Application of leaf size and leafing intensity scaling across subtropical trees

Understanding the scaling between leaf size and leafing intensity (leaf number per stem size) is crucial for comprehending theories about the leaf costs and benefits in the leaf size–twig size spectrum. However, the scaling scope of leaf size versus leafing intensity changes along the twig leaf size variation in different leaf habit species remains elusive. Here, we hypothesize that the numerical value of scaling exponent for leaf mass versus leafing intensity in twig is governed by the minimum leaf mass versus maximum leaf mass (M _min versus M _max) and constrained to be ≤−1.0. We tested this hypothesis by analyzing the twigs of 123 species datasets compiled in the subtropical mountain forest. The standardized major axis regression (SMA) analyses showed the M _min scaled as the 1.19 power of M _max and the ‐α (−1.19) were not statistically different from the exponents of M _min versus leafing intensity in whole data. Across leaf habit groups, the M _max scaled negatively and isometrically with respect to leafing intensity. The pooled data''s scaling exponents ranged from −1.14 to −0.96 for M _min and M _max versus the leafing intensity based on stem volume (LIV). In the case of M _min and M _max versus the leafing intensity based on stem mass (LIM), the scaling exponents ranged from −1.24 to −1.04. Our hypothesis successfully predicts that the scaling relationship between leaf mass and leafing intensity is constrained to be ≤−1.0. More importantly, the lower limit to scaling of leaf mass and leafing intensity may be closely correlated with M _min versus M _max. Besides, constrained by the maximum leaf mass expansion, the broad scope range between leaf size and number may be insensitive to leaf habit groups in subtropical mountain forest.     

Isometric scaling of above- and below-ground biomass at the individual and community levels in the understorey of a sub-tropical forest

Background and Aims Empirical studies and allometric partitioning (AP) theory indicate that plant above-ground biomass (M_A) scales, on average, one-to-one (isometrically) with below-ground biomass (M_R) at the level of individual trees and at the level of entire forest communities. However, the ability of the AP theory to predict the biomass allocation patterns of understorey plants has not been established because most previous empirical tests have focused on canopy tree species or very large shrubs.Methods In order to test the AP theory further, 1586 understorey sub-tropical forest plants from 30 sites in south-east China were harvested and examined. The numerical values of the scaling exponents and normalization constants (i.e. slopes and y-intercepts, respectively) of log–log linear M_A vs. M_R relationships were determined for all individual plants, for each site, across the entire data set, and for data sorted into a total of 19 sub-sets of forest types and successional stages. Similar comparisons of M_A/M_R were also made.Key Results The data revealed that the mean M_A/M_R of understorey plants was 2·44 and 1·57 across all 1586 plants and for all communities, respectively, and M_A scaled nearly isometrically with respect to M_R, with scaling exponents of 1·01 for all individual plants and 0·99 for all communities. The scaling exponents did not differ significantly among different forest types or successional stages, but the normalization constants did, and were positively correlated with M_A/M_R and negatively correlated with scaling exponents across all 1586 plants.Conclusions The results support the AP theory’s prediction that M_A scales nearly one-to-one with M_R (i.e. M_A ∝ M_R ^≈1·0) and that plant biomass partitioning for individual plants and at the community level share a strikingly similar pattern, at least for the understorey plants examined in this study. Furthermore, variation in environmental conditions appears to affect the numerical values of normalization constants, but not the scaling exponents of the M_A vs. M_R relationship. This feature of the results suggests that plant size is the primary driver of the M_A vs. M_R biomass allocation pattern for understorey plants in sub-tropical forests.     

