the Na⁺-dependent P _i -transport observed in NaPi-1 expressing oocytes. The properties of the endogenous P _i -uptake system (K _m for P _i > 1 mm; partial Na⁺- and Cl⁻-dependence; lack of NPPB block) were similar to the NaPi-1 induced P _i -uptake, but no Ip could be recorded at P _i -concentrations ≤3 mm. In summary, the present data suggest that Ip does not reflect charge transfer related to P _i -uptake, but a P _i -mediated modulation of G_Cl. Received: 22 October 1997/Revised: 24 March 1998     

Inhibition by light of CO2 evolution from dark respiration: Comparison of two gas exchange methods

Two approaches to determine the fraction (μ) of mitochondrial respiration sustained during illumination by measuring CO₂ gas exchange are compared. In single leaves, the respiration rate in the light (`day respiration' rate R<sub>d</sub>) is determined as the ordinate of the intersection point of A–c<sub>i</sub> curves at various photon flux densities and compared with the CO<sub>2</sub> evolution rate in darkness (`night respiration' rate R_n). Alternatively, using leaves with varying values of CO₂ compensation concentration (Γ), intracellular resistance (r_i) and R_n, an average number for μ can be derived from the linear regression between Γ and the product r_iċR_n. Both methods also result in a number c* for that intercellular CO₂ concentration at which net CO₂ uptake rate is equal to –R_d. c* is an approximate value of the photocompensation point Γ* (Γ in the absence of mitochondrial respiration), which is related to the CO₂/O₂ specificity factor of Rubisco S_c/o. The presuppositions and limitations for application of both approaches are discussed. In leaves of Nicotiana tabacum, at 22 °C, single leaf measurements resulted in mean values of μ = 0.71 and c_* = 34 μmol mol⁻¹. At the photosynthetically active photon flux density of 960 μmol quanta m⁻² s⁻¹, nearly the same numbers were derived from the linear relationship between Γ and r_iċR_n. c* and R_d determined by single leaf measurements varied between 31 and 41 μmol mol⁻¹ and between 0.37 and 1.22 μmol m⁻² s⁻¹, respectively. A highly significant negative correlation between c* and R_d was found. From the regression equation we obtained estimates for Γ* (39 μmol mol⁻¹), S_c/o (96.5 mol mol⁻¹) and the mesophyll CO₂ transfer resistance (7.0 mol⁻¹ m² s). This revised version was published online in June 2006 with corrections to the Cover Date.     

Mitochondria from Dipodascus (Endomyces) magnusii and Yarrowia lipolytica yeasts did not undergo a Ca2+-dependent permeability transition even under anaerobic conditions

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts. The two yeast strains are good alternatives to Saccharomyces cerevisiae, being aerobes containing well-structured mitochondria (thus ensuring less structural limitation to observe their appreciable swelling) and fully competent respiratory chain with three invariantly functioning energy conservation points, including Complex I, that can be involved in induction of the canonical Ca²⁺/P_i-dependent mitochondrial permeability transition (mPTP pore) with an increased open probability when electron flux increases (Fontaine et al. J Biol Chem 273:25734–25740, 1998; Bernardi et al. FEBS J 273:2077–2099, 2006). High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating pore opening. Previously (Kovaleva et al. J Bioenerg Biomembr 41:239–249, 2009; Kovaleva et al. Biochemistry (Moscow) 75:297–303, 2010) we have shown that mitochondria from Y. lipolytica and D. magnusii were very resistant to the Ca²⁺ overload combined with varying concentrations of P_i, palmitic acid, SH-reagents, carboxyatractyloside (an inhibitor of ADP/ATP translocator), as well as depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high [P_i]. Here we subjected yeast mitochondria to other conditions known to induce an mPTP in animal and plant mitochondria, namely to Ca²⁺ overload under hypoxic conditions (anaerobiosis). We were unable to observe Ca²⁺-induced high permeability of the inner membrane of D. magnusii and Y. lipolytica yeast mitochondria under anaerobic conditions, thus suggesting that an mPTP-like pore, if it ever occurs in yeast mitochondria, is not coupled with the Ca²⁺ uptake. The results provide the first demonstration of ATP-dependent energization of yeast mitochondria under conditions of anaerobiosis.     

