Conformation-Dependent Interaction of α-Lactalbumin with Model and Biological Membranes: A Spin-Label ESR Study

Alpha-lactalbumin (alpha-LA) is biosynthesized and stored at the smooth endoplasmic reticulum (ER), then transferred to the Golgi lumen when prolactin stimulation of lactose biosynthesis and secretion takes place. Because both environments are composed of membranes, it was of interest to examine the interactions of alpha-LA with relevant model and biological membranes. Using the ESR spin-labeled fatty acid analog 5-doxyl stearic acid, we found evidence reflecting the insertion of "acid-shocked" molten globule (MG) alpha-LA into lecithin or phosphatidylserine (PS) multi-lamellar vesicles. An additional approximately 3 G immobilization was observed in the alpha-LA-lecithin sample versus the lipid alone. With PS, the increased immobilization was almost 6 G, reflecting an enhanced effect caused by strong electrostatic interactions between the positively charged protein with the negatively charged headgroup at pH 2.4. This was also reflected in the broadening of the PS:alpha-LA phase transition. Additionally, we have demonstrated that alpha-LA in its apo-form also shows similar insertion characteristics with both model and natural lipid membranes. Upon addition of calcium, the apo-form is released from the membrane as the Ca(2+)-bound protein.     

A Molecular Dynamics Study of DMPC Lipid Bilayers Interacting with Dimethylsulfoxide–Water Mixtures

The diffusion process of dimethylsulfoxide (DMSO) through zwitterionic dimyristoylphosphatidylcholine (DMPC) lipid bilayer was studied by means of molecular dynamics (MD) simulations. To account for the cryoprotectant concentration difference between the inside and the outside of the cell, dual DMPC lipid bilayers which separate two aqueous reservoirs with and without DMSO were modeled. The initial configuration of the simulation model had DMSO molecules present in one of the aqueous phases (outside the cell) at two different concentrations of ~3 and ~6?mol%. MD simulations were performed on the systems for 50?ns at 323?K and 1?bar. Although the simulation time considered in the study was insufficient for the DMSO molecules to reach the other aqueous phase and equilibrium, early stages of the diffusion process indicated that DMSO molecules had a tendency to diffuse towards the other aqueous phase. The effects of DMSO on bilayer structural characteristics during the diffusion process were investigated. Simulations were analyzed to correlate the following properties of lipid bilayers in the presence of two different aqueous phases: area per lipid, lipid thickness, mass density profiles, lipid tail order parameter and water dipole orientation. Area per lipid calculated for the leaflet facing the aqueous DMSO?Cwater mixture did not show any significant difference compared to area per lipid for the DMSO-free pure DMPC bilayer. Mass density profiles revealed that DMSO molecules had a strong tendency to diffuse toward the aqueous phase with pure water. The lipid tail order parameter calculated for the sn-1 tail of the leaflet facing the aqueous DMSO?Cwater mixture showed that the ordering of lipid tails decreased compared to the leaflet exposed to pure water. However, the ordering of lipid tails in a system where a single bilayer is hydrated by an aqueous DMSO?Cwater mixture is far lower.     

A Computational Study of the Development of Epithelial Acini: II. Necessary Conditions for Structure and Lumen Stability

Simple epithelial tissues are organized as single layers of tightly packed cells that surround hollow lumens and form selective barriers separating different internal compartments of the body. The maintenance of epithelial structure and its function requires tight coordination and control of all the life processes of epithelial cells via cell-to-cell communication and signaling. These well-balanced cellular systems are, however, quite often disturbed by genetic or environmental cues that may lead to the formation of epithelial tumors (carcinomas). In fact, more than a half of all diagnosed tumors are initiated from epithelial cells. It is, therefore, important to gain a greater understanding of the factors that form and maintain the epithelial structure, as well as the features of the acinar structure that are modified during cancer development as observable in experimental and clinical research. We address these questions using the bio-mechanical model of the developing hollow epithelial acini introduced in Rejniak and Anderson (Bull. Math. Biol. 70:677–712, 2008). Here, we propose several scenarios involving various bio-mechanical interactions between neighboring cells that result in abnormal acinar development. Whenever possible, we compare our computational results with known experimental cases of mutant acini.     