Effects of Air Pollutants on the Composition of Stable Carbon Isotopes, deltaC, of Leaves and Wood, and on Leaf Injury

Air pollutants are known to cause visible leaf injury as well as impairment of photosynthetic CO₂ fixation. Here we evaluate whether the effects on photosynthesis are large enough to cause changes in the relative composition of stable carbon isotopes, δ¹³C, of plant tissue samples, and, if so, how the changes relate to visual leaf injury. For that purpose, several woody and herbaceous plant species were exposed to SO₂ + O₃ and SO₂ + O₃ + NO₂ for one month (8 hours per day, 5 days per week). At the end of the fumigations, the plants were evaluated for visual leaf lesions, and δ¹³C of leaf tissue was determined. Woody plants generally showed less visual leaf injury and smaller effects on δ¹³C of pollutant exposure than did herbaceous plants. If δ¹³C was affected by pollutants, it became, with few exceptions, less negative. The data from the fumigation experiments were consistent with δ¹³C analyses of whole wood of annual growth rings from two conifer tree species, Pseudotsuga menziesii and Pinus strobus. These trees had been exposed until 1977 to exhaust gases from a gas plant at Lacq, France. Wood of both conifer species formed in the polluted air of 1972 to 1976 had less negative δ¹³C values than had wood formed in the much cleaner air in 1982 to 1986. No similar, time-dependent differences in δ¹³C of wood were observed in trees which had been continuously growing in clean air. Our δ¹³C data from both relatively short-term artificial exposures and long-term natural exposure are consistent with greater stomatal limitation of photosynthesis in polluted air than in clean air.     

Differentiation of Human T Cells Alters Their Repertoire of G Protein α-Subunits

Because T cell differentiation leads to an expanded repertoire of chemokine receptors, a subgroup of G protein-coupled receptors, we hypothesized that the repertoire of G proteins might be altered in parallel. We analyzed the abundance of mRNA and/or protein of six G protein α-subunits in human CD4⁺ and CD8⁺ T cell subsets from blood. Although most G protein α-subunits were similarly expressed in all subsets, the abundance of Gα_o, a protein not previously described in hematopoietic cells, was much higher in memory versus naive cells. Consistent with these data, activation of naive CD4⁺ T cells in vitro significantly increased the abundance of Gα_o in cells stimulated under nonpolarizing or T_H17 (but not T_H1 or T_H2)-polarizing conditions. In functional studies, the use of a chimeric G protein α-subunit, Gα_qo5, demonstrated that chemokine receptors could couple to Gα_o-containing G proteins. We also found that Gα_i1, another α-subunit not described previously in leukocytes, was expressed in naive T cells but virtually absent from memory subsets. Corresponding to their patterns of expression, siRNA-mediated knockdown of Gα_o in memory (but not naive) and Gα_i1 in naive (but not memory) CD4⁺ T cells inhibited chemokine-dependent migration. Moreover, although even in Gα_o- and Gα_i1-expressing cells mRNAs of these α-subunits were much less abundant than Gα_i2 or Gα_i3, knockdown of any of these subunits impaired chemokine receptor-mediated migration similarly. Together, our data reveal a change in the repertoire of Gα_i/o subunits during T cell differentiation and suggest functional equivalence among Gα_i/o subunits irrespective of their relative abundance.     

Subunit Composition Determines the Single Channel Kinetics of the Epithelial Sodium Channel

We have further characterized at the single channel level the properties of epithelial sodium channels formed by coexpression of α with either wild-type β or γ subunits and α with carboxy-terminal truncated β (β_T) or γ (γ_T) subunits in Xenopus laevis oocytes. αβ and αβ_T channels (9.6 and 8.7 pS, respectively, with 150 mM Li⁺) were found to be constitutively open. Only upon inclusion of 1 μM amiloride in the pipette solution could channel activity be resolved; both channel types had short open and closed times. Mean channel open probability (P _o) for αβ was 0.54 and for αβ_T was 0.50. In comparison, αγ and αγ_T channels exhibited different kinetics: αγ channels (6.7 pS in Li⁺) had either long open times with short closings, resulting in a high P _o (0.78), or short openings with long closed times, resulting in a low P _o (0.16). The mean P _o for all αγ channels was 0.48. αγ_T (6.6 pS in Li⁺) behaved as a single population of channels with distinct kinetics: mean open time of 1.2 s and closed time of 0.4 s, with a mean P _o of 0.6, similar to that of αγ. Inclusion of 0.1 μM amiloride in the pipette solution reduced the mean open time of αγ_T to 151 ms without significantly altering the closed time. We also examined the kinetics of amiloride block of αβ, αβ_T (1 μM amiloride), and αγ_T (0.1 μM amiloride) channels. αβ and αβ_T had similar blocking and unblocking rate constants, whereas the unblocking rate constant for αγ_T was 10-fold slower than αβ_T. Our results indicate that subunit composition of ENaC is a main determinant of P _o. In addition, channel kinetics and P _o are not altered by carboxy-terminal deletion in the β subunit, whereas a similar deletion in the γ subunit affects channel kinetics but not P _o.     

