A Comparison between Conductive and Infrared Devices for Measuring Mean Skin Temperature at Rest,during Exercise in the Heat,and Recovery

PurposeSkin temperature assessment has historically been undertaken with conductive devices affixed to the skin. With the development of technology, infrared devices are increasingly utilised in the measurement of skin temperature. Therefore, our purpose was to evaluate the agreement between four skin temperature devices at rest, during exercise in the heat, and recovery.MethodsMean skin temperature (T-sk) was assessed in thirty healthy males during 30 min rest (24.0 ± 1.2°C, 56 ± 8%), 30 min cycle in the heat (38.0 ± 0.5°C, 41 ± 2%), and 45 min recovery (24.0 ± 1.3°C, 56 ± 9%). T-sk was assessed at four sites using two conductive devices (thermistors, iButtons) and two infrared devices (infrared thermometer, infrared camera).ResultsBland–Altman plots demonstrated mean bias ± limits of agreement between the thermistors and iButtons as follows (rest, exercise, recovery): -0.01 ± 0.04, 0.26 ± 0.85, -0.37 ± 0.98°C; thermistors and infrared thermometer: 0.34 ± 0.44, -0.44 ± 1.23, -1.04 ± 1.75°C; thermistors and infrared camera (rest, recovery): 0.83 ± 0.77, 1.88 ± 1.87°C. Pairwise comparisons of T-sk found significant differences (p < 0.05) between thermistors and both infrared devices during resting conditions, and significant differences between the thermistors and all other devices tested during exercise in the heat and recovery.ConclusionsThese results indicate poor agreement between conductive and infrared devices at rest, during exercise in the heat, and subsequent recovery. Infrared devices may not be suitable for monitoring T-sk in the presence of, or following, metabolic and environmental induced heat stress.     

Higher ventilatory responses during and after passive walking-like leg movement in older individuals

Background

Minute ventilation (V·E) during walking has been shown to be higher in older individuals than in young individuals, but the mechanisms underlying the higher ventilatory response is unclear. Central command and peripheral neural reflex are important neural control mechanisms underlying ventilatory response during exercise. Passive leg movement has been used to exclude the influence of central command due to the lack of voluntary activation of muscles. The aim of the present study was to compare the ventilatory response during and after passive walking-like leg movement (PWM) in young and older individuals.

Methods

Eight young subjects (20 ± 2 years) and seven older subjects (70 ± 1 years) participated in this study. Subjects spent 7 minutes in a quiet standing (QS) position. Thereafter, they performed 14-minute rhythmic PWM at 1 Hz and this was followed by 7 minutes of QS.

Results

V·E values during pre-PWM QS were calculated as 1-minute averages using data obtained between 5 and 6 minutes. V·E values at pre-PWM QS in the young and older groups were 8.4 ± 2.1 and 7.5 ± 1.2 l/minute, respectively. V·E values increased significantly at the first minute of PWM to 11.4 ± 2.2 and 10.4 ± 2.5 l/minute in the young and older groups, respectively (P <0.001). In the young group, V·E at the last minute of PWM (9.2 ± 2.0 l/minute) was not significantly different from that at pre-PWM QS due to a decline in V·E, whereas V·E at the last minute of PWM in the older group (9.4 ± 2.2 l/minute) was still significantly higher (P <0.01). On the other hand, V·E at the first minute of post-PWM QS (7.2 ± 1.8 l/minute) was significantly lower than that during pre-PWM QS in the young group (P <0.05) but not in the older group.

Conclusions

Ventilatory response during and after PWM is higher in older individuals than in young individuals. This may be associated with a mechanism(s) other than central command. Our findings may explain part of the higher V·E response while walking in older individuals.     

Effects of abscisic Acid treatment on the thermostability of the photosynthetic apparatus in barley chloroplasts

Thermostability of the photosynthetic apparatus of abscisic acid (ABA)-treated seedlings of barley (Hordeum vulgare) was studied by light-scattering and by fluorescence measurements of isolated chloroplasts. ABA treatment markedly decreased heat damage of the chloroplast ultrastructure; an exogenous ABA concentration of 10⁻⁵ molar was most effective. Heat-induced increase of the 77 kilodalton fluorescence ratio F₇₄₀/F₆₈₅ was also smaller at this ABA concentration. The heat-induced increase of the initial chlorophyll fluorescence level (F_o) was virtually eliminated in ABA-treated (10⁻⁵ molar) chloroplasts up to 45°C and slightly increased at 50°C, relative to control chloroplasts where F_o increased even at 35°C and reached its maximal value at 45°C. In control chloroplasts, F_o increased with a 5-minute pretreatment temperature, an effect observed as low as 35°C. F_o was maximal at 45°C. In contrast, chloroplasts treated with 10⁻⁵ molar ABA did not exhibit a heat-induced increase in F_o until 50°C.     

Erythrocruorin of Marphysa sanguinea. Isolation of some physical, physicochemical and other properties

1. The polychaete worm Marphysa sanguinea has a circulating erythrocruorin of mol.wt. about 2·4×10⁶ (S⁰_20,w 58·2s, D_20,w 2·06×10⁻⁷ cm.²/sec). This is the predominant form existing at pH 6–8 and (non-protein) I 0·10–0·21, and also at approx. pH 6·7 and I 0·15–3·00. 2. The pigment contains 2·24% of protohaem. 3. The 58s protein has an electrophoretic mobility of 8·08×10⁻⁵ cm.²/v/sec. at pH 8·12, I 0·21 and 0°. The isoelectric point of suspended particles is 4·63 at I 0·16 and 21·5°. 4. At very low ionic strength and pH 6·7 (unbuffered) the 58s pigment associates reversibly to 97s and 150s forms, which are probably dimer and tetramer species. 5. At pH 10·0 and I 0·025, it dissociates irreversibly to give a small amount of 2–4s non-haem-containing protein and much 9s haem-enriched protein. These and the 58s pigment may correspond to structures found in Levin's (1963) electron-microscope studies of other erythrocruorins. 6. Absorption spectra of the 58s oxygenated erythrocruorin and the deoxygenated and carbon monoxide derivatives have been obtained.     