Photochemical Efficiency of PSⅡand Membrane Lipid Peroxidation in Leaves of indica and japonica Rice (Oryza sativa) Under Chilling Temperature and Strong Light Stress Conditions

Relationships between fluorescence parameters and membrane lipid peroxidation in leaves of indica and japonica rice (Oryza sativa L.) during later growth stage were studied under chilling temperature and strong light stress conditions. Results showed that D1 protein contents of PSⅡ in photosynthetic app aratus dropped, the generation of antheraxanthin (A) and zeaxanthin (Z)of xanthophyll cycle were inhibited partly, PSⅡ photochemical efficiency （Fv/Fm）and non photochemical quenching (qN) were also decreased obviously. In addition, endogenous active oxygen scavenger—superoxide dismutase (SOD) reduced, superoxide anion radical (O[SX(B-*3)-[]·[SX]]2) and malondialdehyde (MDA) accumulated, as a result, photooxidation of leaves occurred under chilling temperature and strong light stress conditions. Obvious differences in the changes of the above mentioned physiological parameters between indica and japonica rice were observed. Experiments in leaves treated with inhibitors under chilling temperature and strong light conditions showed that indica rice was more sensitive to chilling temperature with strong light and subjected to photooxidation more than japonica rice. Notable positive correlation between D1 protein contents and Fv/Fm or (A+Z)/(A+Z+V), and a marked negative correlation between Fv/Fm and MDA contents were obtained by regression analysis in indica and japonica rice during chilling temperature and strong light conditions. According to the facts mentioned above, it was inferred that PSⅡ photochemical efficiency（Fv/Fm） was the key index to forecast for the prediction of photooxidation under stress circumstances and the physiological basis were the synthetic capacity of D1 protein and the protection of xanthophyll cycle.     

Membrane Docking Geometry and Target Lipid Stoichiometry of Membrane-Bound PKCα C2 Domain: A Combined Molecular Dynamics and Experimental Study

Protein kinase Cα (PKCα) possesses a conserved C2 domain (PKCα C2 domain) that acts as a Ca²⁺-regulated membrane targeting element. Upon activation by Ca²⁺, the PKCα C2 domain directs the kinase protein to the plasma membrane, thereby stimulating an array of cellular pathways. At sufficiently high Ca²⁺ concentrations, binding of the C2 domain to the target lipid phosphatidylserine (PS) is sufficient to drive membrane association; however, at typical physiological Ca²⁺ concentrations, binding to both PS and phosphoinositidyl-4,5-bisphosphate (PIP₂) is required for specific plasma membrane targeting. Recent EPR studies have revealed the membrane docking geometries of the PKCα C2 domain docked to (i) PS alone and (ii) both PS and PIP₂ simultaneously. These two EPR docking geometries exhibit significantly different tilt angles relative to the plane of the membrane, presumably induced by the large size of the PIP₂ headgroup. The present study utilizes the two EPR docking geometries as starting points for molecular dynamics simulations that investigate atomic features of the protein-membrane interaction. The simulations yield approximately the same PIP₂-triggered change in tilt angle observed by EPR. Moreover, the simulations predict a PIP₂:C2 stoichiometry approaching 2:1 at a high PIP₂ mole density. Direct binding measurements titrating the C2 domain with PIP₂ in lipid bilayers yield a 1:1 stoichiometry at moderate mole densities and a saturating 2:1 stoichiometry at high PIP₂ mole densities. Thus, the experiment confirms the target lipid stoichiometry predicted by EPR-guided molecular dynamics simulations. Potential biological implications of the observed docking geometries and PIP₂ stoichiometries are discussed.     

Amphotericin B Lipid Complex (Ablc™): A Molecular Rationale for the Attenuation of Amphotericin B Related Toxicities

Abstract

When the idea of putting together a Forum on the subject of polymer and glycan coated liposomes came across my mind, I thought that this would be an unique opportunity for those of us who argue fiercefully in meetings to do the same in writing. The idea was well received when I contacted my colleagues about a year ago. After some minor arm-twisting by phone and FAX, here is the first Forum published in JLR.     

Architecture and Radiocarbon Dating of the Antelope Creek Focus: A Test of Campbell’S Model

Abstract

Recently, R. G. Campbell has proposed a model accounting for the architectural variability in the Antelope Creek focus of the Texas and Oklahoma panhandles. Although the model was drawn by extension from the adjacent Apishapa focus, it postulates a developmental sequence within Antelope Creek from isolated circular to isolated rectangular to contiguous multiunit structures. Only partial chronological support for the sequence was presented. This paper summarizes Antelope Creek chronological information and tests Campbell’s model. Thirtyeight radiocarbon dates provenienced by the three architectural types are used in the test. The results indicate that the mean date for each of the architectural types yields a sequence opposite of that proposed by Campbell. Although no significant difference was found in the mean age of the isolated circular and isolated rectangular structures, the age of the multiunit structures was significantly earlier at the .05 level. The rejection of Campbell’s model indicates both the dangers of extrapolating data from adjacent cultural manifestations and indicates the complexity of Antelope Creek architectural variation. Alternative factors are proposed but are not explored.     

