Simple Uniaxial and Uniform Biaxial Deformation of Nearly Isotropic Incompressible Tissues

A method is developed for analyzing in a unified manner both uniaxial and uniform biaxial strain data obtained from nearly isotropic tissues. The formulation is a direct application of nonlinear elasticity theory pertaining to large deformations. The general relation between Eulerian stress (σ) and extension ratio (λ) in soft isotropic elastic bodies undergoing uniform deformation takes the simple form: σ = ((λ³ - 1)/λ) f(λ), where f(λ) must be determined for each material. The extension ratio may be either greater than 1.0 (uniaxial elongation), or lie between zero and 1.0 (uniform biaxial extension). Simple analytical functions for f(λ) are most readily found for each tissue by plotting all data as (λ³ - 1)/λσ vs. λ. Of those tissues investigated in this way (dog pericardium and pleura, and cat mesentery and dura), all but pleura could be adequately described by a parabola: 1/f(λ) = 1/k{[(λ_M - λ)(λ - λ_m)]/[λ_M - λ_m}. In these instances, three material constants per tissue (K, λ_M, λ_m) served to predict approximately the stresses attained during both small and large deformations, in strips and sheets alike. It was further found that the uniaxial strain asymptote (λ_M) was linearly related to the biaxial strain asymptote (Λ_M), thus effectively reducing the number of constants by one.     

Purification, Characterization, and Crystallization of the Components of the Nitrobenzene and 2-Nitrotoluene Dioxygenase Enzyme Systems

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an α₃β₃ hexamer. The apparent K_m of 2-nitrotoluene dioxygenase for 2NT was 20 μM, and that for naphthalene was 121 μM. The specificity constants were 7.0 μM⁻¹ min⁻¹ for 2NT and 1.2 μM⁻¹ min⁻¹ for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.     

A study of lipid bilayer membrane stability using precise measurements of specific capacitance

A method is described for measuring the specific capacitance (C_m) of lipid bilayer membranes with an estimated experimental error of only 1%. The gross capacitance was measured with an AC Wheatstone bridge and a photographic technique was used to determine the area of thin membrane. The results of measurements on oxidized cholesterol-decane membranes formed in 1 × 10^-2 M KCl show that C_m depends upon temperature, voltage, time, and the age of the bulk membrane solutions. For a freshly thinned membrane (from 5 week old solution), C_m increases exponentially from an initial value of 0.432 ±0.021 (SD) μF/cm² with a time constant of ~15 min. A 100 mv potential applied across the membrane for 10-20 min prior to making measurements eliminated this time dependence and produced final-state membranes. C_m of final-state membranes depends upon applied voltage (V_a) and obeys the equation C_m = C₀ + βV_a² where V_a V_DC + V^rms_AC. C₀ and β depend upon temperature; C₀ decreases linearly with temperature while β increases linearly. At 20°C, C₀ = 0.559 ±0.01 (SD) μF/cm² and β = 0.0123 ±0.0036 (SD) (μF/cm²)/(mv²) and at 34°C, C₀ = 0.472 ±0.01 and β = 0.0382 ±0.0039. These variations in C_m are interpreted as resulting from thickness changes. The possibility that they result from diffuse layer and/or membrane dielectric phenomena is discussed and found to be unlikely. The results are discussed in terms of membrane stability by constructing hypothetical potential energy vs. thickness curves.     

Single-channel analysis of the anion channel-forming protein from the plant pathogenic bacterium Clavibacter michiganense ssp. nebraskense