CO2 Uptake and Fixation by a Thermoacidophilic Microbial Community Attached to Precipitated Sulfur in a Geothermal Spring

Carbon fixation at temperatures above 73°C, the upper limit for photosynthesis, is carried out by chemosynthetic thermophiles. Yellowstone National Park (YNP), Wyoming possesses many thermal features that, while too hot for photosynthesis, presumably support chemosynthetic-based carbon fixation. To our knowledge, in situ rates of chemosynthetic reactions at these high temperatures in YNP or other high-temperature terrestrial geothermal springs have not yet been reported. A microbial community attached to precipitated elemental sulfur (S^o floc) at the source of Dragon Spring (73°C, pH 3.1) in Norris Geyser Basin, YNP, exhibited a maximum rate of CO₂ uptake of 21.3 ± 11.9 μg of C 10⁷ cells⁻¹ h⁻¹. When extrapolated over the estimated total quantity of S^o floc at the spring''s source, the S^o floc-associated microbial community accounted for the uptake of 121 mg of C h⁻¹ at this site. On a per-cell basis, the rate was higher than that calculated for a photosynthetic mat microbial community dominated by Synechococcus spp. in alkaline springs at comparable temperatures. A portion of the carbon taken up as CO₂ by the S^o floc-associated biomass was recovered in the cellular nucleic acid pool, demonstrating that uptake was coupled to fixation. The most abundant sequences in a 16S rRNA clone library of the S^o floc-associated community were related to chemolithoautotrophic Hydrogenobaculum strains previously isolated from springs in the Norris Geyser Basin. These microorganisms likely contributed to the uptake and fixation of CO₂ in this geothermal habitat.The upper temperature limit for primary production via photosynthesis is ∼73°C (7, 8, 11). At this temperature, photosynthesis is restricted to cyanobacteria of the genus Synechococcus, which generally inhabit alkaline environments (11). In acidic environments (pH < 4.0), the upper temperature limit for photosynthetic-based primary production is ∼56°C. Under these conditions, phototrophic activity is restricted to the unicellular eukaryotic red algae Cyanidium, Galdieria, and Cyanidioschyzon, collectively referred to as “cyanidia” (6, 12, 31, 48). Primary production above this temperature in acidic environments occurs through chemoautotrophy, a metabolism restricted to prokaryotes.Yellowstone National Park (YNP), WY, possesses numerous high-temperature (73 to 93°C) geothermal environments that are thought to support communities of microorganisms through chemoautotrophic-based primary production. Evidence for chemosynthesis in these environments is based on the recovery of 16S rRNA gene sequences that are affiliated with cultivated representatives of the phyla Aquificae and Crenarchaeota, many of which are capable of CO₂ fixation via the oxidation of hydrogen (H₂) and/or sulfide (HS⁻) (15, 17, 21, 24, 26, 28, 41, 46). Surprisingly, CO₂ fixation has yet to be demonstrated in situ in YNP hot spring environments (acidic or alkaline) where temperatures exceed the limits of photosynthesis and where primary production is thought to be driven by chemoautotrophic metabolism (14, 15, 28, 29).Dragon Spring, an acid-sulfate-chloride (ASC) spring located in the Norris Geyser Basin of YNP, is a likely habitat for chemoautotrophic primary production. The pH of the water is ∼3.1, and the temperature of the water at the source fluctuates from 65 to 78°C, which is well above the upper temperature limit for photosynthesis under acidic conditions. Potential electron donors for chemolithoautotrophic growth in the source water include hydrogen (H₂) and sulfide (S²⁻) at concentrations of 13 nM and 65 μM, respectively (15). In addition, submerged substrata at the spring''s source are blanketed by precipitates of elemental sulfur (S°), hereafter referred to as S^o floc (23). Inventories of bacterial and archaeal 16S rRNA genes recovered from S^o floc collected from the source of Dragon Spring indicate the presence of Crenarchaeota and Aquificae (4, 15). The latter are related to chemolithoautotrophic Hydrogenobaculum spp., representatives of which have recently been isolated from the spring (15). In the present study, we demonstrate uptake and fixation of CO₂ at a temperature of 73°C by a Hydrogenobaculum-dominated microbial community associated with S^o floc collected from the source of Dragon Spring. This is the first direct evidence of CO₂ uptake in situ by a thermoacidophilic microbial community at a temperature that precludes photosynthesis in terrestrial geothermal springs.     

Transport of glucose and galactose in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25° was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S]_i) and extracellular ([S]_o) glucose concentrations was increased by 0·4mm-phlorrhizin and 0·3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S]_i/[S]_o fell below 1·0 only at [S]_o higher than 0·5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K_m 1·16mm; V_max. 4·5μmoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0·1mm-2,4-dinitrophenol, 0·4mm-phlorrhizin and by the absence of external Na⁺. 6. The kinetic parameters of galactose entry into the cells were: K_m 1·5mm; V_max 10μmoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0°, was inhibited by 0·4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na⁺-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na⁺-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

The Genome Sequence of Psychrobacter arcticus 273-4, a Psychroactive Siberian Permafrost Bacterium,Reveals Mechanisms for Adaptation to Low-Temperature Growth

Psychrobacter arcticus strain 273-4, which grows at temperatures as low as −10°C, is the first cold-adapted bacterium from a terrestrial environment whose genome was sequenced. Analysis of the 2.65-Mb genome suggested that some of the strategies employed by P. arcticus 273-4 for survival under cold and stress conditions are changes in membrane composition, synthesis of cold shock proteins, and the use of acetate as an energy source. Comparative genome analysis indicated that in a significant portion of the P. arcticus proteome there is reduced use of the acidic amino acids and proline and arginine, which is consistent with increased protein flexibility at low temperatures. Differential amino acid usage occurred in all gene categories, but it was more common in gene categories essential for cell growth and reproduction, suggesting that P. arcticus evolved to grow at low temperatures. Amino acid adaptations and the gene content likely evolved in response to the long-term freezing temperatures (−10°C to −12°C) of the Kolyma (Siberia) permafrost soil from which this strain was isolated. Intracellular water likely does not freeze at these in situ temperatures, which allows P. arcticus to live at subzero temperatures.Temperature is one of the most important parameters that determine the distribution and extent of life on earth, and it does this by affecting cell structure and function. High temperatures break covalent bonds and ionic interactions between molecules, inactivating proteins and disrupting cell structures. Low temperatures reduce biochemical reaction rates and substrate transport and induce the formation of ice that damages cell structures. Not surprisingly, an organism''s compatibility with the temperature of its habitat is ultimately determined by its underlying genetic architecture.The strong emphasis in research on mesophile biology (temperatures in the 20°C to 37°C range) has given us a misimpression of the importance of cold on earth. However, 70% of the Earth''s surface is covered by oceans with average temperatures between 1°C and 5°C (11), 20% of the Earth''s terrestrial surface is permafrost (47), and a larger portion of the surface undergoes seasonal freezing, making our planet a predominantly cold environment. Hence, cold adaptation in the microbial world should be expected (55).Permafrost is defined as soils or sediments that are continuously exposed to a temperature of 0°C or less for at least 2 years (44). Permafrost temperatures range from −10°C to −20°C in the Arctic and from −10°C to −65°C in the Antarctic, and permafrost has low water activity, often contains small amounts of carbon (0.85 to 1%), and is subjected to prolonged exposure to damaging gamma radiation from ⁴⁰K in soil minerals (49). Liquid water occurs as a very thin, salty layer surrounding the soil particles in the frozen layer. Despite the challenges of the permafrost, a variety of microorganisms successfully colonize this environment, and many microorganisms have been isolated from it (54, 70). The bacterial taxa most frequently isolated from the Kolyma permafrost of northeast Siberia include Arthrobacter, Exiguobacterium, Flavobacterium, Sphingomonas, and Psychrobacter (71). Rhode and Price (56) proposed that microorganisms can survive in frozen ice for very long periods due to the very thin film of water surrounding each cell that serves as a reserve of substrates. Permafrost is a more favorable environment than ice as a result of its heterogeneous soil particles and larger reservoirs of nutrients.The genus Psychrobacter comprises a group of Gram-negative, rod-shaped, heterotrophic bacteria, and many Psychrobacter species are capable of growth at low temperatures. Members of this genus can grow at temperatures between −10°C and 42°C, and they have frequently been isolated from various cold environments, including Antarctic sea ice, ornithogenic soil and sediments, the stomach contents of Antarctic krill (Euphausia), deep seawater, and permafrost (9, 36, 57, 70, 71, 76; http://www.bacterio.cict.fr/p/psychrobacter.html). Psychrobacter arcticus 273-4 is a recently described species (4) that was isolated from a 20,000- to 30,000-year-old continuously frozen permafrost horizon in the Kolyma region in Siberia that was not exposed to temperatures higher than 4°C during isolation (70). This strain, the type strain of the species, grows at temperatures ranging from −10°C to 28°C, has a generation time of 3.5 days at −2.5°C, exhibits excellent long-term survival under freezing conditions, and has temperature-dependent physiological modifications in membrane composition and carbon source utilization (50). The fact that Psychrobacter has been found to be an indicator genus for permafrost and other polar environments (66) suggests that many of its members are adapted to low temperatures and increased levels of osmotica and have evolved molecular-level changes that aid survival at low temperatures.Early studies on cold adaptation in microorganisms revealed physiological strategies to deal with low temperatures, such as changes in membrane saturation, accumulation of compatible solutes, and the presence of cold shock proteins (CSPs) and many other proteins with general functions (62). However, many of the studies were conducted with mesophilic microorganisms, which limits the generality of the conclusions. We addressed the question of cold adaptation by studying microorganisms isolated from subzero environments using physiologic and genomic methods. We chose P. arcticus as our model because of its growth at subzero temperatures and widespread prevalence in permafrost. This paper focuses on the more novel potential adaptations.     