Direct measurement of turgor and osmotic potential in individual epidermal cells : independent confirmation of leaf water potential as determined by in situ psychrometry

The pressure probe, which is routinely used to measure the turgor potential (Ψ_p) of individual epidermal cells in Tradescantia virginiana (L.), has also been used to sample small volumes of vacuolar fluid from these same cells (as low as 0.02 nl) for measurement of cellular solute (osmotic) potential (Ψ_s) in a micro freezing point osmometer. The water potential components Ψ_p and Ψ_o have been used to calculate the total water potential of individual epidermal cells (Ψ_cell) which has then been directly compared to the total leaf water potential (Ψ_leaf) measured psychrometrically. The relation of Ψ_leaf and Ψ_cell to leaf transpiration indicates that in T. virginiana, a relatively straightforward relation exists between the level of water flow through the leaf tissue, and the ΔΨ within the leaf, between two points along the water flow pathway. Substantial agreement was found between the two independent, in situ methods of measuring Ψ when extrapolated to zero transpiration conditions. These results are discussed with respect to the thermodynamics of water transport in plant tissues.     

The scaling of ground reaction forces and duty factor in monitor lizards: implications for locomotion in sprawling tetrapods

Geometric scaling predicts a major challenge for legged, terrestrial locomotion. Locomotor support requirements scale identically with body mass (α M¹), while force-generation capacity should scale α M^2/3 as it depends on muscle cross-sectional area. Mammals compensate with more upright limb postures at larger sizes, but it remains unknown how sprawling tetrapods deal with this challenge. Varanid lizards are an ideal group to address this question because they cover an enormous body size range while maintaining a similar bent-limb posture and body proportions. This study reports the scaling of ground reaction forces and duty factor for varanid lizards ranging from 7 g to 37 kg. Impulses (force×time) (α M^0.99−1.34) and peak forces (α M^0.73−1.00) scaled higher than expected. Duty factor scaled α M^0.04 and was higher for the hindlimb than the forelimb. The proportion of vertical impulse to total impulse increased with body size, and impulses decreased while peak forces increased with speed.     

Carbon Isotope Composition of Nighttime Leaf-Respired CO2 in the Agricultural-Pastoral Zone of the Songnen Plain,Northeast China

Variations in the carbon isotope signature of leaf dark-respired CO₂ (δ¹³C_R) within a single night is a widely observed phenomenon. However, it is unclear whether there are plant functional type differences with regard to the amplitude of the nighttime variation in δ¹³C_R. These differences, if present, would be important for interpreting the short-term variations in the stable carbon signature of ecosystem respiration and the partitioning of carbon fluxes. To assess the plant functional type differences relating to the magnitude of the nighttime variation in δ¹³C_R and the respiratory apparent fractionation, we measured the δ¹³C_R, the leaf gas exchange, and the δ¹³C of the respiratory substrates of 22 species present in the agricultural-pastoral zone of the Songnen Plain, northeast China. The species studied were grouped into C₃ and C₄ plants, trees, grasses, and herbs. A significant nocturnal shift in δ¹³C_R was detected in 20 of the studied species, with the magnitude of the shift ranging from 1‰ to 5.8‰. The magnitude of the nighttime variation in δ¹³C_R was strongly correlated with the daytime cumulative carbon assimilation, which suggests that variation in δ¹³C_R were influenced, to some extent, by changes in the contribution of malate decarboxylation to total respiratory CO₂ flux. There were no differences in the magnitude of the nighttime variation in δ¹³C_R between the C₃ and C₄ plants, as well as among the woody plants, herbs and graminoids. Leaf respired CO₂ was enriched in ¹³C compared to biomass, soluble carbohydrates and lipids; however the magnitude of enrichment differed between 8 pm and 4 am, which were mainly caused by the changes in δ¹³C_R. We also detected the plant functional type differences in respiratory apparent fractionation relative to biomass at 4 am, which suggests that caution should be exercised when using the δ¹³C of bulk leaf material as a proxy for the δ¹³C of leaf-respired CO₂.     

