Synthetic cells and organelles: compartmentalization strategies

The recent development of RNA replicating protocells and capsules that enclose complex biosynthetic cascade reactions are encouraging signs that we are gradually getting better at mastering the complexity of biological systems. The road to truly cellular compartments is still very long, but concrete progress is being made. Compartmentalization is a crucial natural methodology to enable control over biological processes occurring within the living cell. In fact, compartmentalization has been considered by some theories to be instrumental in the creation of life. With the advancement of chemical biology, artificial compartments that can mimic the cell as a whole, or that can be regarded as cell organelles, have recently received much attention. The membrane between the inner and outer environment of the compartment has to meet specific requirements, such as semi‐permeability, to allow communication and molecular transport over the border. The membrane can either be built from natural constituents or from synthetic polymers, introducing robustness to the capsule.     

Overexpression of γ-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress: Physiological and chlorophyll a fluorescence measurements

Tocopherols (vitamin E) are lipid soluble antioxidants synthesized by plants and some cyanobacteria. We have earlier reported that overexpression of the γ-tocopherol methyl transferase (γ-TMT) gene from Arabidopsis thaliana in transgenic Brassica juncea plants resulted in an over six-fold increase in the level of α-tocopherol, the most active form of all the tocopherols. Tocopherol levels have been shown to increase in response to a variety of abiotic stresses. In the present study on Brassica juncea, we found that salt, heavy metal and osmotic stress induced an increase in the total tocopherol levels. Measurements of seed germination, shoot growth and leaf disc senescence showed that transgenic Brassica juncea plants overexpressing the γ-TMT gene had enhanced tolerance to the induced stresses. Analysis of the chlorophyll a fluorescence rise kinetics, from the initial “O” level to the “P” (the peak) level, showed that there were differential effects of the applied stresses on different sites of the photosynthetic machinery; further, these effects were alleviated in the transgenic (line 16.1) Brassica juncea plants. We show that α-tocopherol plays an important role in the alleviation of stress induced by salt, heavy metal and osmoticum in Brassica juncea.     

Redox potential of the Rieske iron-sulfur protein: Quantum-chemical and electrostatic study

Quantum-chemical study of structures, energies, and effective partial charge distribution for several models of the Rieske protein redox center is performed in terms of the B3LYP density functional method in combination with the broken symmetry approach using three different atomic basis sets. The structure of the redox complex optimized in vacuum differs markedly from that inside the protein. This means that the protein matrix imposes some stress on the active site resulting in distortion of its structure. The redox potentials calculated for the real active site structure are in a substantially better agreement with the experiment than those calculated for the idealized structure. This shows an important role of the active site distortion in tuning its redox potential. The reference absolute electrode potential of the standard hydrogen electrode is used that accounts for the correction caused by the water surface potential. Electrostatic calculations are performed in the framework of the polarizable solute model. Two dielectric permittivities of the protein are employed: the optical permittivity for calculation of the intraprotein electric field, and the static permittivity for calculation of the dielectric response energy. Only this approach results in a reasonable agreement of the calculated and experimental redox potentials.     

More food,less habitat: how necromass and leaf litter decomposition combine to regulate a litter ant community

1. In brown food webs of the forest floor, necromass (e.g. insect carcasses and frass) falling from the canopy feeds both microbes and ants, with the former decomposing the homes of the latter. In a tropical litter ant community, we added necromass to 1 m² plots, testing if it added as a net food (increasing ant colony growth and recruitment) or destroyer of habitat (by decomposing leaf litter). 2. Maximum, but not mean, colony growth rates were higher on +food plots. However, neither average colony size, nor density was higher on +food plots. In contrast, +food plots saw diminished availability of leaf litter and higher microbial decomposition of cellulose, a main component of the organic substrate that comprises litter habitat. 3. Furthermore, necromass acted as a limiting resource to the ant community only when nest sites were supplemented on +food plots in a second experiment. Many of these +food +nest plots were colonised by the weedy species Wasmannia auropunctata. 4. Combined, these results support the more food–less habitat hypothesis and highlight the importance of embedding studies of litter ant ecology within broader decomposer food web dynamics.     

The size-grain hypothesis: a phylogenetic and field test

Abstract.  1. The size-grain hypothesis predicts that environmental rugosity results in positive allometric scaling of leg length on body length because of changes in locomotion costs.
2. The scaling of leg length and body length in ants was re-examined using phylogenetic independent contrast methods, and the allometric relationship found by Kaspari and Weiser ( Functional Ecology , 13 , 530–538, 1999) was supported.
3. The size-grain hypothesis was tested further by comparing the body sizes of ants from areas of contrasting habitat complexity in two different savanna habitats. No support for the size-grain hypothesis was found. Small body size classes were no more speciose in the rugose than in the more planar environment, and small ants were more abundant in the planar environment.     