Kinetics and Energetics of Biomolecular Folding and Binding

The ability of biomolecules to fold and to bind to other molecules is fundamental to virtually every living process. Advanced experimental techniques can now reveal how single biomolecules fold or bind against mechanical force, with the force serving as both the regulator and the probe of folding and binding transitions. Here, we present analytical expressions suitable for fitting the major experimental outputs from such experiments to enable their analysis and interpretation. The fit yields the key determinants of the folding and binding processes: the intrinsic on-rate and the location and height of the activation barrier.Dynamic processes in living cells are regulated through conformational changes in biomolecules—their folding into a particular shape or binding to selected partners. The ability of biomolecules to fold and to bind enables them to act as switches, assembly factors, pumps, or force- and displacement-generating motors (1). Folding and binding transitions are often hindered by a free energy barrier. Overcoming the barrier requires energy-demanding rearrangements such as displacing water from the sites of native contacts and breaking nonnative electrostatic contacts, as well as loss of configurational entropy. Once the barrier is crossed, the folded and bound states are stabilized by short-range interactions: hydrogen bonds, favorable hydrophobic effects, and electrostatic and van der Waals attractions (2).Mechanistic information about folding and binding processes is detailed in the folding and binding trajectories of individual molecules: observing an ensemble of molecules may obscure the inherent heterogeneity of these processes. Single-molecule trajectories can be induced, and monitored, by applying force to unfold/unbind a molecule and then relaxing the force until folding or binding is observed (3–5) (Fig. 1). Varying the force relaxation rate shifts the range of forces at which folding or binding occurs, thus broadening the explorable spectrum of molecular responses to force and revealing conformational changes that are otherwise too fast to detect. The measured force-dependent kinetics elucidates the role of force in physiological processes (6) and provides ways to control the timescales, and even the fate, of these processes. The force-dependent data also provides a route to understanding folding and binding in the absence of force—by extrapolating the data to zero force via a fit to a theory.Open in a separate windowFigure 1Schematic of the output from a force-relaxation experiment. The applied force is continuously relaxed from the initial value F₀ until the biomolecule folds or binds, as signified by a sharp increase in the measured force. From multiple repeats of this experiment, distributions of the folding or binding forces are collected (inset). Fitting the force distributions with the derived analytical expression yields the key parameters that determine the kinetics and energetics of folding or binding.In this letter, we derive an analytical expression for the distribution of transition forces, the major output of force-relaxation experiments that probe folding and binding processes. The expression extracts the key determinants of these processes: the on-rate and activation barrier in the absence of force. The theory is first developed in the context of biomolecular folding, and is then extended to cover the binding of a ligand tethered to a receptor. In contrast to unfolding and unbinding, the reverse processes of folding and binding require a theory that accounts for the compliance of the unfolded state, as well as the effect of the tether, to recover the true kinetic parameters of the biomolecule of interest.In a force-relaxation experiment, an unfolded biomolecule or unbound ligand-receptor complex is subject to a stretching force, which is decreased from the initial value F₀ as the pulling device approaches the sample at speed V until a folding or binding transition is observed (Fig. 1) (3–5). Define S(t) as the probability that the molecule has not yet escaped from the unfolded (implied: or unbound) state at time t. When escape is limited by one dominant barrier, S(t) follows the first-order rate equationS˙(t)≡dS(t)dt=−k←(F(t))S(t),where k_←(F(t)) is the on-rate at force F at time t. Because, prior to the transition, the applied force decreases monotonically with time, the distribution of transition forces, p(F), is related to S(t) through p(F)dF=S˙(t)dt, yieldingp(F)=−k←(F)F˙(F)e−∫F0Fk←(F′)F˙(F′)dF′.(1)Here F˙(F)≡dF(t)/dt<0 is the force relaxation rate. The proper normalization of p(F) is readily confirmed by integrating Eq. 1 from the initial force F₀ to negative infinity, the latter accounting for transitions that do not occur by the end of the experiment. Note that the expression for the distribution of folding/binding forces in Eq. 1 differs from its analog for the unfolding process (7) by the limits of integration and a negative sign, reflecting the property of a relaxation experiment to decrease the survival probability S(t) by decreasing the force. Converting the formal expression in Eq. 1 into a form suitable for fitting experimental data requires establishing functional forms for k_←(F) and F˙(F) and analytically solving the integral. These steps are accomplished below.The on-rate k_←(F) is computed by treating the conformational dynamics of the molecule as a random walk on the combined free energy profile G(x,t) = G₀(x) + G_pull(x,t) along the molecular extension x. Here G₀(x) is the intrinsic molecular potential and G_pull(x,t) is the potential of the pulling device. When G(x,t) features a high barrier on the scale of k_BT (k_B is the Boltzmann constant and T the temperature), the dynamics can be treated as diffusive. The unfolded region of the intrinsic potential for a folding process, unlike that for a barrierless process (8), can be captured by the functionG0(x)=ΔG‡ν1−ν(xx‡)11−ν−ΔG‡ν(xx‡),which has a sharp (if ν = 1/2, Fig. 2, inset) or smooth (if ν = 2/3) barrier of height ΔG^‡ and location x^‡. The potential of a pulling device of stiffness κ_S is G_pull(x,t) = κ_S/2(X₀ – Vt – x)² with an initial minimum at X₀ (corresponding to F₀). Applying Kramers formalism (9) to the combined potential G(x,t), we establish the analytical form of the on-rate at force F(t),k←(F)=k0(1+κSκU(F))1ν−12(1+νFx‡ΔG‡)1ν−1×eβΔG‡[1−(1+κSκU(F))2ν1−ν−1(1+νFx‡ΔG‡)1ν],where k₀ is the intrinsic on-rate, β ≡ (k_BT)⁻¹, andκU(F)=ν(1−ν)2ΔG‡x‡2(1+νFx‡ΔG‡)2−1νis the stiffness of the unfolded biomolecule under force F (see the Supporting Material for details on all derivations). The full nonlinear form of G_pull(x,t) was necessary in the derivation because, in contrast to the typically stiff folded state, the unfolded state may be soft (to be exact, 1/2κ_S x^‡2(F) << k_BT may not be satisfied) and thus easily deformed by the pulling device. Because of this deformation, the folding transition faces an extra contribution (regulated by the ratio κ_S/κ_U(F)) to the barrier height, typically negligible for unfolding, that decreases the on-rate in addition to the applied force F.Open in a separate windowFigure 2Contributions to the free energy profile for folding (inset) and binding (main figure). The derived expression (Eq. 2) extracts the on-rate and the location and height of the activation barrier to folding. When applied to binding data, the expression extracts the parameters of the ligand-tether-receptor (LTR) potential G˜0 (x); the proposed algorithm (Eqs. 3 and 4) removes the contribution of the tether potential G_teth(x) to recover the parameters of the intrinsic ligand-receptor (LR) potential G₀(x).The last piece required for Eq. 1, the loading rate F˙(F), is computed as the time derivative of the force F(t) on the unfolded molecule at its most probable extension at time t:F˙(F)=−κSV1+κS/κU(F).Finally, we realize that the integral in Eq. 1 can be solved analytically exactly, both for ν = 1/2 and ν = 2/3, resulting in the analytical expression for the distribution of folding forces:p(F)=k←(F)|F˙(F)|e−k←(F)β|F˙(F)|x‡(1+κSκU(F))νν−1(1+νFx‡ΔG‡)1−1ν.(2)Equation 2 can be readily applied to (normalized) histograms from force-relaxation experiments to extract the parameters of the intrinsic kinetics and energetics of folding. Being exact for ν = 1/2 and ν = 2/3, Eq. 2 is also an accurate approximation for any ν in the interval 1/2 < ν < 2/3 as long as κ_S ≲ κ_U (F) (see Fig. S1 in the Supporting Material). For simplicity, in Eq. 2 we have omitted the term containing F₀ as negligible if F₀ is large enough to prevent folding events.The solution in Eq. 2 reveals properties of the distribution of folding forces that distinguish it from its unfolding counterpart (7):