Relationships between C3 Plant Foliar Carbon Isotope Composition and Element Contents of Grassland Species at High Altitudes on the Qinghai-Tibet Plateau,China

Relationships of foliar carbon isotope composition (δ¹³C) with foliar C, N, P, K, Ca, Mg contents and their ratios of 219 C₃ species leaf samples, obtained in August in 2004 to 2007 from 82 high altitude grassland sites on the Qinghai-Tibet Plateau China, were examined. This was done with reference to the proposition that foliar δ¹³C increases with altitude and separately for the life-form groups of graminoids, forbs and shrubs and for the genera Stipa and Kobresia. For all samples, foliar δ¹³C was negatively related to foliar K, P and ∑_{K+ Ca+ Mg}, and positively correlated to foliar C, C/N and C/P. The significance of these correlations differed for the taxonomic and life-form groups. Lack of a relationship of foliar δ¹³C with foliar N was inconsistent with the majority of studies that have shown foliar δ¹³C to be positively related to foliar N due to a decrease of C_i/C_a (the ratio between intercellular and atmospheric concentration of CO₂) and explained as a result of greater photosynthetic capacity at higher foliar N concentration. However this inconsistency relates to other high altitude studies that have found that photosynthetic capacity remains constant as foliar N increases. After accounting for the altitudinal relationship with foliar δ¹³C, of the elements only the K effect was significant and was most strongly expressed for Kobresia. It is concluded that factors critical to plant survival and growth at very high altitudes, such as low atmospheric pressure and low temperatures, may preclude expression of relationships between foliar δ¹³C and foliar elements that have been observed at lower altitudes.     

Novel β-1,4-Mannanase Belonging to a New Glycoside Hydrolase Family in Aspergillus nidulans

Many filamentous fungi produce β-mannan-degrading β-1,4-mannanases that belong to the glycoside hydrolase 5 (GH5) and GH26 families. Here we identified a novel β-1,4-mannanase (Man134A) that belongs to a new glycoside hydrolase (GH) family (GH134) in Aspergillus nidulans. Blast analysis of the amino acid sequence using the NCBI protein database revealed that this enzyme had no similarity to any sequences and no putative conserved domains. Protein homologs of the enzyme were distributed to limited fungal and bacterial species. Man134A released mannobiose (M₂), mannotriose (M₃), and mannotetraose (M₄) but not mannopentaose (M₅) or higher manno-oligosaccharides when galactose-free β-mannan was the substrate from the initial stage of the reaction, suggesting that Man134A preferentially reacts with β-mannan via a unique catalytic mode. Man134A had high catalytic efficiency (k_cat/K_m) toward mannohexaose (M₆) compared with the endo-β-1,4-mannanase Man5C and notably converted M₆ to M₂, M₃, and M₄, with M₃ being the predominant reaction product. The action of Man5C toward β-mannans was synergistic. The growth phenotype of a Man134A disruptant was poor when β-mannans were the sole carbon source, indicating that Man134A is involved in β-mannan degradation in vivo. These findings indicate a hitherto undiscovered mechanism of β-mannan degradation that is enhanced by the novel β-1,4-mannanase, Man134A, when combined with other mannanolytic enzymes including various endo-β-1,4-mannanases.     