Short-range spatial variability of soil δ15N natural abundance – effects on symbiotic N2-fixation estimates in pea

The δ¹⁵N natural abundance (‰) of the total soil N pool varies at the landscape level, but knowledge on short-range variability and consequences for the reliability of isotopic methods are poorly understood. The short-range spatial variability of soil δ¹⁵N natural abundance as revealed by the ¹⁵N abundance in spring barley and N₂-fixing pea was measured within the 0.15–4 m scale at flowering and at maturity. The short-range spatial variability of soil δ¹⁵N natural abundance and symbiotic nitrogen fixation were high at both growth stages. Along a 4-m row, the δ¹⁵N natural abundance in barley reference plants varied up to 3.9‰, and sometimes this variability was observed even between plants grown only 30 cm apart. The δ¹⁵N natural abundance in pea varied up to 1.4‰ within the 4-m row. The estimated percentage of nitrogen derived from the atmosphere (%Ndfa) varied from 73–89% at flowering and from 57–95% at maturity. When increasing the sampling area from 0.01 m² (single plants) and up to 0.6 m² (14 plants) the %Ndfa coefficient of variation (CV) declined from 5 to 2% at flowering and from 12 to 2% at maturity. The implications of the short-range variability in δ¹⁵N natural-abundance are that estimates of symbiotic N₂-fixation can be obtained from the natural abundance method if at least half a square meter of crop and reference plants is sampled for the isotopic analysis. In fields with small amounts of representative reference crops (weeds) it might be necessary to sow in reference crop species to secure satisfying N₂-fixation estimates.     

Periodic orbits near heteroclinic cycles in a cyclic replicator system

A species is semelparous if every individual reproduces only once in its life and dies immediately after the reproduction. While the reproduction opportunity is unique per year and the individual’s period from birth to reproduction is just n years, the individuals that reproduce in the ith year (modulo n) are called the ith year class, i = 1, 2, . . . , n. The dynamics of the n year-class system can be described by a differential equation system of Lotka–Volterra type. For the case n = 4, there is a heteroclinic cycle on the boundary as shown in previous works. In this paper, we focus on the case n = 4 and show the existence, growth and disappearance of periodic orbits near the heteroclinic cycle, which is a part of the conjecture by Diekmann and van Gils (SIAM J Appl Dyn Syst 8:1160–1189, 2009). By analyzing the Poincaré map near the heteroclinic cycle and introducing a metric to measure the size of the periodic orbit, we show that (i) when the average competitive degree among subpopulations (year classes) in the system is weak, there exists an asymptotically stable periodic orbit near the heteroclinic cycle which is repelling; (ii) the periodic orbit grows in size when some competitive degree increases, and converges to the heteroclinic cycle when the average competitive degree tends to be strong; (iii) when the average competitive degree is strong, there is no periodic orbit near the heteroclinic cycle which becomes asymptotically stable. Our results provide explanations why periodic solutions expand and disappear and why all but one subpopulation go extinct.     

Intensity of nitrification in soil as a function of time and soil moisture

Proceeding from three previously derived expressions for the intensity of nitrification in soil as a function of time (logΣN=K.logt+q), as a function of incubation moisture (logΣN=A.pF _i+B), as a function of initial moisture (logΣN=C.pF _v+D), it was shown that the nitrification intensity as a function of time and of moisture can be expressed by the bilinear function log ΣN=a.pF _i.logT+b.pF _i+c.logt+d; as a function of time and of initial moisture by the bilinear function logΣ=N=a.pF _v.logt+b.pF _v+c.logt+d; as a function of initial and incubation moisture by the bilinear function log ΣN=a.pF _ipF _v+b.pF _i+c.pF _v+d. The intensity of nitrification as a function of time, incubation moisture and initial moisture may be expressed by the multilinear function log ΣN=a.pF _i.pF _v.logt+b.pF _i.pF _v+c.pF _i.logt+d.pF _v.logt+e .pF _i+f.pF _v=g.logt+h. This function is valid for all the incubation moistures lying between pF _i 3.0 and 4.0 and for all initial moistures between 3.5 and 5.9 provided that the incubation temperature remains constant.     