• 1.The distribution has a positive skew (Fig. 3), as intuitively expected: the rare folding events occur at high forces when the barrier is still high.Open in a separate windowFigure 3Force histograms from folding (left) and binding (right) simulations at several values of the force-relaxation speed (in nanometers per second, indicated at each histogram). Fitting the histograms with the analytical expression in Eq. 2 (lines) recovers the on-rate and activation barrier for folding or binding (2.Increasing the relaxation speed shifts the distribution to lower forces (Fig. 3): faster force relaxation leaves less time for thermal fluctuations to push the system over a high barrier, causing transitions to occur later (i.e., at lower forces), when the barrier is lower.
• 3.The stiffness κ_S and speed V enter Eq. 2 separately, providing independent routes to control the range of folding forces and thus enhance the robustness of a fit.

The application of the above framework to binding experiments on a ligand and receptor connected by a tether (3) involves an additional step—decoupling the effect of the tether—to reconstruct the parameters of ligand-receptor binding. Indeed, the parameters extracted from a fit of experimental histograms to Eq. 2 characterize the ligand-tether-receptor (LTR) potential (k˜0, x˜‡, ΔG˜‡, ν) (Fig. 2). The parameters of the natural ligand-receptor (LR) potential (k₀, x^‡, ΔG^‡) can be recovered using three characteristics of the tether: contour length L; persistence length p; and extension Δℓ of the tether along the direction of the force in the LTR transition state. The values of L and p can be determined from the force-extension curve of the tether (10); these define the tether potential G_teth(x) (Fig. 2). The value of Δℓ can be found from an unbinding experiment (7) on LTR and the geometry of the tether attachment points (see Fig. S3). Approximating the region of the LR potential between the transition and unbound states as harmonic, with no assumptions about the shape of the potential beyond x^‡, the ligand-receptor barrier parameters are thenx‡=α−1α−2x˜‡,ΔG‡=(α−1)22(α−2)x˜‡Fteth(Δℓ+x˜‡),(3)and the intrinsic unimolecular association rate isk0≈k˜0(βΔG‡)32(βΔG˜‡)1ν−12(x˜‡x‡)2eβ(ΔG˜‡−ΔG‡).(4)Here, the force value Fteth(Δℓ+x˜‡) is extracted from the force-extension curve of the tether at extension Δℓ+x˜‡ andα=2(ΔG˜‡−Gteth(Δℓ)+Gteth(Δℓ+x˜‡))x˜‡Fteth(Δℓ+x˜‡),where G_teth(x) is the wormlike-chain potential (see Eq. S13 in the Supporting Material). Equations 3–4 confirm that a tether decreases the height and width of the barrier (see Fig. 2), thus increasing the on-rate.In Fig. 3, the developed analytical framework is applied to folding and binding force histograms from Brownian dynamics simulations at parameters similar to those in the analogous experimental and computational studies (3,5,11) (for details on simulations and fitting procedure, see the Supporting Material). For the stringency of the test, the simulations account for the wormlike-chain nature of the molecular unfolded and LTR unbound states that is not explicitly accounted for in the theory. With optimized binning (12) of the histograms and a least-squares fit, Eqs. 2–4 recover the on-rate, the location and the height of the activation barrier, and the value of ν that best captures how the kinetics scale with force (

1.Multiple relaxation speeds,

2.Folding/binding events at low forces, and

3.A large number of events at each speed.

Table 1

On-rate and the location and height of the activation barrier from the fit of simulated data to the theory in Eq. 2

Saturation Mutagenesis of Toluene ortho-Monooxygenase of Burkholderia cepacia G4 for Enhanced 1-Naphthol Synthesis and Chloroform Degradation

Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase α-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (V_max of 9.3 versus 4.5 nmol·min⁻¹·mg of protein⁻¹ and unchanged K_m), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 ± 0.07 versus 1.30 ± 0.06 nmol·min⁻¹·mg of protein⁻¹ [mean ± standard deviation]) at 91 μM and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (V_max of 2.61 versus 0.95 nmol·min⁻¹·mg of protein⁻¹ and unchanged K_m) and chloride release (0.034 ± 0.002 versus 0.012 ± 0.001 nmol·min⁻¹·mg of protein⁻¹). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 ± 0.3 versus 1.30 ± 0.06 nmol·min⁻¹·mg of protein⁻¹). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.     

