Multiple Modes of Protein–Protein Interactions Promote RNP Granule Assembly

Eukaryotic cells are known to contain a wide variety of RNA–protein assemblies, collectively referred to as RNP granules. RNP granules form from a combination of RNA–RNA, protein–RNA, and protein–protein interactions. In addition, RNP granules are enriched in proteins with intrinsically disordered regions (IDRs), which are frequently appended to a well-folded domain of the same protein. This structural organization of RNP granule components allows for a diverse set of protein–protein interactions including traditional structured interactions between well-folded domains, interactions of short linear motifs in IDRs with the surface of well-folded domains, interactions of short motifs within IDRs that weakly interact with related motifs, and weak interactions involving at most transient ordering of IDRs and folded domains with other components. In addition, both well-folded domains and IDRs in granule components frequently interact with RNA and thereby can contribute to RNP granule assembly. We discuss the contribution of these interactions to liquid–liquid phase separation and the possible role of phase separation in the assembly of RNP granules. We expect that these principles also apply to other non-membrane bound organelles and large assemblies in the cell.     

Zone Precipitation Chromatography: Its Use in the Isolation of Different Collagen Types

Zone Precipitation Chromatography is useful tech-nique for the initial isolation of the different colla-gen types in their native configuration. Small quan-tities of collagen mixtures can be rapidly separated into different collagen types with relatively high degree of purity, based upon stained protein patterns on sodium dodecyl sulfate polyacrylamide gel electro-phoresis (SDS-PAGE) slab gels. Tn the commonly used bulk salt preparative method for isolating the different collagens, 50 mg of starting material was needed. Three days were required to complete the procedure. The stained protein patterns on SDS-PAGE slab gels showed about 25% contamination with the bulk purified Type III fraction and 20% contamination with the bulk purified type AB collagen. With Zone Precipitation Chromatography 5 mg of starting material was used and in less than 4 hours the mixture was separated with Types III and AB fractions showing less than 10% contamination from other collagen types. The technique is patterned after the Zone Precinitation method reported by Porath seventeen years ago and utilizes a step-wise sodium chloride gradient to precipitate and redissolve the collagens, eluting from the interbead spaces of a molecular sieve column.     

Benzoyl cellulose beads in the pure polymeric form as a new powerful sorbent for the chromatographic resolution of racemates

Cellulose-based stationary phases are known to be very efficient and versatile chiral sorbents for the chromatographic resolution of racemates. Except for microcrystalline cellulose triacetate (CTA I), basically all other cellulose-based phases have been prepared by coating of ca. 20% weight polymer on a wide pore silica gel used as a carrier. In this work we describe the preparation of benzoylcellulose (TBC) beads in the pure polymeric form (without inorganic carrier) from an emulsion of the organic polymer. The new material has been fully characterized and used as a chiral stationary phase for the resolution of various classes of racemic compounds such as benzylic alcohols or acetate derivatives of aliphatic alcohols and diols. The structural variety of the separated solutes as well as the irrational influence of the aromatic substituent in different classes of aryl compounds suggest that multiple interaction sites are involved in the complexation, making a prediction of the separation difficult. The benzoyl cellulose beads exhibit a very high loading capacity, which is particularly useful for preparative purposes as demonstrated for selected examples.     

Comparative study on the properties of saturated phosphatidylethanolamine and phosphatidylcholine bilayers: Barrier characteristics and susceptibility to phospholipase A2 degradation

Comparative studies on bilayer systems of saturated phosphatidylcholines and phosphatidylethanolamines revealed a maximum in ionic permeability in phosphatidylcholine bilayers at the temperature of the gel to liquid-crystalline phase transition but such an increase in permeability was not detectable in bilayers of phosphatidylethanolamine. Furthermore, it was found that at the phase transition temperature the phosphatidylcholine bilayers are subject to rapid hydrolysis by pancreatic phospholipase A₂ whereas phosphatidylethanolamine bilayers are not. These differences are discussed in view of detailed information on the molecular organization in the gel and liquid crystalline phases of the two phospholipid classes.     

Temperature impact on the midsummer decline of Daphnia galeata: an analysis of long-term data from the biomanipulated Bautzen Reservoir (Germany)

