Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.     

Substrate water exchange in photosystem II core complexes of the extremophilic red alga Cyanidioschyzon merolae

The binding affinity of the two substrate–water molecules to the water-oxidizing Mn₄CaO₅ catalyst in photosystem II core complexes of the extremophilic red alga Cyanidioschyzon merolae was studied in the S₂ and S₃ states by the exchange of bound ¹⁶O-substrate against ¹⁸O-labeled water. The rate of this exchange was detected via the membrane-inlet mass spectrometric analysis of flash-induced oxygen evolution. For both redox states a fast and slow phase of water-exchange was resolved at the mixed labeled m/z 34 mass peak: k_f = 52 ± 8 s^− 1 and k_s = 1.9 ± 0.3 s^− 1 in the S₂ state, and k_f = 42 ± 2 s^− 1 and k_slow = 1.2 ± 0.3 s^− 1 in S₃, respectively. Overall these exchange rates are similar to those observed previously with preparations of other organisms. The most remarkable finding is a significantly slower exchange at the fast substrate–water site in the S₂ state, which confirms beyond doubt that both substrate–water molecules are already bound in the S₂ state. This leads to a very small change of the affinity for both the fast and the slowly exchanging substrates during the S₂ → S₃ transition. Implications for recent models for water-oxidation are briefly discussed.     

Collagen functionalized with unsaturated cyclic anhydrides—interactions in solution and solid state

Maleic anhydride (CMA) and itaconic anhydride modified collagen (CITA) were prepared as precursors for production of interpenetrated polymer networks (IPN). Calculated values for Huggins coefficient in aqueous diluted and semi‐diluted solutions of modified collagen indicated a slightly tendency of aggregation for itaconic anhydride‐modified collagen. In semi‐diluted solution collagen (Coll) and CMA present slightly differences in the thixotropic behavior, while CITA has a pronounced thixotropic behavior. Flow and oscillatory measurements revealed an elastic behavior of the collagen solutions, pure and modified with MA or ITA, as the storage modulus (G′) has always a superior value compared with the loss modulus (G″). The denaturation temperature (T_d) of unmodified collagen increased from 34^oC to 40^oC for CMA and to 39^oC for CITA respectively, by formation of covalent bonds that stabilize the triple helix. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 228–236, 2014.     

Stabilization of collagen through bioconversion: An insight in protein–protein interaction

Collagen is a natural protein, which is used as a vital biomaterial in tissue engineering. The major concern about native collagen is lack of its thermal stability and weak resistance to proteolytic degradation. In this scenario, the crosslinking compounds used for stabilization of collagen are mostly of chemical nature and exhibit toxicity. The enzyme mediated crosslinking of collagen provides a novel alternative, nontoxic method for stabilization. In this study, aldehyde forming enzyme (AFE) is used in the bioconversion of hydroxylmethyl groups of collagen to formyl groups that results in the formation of peptidyl aldehyde. The resulted peptidyl aldehyde interacts with bipolar ions of basic amino acid residues of collagen. Further interaction leads to the formation of conjugated double bonds (aldol condensation involving the aldehyde group of peptidyl aldehyde) within the collagen. The enzyme modified collagen matrices have shown an increase in the denaturation temperature, when compared with native collagen. Enzyme modified collagen membranes exhibit resistance toward collagenolytic activity. Moreover, they exhibited a nontoxic nature. The catalytic activity of AFE on collagen as a substrate establishes an efficient modification, which enhances the structural stability of collagen. This finds new avenues in the context of protein–protein stabilization and discovers paramount application in tissue engineering. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 903–911, 2014.     

Altitudinal Patterns in Two Syntopic Species of Sturnira (Mammalia: Chiroptera: Phyllostomidae) in the Montane Rain Forests of Argentina

We evaluated altitudinal segregation in two Sturnira species as a mechanism allowing their coexistence. Tests were devised to discern between interspecific interactions and regional responses to geographic and environmental variables, using extensive capture data from 18 montane rain forest sites. No significant correlation between captures was found. Additionally and according to the hypothesis of historical occupation of highland versus lowland forests, each species has shown specificity in their response to the geographical and environmental variables. Our results indicated that abundance patterns in these bats likely result from the regional distribution of each species rather than local interactions between them, a pattern that may have ancient roots within the group.     