Phage Resistance in a Phage-Insensitive Strain of Streptococcus lactis: Temperature-Dependent Phage Development and Host-Controlled Phage Replication

Streptococcus lactis ME2 is a dairy starter strain that is insensitive to a variety of phage, including 18. The efficiency of plating of 18 on ME2 and N1 could be increased from <1 × 10⁻⁹ to 5.0 × 10⁻² and from 7.6 × 10⁻⁷ to 2.1 × 10⁻², respectively, when the host strains were subcultured at 40°C before plating the phage and the phage assay plates were incubated at 40°C. Host-dependent replication was demonstrated in N1 at 30°C and in N1 and ME2 at 40°C, suggesting the operation of a temperature-sensitive restriction and modification system in ME2 and N1. The increased sensitivity of ME2 and N1 to 18 at 40°C was also demonstrated by lysis of broth cultures and increased plaque size. ME2 grown at 40°C showed an increased ability to adsorb 18, indicating a second target for temperature-dependent phage sensitivity in ME2. Challenge of N1 with a 18 preparation that had been previously modified for growth on N1 indicated that at 40°C phage development was characterized by a shorter latent period and larger burst size than at 30°C. The evidence presented suggests that the high degree of phage insensitivity expressed by ME2 consists of a variety of temperature-sensitive mechanisms, including (i) the prevention of phage adsorption, (ii) host-controlled restriction of phage, and (iii) suppression of phage development. At 30°C these factors appear to act cooperatively to prevent the successful emergence of lytic phage active against S. lactis ME2.     

Demographic effects of a megafire on a declining prairie grouse in the mixed‐grass prairie

Recent studies have documented benefits of small, prescribed fire and wildfire for grassland‐dependent wildlife, such as lesser prairie‐chickens (Tympanuchus pallidicintus), but wildlife demographic response to the scale and intensity of megafire (wildfire >40,000 ha) in modern, fragmented grasslands remains unknown. Limited available grassland habitat makes it imperative to understand if increasing frequency of megafires could further reduce already declining lesser prairie‐chicken populations, or if historical evolutionary interactions with fire make lesser prairie‐chickens resilient. To evaluate lesser prairie‐chicken demographic response to megafires, we compared lek counts, nest density, and survival rates of adults, nests, and chicks before (2014–2016) and after (2018–2020) a 2017 megafire in the mixed‐grass prairie of Kansas, USA (Starbuck fire ~254,000 ha). There was a 67% decline in attending males on leks post‐fire and a 57% decline in occupied leks post‐fire. Despite population declines as indicated by lek counts, adult female breeding season survival (S^) was similar pre‐ (S^ = 0.65 ± 0.08 [SE]) and post‐fire (0.61 ± 0.08), as was chick survival (pre‐fire: 0.23 ± 0.07; post‐fire: 0.27 ± 0.11). Nest survival appeared lower post‐fire (pre‐fire: 0.38 ± 0.06; post‐fire: 0.20 ± 0.06), but did not differ at the 95% confidence interval. Nest density of marked females declined 73% in areas burned by megafire. Although lesser prairie‐chickens persisted in the study area and we documented minimal effects on most demographic rates, reduced lesser prairie‐chicken abundance and reproductive output suggests full recovery may take >3 years. Increased propensity for megafire resulting from suppression of smaller fires, compounded by climate change and woody encroachment, may impose a short‐term (3–5 year) threat to already declining lesser prairie‐chicken populations.     

Effect of training in minimalist footwear on oxygen consumption during walking and running

The present study sought to examine the effect of 5 weeks of training with minimalist footwear on oxygen consumption during walking and running. Thirteen college-aged students (male n = 7, female n = 6, age: 21.7±1.4 years, height: 168.9±8.8 cm, weight: 70.4±15.8 kg, VO₂max: 46.6±6.6 ml·kg⁻¹·min⁻¹) participated in the present investigation. The participants did not have experience with minimalist footwear. Participants underwent metabolic testing during walking (5.6 km·hr⁻¹), light running (7.2 km·hr⁻¹), and moderate running (9.6 km·hr⁻¹). The participants completed this assessment barefoot, in running shoes, and in minimalist footwear in a randomized order. The participants underwent 5 weeks of training with the minimalist footwear. Afterwards, participants repeated the metabolic testing. Data was analyzed via repeated measures ANOVA. The analysis revealed a significant (F_4,32= 7.576, ηp2=0.408, p ≤ 0.001) interaction effect (time × treatment × speed). During the initial assessment, the minimalist footwear condition resulted in greater oxygen consumption at 9.6 km·hr⁻¹ (p ≤ 0.05) compared to the barefoot condition, while the running shoe condition resulted in greater oxygen consumption than both the barefoot and minimalist condition at 7.2 and 9.6 km·hr⁻¹. At post-testing the minimalist footwear was not different at any speed compared to the barefoot condition (p> 0.12). This study suggests that initially minimalist footwear results in greater oxygen consumption than running barefoot, however; with utilization the oxygen consumption becomes similar.     

The Arabidopsis Protein CONSERVED ONLY IN THE GREEN LINEAGE160 Promotes the Assembly of the Membranous Part of the Chloroplast ATP Synthase