1. The influence of water temperature on occurrence and duration of a midsummer decline (MSD) of Daphnia galeata was studied in the biomanipulated Bautzen Reservoir in Germany. The proportion of piscivores in the fish community of the reservoir has been enhanced experimentally since 1981. As a consequence, Daphnia galeata has dominated the zooplankton. Over 18 years of study (1981–1998), a long‐lasting MSD (longer than 30 days) occurred in 7 years, whereas a short MSD (shorter than 30 days) was observed in 6 years. During the remaining 5 years, an MSD was not observed.
2. Two hypotheses were examined to explain the observed patterns. First, we postulated that high water temperature during winter and early spring (January–April) leads to an MSD after an early and high spring peak of daphnids. On the other hand, low temperature during winter and early spring should not cause an MSD owing to a slower increase of the population, resulting in a later peak of daphnids. Second, we hypothesized that the mean water temperature during early summer (May and June) influences the occurrence of an MSD (by controlling young‐of‐the‐year (YOY) fish predation on daphnids).
3. The water temperature during winter and early spring explains 83%, and the early summer water temperature 55%, of interannual variation in the occurrence of an MSD.
4. The interannual variation in duration of an MSD was neither explained by temperature during winter and early spring nor by early summer temperature alone, but in 14 of the 18 years (78%) by a combination of both.
5. We conclude that water temperature during winter and early spring had a strong impact on Daphnia mortality by influencing height and timing of the spring peak which, in turn, influenced the extent of overexploitation of their food resources. By contrast, the water temperature during early summer probably influenced the mortality of daphnids caused by predation of YOY fish. The relative timing of both sources of mortality, which depends on the temperature regime during the first 6 months of the year, is the key process in controlling the occurrence and duration of an MSD. A long‐lasting MSD, therefore, is likely in Bautzen Reservoir only if temperatures are high during winter and early spring, as well as during early summer.
6. As a consequence of climate warming, recent climate records reveal warming during winter, spring and early summer in middle Europe, rather than an increase in mean annual temperatures. If our findings and conclusions are related to this regional and temporal pattern of climate warming, an increasing frequency of years with a long‐lasting MSD and, consequently, a decreasing efficiency of biomanipulation can be predicted.     

Hydration, structure, and molecular interactions in the headgroup region of dioleoylphosphatidylcholine bilayers: an electron spin resonance study

The relationship between bilayer hydration and the dynamic structure of headgroups and interbilayer water in multilamellar vesicles is investigated by electron spin resonance methods. Temperature variations of the order parameter of a headgroup spin label DPP-Tempo in DOPC in excess water and partially dehydrated (10 wt % water) show a cusp-like pattern around the main phase transition, Tc. This pattern is similar to those of temperature variations of the quadrupolar splitting of interbilayer D2O in PC and PE bilayers previously measured by 2H NMR, indicating that the ordering of the headgroup and the interbilayer water are correlated. The cusp-like pattern of these and other physical properties around Tc are suggestive of quasicritical fluctuations. Also, an increase (a decrease) in ordering of DPP-Tempo is correlated with water moving out of (into) interbilayer region into (from) the bulk water phase near the freezing point, Tf. Addition of cholesterol lowers Tf, which remains the point of increasing headgroup ordering. Using the small water-soluble spin probe 4-PT, it is shown that the ordering of interbilayer water increases with bilayer dehydration. It is suggested that increased ordering in the interbilayer region, implying a lowering of entropy, will itself lead to further dehydration of the interbilayer region until its lowered pressure resists further flow, i.e., an osmotic phenomenon.     

Phallus impudicus loaded with γ-Fe2O3 as solid phase bioextractor for the preconcentrations of Zn(II) and Cr(III) from water and food samples

We investigated the application of fungus Phallus impudicus loaded γ-Fe₂O₃ nanoparticles as a biosorbent for magnetic solid phase extractions of trace levels of Zn(II) and Cr(III) ions from natural samples before their measurements by inductively coupled plasma optical emission spectrometry. The characterization of magnetized P. impudicus was performed using the scanning electron microscope, the energy dispersive X-ray and Fourier transform infrared spectroscopy. Important experimental factors were investigated. The experimental results fitted well to the Langmuir adsorption model and pseudo-second order kinetic model. Limit of detections of targeted ions by magnetic solid phase extraction method based on use of P. impudicus were found as 10.5 ngL⁻¹ and 12.6 ngL⁻¹ respectively for Cr(III) and Zn(II). The sorption capacities of the biosorbent were 22.8 mgg⁻¹ for Cr(III) and 25.6 mgg⁻¹ for Zn(II). The preconcentration factors were achieved as 100 for both of ions. RSDs for inter- and intraday precisions were found as lower than 2.0% and 2.1% respectively for both of targeted ions. The accuracy of the recommended process was tested by recovery measurements on the certificated reference materials and successfully applied for quantification recoveries of Cr(III) and Zn(II) ions from water and food samples.     