The anion channel protein from Clavibacter michiganense ssp. nebraskense (Schürholz, Th. et al. 1991, J. Membrane Biol. 123: 1-8) was analyzed at different concentrations of KCl and KF. At 0.8 M KCl the conductance G(V_m) increases exponentially from 21 pS at 50 mV up to 53 pS at V_m = 200 mV, 20°C. The concentration dependence of G(V_m) corresponds to a Michaelis-Menten type saturation function at all membrane voltage values applied (0-200 mV). The anion concentration K_0.5, where G(V_m) has its half-maximum value, increases from 0.12 M at 50 mV to 0.24 M at 175 mV for channels in a soybean phospholipid bilayer. The voltage dependence of the single channel conductance, which is different for charged and neutral lipid bilayers, can be described either by a two-state flicker (2SF) model and the Nernst-Planck continuum theory, or by a two barrier, one-site (2B1S) model with asymmetric barriers. The increase in the number of open channels after a voltage jump from 50 mV to 150 mV has a time constant of 0.8 s. The changes of the single-channel conductance are much faster (<1 ms). The electric part of the gating process is characterized by the (reversible) molar electrical work ΔG^θ_el = ρZ_gFV_m ≈ -1.3 RT, which corresponds to the movement of one charge of the gating charge number |Z_g| = 1 across the fraction ρ = ΔV_m/V_m = 0.15 of the membrane voltage V_m = 200 mV. Unlike with chloride, the single channel conductance of fluoride has a maximum at about 150 mV in the presence of the buffer PIPES (≥5 mM, pH 6.8) with K_0.5 ≈ 1 M. It is shown that the decrease in conductance is due to a blocking of the channel by the PIPES anion. In summary, the results indicate that the anion transport by the Clavibacter anion channel (CAC) does not require a voltage dependent conformation change of the CAC.     

Simulation of Interstitial Fluid Flow in Ligaments: Comparison among Stokes,Brinkman and Darcy Models

In this paper, we use Stokes, Brinkman and Darcy equations to approximate the porous continuum media of ligament tissues respectively, simulate the flow field with FLUENT software, and study the shear stress on the cell surface due to the interstitial fluid flow. Since the Brinkman equation approaches Stokes equation well in high hydraulic permeability (k_p) condition (k_p ≥1.0×10^-8 m² in our numerical simulation), and it is an approximation to Darcy model in low k_p condition (k_p ≤5.0×10^-12 m² in our numerical simulation), we used the Brinkman model to simulate the interstitial fluid flow in the ligament where k_p is approximately 1.0×10^-16 m². It shows k_p and anisotropic property have a little effect on the flow field, but have a great effect on the shear stress on the membrane of interstitial cells (τ_cell). There is a linear relationship between τ_cell and , when k_p =1.0×10^-16 m² and the maximum τ_cell (τ_cell,max) is approximately 10 Pa. The anisotropic property will affect τ_cell''s distribution on the cell surface. When k_x/k_y>1, low τ_cell dominates the cell, while when k_x/k_y<1, high τ_cell dominants the cell.     

Estimating Finite Rate of Population Increase for Sharks Based on Vital Parameters

The vital parameter data for 62 stocks, covering 38 species, collected from the literature, including parameters of age, growth, and reproduction, were log-transformed and analyzed using multivariate analyses. Three groups were identified and empirical equations were developed for each to describe the relationships between the predicted finite rates of population increase (λ’) and the vital parameters, maximum age (T_max), age at maturity (T_m), annual fecundity (f/R_c)), size at birth (L_b), size at maturity (L_m), and asymptotic length (L_∞). Group (1) included species with slow growth rates (0.034 yr^-1 < k < 0.103 yr^-1) and extended longevity (26 yr < T_max < 81 yr), e.g., shortfin mako Isurus oxyrinchus, dusky shark Carcharhinus obscurus, etc.; Group (2) included species with fast growth rates (0.103 yr^-1 < k < 0.358 yr^-1) and short longevity (9 yr < T_max < 26 yr), e.g., starspotted smoothhound Mustelus manazo, gray smoothhound M. californicus, etc.; Group (3) included late maturing species (L_m/L_∞ ≧ 0.75) with moderate longevity (T_max < 29 yr), e.g., pelagic thresher Alopias pelagicus, sevengill shark Notorynchus cepedianus. The empirical equation for all data pooled was also developed. The λ’ values estimated by these empirical equations showed good agreement with those calculated using conventional demographic analysis. The predictability was further validated by an independent data set of three species. The empirical equations developed in this study not only reduce the uncertainties in estimation but also account for the difference in life history among groups. This method therefore provides an efficient and effective approach to the implementation of precautionary shark management measures.     