The chloroplast F₁F_o-ATP synthase/ATPase (cpATPase) couples ATP synthesis to the light-driven electrochemical proton gradient. The cpATPase is a multiprotein complex and consists of a membrane-spanning protein channel (comprising subunit types a, b, b′, and c) and a peripheral domain (subunits α, β, γ, δ, and ε). We report the characterization of the Arabidopsis (Arabidopsis thaliana) CONSERVED ONLY IN THE GREEN LINEAGE160 (AtCGL160) protein (AtCGL160), conserved in green algae and plants. AtCGL160 is an integral thylakoid protein, and its carboxyl-terminal portion is distantly related to prokaryotic ATP SYNTHASE PROTEIN1 (Atp1/UncI) proteins that are thought to function in ATP synthase assembly. Plants without AtCGL160 display an increase in xanthophyll cycle activity and energy-dependent nonphotochemical quenching. These photosynthetic perturbations can be attributed to a severe reduction in cpATPase levels that result in increased acidification of the thylakoid lumen. AtCGL160 is not an integral cpATPase component but is specifically required for the efficient incorporation of the c-subunit into the cpATPase. AtCGL160, as well as a chimeric protein containing the amino-terminal part of AtCGL160 and Synechocystis sp. PCC6803 Atp1, physically interact with the c-subunit. We conclude that AtCGL160 and Atp1 facilitate the assembly of the membranous part of the cpATPase in their hosts, but loss of their functions provokes a unique compensatory response in each organism.The majority of cellular energy is stored in the form of ATP synthesized by the ubiquitous F₁F_o-ATP synthase (F₁ stands for coupling factor 1, F_o for coupling factor o), which is found in the energy-transducing membranes of bacteria, mitochondria, and chloroplasts. The chloroplast F₁F_o-ATP synthase/ATPase (cpATPase) is a rotary motor that is responsible for coupling ATP synthesis (and hydrolysis) to the light-driven electrochemical proton gradient. The cpATPase comprises two physically separable parts, chloroplast coupling factor o (CF_o), which is an integral membrane-spanning proton channel, and chloroplast coupling factor 1 (CF₁), which is located peripheral to the membrane and contains the catalytic site(s) for reversible ATP synthesis (for review, see von Ballmoos et al., 2009). CF_o comprises four different subunit types, designated b (synonymously, I or AtpF), b′ (II or AtpG), c (III or AtpH), and a (IV or AtpI), and contains one each of subunits a, b, and b′ and a ring made up of 14 copies of subunit c. CF₁ comprises five different subunits, α (AtpA), β (AtpB), γ (AtpC), δ (AtpD), and ε (AtpE), and its subunit composition is α₃β₃γδε (for review, see von Ballmoos et al., 2009).The passage of protons through the CF_o motor drives rotation of the ring of c-subunits, which together form a rotor. The c-ring is connected to subunit γ, and rotation of γ causes conformational changes in the catalytic nucleotide-binding sites of the CF₁ motor, resulting in the synthesis and release of ATP (for review, see Okuno et al., 2011). This process is made possible by the fact that CF₁ and CF_o are physically connected by two stalks, a central one containing the ε- and γ-subunits and a peripheral one made up of δ, b, and b′ (for review, see Böttcher and Gräber, 2000; Weber, 2007). There are six nucleotide-binding sites in CF₁, one at each of the αβ-subunit interfaces about halfway along the vertical axis of the hexamer. Three of the sites are located primarily on the β-subunits and are catalytic; the other three are noncatalytic and probably regulatory. While the three-dimensional structure of the α₃β₃ hexamer in chloroplasts has been solved to a resolution of 3.2 Å (Groth and Pohl, 2001), the structure of the entire CF_o has not yet been determined. However, the conformation of the ring-forming part of CF_o from spinach (Spinacia oleracea) chloroplasts has been defined and found to consist of 14 c-units (Vollmar et al., 2009), whereas the c-ring of the ATP synthase from the cyanobacterium Spirulina platensis contains 15 units (Pogoryelov et al., 2009).Similar to other thylakoid multiprotein complexes like PSII and PSI as well as the cytochrome b₆f complex (Cyt b₆f), the assembly of the ATP synthase must be tightly regulated. Moreover, the variable stoichiometry of the constituents of F₁ (three α/β-subunits versus one each of γ, δ, and ε) and F_o (10–15 c-subunits versus one each of a, b, and b′) requires coordination of the expression of the corresponding genes. This is particularly important in eukaryotes, where the genes are located in different compartments, for instance, in the case of the cpATPase, in the plastid (for α, β, ε, a, b, and c) and the nucleus (for b′, γ, and δ).The assembly of ATP synthase has been most extensively studied in Saccharomyces cerevisiae mitochondria, leading to the identification of several factors involved in this process (for review, see Rak et al., 2009). Thus, three proteins in yeast are known to be involved in the assembly of the α₃β₃ hexamer of F₁. Atp11p (Ackerman and Tzagoloff, 1990a; Wang and Ackerman, 1996) and Atp12p (Ackerman and Tzagoloff, 1990a; Wang and Ackerman, 1998) code for mitochondrial proteins that interact with the β- and α-subunits, respectively, to promote their assembly into the oligomeric F₁-ATPase, and the absence of either protein causes the α- and β-subunits to aggregate into insoluble inclusion bodies in the mitochondrial matrix. Lack of the third protein, FORMATION OF MITOCHONDRIAL COMPLEXES1 (Fmc1p), is associated with aggregation of the α- and β-subunits under heat stress, suggesting that Fmc1p is required for correct folding of Atp12p at elevated temperatures (Lefebvre-Legendre et al., 2001). Originally, the c-ring was assumed to form spontaneously (Arechaga et al., 2002), but subsequent studies have indicated that the assembly of this structural component is also a protein-assisted process. Thus, Atp25p is required for both the synthesis of the c-subunit and its oligomerization into a ring structure of the proper size (Zeng et al., 2008). Moreover, Atp10p (Ackerman and Tzagoloff, 1990b), Atp23p (Osman et al., 2007), and OXIDASE ASSEMBLY1 (Oxa1p) (Jia et al., 2007) are involved in F_o assembly in yeast mitochondria.In prokaryotes, two ATP synthase assembly factors have been described in detail. The membrane protein insertase YidC belongs to the Oxa1 family, is required in vitro for the membrane insertion of subunit c, and assists in the formation of the c-ring from monomers (van der Laan et al., 2004; Kol et al., 2008). In bacterial genomes, the atp1/uncI genes typically precede the genes encoding the structural subunits of the F₁F_o-ATP synthase (for review, see Kol et al., 2008). Moreover, in Synechocystis sp. PCC6803, sll1321/atp1 is coordinately expressed with the seven other genes in the ATP synthase operon (Grossman et al., 2010), implying that Sll1321/Atp1 might have a function associated with the ATP synthase. The genes atp1 and uncI code for small proteins; for instance, Synechocystis sp. PCC6803 Sll1321 has 117 amino acids, and Escherichia coli UncI has 130 amino acids. The function of Atp1/UncI has long remained elusive because deletion of uncI in E. coli results merely in a slightly reduced growth yield (Gay, 1984), indicating that the protein is not essential for the formation of the F₁F_o-ATP synthase complex. Similarly, in the alkaliphilic Bacillus pseudofirmus OF4, Atp1/UncI is not absolutely required for ATP synthase function, and a B. pseudofirmus strain deleted for the atp1 gene could still grow nonfermentatively and its purified ATP synthase had a c-ring of normal size (Liu et al., 2013). Recently, a hybrid F₁F_o (F₁ from Bacillus PS3 and F_o from Propionigenium modestum) was expressed in E. coli. In this system, P. modestum Atp1/UncI was found to be indispensable for c-ring formation and coupled ATPase activity (Suzuki et al., 2007). Similarly, functional production of the Na⁺ F₁F_o-ATP synthase from Acetobacterium woodii in E. coli required the A. woodii atp1/uncI gene for proper assembly (Brandt et al., 2013). Moreover, because subunit c monomers, as well as assembled c-rings, can be copurified together with P. modestum UncI/Atp1 (Suzuki et al., 2007) and the oligomerization of P. modestum c-subunits into c₁₁-rings is mediated by Atp1/UncI in vitro (Ozaki et al., 2008), Atp1/UncI seems to play a role in c-ring assembly for some bacterial ATP synthases.In plants and green algae, regulation of the biogenesis of the cpATPase is well understood at the level of translation of CF₁ subunits (Drapier et al., 2007). Thus, synthesis of the nucleus-encoded subunit γ is required for sustained translation of the chloroplast-encoded subunit β, which in turn transactivates the translation of chloroplast-encoded subunit α. Translational down-regulation of subunit β or α, when not assembled, involves the 5′ untranslated regions (UTRs) of their own mRNAs, pointing to control at the level of translation initiation. In addition, a negative feedback exerted by α/β assembly intermediates on the translation of subunit β can be released when subunit γ assembles with α₃β₃ hexamers.Our knowledge of the nature of true assembly factors for the cpATPase is scarce. So far, only the ALBINO3 homolog Alb4 protein, which can functionally substitute for YidC in E. coli, has been shown to play a role in the biogenesis of the cpATPase, possibly by stabilizing or promoting the assembly of CF₁ during its attachment to the CF_o portion (Benz et al., 2009). Thus, Alb4-Oxa1p-YidC represents an ATP synthase assembly factor family that is conserved between prokaryotes, yeast, and plants. For the bacterial Atp1/UncI protein, one homolog exists in yeast, Vma21p, which is an integral membrane protein localized to the endoplasmic reticulum and is required for vacuolar H⁺-ATPase biogenesis (Graham et al., 1998).In this study, we have identified and characterized a knockout mutant for Arabidopsis (Arabidopsis thaliana) CGL160, a protein that displays moderate similarity to prokaryotic Atp1/UncI proteins in its C-terminal domain. AtCGL160 is required for the efficient assembly of the cpATPase, but lack of AtCGL160 in Arabidopsis has more severe effects on cpATPase assembly than those reported in the literature for inactivation of its prokaryotic relatives and can be located to the assembly of c-subunits into the membranous subcomplex. AtCGL160 physically interacts with the c-subunit of CF_o, and, interestingly, Atp1 can replace the C-terminal part of AtCGL160 in such interactions, indicating that the function of Atp1 and CGL160 proteins is conserved.     