Alzheimer's Aβ1–40 Peptide Modulates Lipid Synthesis in Neuronal Cultures and Intact Rat Fetal Brain Under Normoxic and Oxidative Stress Conditions

The effect of amyloid (A), the major constituent of the Alzheimer's (AD) brain on lipid metabolism was investigated in cultured nerve cells and in a fetal rat brain model. Differentiated (NGF) and undifferentiated PC12 cells or primary cerebral cell cultures were incubated with [¹⁴C]acetate in the absence or presence of A_1–40. Incorporation of label into lipid species was determined after lipid extraction and TLC separation. Phosphatidylcholine (PC) and phosphatidylserine (PS) synthesis was increased by A_1–40, in a dose dependent manner, an effect which was more pronounced in differentiated PC12 cells. A significant proportion of radioactivity (5–6%) was released into the medium with a radioactivity distribution similar to that of the cellular lipids. Cholesterol and PC were the highest labeled medium lipids. Increasing A_1–40 concentration up to 0.1 g/ml in cerebral cells but not in PC12 cells, caused a relative increase (1.5 fold) in release of PS, while that of PE decreased. Stimulation of PS release may possibly be associated with apoptotic cell death. A_1–40 peptide (5 g) was administered intraperitonealy into rat fetuses (18 days gestation) along with [¹⁴C]acetate (2Ci/fetus). After 24 h, the maternal-fetal blood supply was occluded for 20 min (ischemia) followed by 15 min reperfusion. Fetuses were killed and liver and brain tissue subjected to lipid extraction and radioactivity determination after TLC. A_1–40 peptide increased synthesis of different classes of lipids up to 20–40% in brain tissue compared to controls. Labeling of liver lipids was decreased by A_1–40 by 20–30%. A general decrease in synthesis of lipids was observed after ischemia/reperfusion. Our data suggest that A_1–40 peptide regulates normal lipid biosynthesis but under ischemia it compromises it. The latter finding may confirm the oxidative stress etiology in AD and suggests that A_1–40 modulation of lipid metabolism may have Alzheimer's pathological relevance, particularly at high peptide concentrations.     

Prey Habitat Location by the Cassava Mealybug Predator Exochomus flaviventris: Olfactory Responses to Odor of Plant, Mealybug, Plant–Mealybug Complex, and Plant–Mealybug–Natural Enemy Complex

Exochomus flaviventris Mader is considered to be the most active predator of the cassava mealybug Phenacoccus manihoti Matile–Ferrero in Central Africa. The response of experienced gravid female coccinellids to the odor of cassava plant (var. Zanaga), unparasitized mealybugs, plant–mealybug complex with or without feeding prey (parasitized or not), and plant–mealybug complex with or without conspecific coccinellids was investigated in a Y-tube olfactometer. The odor of uninfested cassava plants was not more attractive than clean air. Dual-choice tests revealed that mealybug-infested plants were preferred to mealybugs alone and mealybug-damaged plants and were the major sources of volatiles that attract females coccinellids to the microhabitat of its prey. The emission of volatile chemicals did not appear to be limited to the infested parts of the plant but did occur systemically throughout the plant. The presence of conspecific coccinellid larvae or adult males did not modify the attractiveness of the mealybug-infested plants. However, when an infested plant with conspecific predator females (alone or with conspecific males) was compared to an infested plant or infested plant with conspecific males, E. flaviventris females showed a preference for the last two sources of odor. The uninfested plant with conspecific males was also preferred to the uninfested plant with conspecific females. In addition, the odor of conspecific males was preferred over that of conspecific females. Female predators preferred the plant infested with unparasitized mealybugs over the plant infested with mealybugs previously parasitized. These results showed that E. flaviventris females use herbivore-induced plant volatiles during foraging and can detect via olfaction the presence of conspecific gravid females and parasitized prey, thus assessing patch suitability from a distance.     

A Study on the Transfer of Iron in Soil–Plant–Animal Continuum Under Semi-arid Environmental Conditions in Sargodha,Pakistan

The present investigation on the iron (Fe) transfer from soil to plant and in turn to animal (cows), as a function of sampling periods was conducted at the Livestock Experimental Station Sargodha, Pakistan which falls under semi-arid conditions. Although the iron transfer from soil to forage increased consistently, the forage Fe content decreased progressively with increase in sampling period. Highest Fe transfer from forage to cow blood plasma was observed during October and lowest during January. The transfer of Fe from forage to animal milk was maximum during the months of October and January and minimum during December. The transfer of Fe to plasma and milk was found to be dependent variably on the growth stage of forage in this investigation. Based on the findings of the present study, it is evident that mineral supplementation with higher Fe availability is urgently warranted to the animals particularly during the months of December and January to enhance plasma Fe in the cows being reared at that livestock farm during the entire grazing period. Thus, obligatory supplementation of Fe to the ruminants is highly recommended. Since the processes involved in iron management system in humans, animals, and plants are basically similar, appropriate elemental management must be provided to the living organisms, otherwise deficient or excessive levels of iron may deteriorate the developing cells of the organisms.     