Exploring the interactions of gliadins with model membranes: Effect of confined geometry and interfaces

Mechanisms leading to the assembly of wheat storage proteins into proteins bodies within the endoplasmic reticulum (ER) of endosperm cells are unresolved today. In this work, physical chemistry parameters which could be involved in these processes were explored. To model the confined environment of proteins within the ER, the dynamic behavior of γ‐gliadins inserted inside lyotropic lamellar phases was studied using FRAP experiments. The evolution of the diffusion coefficient as a function of the lamellar periodicity enabled to propose the hypothesis of an interaction between γ‐gliadins and membranes. This interaction was further studied with the help of phospholipid Langmuir monolayers. γ‐ and ω‐gliadins were injected under DMPC and DMPG monolayers and the two‐dimensional (2D) systems were studied by Brewster angle microscopy (BAM), polarization modulation infrared reflection‐absorption spectroscopy (PM‐IRRAS), and surface tension measurements. Results showed that both gliadins adsorbed under phospholipid monolayers, considered as biological membrane models, and formed micrometer‐sized domains at equilibrium. However, their thicknesses, probed by reflectance measurements, were different: ω‐gliadins aggregates displayed a constant thickness, consistent with a monolayer, while the thickness of γ‐gliadins aggregates increased with the quantity of protein injected. These different behaviors could find some explanations in the difference of aminoacid sequence distribution: an alternate repeated ‐ unrepeated domain within γ‐gliadin sequence, while one unique repeated domain was present within ω‐gliadin sequence. All these findings enabled to propose a model of gliadins self‐assembly via a membrane interface and to highlight the predominant role of wheat prolamin repeated domain in the membrane interaction. In the biological context, these results would mean that the repeated domain could be considered as an anchor for the interaction with the ER membrane and a nucleus point for the formation and growth of protein bodies within endosperm cells. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 610–622, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Comparative studies between covalently immobilized and coated chiral stationary phases based on polysaccharide derivatives for enantiomer separation of N‐protected α‐amino acids and their ester derivatives

Liquid chromatographic enantiomer separation of several N‐benzyloxycarbonyl (CBZ) and N‐tert‐butoxycarbonyl (BOC) α‐amino acids and their corresponding ethyl esters was performed on covalently immobilized chiral stationary phases (CSPs) (Chiralpak IA and Chiralpak IB) and coated‐type CSPs (Chiralpak AD and Chiralcel OD) based on polysaccharide derivatives. The solvent versatility of the covalently immobilized CSPs in enantiomer separation of N‐CBZ and BOC‐α‐amino acids and their ester derivatives was shown and the chromatographic parameters of their enantioselectivities and resolution factors were greatly influenced by the nature of the mobile phase. In general, standard mobile phases using 2‐propanol and hexane on Chiralpak IA showed fairly good enantioselectivities for resolution of N‐CBZ and BOC‐α‐amino acids and their esters. However, 50% MTBE/hexane (v/v) for resolution of N‐CBZ‐α‐amino acids ethyl esters and 20% THF/hexane (v/v) for resolution of N‐BOC‐α‐amino acids ethyl esters afforded the greatest enantioselectivities on Chiralpak IA. Also, liquid chromatographic comparisons of the enantiomer resolution of these analytes were made on amylose tris(3,5‐dimethylphenylcarbamate)‐derived CSPs (Chiralpak IA and Chiralpak AD) and cellulose tris(3,5‐dimethylphenylcarbamate)‐derived CSPs (Chiralpak IB and Chiralcel OD). Chiralpak AD and/or Chiralcel OD showed the highest enantioselectivities for resolution of N‐CBZ‐α‐amino acids and esters, while Chiralpak AD or Chiralpak IA showed the highest resolution of N‐BOC‐α‐amino acids and esters. Chirality 2009. © 2008 Wiley‐Liss, Inc.     

O‐glycoside sequence of pentacyclic triterpene saponins from Phytolacca bogotensis using HPLC‐ESI/multi‐stage tandem mass spectrometry

Introduction – The lack of pharmacopoeial methodologies for the quality control of plants used for therapeutic purposes is a huge problem that impacts directly upon public health. In the case of saponins, their great structural complexity, weak glycoside bonds and high polarity hinder their identification by conventional techniques. Objective – To apply high‐performance liquid chromatography–electrospray tandem mass spectrometry (HPLC‐ESI/MSⁿ) to identify the O‐glycoside sequence of saponins from the roots of Phytolacca bogotensis. Methodology – Saponins were isolated by preparative HPLC and characterised by NMR spectroscopic experiments. Collision‐induced dissociation (CID) of isolated saponins was performed producing typical degradation reactions that can be associated with several glycosidic bonds as empirical criteria. A method using solid‐phase extraction (SPE) and HPLC/ESI‐MSⁿ for the characterisation of saponins and identification of novel molecules is described. Results – Three saponins reported for the first time in P. bogotensis were isolated and characterised by NMR spectroscopy. Characteristic cross ring cleavage reactions have been used as empirical criteria for the characterisation of the glycosidic bonds most frequently reported for Phytolacca saponins. One new saponin was proposed on the basis of empirical criteria, and other five saponins were identified for the first time for P. bogotensis using HPLC‐ESI/MSⁿ. Conclusion – Electrospray ionisation in combination with tandem mass spectrometry has been established as a powerful tool for the profiling of saponins from roots of P. bogotensis. CID proved to be a useful tool for the characterisation and identification of known and novel saponins from the plant family Phytolaccaceae and can be used for quality control purposes of crude plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.