Direct-Imaging-Based Quantification of Bacillus cereus ATCC 14579 Population Heterogeneity at a Low Incubation Temperature

Bacillus cereus ATCC 14579 was cultured in microcolonies on Anopore strips near its minimum growth temperature to directly image and quantify its population heterogeneity at an abusive refrigeration temperature. Eleven percent of the microcolonies failed to grow during low-temperature incubation, and this cold-induced population heterogeneity could be partly attributed to the loss of membrane integrity of individual cells.Bacillus cereus is a food poisoning- and food spoilage-causing organism that can be found in a large variety of foods (4, 23). There are two illnesses associated with B. cereus, namely, emetic and diarrheal intoxication (17, 24). Most of the strains related to cases or outbreaks of B. cereus food-borne poisoning were shown to be unable to grow at 7°C (1, 12). The average temperatures of domestic refrigerators have been investigated in various surveys around the world and often ranged from 5°C to 7°C, but extreme values exceeded 10°C to 12°C (5, 16). Inadequate chilling was indeed reported in various incidents of B. cereus food-borne illness (7, 8, 18, 19), pointing to the importance of appropriate refrigeration of foods contaminated with B. cereus to control its growth and toxin production in foods (9).Several studies have demonstrated that microorganisms can show diversity in their population stress response, even in an apparently homogeneous stress environment (6, 11, 21, 22). However, only very limited data describing the heterogeneity in growth performance of individual cells from food-borne pathogens cultured at low temperatures are available (10). Because inadequate chilling of food is one of the factors that contribute to the number of incidents of B. cereus food-borne illness, there is a need for better understanding of its growth performance at lowered incubation temperatures. In this study, we used the direct-imaging-based Anopore technology (6, 13-15) to quantitatively describe the population heterogeneity of B. cereus ATCC 14579 cells at 12°C. The minimum temperature for the growth of B. cereus ATCC 14579 in brain heart infusion (BHI) broth is 7.5°C (personal communication from F. Carlin), but various food-borne-associated B. cereus isolates were shown to be unable to grow at 10°C (1). Therefore, in this study, a culturing temperature of 12°C was chosen, to mimic temperature abuse of refrigerated foods. In addition, the membrane integrity of individual cells was assessed using both membrane permeant and impermeant nucleic acid dyes in order to get more insight into cellular characteristics that may contribute to heterogeneity in growth response.     

Whole-Body Insulin Sensitivity Rather than Body-Mass-Index Determines Fasting and Post-Glucose-Load Growth Hormone Concentrations

Background

Obese, non-acromegalic persons show lower growth hormone (GH) concentrations at fasting and reduced GH nadir during an oral glucose tolerance test (OGTT). However, this finding has never been studied with regard to whole-body insulin-sensitivity as a possible regulator.

Methods

In this retrospective analysis, non-acromegalic (NonACRO, n = 161) and acromegalic (ACRO, n = 35), non-diabetic subjects were subdivided into insulin-sensitive (IS) and –resistant (IR) groups according to the Clamp-like Index (CLIX)-threshold of 5 mg·kg⁻¹·min⁻¹ from the OGTT.

Results

Non-acromegalic IS (CLIX: 8.8±0.4 mg·kg⁻¹·min⁻¹) persons with similar age and sex distribution, but lower (p<0.001) body-mass-index (BMI = 25±0 kg/m², 84% females, 56±1 years) had 59% and 70%, respectively, higher (p<0.03) fasting GH and OGTT GH area under the curve concentrations than IR (CLIX: 3.5±0.1 mg·kg⁻¹·min⁻¹, p<0.001) subjects (BMI = 29±1 kg/m², 73% females, 58±1 years). When comparing on average overweight non-acromegalic IS and IR with similar anthropometry (IS: BMI: 27±0 kg/m², 82% females, 58±2 years; IR: BMI: 27±0 kg/m², 71% females, 60±1 years), but different CLIX (IS: 8.7±0.9 vs. IR: 3.8±0.1 mg·kg⁻¹·min⁻¹, p<0.001), the results remained almost the same. In addition, when adjusted for OGTT-mediated glucose rise, GH fall was less pronounced in IR. In contrast, in acromegalic subjects, no difference was found between IS and IR patients with regard to fasting and post-glucose-load GH concentrations.

Conclusions

Circulating GH concentrations at fasting and during the OGTT are lower in non-acromegalic insulin-resistant subjects. This study seems the first to demonstrate that insulin sensitivity rather than body-mass modulates fasting and post-glucose-load GH concentrations in non-diabetic non–acromegalic subjects.     

Polyphenol oxidase-mediated protection against oxidative stress is not associated with enhanced photosynthetic efficiency

Background and Aims Polyphenol oxidases (PPOs) catalyse the oxidation of monophenols and/or o-diphenols to highly reactive o-quinones, which in turn interact with oxygen and proteins to form reactive oxygen species (ROS) and typical brown-pigmented complexes. Hence PPOs can affect local levels of oxygen and ROS. Although the currently known substrates are located in the vacuole, the enzyme is targeted to the thylakoid lumen, suggesting a role for PPOs in photosynthesis. The current study was designed to investigate the potential involvement of PPOs in the photosynthetic response to oxidative stress.Methods Photosynthesis (A, F_v/F_m, ΦPSII, q_N, q_P, NPQ) was measured in leaves of a wild-type and a low-PPO mutant of red clover (Trifolium pratense ‘Milvus’) under control conditions and under a stress treatment designed to induce photooxidative stress: cold/high light (2 °C/580 µmol m²s^–1) or 0–10 µm methyl viologen. Foliar protein content and oxidation state were also determined.Key Results Photosynthetic performance, and chlorophyll and protein content during 4 d of cold/high light stress and 3 d of subsequent recovery under control growth conditions showed similar susceptibility to stress in both lines. However, more extensive oxidative damage to protein in mutants than wild-types was observed after treatment of attached leaves with methyl viologen. In addition, PPO activity could be associated with an increased capacity to dissipate excess energy, but only at relatively low methyl viologen doses.Conclusions The presence of PPO activity in leaves did not correspond to a direct role for the enzyme in the regulation or protection of photosynthesis under cold stress. However, an indication that PPO could be involved in cellular protection against low-level oxidative stress requires further investigation.     