Controls of Evapotranspiration and CO2 Fluxes from Scots Pine by Surface Conductance and Abiotic Factors

Evapotranspiration (E) and CO₂ flux (F_c) in the growing season of an unusual dry year were measured continuously over a Scots pine forest in eastern Finland, by eddy covariance techniques. The aims were to gain an understanding of their biological and environmental control processes. As a result, there were obvious diurnal and seasonal changes in E, F_c, surface conductance (g_c), and decoupling coefficient (Ω), showing similar trends to those in radiation (PAR) and vapour pressure deficit (δ). The maximum mean daily values (24-h average) for E, F_c, g_c, and Ω were 1.78 mmol m⁻² s⁻¹, −11.18 µmol m⁻² s⁻¹, 6.27 mm s⁻¹, and 0.31, respectively, with seasonal averages of 0.71 mmol m⁻² s⁻¹, −4.61 µmol m⁻² s⁻¹, 3.3 mm s⁻¹, and 0.16. E and F_c were controlled by combined biological and environmental variables. There was curvilinear dependence of E on g_c and F_c on g_c. Among the environmental variables, PAR was the most important factor having a positive linear relationship to E and curvilinear relationship to F_c, while vapour pressure deficit was the most important environmental factor affecting g_c. Water use efficiency was slightly higher in the dry season, with mean monthly values ranging from 6.67 to 7.48 μmol CO₂ (mmol H₂O)⁻¹ and a seasonal average of 7.06 μmol CO₂ (μmol H₂O)⁻¹. Low Ω and its close positive relationship with g_c indicate that evapotranspiration was sensitive to surface conductance. Mid summer drought reduced surface conductance and decoupling coefficient, suggesting a more biotic control of evapotranspiration and a physiological acclimation to dry air. Surface conductance remained low and constant under dry condition, supporting that a constant value of surface constant can be used for modelling transpiration under drought condition.     

Effect of ENaC Modulators on Rat Neural Responses to NaCl

The effects of small molecule ENaC activators N,N,N-trimethyl-2-((4-methyl-2-((4-methyl-1H-indol-3-yl)thio)pentanoyl)oxy)ethanaminium iodide (Compound 1) and N-(2-hydroxyethyl)-4-methyl-2-((4-methyl-1H-indol-3-yl)thio)pentanamide (Compound 2), were tested on the benzamil (Bz)-sensitive NaCl chorda tympani (CT) taste nerve response under open-circuit conditions and under ±60 mV applied lingual voltage-clamp, and compared with the effects of known physiological activators (8-CPT-cAMP, BAPTA-AM, and alkaline pH), and an inhibitor (ionomycin+Ca²⁺) of ENaC. The NaCl CT response was enhanced at −60 mV and suppressed at +60 mV. In every case the CT response (r) versus voltage (V) curve was linear. All ENaC activators increased the open-circuit response (r_o) and the voltage sensitivity (κ, negative of the slope of the r versus V curve) and ionomycin+Ca²⁺ decreased r_o and κ to zero. Compound 1 and Compound 2 expressed a sigmoidal-saturating function of concentration (0.25–1 mM) with a half-maximal response concentration (k) of 0.49 and 1.05 mM, respectively. Following treatment with 1 mM Compound 1, 8-CPT-cAMP, BAPTA-AM and pH 10.3, the Bz-sensitive NaCl CT response to 100 mM NaCl was enhanced and was equivalent to the Bz-sensitive CT response to 300 mM NaCl. Plots of κ versus r_o in the absence and presence of the activators or the inhibitor were linear, suggesting that changes in the affinity of Na⁺ for ENaC under different conditions are fully compensated by changes in the apical membrane potential difference, and that the observed changes in the Bz-sensitive NaCl CT response arise exclusively from changes in the maximum CT response (r_m). The results further suggest that the agonists enhance and ionomycin+Ca²⁺ decreases ENaC function by increasing or decreasing the rate of release of Na⁺ from its ENaC binding site to the receptor cell cytosol, respectively. Irrespective of agonist type, the Bz-sensitive NaCl CT response demonstrated a maximum response enhancement limit of about 75% over control value.     