Application of the Kwei equation to model the Tg behavior of binary blends of sugars and salts

Vitrification of sugar-based solutions plays an important role in cryopreservation, lyophilization, and the emerging field of anhydrous preservation. An understanding of the glass transition characteristics of such formulations is essential for determining an appropriate storage temperature to ensure an extended shelf life of vitrified products. To better understand the effect of salts on the glass transition temperature (T_g) of glass-forming sugars, we investigated several data-fitting models (Fox, Gordon–Taylor and Kwei) for sugar–salt formulations using data from the literature, as well as new data generated on blends of trehalose and choline dihydrogen phosphate (CDHP). CDHP has recently been shown to have promise as a stabilizing agent for proteins and DNA. The Kwei equation, which has a specific parameter characterizing intermolecular interactions, provides good fits to the T_g data for sugar–salt blends, and complements other commonly used models that are frequently used to model T_g data.     

Temperature adaptation in Chrysomya megacephala and Chrysomya pinguis,two blow fly species of forensic significance

Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) is a common and forensically important blow fly species in the Oriental region. However, in the higher mountain regions and on winter days, its habitats are occupied by a closely related species, Chrysomya pinguis (Walker). The resources that the two species employ to survive are very similar and competition between the species may be one of the factors that trigger differentiation of their behaviors. We conducted experiments to examine how these two closely related species may have adapted to different temperature regimes to avoid competition. Several adult and immature parameters were assessed, such as fecundity, locomotor ability, hatching ratio, larval survivorship, and eclosion ratio. Results indicate that species show specific diapause at high temperature (38 °C), larval survivorship of Ch. megacephala was significantly better than that of Ch. pinguis. Conversely, at low temperature (15 °C), adult locomotor ability was better for Ch. pinguis than for Ch. megacephala. The results indicate that the two species may have evolved different temperature adaptation strategies to avoid competition. In mixed‐species larval rearing experiments, competition between Ch. pinguis and Ch. megacephala was observed: at higher temperature (30 °C), the immature performance index of Ch. megacephala was significantly increased when compared to that in single‐species culture, whereas the index of Ch. pinguis was decreased. These data are consistent with the idea that tolerance for higher temperature conditions would allow larvae of Ch. megacephala to gain a competitive advantage over Ch. pinguis in certain habitats. These results may help to explain their current distribution in the environment and provide more biological information on these forensically important species.     

Climate Change Implications for River Restoration in Global Biodiversity Hotspots

Global biodiversity hotspots contain exceptional concentrations of endemic species in areas of escalating habitat loss. However, most hotspots are geographically constrained and consequently vulnerable to climate change as there is limited ability for the movement of species to less hostile conditions. Predicted changes to rainfall and temperature will undoubtedly further impact on freshwater ecosystems in these hotspots. Southwestern Australia is a biodiversity hotspot and, as one of the first to experience significant climate change, is an example and potentially a global bellwether for issues associated with river restoration. In this hotspot, current and predicted water temperatures may exceed thermal tolerances of aquatic fauna. Gondwanic aquatic fauna, characteristic of southwestern Australia, are typically cold stenotherms and consequently intolerant of elevated temperatures. The hotspot in southwestern Australia is geographically restricted being surrounded by ocean and desert, and many important national parks are located on the extreme south coast, where the landscape is relatively flat. Consequently, fauna cannot change their distribution southwards or with altitude as a response to increasing temperatures. Therefore, any mitigation responses need to be in situ to produce a suitable biophysical envelope to enhance species' resilience. This could be through “over restoration” by increased riparian replanting at a catchment scale. A rule‐of‐thumb of a 10% increase in riparian cover would be required to reduce water temperatures by 1°C. These restoration techniques are considered applicable to other global biodiversity hotspots where geography constrains species' movement and the present condition is the desired restoration endpoint.     

Mechanism of low‐temperature‐induced pollen failure in rice

Low‐temperature stress during microspore development alters cellular organization in rice anthers. The major cellular damage includes unusual starch accumulation in the plastids of the endothecium in postmeiotic anthers, abnormal vacuolation and hypertrophy of the tapetum, premature callose (1,3‐β‐glucan) breakdown and lack of normal pollen wall formation. These cellular lesions arise from damage to critical biochemical processes that include sugar metabolism in the anthers and its use by the microspores. Failure of utilization of the callose breakdown product and other microspore wall components like sporopollenin can also be considered as critical. In recent years, considerable progress has been made in the understanding of major biochemical processes including the expression of critical genes that are sensitive to low temperature in rice and cause male sterility. This paper combines a discussion of cellular organization and associated biochemical processes that are sensitive to low temperatures and provides an overview of the potential mechanisms of low‐temperature‐induced male sterility in rice.