7 The Molecular Biology of Tryptophan Synthase: A Model for Protein–Protein Interaction

Abstract

The use of plastic produced from non-renewable resources constitutes a major environmental problem of the modern society. Polylactide polymers (PLA) have recently gained enormous attention as one possible substitution of petroleum derived polymers. A prerequisite for high quality PLA production is the provision of optically pure lactic acid, which cannot be obtained by chemical synthesis in an economical way. Microbial fermentation is therefore the commercial option to obtain lactic acid as monomer for PLA production. However, one major economic hurdle for commercial lactic acid production as basis for PLA is the costly separation procedure, which is needed to recover and purify the product from the fermentation broth. Yeasts, such as Saccharomyces cerevisiae (bakers yeast) offer themselves as production organisms because they can tolerate low pH and grow on mineral media what eases the purification of the acid. However, naturally yeasts do not produce lactic acid. By metabolic engineering, ethanol was exchanged with lactic acid as end product of fermentation. A vast amount of effort has been invested into the development of yeasts for lactic acid production since the first paper on this topic by Dequin and process insight. If pH stress is used as basis for DNA microarray analyses, in order to improve the host, what exactly is addressed? Growth? Or productivity? They might be connected, but can be negatively correlated. A better growing strain might not be a better producer. So if the question was growth, the answer might not be what was initially intended (productivity).

A major task for the future is to learn to ask the right questions – a lot of studies intended to lead to better productivity, did lead to interesting results, but NOT to better production strains.

Taking together what we learned from lactic acid production with yeasts, we see a bright future for bulk and fine chemical production with these versatile hosts.     

The Phase Behavior and Organization of Sphingomyelin/Cholesterol Membranes: A Deuterium NMR Study

We have studied the dependence of the phase and domain characteristics of sphingomyelin (SM)/cholesterol model membranes on sterol content and temperature using deuterium nuclear magnetic resonance. NMR spectra of N-palmitoyl(D31)-D-erythro-sphingosylphosphorylcholine (PSM-d31) were taken for temperatures from 25 to 70°C and cholesterol concentrations of 0–40%. Analogous experiments were performed using 1-palmitoyl,2-palmitoyl(D31)-sn-glycero-3-phosphocholine (DPPC-d31)/cholesterol membranes to carefully compare the data obtained using palmitoyl chains that have similar “kinked” conformations. The constructed phase diagrams exhibit both solid-ordered (so) + liquid-ordered (lo) and liquid-disordered (ld) + lo phase-coexistence regions with a clear three-phase line. Macroscopic (micron-sized) coexistence of ld and lo phases was not observed; instead, line-broadening in the ld+lo region was characterized by intermediate exchange of lipids between the two types of domains. The length scales associated with the domains were estimated to be 75–150 nm for PSM-d31/cholesterol and DPPC-d31/cholesterol model membranes.     

Localization of Cholesterol and Fatty Acid in a Model Lipid Membrane: A?Neutron Diffraction Approach

The intercellular lipid matrix of the skin’s stratum corneum serves to protect the body against desiccation and simultaneously limits the passage of drugs and other xenobiotics into the body. The matrix is made up of ceramides, free fatty acids, and cholesterol, which are organized as two coexisting crystalline lamellar phases. In studies reported here, we sought to use the technique of neutron diffraction, together with the device of isotopic (H/D) substitution, to determine the molecular architecture of the lamellar phase having a repeat distance of 53.9 ± 0.3 Å. Using hydrogenous samples as well as samples incorporating perdeuterated (C24:0) fatty acids and selectively deuterated cholesterol, the diffraction data obtained were used to construct neutron scattering length density profiles. By this means, the locations within the unit cell were determined for the cholesterol and fatty acids. The cholesterol headgroup was found to lie slightly inward from the unit cell boundary and the tail of the molecule located 6.2 ± 0.2 Å from the unit cell center. The fatty acid headgroups were located at the unit cell boundary with their acyl chains straddling the unit cell center. Based on these results, a molecular model is proposed for the arrangement of the lipids within the unit cell.     