Microbial oxidation of amines. Distribution, purification and properties of two primary-amine oxidases from the yeast Candida boidinii grown on amines as sole nitrogen source

1. The yeast Candida boidinii was grown on glucose as carbon source with a range of amines and amino acids as nitrogen sources. Cells grown on amines contained elevated activities of catalase. If the amines contained N-methyl groups, formaldehyde dehydrogenase, formate dehydrogenase and S-formylglutathione hydrolase were also elevated in activity compared with cells grown on (NH₄)₂SO₄. 2. Cells grown on all the amines tested, but not those grown on urea or amino acids, contained an oxidase attacking primary amines, which is referred to as methylamine oxidase. In addition, cells grown on some amines contained a second amine oxidase, which is referred to as benzylamine oxidase. 3. Both amine oxidases were purified to near homogeneity. 4. Benzylamine oxidase was considerably more stable at 45 and 50°C than was methylamine oxidase. 5. Both enzymes had a pH optimum in the region of 7.0, and had a considerable number of substrates in common. There were, however, significant differences in the substrate specificity of the two enzymes. The ratio V/K^app._m increased with increasing n-alkyl carbon chain length for benzylamine oxidase, but decreased for methylamine oxidase. 6. Both enzymes showed similar sensitivity to carbonyl-group reagents, copper-chelating agents and other typical `diamine oxidase inhibitors'. 7. The stoicheiometry for the reaction catalysed by each enzyme was established. 8. The kinetics of methylamine oxidase were examined by varying the methylamine and oxygen concentrations in turn. A non-Ping Pong kinetic pattern with intersecting double-reciprocal plots was obtained, giving K_m values of 10μm for O₂ and 198μm for methylamine. The significance of this unusual kinetic behaviour is discussed. Similar experiments were not possible with the benzylamine oxidase, because it seemed to have an even lower K_m for O₂. 9. Both enzymes had similar subunit M_r values of about 80000, but the benzylamine oxidase behaved as if it were usually a dimer, M_r 136000, which under certain conditions aggregated to a tetramer, M_r 288000. Methylamine oxidase was mainly in the form of an octamer, M_r 510000, which gave rise quite readily to dimers of M_r 150000, and on gel filtration behaved as if the M_r was 286000.     

The Activity of σV,an Extracytoplasmic Function σ Factor of Bacillus subtilis,Is Controlled by Regulated Proteolysis of the Anti-σ Factor RsiV

During growth in the environment, bacteria encounter stresses which can delay or inhibit their growth. To defend against these stresses, bacteria induce both resistance and repair mechanisms. Many bacteria regulate these resistance mechanisms using a group of alternative σ factors called extracytoplasmic function (ECF) σ factors. ECF σ factors represent the largest and most diverse family of σ factors. Here, we demonstrate that the activation of a member of the ECF30 subfamily of ECF σ factors, σ^V in Bacillus subtilis, is controlled by the proteolytic destruction of the anti-σ factor RsiV. We will demonstrate that the degradation of RsiV and, thus, the activation of σ^V requires multiple proteolytic steps. Upon exposure to the inducer lysozyme, the extracellular domain of RsiV is removed by an unknown protease, which cleaves at site 1. This cleavage is independent of PrsW, the B. subtilis site 1 protease, which cleaves the anti-σ factor RsiW. Following cleavage by the unknown protease, the N-terminal portion of RsiV requires further processing, which requires the site 2 intramembrane protease RasP. Our data indicate that the N-terminal portion of RsiV from amino acid 1 to 60, which lacks the extracellular domain, is constitutively degraded unless RasP is absent, indicating that RasP cleavage is constitutive. This suggests that the regulatory step in RsiV degradation and, thus, σ^V activation are controlled at the level of the site 1 cleavage. Finally, we provide evidence that increased resistance to lysozyme decreases σ^V activation. Collectively, these data provide evidence that the mechanism for σ^V activation in B. subtilis is controlled by regulated intramembrane proteolysis (RIP) and requires the site 2 protease RasP.     

Multiple Propofol-binding Sites in a γ-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog

Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABA_ARs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABA_AR modulation. [³H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[³H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABA_AR transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([³H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1β3 GABA_ARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [³H]AziPm photoincorporation into GABA_AR subunits maximally by ∼50%. When the amino acids photolabeled by [³H]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the β3-α1 subunit interface (β3Met-286 in β3M3 and α1Met-236 in α1M1), previously photolabeled by [³H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [³H]AziPm photolabeling of β3Met-227 in βM1, the amino acid in the α1-β3 subunit interface photolabeled by R-[³H]mTFD-MPAB. The propofol-inhibitable [³H]AziPm photolabeling in the GABA_AR β3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-mTFD-MPAB also establish that each anesthetic binds to the homologous site at the β3-β3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the GABA_AR transmembrane domain that binds etomidate or R-mTFD-MPAB with high affinity.     