Veratridine-Mediated Ca2+ Dynamics and Exocytosis in Paramecium Cells

We analyzed [Ca²⁺] _i transients in Paramecium cells in response to veratridine for which we had previously established an agonist effect for trichocyst exocytosis (Erxleben & Plattner, 1994. J. Cell Biol. 127:935–945; Plattner et al., 1994. J. Membrane Biol. 158:197–208). Wild-type cells (7S), nondischarge strain nd9–28°C and trichocyst-free strain ``trichless' (tl), respectively, displayed similar, though somewhat diverging time course and plateau values of [Ca<sup>2+</sup>] <sub>i</sub> transients with moderate [Ca<sup>2+</sup>] <sub>o</sub> in the culture/assay fluid (50 μm or 1 mm). In 7S cells which are representative for a normal reaction, at [Ca<sup>2+</sup>] <sub>o</sub> = 30 nm (c.f. [Ca<sup>2+</sup>] <sup>rest</sup> <sub>i</sub> =∼50 to 100 nm), veratridine produced only a small cortical [Ca<sup>2+</sup>] <sub>i</sub> transient. This increased in size and spatial distribution at [Ca<sup>2+</sup>] <sub>o</sub> = 50 μm of 1 mm. Interestingly with unusually high yet nontoxic [Ca<sup>2+</sup>] <sub>o</sub> = 10 mm, [Ca<sup>2+</sup>] <sub>i</sub> transients were much delayed and also reduced, as is trichocyst exocytosis. We interpret our results as follows. (i) With [Ca<sup>2+</sup>] <sub>o</sub> = 30 nm, the restricted residual response observed is due to Ca<sup>2+</sup> mobilization from subplasmalemmal stores. (ii) With moderate [Ca<sup>2+</sup>] <sub>o</sub> = 50 μm to 1 mm, the established membrane labilizing effect of veratridine may activate not only subplasmalemmal stores but also Ca<sup>2+</sup> <sub>o</sub> influx from the medium via so far unidentified (anteriorly enriched) channels. Visibility of these phenomena is best in tl cells, where free docking sites allow for rapid Ca<sup>2+</sup> spread, and least in 7S cells, whose perfectly assembled docking sites may ``consume' a large part of the [Ca²⁺] _i increase. (iii) With unusually high [Ca²⁺] _o , mobilization of cortical stores and/or Ca²⁺ _o influx may be impeded by the known membrane stabilizing effect of Ca²⁺ _o counteracting the labilizing/channel activating effect of veratridine. (iv) We show these effects to be reversible, and, hence, not to be toxic side-effects, as confirmed by retention of injected calcein. (v) Finally, Mn²⁺ entry during veratridine stimulation, documented by Fura-2 fluorescence quenching, may indicate activation of unspecific Me²⁺ channels by veratridine. Our data have some bearing on analysis of other cells, notably neurons, whose response to veratridine is of particular and continous interest. Received: 8 December 1998/Revised: 2 March 1999     

Within-branch heterogeneity of the light environment and leaf temperature in a Fagus crenata crown and its significance for photosynthesis calculations

On days with clear skies in late August 2002 diurnal changes in the within-branch heterogeneity of photosynthetic photon flux density at the leaf surface (PPFD_s) and leaf temperature (T _leaf) were measured at natural leaf orientations in the upper and lower layers of a Fagus crenata crown. The PPFD_s and T _leaf measurements were converted to branch photosynthesis rates (P _B; μmol s⁻¹) using a photosynthetic model proposed by Farquhar et al. (Planta 149:78–90, 1980), an empirical stomatal conductance model suggested by Leuning et al. (Plant Cell Environ 18:339–335, 1995), and the total leaf area of the branches. To evaluate the importance of the variation in PPFD_s and T _leaf on photosynthesis calculations, P _B calculated with the observed variation in PPFD_s and T _leaf was compared with estimates, based on the average (variation-free) values of PPFD_s and T _leaf, respectively. In both the layers, daily total P _B values obtained with T _leaf averaging were very close to those obtained with no averaging because of the weak inflection of the net photosynthesis rate (P _n) to T _leaf curves in the observed T _leaf ranges (24.4–36.5 and 21.9–29.1°C in the upper and lower layers, respectively) and relatively small variation in within-branch T _leaf at each time of day. This finding applied across potential climate conditions on fine days in August (T _leaf range of 19.4–41.5 and 16.9–34.1°C in the upper and lower layers, respectively) and when the spatial scale was increased from branch to leaf layer, which increased the maximum variation in within-branch T _leaf from 7.8 to 9.5°C and 4.5 to 5.5°C in the upper and lower layers, respectively. In contrast, averaging PPFD_s caused 25–50% and 41–90% overestimation of daily total P _B in the upper and lower layers, respectively, due to the sharp curvature in the PPFD_s response curve to P _n, and relatively large variation in within-branch PPFD_s. Further, it led to overestimation of midday depression of P _B in the upper layer, possibly because branch structural acclimation to incident light was neglected. Our results indicate that averaged values of T _leaf could be used for the estimation of carbon gain at layer scale throughout August, but spatial variations in PPFD_s need to be considered in detail for reliable estimates of carbon gain.     