DNA,Flexibly Flexible

Investigators have constructed dsDNA molecules with several different base modifications and have characterized their bending and twisting flexibilities using atomic force microscopy, DNA ring closure, and single-molecule force spectroscopy with optical tweezers. The three methods provide persistence length measurements that agree semiquantitatively, and they show that the persistence length is surprisingly similar for all of the modified DNAs. The circular dichroism spectra of modified DNAs differ substantially. Simple explanations based on base stacking strength, polymer charge, or groove occupancy by functional groups cannot explain the results, which will guide further high-resolution theory and experiments.Real double-stranded DNA molecules differ from the idealized zero-Kelvin A, B, and Z forms. They can adopt deformed average conformations, as in bent A-tract DNA or protein-DNA complexes. The path of the DNA helix axis also varies due to thermal energy, so at very long lengths DNA behaves as a random coil. The term “long lengths” is relative to the persistence length P of the wormlike chain model. P is the average offset of the end of a chain along its initial direction, or alternatively the length over which the unit vectors μ1→ and μ2→ tangent to the helix axis lose colinearity according to〈μ1→⋅μ2→〉=〈cosθ〉=e−d12/P,where d₁₂ is the contour length from point 1 to point 2, as in Fig. 1. P can be measured by hydrodynamics (1), atomic force microscopy (AFM) (2), DNA ring closure (3) or protein-DNA looping (4), tethered particle microscopy (5), or single-molecule optical tweezers experiments (6). The long-range loss of memory of DNA direction grows out of local variations in the helix axis direction specified by roll, tilt, and twist angles that parameterize changes in the helix axis direction. For harmonic bending potentials, the bending persistence length is related to roll and tilt according toσroll2+σtilt2=2ℓ/P,where ℓ = 3.4 Å, so for P ∼ 50 nm (147 bp) the average standard deviations in the roll and tilt angles σ_roll and σ_tilt are ∼4.7°, although in real DNA, roll varies more than tilt. Similar relationships hold for twist flexibility (7).Open in a separate windowFigure 1The base modifications studied by Peters et al. (13,14) affect both Watson-Crick hydrogen bonding and groove occupancy. They used AFM, DNA ring closure, single-molecule force spectroscopy, and circular dichroism spectroscopy (not shown) to characterize the resulting changes in bending and twisting flexibility. DNA molecules are not shown to scale. To see this figure in color, go online.DNA flexibility can be studied at contour length scales from Ångstroms to microns. Flexibility at the atomic scale accessed by nuclear magnetic resonance, x-ray crystallography, cryo-electron microscopy, and molecular dynamics simulations (8) refers to many aspects of conformational variability. One active thread of research at this scale concerns interconversion among helical forms, base flipping, DNA kinking, changes in backbone torsion angles, and the sequence dependence of all of these local properties. Local fluctuations in the basepair roll, tilt, and twist angles do seem to predict the correct long-range behavior (9). A second thread asks whether the wormlike chain model holds at DNA lengths shorter than P (2,10); the active controversy concerning enhanced bendability at short lengths has recently been reviewed by Vologodskii and Frank-Kamenetskii (11). A third thread asks whether we can understand the underlying biophysical causes of long-range DNA flexibility. These presumably include base stacking, electrostatic repulsion along the backbone, changes in the counterion atmosphere (12), occupancy of the major and minor grooves by functional groups, conformational entropy, the strength of Watson-Crick hydrogen bonding, and water structure. Helical polymorphisms and the junctions between polymorphs presumably affect the sequence dependence of the persistence length.Peters et al. (13,14) have attempted to understand bending and twisting flexibility by characterizing a variety of modified nucleic acids using DNA ring closure, AFM, and optical tweezer methods, sketched in Fig. 1. In previous work (13), they used ring closure to show that major groove substituents that alter the charge on the polymer do not have substantial effects on the bending persistence length, and that the effects were not correlated in an obvious way to the stacking propensity of the modified bases. The work described in this issue of the Biophysical Journal (14) uses all three methods to demonstrate that DNA with 2-amino-adenosine (a.k.a., 2,6-diaminopurine) substituted for adenosine has an increased persistence length, whereas inosine substitution for guanosine reduces the persistence length, as would be expected if groove occupancy (or the number of Watson-Crick hydrogen bonds) affects flexibility. However, the authors did one experiment too many—when they measured the effects of the earlier major groove substituents (13) using AFM, the correlation with groove occupancy disappeared. This could be because changes in helical geometry, as evidenced by the circular dichroism spectroscopy also reported in the article, alter the grooves sufficiently to prevent a straightforward connection to flexibility.The magnitude of the effect of base modifications on P is the largest for the optical tweezers and the smallest for DNA ring closure, showing that no more than one of the experiments is perfect. The Supporting Material for both articles (13,14) offers valuable resources for the careful evaluation of experimental results and possible sources of error within and between experiments. For example, the DNA lengths and the ionic conditions required by the different methods differ. Ring closure results depend critically on the purity of the DNA and appropriate ligation conditions. Analysis of AFM results averaged several different statistical measures of decaying angular correlations and end-to-end distance, which did not individually always agree. In force spectroscopy there are variations in the bead attachment for each molecule, errors in the stretch modulus can affect the measured persistence length, force can induce DNA melting, and very few molecules can be observed. Rare kinking events proposed to explain enhanced bendability should affect the cyclization experiment most markedly; no evidence for enhanced flexibility was seen. Finally, Peters et al. (14) have observed that DNA twist and twisting flexibility seem to be more sensitive than the persistence length to base modifications.Taken as a whole, this extremely thorough series of experiments shows that we still do not understand the fundamental origins of the remarkable stiffness of double-stranded DNA. There may be compensating effects that make the dissection difficult. For example, changing the charge on the polymer may induce a corresponding adjustment in the counterion condensation atmosphere, leading to a relatively constant residual charge. Groove substituents that enhance basepair stability could enhance bendability for steric reasons. Stacking thermodynamics may not change very much for the very small bend angles at any individual basepair. Locally stiff regions may introduce nearby junctions that are flexible.The stiffness of DNA relative to other biopolymers inspired the development of DNA nanotechnology (although that field has adopted bridged synthetic constructs that are even more rigid). Further research on the biophysics, and specifically the long-range mechanical properties of DNA, will be essential as we build better models of DNA in the cell, which has evolved many proteins that act to increase apparent flexibility. The various aspects of DNA flexibility influence the protein-DNA complexes that mediate DNA’s informational role, the induction of and responses to supercoiling used for long-range communication among sites (15), and chromosome structure and genome organization.