Glioma Cell and Astrocyte Co-cultures As a Model to Study Tumor–Tissue Interactions: A Review of Methods

Astrocytes are a dominant cell type that envelopes the glioma bed. Typically, that is followed by formation of contacts between astrocytes and glioma cells and accompanied by change in astrocyte phenotype, a phenomenon known as a ‘reactive astrogliosis.’ Generally considered glioma-promoting, astrocytes have many controversial peculiarities in communication with tumor cells, which need thorough examination in vitro. This review is devoted to in vitro co-culture studies of glioma cells and astrocytes. Firstly, we list several fundamental works which allow understanding the modalities of co-culturing. Cell-to-cell interactions between astrocytes and glioma cells, the roles of astrocytes in tumor metabolism, and glioma-related angiogenesis are reviewed. In the review, we also discuss communications between glioma stem cells and astrocytes. Co-cultures of glioma cells and astrocytes are used for studying anti-glioma treatment approaches. We also enumerate surgical, chemotherapeutic, and radiotherapeutic methods assessed in co-culture experiments. In conclusion, we underline collisions in the field and point out the role of the co-cultures for neurobiological studies.     

Metabolism of Yarrowia lipolytica Grown on Ethanol under Conditions Promoting the Production of α-Ketoglutaric and Citric Acids: A Comparative Study of the Central Metabolism Enzymes

A comparative study of the enzymes of tricarboxylic acid (TCA) and glyoxylate cycles in the mutant Yarrowia lipolytica strain N1 capable of producing -ketoglutaric acid (KGA) and citric acid showed that almost all enzymes of the TCA cycle are more active under conditions promoting the production of KGA. The only exception was citrate synthase, whose activity was higher in yeast cells producing citric acid. The production of both acids was accompanied by suppression of the glyoxylate cycle enzymes. The activities of malate dehydrogenase, aconitase, NADP-dependent isocitrate dehydrogenase, and fumarase were higher in cells producing KGA than in cells producing citric acid.     

Augmented Phytoextraction of Lead (Pb2+)-Polluted Soils: A Comparative Study of the Effectiveness of Plant Growth Regulators,EDTA, and Plant Growth–Promoting Rhizobacteria

Abstract

The role of gibberellic acid (GA3), indole-3-acetic acid (IAA), plant growth–promoting bacteria (Rhizobium and Azotobacter), and a synthetic chelator (EDTA; ethylenediaminetetraacetic acid) in lead (Pb) phytoextraction was evaluated using Parthenium hysterophorus (dicot, unpalatable noncrop) and Zea mays (monocot food/forage crop) plants at the flowering stage. Various plant parts were analyzed by atomic absorption/flame spectrophotometer for their Pb content. Both plant growth regulators and both growth-promoting bacteria significantly increased the plant growth in Pb-polluted soils, whereas EDTA significantly decreased growth and biomass of both plants. EDTA increased the Pb uptake (μg g^?1 dry biomass), but the total plant Pb accumulation was decreased. GA3 and IAA significantly increased both uptake and translocation, and the maximum total Pb in the entire plant of Parthenium was found with GA3 foliar spray, whereas in Z. mays the total Pb was maximum in the plant treated with GA3 in combination with EDTA, followed by the GA3 foliar spray treatment. Overall, the GA3 foliar application showed superior response compared with all other treatments. Further research is recommended to observe the role of endogenous GA3 levels in correlation with metal phytoextraction in different plants.     

Amphipol-Trapped ExbB–ExbD Membrane Protein Complex from Escherichia coli: A Biochemical and Structural Case Study

Nutrient import across Gram-negative bacteria’s outer membrane is powered by the proton-motive force, delivered by the cytoplasmic membrane protein complex ExbB–ExbD–TonB. Having purified the ExbB₄–ExbD₂ complex in the detergent dodecyl maltoside, we substituted amphipol A8-35 for detergent, forming a water-soluble membrane protein/amphipol complex. Properties of the ExbB₄–ExbD₂ complex in detergent or in amphipols were compared by gel electrophoresis, size exclusion chromatography, asymmetric flow field-flow fractionation, thermal stability assays, and electron microscopy. Bound detergent and fluorescently labeled amphipol were assayed quantitatively by 1D NMR and analytical ultracentrifugation, respectively. The structural arrangement of ExbB₄–ExbD₂ was examined by EM, small-angle X-ray scattering, and small-angle neutron scattering using a deuterated amphipol. The amphipol-trapped ExbB₄–ExbD₂ complex is slightly larger than its detergent-solubilized counterpart. We also investigated a different oligomeric form of the two proteins, ExbB₆–ExbD₄, and propose a structural arrangement of its transmembrane α-helical domains.