Intracellular Mg<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Influences Both Open and Closed Times of a Native Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-activated BK Channel in Cultured Human Renal Proximal Tubule Cells

Effects of intracellular Mg²⁺ on a native Ca²⁺-and voltage-sensitive large-conductance K⁺ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca²⁺ ([Ca²⁺]_i) of 10⁻⁵–10⁻⁴ M, addition of 1–10 mM Mg²⁺ increased the open probability (P_o) of the channel, which shifted the P_o –membrane potential (V_m) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg²⁺-induced increase in P_o was suppressed at a relatively low [Ca²⁺]_i (10^−5.5–10⁻⁶ M). Dwell-time histograms have revealed that addition of Mg²⁺ mainly increased P_o by extending open times at 10⁻⁵ M Ca²⁺ and extending both open and closed times simultaneously at 10^−5.5 M Ca²⁺. Since our data showed that raising the [Ca²⁺]_i from 10⁻⁵ to 10⁻⁴ M increased P_o mainly by shortening the closed time, extension of the closed time at 10^−5.5 M Ca²⁺ would result from the Mg²⁺-inhibited Ca²⁺-dependent activation. At a constant V_m, adding Mg²⁺ enhanced the sigmoidicity of the P_o–[Ca²⁺]_i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg²⁺ on this channel is to elevate P_o by lengthening the open time, while extension of the closed time at a relatively low [Ca²⁺]_i results from a lowering of the sensitivity to Ca²⁺ of the channel by Mg²⁺, which causes the increase in the Hill coefficient. M. Kubokawa and Y. Sohma contributed equally to this work.     

A method for numbering binary rooted and unrooted phylogenies and their hypothetical taxonomic units

A set of 2n−2 relations (edges) and a set ofn−1 hypothetical taxonomic units (HTUs) derive from the estimation of a binary phylogeny of a set ofn operational taxonomic units (OTUs). We propose an easy way for numbering thesen−1 hypothetical taxonomic units, as well as for then−2 interior points of an unrooted binary phylogeny. We also present an alternative method to the one proposed by Rohlf (Bull. math. Biol. 45, 33–40, 1983) for numbering the π _i=1 ⁿ (2i−3) possible rooted binary phylogenies and the π _i=1 ⁿ⁻¹ (2i−3) possible unrooted binary phylogenies conerning a set ofn operational taxonomic units. An illustrative example of the method is presented. It is hoped that some studies in phylogenetics will become more accessible, from the viewpoint of computational economy, by the use of this method.     

Improved accuracy in measuring one-bond and two-bond <Superscript>15</Superscript>N,<Superscript>13</Superscript>C<Superscript>α</Superscript> coupling constants in proteins by double-inphase/antiphase (DIPAP) spectroscopy