The Kinetics of Osmotic Transport through Pores of Molecular Dimensions

This paper presents a theoretical analysis of the kinetics of osmotic transport across a semipermeable membrane. There is a thermodynamic connection between the rate of flow under a hydrostatic pressure difference and the rate of flow due to a difference in solute concentration on the two sides. One might therefore attempt to calculate the osmotic transport coefficient by applying Poiseuille's equation to the flow produced by a difference in hydrostatic pressure. Such a procedure is, however, inappropriate if the pores in the membrane are too small to allow molecules to “overtake.” It then becomes necessary to perform a statistical calculation of the transport coefficient, and such a calculation is described in this paper. The resulting expression for the number of solvent molecules passing through a pore per second is J = m D₁ δn₁/l² where m is the number of solvent molecules in the pore, l is the length of the pore, D₁ is the self-diffusion coefficient of the solute, and δn₁ the difference in solvent mole fraction on the two sides of the membrane. This equation is used for estimating the number of pores per unit area of the squid axon membrane; the result is 6 × 10⁹ pores/cm².     

Effects of Limiting Extension at the αIIb Genu on Ligand Binding to Integrin αIIbβ3

Structural data of integrin αIIbβ3 have been interpreted as supporting a model in which: 1) the receptor exists primarily in a “bent,” low affinity conformation on unactivated platelets and 2) activation induces an extended, high affinity conformation prior to, or following, ligand binding. Previous studies found that “clasping” the αIIb head domain to the β3 tail decreased fibrinogen binding. To study the role of αIIb extension about the genu, we introduced a disulfide “clamp” between the αIIb thigh and calf-1 domains. Clamped αIIbβ3 had markedly reduced ability to bind the large soluble ligands fibrinogen and PAC-1 when activated with monoclonal antibody (mAb) PT25-2 but not when activated by Mn²⁺ or by coexpressing the clamped αIIb with a β3 subunit containing the activating mutation N339S. The clamp had little effect on the binding of the snake venom kistrin (M_r 7,500) or αIIbβ3-mediated adhesion to immobilized fibrinogen, but it did diminish the enhanced binding of mAb AP5 in the presence of kistrin. Collectively, our studies support a role for αIIb extension about the genu in the binding of ligands of 340,000 and 900,000 M_r with mAb-induced activation but indicate that it is not an absolute requirement. Our data are consistent with αIIb extension resulting in increased access to the ligand-binding site and/or facilitating the conformational change(s) in β3 that affect the intrinsic affinity of the binding pocket for ligand.     

Activation of αVβ3 on Vascular Cells Controls Recognition of Prothrombin

Regulation of vascular homeostasis depends upon collaboration between cells of the vessel wall and blood coagulation system. A direct interaction between integrin α_Vβ₃ on endothelial cells and smooth muscle cells and prothrombin, the pivotal proenzyme of the blood coagulation system, is demonstrated and activation of the integrin is required for receptor engagement. Evidence that prothrombin is a ligand for α_Vβ₃ on these cells include: (a) prothrombin binds to purified α_Vβ₃ via a RGD recognition specificity; (b) prothrombin supports α_Vβ₃-mediated adhesion of stimulated endothelial cells and smooth muscle cells; and (c) endothelial cells, either in suspension and in a monolayer, recognize soluble prothrombin via α_Vβ₃. α_Vβ₃-mediated cell adhesion to prothrombin, but not to fibrinogen, required activation of the receptor. Thus, the functionality of the α_Vβ₃ receptor is ligand defined, and prothrombin and fibrinogen represent activation- dependent and activation-independent ligands. Activation of α_Vβ₃ could be induced not only by model agonists, PMA and Mn²⁺, but also by a physiologically relevant agonist, ADP. Inhibition of protein kinase C and calpain prevented activation of α_Vβ₃ on vascular cells, suggesting that these molecules are involved in the inside-out signaling events that activate the integrin. The capacity of α_Vβ₃ to interact with prothrombin may play a significant role in the maintenance of hemostasis; and, at a general level, ligand selection by α_Vβ₃ may be controlled by the activation state of this integrin.     