Effects of Ontogeny on δ13C of Plant- and Soil-Respired CO2 and on Respiratory Carbon Fractionation in C3 Herbaceous Species

Knowledge gaps regarding potential ontogeny and plant species identity effects on carbon isotope fractionation might lead to misinterpretations of carbon isotope composition (δ¹³C) of respired CO₂, a widely-used integrator of environmental conditions. In monospecific mesocosms grown under controlled conditions, the δ¹³C of C pools and fluxes and leaf ecophysiological parameters of seven herbaceous species belonging to three functional groups (crops, forage grasses and legumes) were investigated at three ontogenetic stages of their vegetative cycle (young foliage, maximum growth rate, early senescence). Ontogeny-related changes in δ¹³C of leaf- and soil-respired CO₂ and ¹³C/¹²C fractionation in respiration (Δ_R) were species-dependent and up to 7‰, a magnitude similar to that commonly measured in response to environmental factors. At plant and soil levels, changes in δ¹³C of respired CO₂ and Δ_R with ontogeny were related to changes in plant physiological status, likely through ontogeny-driven changes in the C sink to source strength ratio in the aboveground plant compartment. Our data further showed that lower Δ_R values (i.e. respired CO₂ relatively less depleted in ¹³C) were observed with decreasing net assimilation. Our findings highlight the importance of accounting for ontogenetic stage and plant community composition in ecological studies using stable carbon isotopes.     

Elucidation of a Novel Extracellular Calcium-binding Site on Metabotropic Glutamate Receptor 1α (mGluR1α) That Controls Receptor Activation

Metabotropic glutamate receptor 1α (mGluR1α) exerts important effects on numerous neurological processes. Although mGluR1α is known to respond to extracellular Ca²⁺ ([Ca²⁺]_o) and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca²⁺-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca²⁺-binding site in the mGluR1α ECD using a recently developed computational algorithm. This predicted site (comprising Asp-318, Glu-325, and Asp-322 and the carboxylate side chain of the receptor agonist, Glu) is situated in the hinge region in the ECD of mGluR1α adjacent to the reported Glu-binding site, with Asp-318 involved in both Glu and calcium binding. Mutagenesis studies indicated that binding of Glu and Ca²⁺ to their distinct but partially overlapping binding sites synergistically modulated mGluR1α activation of intracellular Ca²⁺ ([Ca²⁺]_i) signaling. Mutating the Glu-binding site completely abolished Glu signaling while leaving its Ca²⁺-sensing capability largely intact. Mutating the predicted Ca²⁺-binding residues abolished or significantly reduced the sensitivity of mGluR1α not only to [Ca²⁺]_o and [Gd³⁺]_o but also, in some cases, to Glu. The dual activation of mGluR1α by [Ca²⁺]_o and Glu has important implications for the activation of other mGluR subtypes and related receptors. It also opens up new avenues for developing allosteric modulators of mGluR function that target specific human diseases.     

Desert Tortoise (Gopherus agassizii) Dietary Specialization Decreases across a Precipitation Gradient

We studied the plant resource use between and within populations of desert tortoise (Gopherus agassizii) across a precipitation gradient in the Sonoran Desert of Arizona. The carbon and nitrogen stable isotope values in animal tissues are a reflection of the carbon and nitrogen isotope values in diet, and consequently represent a powerful tool to study animal feeding ecology. We measured the δ¹³C and δ¹⁵N values in the growth rings on the shells of tortoises in different populations to characterize dietary specialization and track tortoise use of isotopically distinct C₄/CAM versus C₃ plant resources. Plants using C₃ photosynthesis are generally more nutritious than C₄ plants and these trait differences can have important growth and fitness consequences for consumers. We found that dietary specialization decreases in successively drier and less vegetated sites, and that broader population niche widths are accompanied by an increase in the dietary variability between individuals. Our results highlight how individual consumer plant resource use is bounded under a varying regime of precipitation and plant productivity, lending insight into how intra-individual dietary specialization varies over a spatial scale of environmental variability.     