An extension to HN(CO-α/β-N,C^α-J)-TROSY (Permi and Annila in J Biomol NMR 16:221–227, 2000) is proposed that permits the simultaneous determination of the four coupling constants ¹ J _{N′(i)Cα(i)}, ² J _HN(i)Cα(i), ² J _{Cα(i−1)N′(i)}, and ³ J _{Cα(i−1)HN(i)} in ¹⁵N,¹³C-labeled proteins. Contrasting the original scheme, in which two separate subspectra exhibit the ² J _CαN′ coupling as inphase and antiphase splitting (IPAP), we here record four subspectra that exhibit all combinations of inphase and antiphase splittings possible with respect to both ² J _CαN′ and ¹ J _N′Cα (DIPAP). Complementary sign patterns in the different spectrum constituents overdetermine the coupling constants which can thus be extracted at higher accuracy than is possible with the original experiment. Fully exploiting data redundance, simultaneous 2D lineshape fitting of the E.COSY multiplet tilts in all four subspectra provides all coupling constants at ultimate precision. Cross-correlation and differential-relaxation effects were taken into account in the evaluation procedure. By applying a four-point Fourier transform, the set of spectra is reversibly interconverted between DIPAP and spin-state representations. Methods are exemplified using proteins of various size.     

Accuracy of dominant markers for estimation of relatedness and heritability in an experimental stand of Prosopis alba (Leguminosae)

The estimation of genetic components of phenotypic variance is based on the resemblance between relatives. In natural populations of most forest tree species without genealogical information, a possible alternative approach is the use of relatedness estimates obtained indirectly from molecular marker data. Heritability (h ²) is then estimated from the covariance of estimated relatedness and phenotypic resemblance. In a stand of Prosopis alba planted in 1991 in Argentina, relatedness was estimated for all individual pairs of trees by means of the information proceeding from 128 dominant markers (57 AFLPs and 71 ISSRs) and compared with estimates obtained from six microsatellite loci previously studied. We empirically compared the accuracy of different relatedness estimators based on dominant markers proposed by Lynch and Milligan (Mol Ecol 3:91–99, 1994), Hardy (Mol Ecol 12:1577–1588, 2003), Wang (Mol Ecol 13:3169–3178, 2004), and Ritland (Mol Ecol 14:3157–3165, 2005). Heritabilities of 13 quantitative traits were then estimated from the regression of pairwise phenotypic distances on pairwise relatedness according to Ritland (Genet Res 67:175–185, 1996a). Relatedness inferred from molecular markers was in all cases significantly correlated with actual relatedness, although Ritland's estimator showed the highest bias but the lowest variance. Dominant marker-based h ² estimates were evidently downwards biased and showed poor correlation with those based on family records. In conclusion, the use of dominant molecular markers evidently produces much greater underestimates of h ² than from using co-dominant ones, attributable to the lower accuracy in the indirect estimation of relatedness coefficient. Many traits with enough genetic variability as to respond readily to selection would remain undetected; only those with very high heritability would show significant h ² estimates.     

A role for anions in ATP synthesis and its molecular mechanistic interpretation

ATP, the ‘universal biological energy currency’, is synthesized by utilizing energy either from oxidation of fuels or from light, via the process of oxidative and photo-phosphorylation respectively. The process is mediated by the enzyme F₁F₀-ATP synthase, using the free energy of ion gradients in the final energy catalyzing step, i.e., the synthesis of ATP from ADP and inorganic phosphate (P_i). The details of the molecular mechanism of ATP synthesis are among the most important fundamental issues in biology and hence need to be properly understood. In this work, a role for anions in making ATP has been found. New experimental data has been reported on the inhibition of ATP synthesis at nanomolar concentrations by the potent, specific anion channel blockers 4,4′-diisothiocyanostilbene-2, 2′-disulphonic acid (DIDS) and tributyltin chloride (TBTCl). Based on these inhibition studies, attention has been drawn to anion translocation (in addition to proton translocation) as a requirement for ATP synthesis. The type of inhibition has been quantified and an overall kinetic scheme for mixed inhibition that explains the data has been evolved. The experimental data and the type of inhibition found have been interpreted in the light of the torsional mechanism of energy transduction and ATP synthesis (Nath J Bioenerg Biomembr 42:293–300, 2010a; J Bioenerg Biomembr 42:301–309, 2010b). This detailed and unified mechanism resolves long-standing problems and inconsistencies in the first theories (Slater Nature 172:975–978, 1953; Williams J Theor Biol 1:1–17, 1961; Mitchell Nature 191:144–148, 1961; Mitchell Biol Rev 41:445–502, 1966), makes several novel predictions that are experimentally verifiable (Nath Biophys J 90:8–21, 2006a; Process Biochem 41:2218–2235, 2006b), and provides us with a new and fruitful paradigm in bioenergetics. The interpretation presented here provides intelligent answers to the unexplained existing results in the literature. It is shown that mechanistic interpretation of the experimental data requires substantial addition to available conceptual foundations such that present concepts, theories, and mechanisms must be revised.     