Membrane Permeability: Generalization of the Reflection Coefficient Method of Describing Volume and Solute Flows

The reflection coefficient method for describing volume and solute fluxes through membranes is generalized to take into account the nonideality of the solutions bathing the membrane and/or multicomponent systems. The reflection coefficient of the impermeable species in these systems is less than unity by a coefficient γ. The reflection coefficient obtained solely from the volume flow equation, σ^v, will always be less than the reflection coefficient obtained from the solute flow equation, σ^8v. These two coefficients are related by σ^8v = σ^v + γ.     

Determination of Bacterial Cell Dry Mass by Transmission Electron Microscopy and Densitometric Image Analysis

We applied transmission electron microscopy and densitometric image analysis to measure the cell volume (V) and dry weight (DW) of single bacterial cells. The system was applied to measure the DW of Escherichia coli DSM 613 at different growth phases and of natural bacterial assemblages of two lakes, Piburger See and Gossenköllesee. We found a functional allometric relationship between DW (in femtograms) and V (in cubic micrometers) of bacteria (DW = 435 · V^0.86); i.e., smaller bacteria had a higher ratio of DW to V than larger cells. The measured DW of E. coli cells ranged from 83 to 1,172 fg, and V ranged from 0.1 to 3.5 μm³ (n = 678). Bacterial cells from Piburger See and Gossenköllesee (n = 465) had DWs from 3 fg (V = 0.003 μm³) to 1,177 fg (V = 3.5 μm³). Between 40 and 50% of the cells had a DW of less than 20 fg. By assuming that carbon comprises 50% of the DW, the ratio of carbon content to V of individual cells varied from 466 fg of C μm⁻³ for Vs of 0.001 to 0.01 μm³ to 397 fg of C μm⁻³ (0.01 to 0.1 μm³) and 288 fg of C μm⁻³ (0.1 to 1 μm³). Exponentially growing and stationary cells of E. coli DSM 613 showed conversion factors of 254 fg of C μm⁻³ (0.1 to 1 μm³) and 211 fg of C μm⁻³ (1 to 4 μm³), respectively. Our data suggest that bacterial biomass in aquatic environments is higher and more variable than previously assumed from volume-based measurements.     

The α-Subunit Regulates Stability of the Metal Ion at the Ligand-associated Metal Ion-binding Site in β3 Integrins

The aspartate in the prototypical integrin-binding motif Arg-Gly-Asp binds the integrin βA domain of the β-subunit through a divalent cation at the metal ion-dependent adhesion site (MIDAS). An auxiliary metal ion at a ligand-associated metal ion-binding site (LIMBS) stabilizes the metal ion at MIDAS. LIMBS contacts distinct residues in the α-subunits of the two β₃ integrins α_IIbβ₃ and α_Vβ₃, but a potential role of this interaction on stability of the metal ion at LIMBS in β₃ integrins has not been explored. Equilibrium molecular dynamics simulations of fully hydrated β₃ integrin ectodomains revealed strikingly different conformations of LIMBS in unliganded α_IIbβ₃ versus α_Vβ₃, the result of stronger interactions of LIMBS with α_V, which reduce stability of the LIMBS metal ion in α_Vβ₃. Replacing the α_IIb-LIMBS interface residue Phe¹⁹¹ in α_IIb (equivalent to Trp¹⁷⁹ in α_V) with Trp strengthened this interface and destabilized the metal ion at LIMBS in α_IIbβ₃; a Trp¹⁷⁹ to Phe mutation in α_V produced the opposite but weaker effect. Consistently, an F191/W substitution in cellular α_IIbβ₃ and a W179/F substitution in α_Vβ₃ reduced and increased, respectively, the apparent affinity of Mn²⁺ to the integrin. These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in α_Vβ₃ structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation.     