Presynaptic Gαo (GOA-1) signals to depress command neuron excitability and allow stretch-dependent modulation of egg laying in Caenorhabditis elegans

Egg laying in the nematode worm Caenorhabditis elegans is a two-state behavior modulated by internal and external sensory input. We have previously shown that homeostatic feedback of embryo accumulation in the uterus regulates bursting activity of the serotonergic HSN command neurons that sustains the egg-laying active state. How sensory feedback of egg release signals to terminate the egg-laying active state is less understood. We find that Gα_o, a conserved Pertussis Toxin-sensitive G protein, signals within HSN to inhibit egg-laying circuit activity and prevent entry into the active state. Gα_o signaling hyperpolarizes HSN, reducing HSN Ca²⁺ activity and input onto the postsynaptic vulval muscles. Loss of inhibitory Gα_o signaling uncouples presynaptic HSN activity from a postsynaptic, stretch-dependent homeostat, causing precocious entry into the egg-laying active state when only a few eggs are present in the uterus. Feedback of vulval opening and egg release activates the uv1 neuroendocrine cells which release NLP-7 neuropeptides which signal to inhibit egg laying through Gα_o-independent mechanisms in the HSNs and Gα_o-dependent mechanisms in cells other than the HSNs. Thus, neuropeptide and inhibitory Gα_o signaling maintain a bi-stable state of electrical excitability that dynamically controls circuit activity in response to both external and internal sensory input to drive a two-state behavior output.     

Two β-Galactosidases from the Human Isolate Bifidobacterium breve DSM 20213: Molecular Cloning and Expression,Biochemical Characterization and Synthesis of Galacto-Oligosaccharides

Two β-galactosidases, β-gal I and β-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both β-gal I and β-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose were determined at pH 7.0 and 50°C for β-gal I, and at pH 6.5 and 55°C for β-gal II, respectively. The k _cat/K _m values for oNPG and lactose hydrolysis are 722 and 7.4 mM⁻¹s⁻¹ for β-gal I, and 543 and 25 mM⁻¹s⁻¹ for β-gal II. Both β-gal I and β-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with β-gal I and β-gal II, respectively. The predominant transgalactosylation products are β-D-Galp-(1→6)-D-Glc (allolactose) and β-D-Galp-(1→3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by β-gal I and β-gal II, respectively, indicating that both enzymes have a propensity to synthesize β-(1→6) and β-(1→3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide.     

Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F1-ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

F₁-ATPase is a rotary molecular machine with a subunit stoichiometry of α₃β₃γ₁δ₁ε₁. It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α₃β₃ core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F₁-ATPase have suggested that the α₃β₃ core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F₁-ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ^o) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F₁ complexes retained 21.7 to 44.6% of the native F₁-ATPase activity. The γ-less F₁ subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ^o conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α₃β₃ subcomplex.     

Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19_Mtb) transformed 3-oxo-4-cholesten-26-oate (k_cat/K_m = 0.33 × 10⁵ ± 0.03 × 10⁵ M⁻¹ s⁻¹) and represents orthologs that activate the C₈ side chain of cholesterol. Both CasG_RHA1 and FadD17_Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C₅ side chain of cholate, while FadD17 and its orthologs appear to activate the C₅ side chain of one or more cholesterol metabolites. CasI_RHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C₃ side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2′-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (k_cat/K_m = 2.4 × 10⁵ ± 0.1 × 10⁵ M⁻¹ s⁻¹ and 3.2 × 10⁵ ± 0.3 × 10⁵ M⁻¹ s⁻¹, respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and a phylogenetic classification enabling prediction of specific functions of related enzymes.     