Seasonal changes in abundance and spatial distribution of the soil arthropods in a Japanese cedar (Cryptomeria japonica D.Don) plantation,with special reference to collembola and acarina

Seasonal changes in abundance and distribution pattern of soil micro-arthropods were studied in connection with a few environmental factors in a Japanese cedar (Cryptomeria japonica D. Don) plantation. The soil arthropods were sampled from three different depths at intervals of two months for two years. Of the collected animals (total 51000–155000 m⁻²), Collembola (20000–76000 m⁻²), oribatid mites (19000–55000 m⁻²) and carnivorous mites (6200–21000 m⁻²) were the numerically dominant animal groups. Low seasonal variations in abundance indicated their seasonal stability in population levels. The trends in seasonal fluctuation were similar among these groups and between the two years, showing bimodal pattern with little peaks in early summer and winter. The pattern of seasonal fluctuation in abundance of carnivorous mites (P _d) was significantly synchronized with that in the total abundance of Collembola and oribatid mites (P _τ). Thus, the number-ratios (P _d/P _τ) were fairly constant, ranging from 0.10 to 0.25. Seasonal changes in vertical distribution of the three animal groups showed a similar pattern for both years. The downward migrations were shown to be more affected by low temperatures in winter accompanied by snow coverage rather than by the desiccation of the surface soil in summer. All the three groups demonstrated as a whole slightly aggregated patterns of horizontal distribution throughout the two years. Temporal increases in the patchiness indices were observed from summer to autumn when the moisture content of the surface soil was low.     

<Emphasis Type="Italic">Paracoccus denitrificans</Emphasis> proton-translocating ATPase: Kinetics of oxidative phosphorylation

The initial rates of ATP synthesis catalyzed by tightly coupled Paracoccus denitrificans plasma membrane were measured. The reaction rate was hyperbolically dependent on the substrates, ADP and inorganic phosphate (P_i). Apparent K _m values for ADP and P_i were 7–11 and 60–120 μM, respectively, at saturating concentration of the second substrate (pH 8.0, saturating Mg²⁺). These values were dependent on coupling efficiency. The substrate binding in the ATP synthesis reaction proceeds randomly: K _m value for a given substrate was independent of the concentration of the other one. A decrease of electrochemical proton gradient by the addition of malonate (when succinate served as the respiratory substrate) or by a decrease of steady-state level of NADH (when NADH served as the respiratory substrate) resulted in a proportional decrease of the maximal rates and apparent K _m values for ADP and P_i (double substitution, ping-pong mechanism). The kinetic scheme for ATP synthesis was compared with that described previously for the proton-translocating ATP hydrolysis catalyzed by the same enzyme preparation (T. V. Zharova and A. D. Vinogradov (2006) Biochemistry, 45, 14552–14558).     

On the Inference of Large Phylogenies with Long Branches: How Long Is Too Long?

The accurate reconstruction of phylogenies from short molecular sequences is an important problem in computational biology. Recent work has highlighted deep connections between sequence-length requirements for high-probability phylogeny reconstruction and the related problem of the estimation of ancestral sequences. In Daskalakis et al. (in Probab. Theory Relat. Fields 2010), building on the work of Mossel (Trans. Am. Math. Soc. 356(6):2379–2404, 2004), a tight sequence-length requirement was obtained for the simple CFN model of substitution, that is, the case of a two-state symmetric rate matrix Q. In particular the required sequence length for high-probability reconstruction was shown to undergo a sharp transition (from O(log n) to poly(n), where n is the number of leaves) at the “critical” branch length g _ML(Q) (if it exists) of the ancestral reconstruction problem defined roughly as follows: below g _ML(Q) the sequence at the root can be accurately estimated from sequences at the leaves on deep trees, whereas above g _ML(Q) information decays exponentially quickly down the tree.