Kinetics of Formate Metabolism in Methanobacterium formicicum and Methanospirillum hungatei

The kinetics of formate metabolism in Methanobacterium formicicum and Methanospirillum hungatei were studied with log-phase formate-grown cultures. The progress of formate degradation was followed by the formyltetrahydrofolate synthetase assay for formate and fitted to the integrated form of the Michaelis-Menten equation. The K_m and V_max values for Methanobacterium formicicum were 0.58 mM formate and 0.037 mol of formate h⁻¹ g⁻¹ (dry weight), respectively. The lowest concentration of formate metabolized by Methanobacterium formicicum was 26 μM. The K_m and V_max values for Methanospirillum hungatei were 0.22 mM and 0.044 mol of formate h⁻¹ g⁻¹ (dry weight), respectively. The lowest concentration of formate metabolized by Methanospirillum hungatei was 15 μM. The apparent K_m for formate by formate dehydrogenase in cell-free extracts of Methanospirillum hungatei was 0.11 mM. The K_m for H₂ uptake by cultures of Methanobacterium formicicum was 6 μM dissolved H₂. Formate and H₂ were equivalent electron donors for methanogenesis when both substrates were above saturation; however, H₂ uptake was severely depressed when formate was above saturation and the dissolved H₂ was below 6 μM. Formate-grown cultures of Methanobacterium formicicum that were substrate limited for 57 h showed an immediate increase in growth and methanogenesis when formate was added to above saturation.     

Biochemical Characterization of Two Thermostable Xylanolytic Enzymes Encoded by a Gene Cluster of Caldicellulosiruptor owensensis

The xylanolytic extremely thermophilic bacterium Caldicellulosiruptor owensensis provides a promising platform for xylan utilization. In the present study, two novel xylanolytic enzymes, GH10 endo-β-1,4-xylanase (Coxyn A) and GH39 β-1,4-xylosidase (Coxyl A) encoded in one gene cluster of C.owensensis were heterogeneously expressed and biochemically characterized. The optimum temperature of the two xylanlytic enzymes was 75°C, and the respective optimum pH for Coxyn A and Coxyl A was 7.0 and 5.0. The difference of Coxyn A and Coxyl A in solution was existing as monomer and homodimer respectively, it was also observed in predicted secondary structure. Under optimum condition, the catalytic efficiency (k _cat/K _m) of Coxyn A was 366 mg ml⁻¹ s⁻¹ on beechwood xylan, and the catalytic efficiency (k _cat/K _m) of Coxyl A was 2253 mM⁻¹ s⁻¹ on pNP-β-D-xylopyranoside. Coxyn A degraded xylan to oligosaccharides, which were converted to monomer by Coxyl A. The two intracellular enzymes might be responsible for xylooligosaccharides utilization in C.owensensis, also provide a potential way for xylan degradation in vitro.     

4-Methylumbelliferyl-β-N-Acetylglucosaminide Hydrolysis by a High-Affinity Enzyme, a Putative Marker of Protozoan Bacterivory

Hydrolysis of an artificial fluorogenic substrate, 4-methylumbelliferyl-β-N-acetylglucosaminide, has been studied in a monoculture predator-prey system with either a flagellate (Bodo saltans) or a ciliate (Cyclidium sp.) fed upon pure bacterial culture (Aeromonas hydrophila or Alcaligenes xylosoxidans). Aeromonas hydrophila produced a low-affinity β-N-acetylglucosaminidase-like enzyme (K_m, 100 μmol liter^-1) but Alcaligenes xylosoxidans did not. Inoculation of both bacterial strains with bacterivorous protozoa induced the occurrence of another, high-affinity, β-N-acetylglucosaminidase-like enzyme (K_m, <0.5 μmol liter^-1). The latter enzyme showed significant, close correlations with total grazing rates of both B. saltans (r² = 0.96) and Cyclidium sp. (r² = 0.89) estimated by using uptake of fluorescently labelled bacteria. Further significant correlations between several protozoan parameters and kinetic parameters of this enzyme suggest its likely protozoan origin. If both types of enzyme occurred together, they could be satisfactorily distinguished by using kinetic data analysis. Hence, measurements of β-N-acetylglucosaminidase-like activities might be promising to use to improve estimations of protozoan bacterivory.