Structural insights into the function-modulating effects of nanobody binding to the integrin receptor αMβ2

The integrin receptor α_Mβ₂ mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix, and transendothelial migration of leukocytes. However, the mechanistic aspects of α_Mβ₂ signaling upon ligand binding are unclear. Here, we present the first atomic structure of the human α_Mβ₂ headpiece fragment in complex with the nanobody (Nb) hCD11bNb1 at a resolution of 3.2 Å. We show that the receptor headpiece adopts the closed conformation expected to exhibit low ligand affinity. The crystal structure indicates that in the R77H α_M variant, associated with systemic lupus erythematosus, the modified allosteric relationship between ligand binding and integrin outside–inside signaling is due to subtle conformational effects transmitted over a distance of 40 Å. Furthermore, we found the Nb binds to the αI domain of the α_M subunit in an Mg²⁺-independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins demonstrated that the Nb acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of complement component iC3b attempting to bind the α_M subunit. Surprisingly, we show that the Nb stimulates the interaction of cell-bound α_Mβ₂ with iC3b, suggesting that it may represent a novel high-affinity proteinaceous α_Mβ₂-specific agonist. Taken together, our data suggest that the iC3b–α_Mβ₂ complex may be more dynamic than predicted from the crystal structure of the core complex. We propose a model based on the conformational spectrum of the receptor to reconcile these observations regarding the functional consequences of hCD11bNb1 binding to α_Mβ₂.     

The influence of external potassium on the inactivation of sodium currents in the giant axon of the squid, Loligo pealei

Isolated giant axons were voltage-clamped in seawater solutions having constant sodium concentrations of 230 mM and variable potassium concentrations of from zero to 210 mM. The inactivation of the initial transient membrane current normally carried by Na⁺ was studied by measuring the Hodgkin-Huxley h parameter as a function of time. It was found that h reaches a steady-state value within 30 msec in all solutions. The values of h _∞, τ_h, α_h,and β_h as functions of membrane potential were determined for various [K _o]. The steady-state values of the h parameter were found to be inversely related, while the time constant, τ_h, was directly related to external K⁺ concentration. While the absolute magnitude as well as the slopes of the h _∞ vs. membrane potential curves were altered by varying external K⁺, only the magnitude and not the shape of the corresponding τ_h curves was altered. Values of the two rate constants, α_h and β_h, were calculated from h_∞ and τ_h values. α_h is inversely related to [K_o] while β_h is directly related to [K_o] for hyperpolarizing membrane potentials and is independent of [K_o] for depolarizing membrane potentials. Hodgkin-Huxley equations relating α_h and β_h to E_m were rewritten so as to account for the observed effects of [K_o]. It is concluded that external potassium ions have an inactivating effect on the initial transient membrane conductance which cannot be explained solely on the basis of potassium membrane depolarization.     

Active-Site Structure of the Thermophilic Foc-Subunit Ring in Membranes Elucidated by Solid-State NMR

F_oF₁-ATP synthase uses the electrochemical potential across membranes or ATP hydrolysis to rotate the F_oc-subunit ring. To elucidate the underlying mechanism, we carried out a structural analysis focused on the active site of the thermophilic c-subunit (TF_oc) ring in membranes with a solid-state NMR method developed for this purpose. We used stereo-array isotope labeling (SAIL) with a cell-free system to highlight the target. TF_oc oligomers were purified using a virtual ring His tag. The membrane-reconstituted TF_oc oligomer was confirmed to be a ring indistinguishable from that expressed in E. coli on the basis of the H⁺-translocation activity and high-speed atomic force microscopic images. For the analysis of the active site, 2D ¹³C-¹³C correlation spectra of TF_oc rings labeled with SAIL-Glu and -Asn were recorded. Complete signal assignment could be performed with the aid of the C^α_i+1-C^α_i correlation spectrum of specifically ¹³C,¹⁵N-labeled TF_oc rings. The C^δ chemical shift of Glu-56, which is essential for H⁺ translocation, and related crosspeaks revealed that its carboxyl group is protonated in the membrane, forming the H⁺-locked conformation with Asn-23. The chemical shift of Asp-61 C^γ of the E. coli c ring indicated an involvement of a water molecule in the H⁺ locking, in contrast to the involvement of Asn-23 in the TF_oc ring, suggesting two different means of proton storage in the c rings.