Sensitivity of volatile monoterpene emission to changes in canopy structure: a model-based exercise with a process-based emission model

This paper investigates the dependence of monoterpene emissions at the canopy scale on total leaf area and leaf distribution. Simulations were carried out for a range of hypothetical but realistic forest canopies of the evergreen Quercus ilex (holm oak). Two emission models were applied that either did (SIM-BIM2) or did not (G93) account for cumulative responses to temperature and light. Both were embedded into a canopy model that considered spatial and temporal variations of foliage properties. This canopy model was coupled to a canopy climate model (CANOAK) to determine the micrometeorological conditions at the leaf scale. Structural properties considerably impacted monoterpene emission. The sensitivities to changes in total leaf area and to leaf area distribution were found to be of similar magnitude. The two different models performed similarly on a whole-year basis but showed clear differences during certain episodes. The analysis showed that structural indices have to be carefully evaluated for proper scaling of emission from leaves to canopy. Further research is encouraged on seasonal dynamics of emission potentials.     

Crystal structure of tryptophanyl-tRNA synthetase complexed with adenosine-5' tetraphosphate: evidence for distributed use of catalytic binding energy in amino acid activation by class I aminoacyl-tRNA synthetases

Tryptophanyl-tRNA synthetase (TrpRS) is a functionally dimeric ligase, which specifically couples hydrolysis of ATP to AMP and pyrophosphate to the formation of an ester bond between tryptophan and the cognate tRNA. TrpRS from Bacillus stearothermophilus binds the ATP analogue, adenosine-5' tetraphosphate (AQP) competitively with ATP during pyrophosphate exchange. Estimates of binding affinity from this competitive inhibition and from isothermal titration calorimetry show that AQP binds 200 times more tightly than ATP both under conditions of induced-fit, where binding is coupled to an unfavorable conformational change, and under exchange conditions, where there is no conformational change. These binding data provide an indirect experimental measurement of +3.0 kcal/mol for the conformational free energy change associated with induced-fit assembly of the active site. Thermodynamic parameters derived from the calorimetry reveal very modest enthalpic changes, consistent with binding driven largely by a favorable entropy change. The 2.5 A structure of the TrpRS:AQP complex, determined de novo by X-ray crystallography, resembles that of the previously described, pre-transition state TrpRS:ATP complexes. The anticodon-binding domain untwists relative to the Rossmann-fold domain by 20% of the way toward the orientation observed for the Products complex. An unexpected tetraphosphate conformation allows the gamma and deltad phosphate groups to occupy positions equivalent to those occupied by the beta and gamma phosphates of ATP. The beta-phosphate effects a 1.11 A extension that relocates the alpha-phosphate toward the tryptophan carboxylate while the PPi mimic moves deeper into the KMSKS loop. This configuration improves interactions between enzyme and nucleotide significantly and uniformly in the adenosine and PPi binding subsites. A new hydrogen bond forms between S194 from the class I KMSKS signature sequence and the PPi mimic. These complementary thermodynamic and structural data are all consistent with the conclusion that the tetraphosphate mimics a transition-state in which the KMSKS loop develops increasingly tight bonds to the PPi leaving group, weakening linkage to the Palpha as it is relocated by an energetically favorable domain movement. Consistent with extensive mutational data on Tyrosyl-tRNA synthetase, this aspect of the mechanism develops high transition-state affinity for the adenosine and pyrophosphate moieties, which move significantly, relative to one another, during the catalytic step.     

Shrinking to fit: fluid jettison from a haemocoelic hydrostatic skeleton during defensive withdrawals of a gastropod larva

Although most of the basic animal body plans are supported by hydrostatic skeletons consisting of fluid maintained at constant volume, studies on how animals have solved biomechanical scaling dilemmas during evolution of large body size have emphasized cases where skeletons are formed by rigid solids. Larvae of gastropod molluscs swim using ciliated velar lobes supported by a constant volume hydrostatic skeleton. Defensive behaviour involves rapid withdrawal of the velar lobes and foot into a protective biomineralized shell. Some gastropod larvae grow to giant size and the velar lobes enlarge allometrically, but the lobes and foot of many can still withdraw completely into the mineral-stiffened shell. I dyed internal fluid of a large gastropod larva with fluorescein to show that fluid supporting the extended velar lobes is expelled from discrete release sites during defensive withdrawals. Scanning electron microscopy suggested that release sites are distinctive papillae on the upper velar epidermis. Ultrathin sections revealed that branched tracks of microvilli-free membrane on the surface of these papillae were formed by very thin epithelial cells, which may rupture and re-anneal during and after defensive withdrawals. Behaviours facilitated by fluid discharge from a haemocoelic (non-coelomic) body compartment have been rarely reported among aquatic invertebrates, but may be more widespread than currently recognized.