Dielectric spectroscopy of plant protoplasts

The relative permittivity and conductivity of the mesophyll protoplasts isolated from Brassica campestris leaves and Tulipa gesneriana petals were measured over a frequency range from 1kHz to 500 MHz.These protoplasts showed a broad dielectric dispersion, which was composed of three subdispersions, termed β₁-, β₂-, and β₃-dispersion in increasing order of frequency.The three subdispersions were assigned to the Maxwell-Wagner dispersion caused by charging processes at the interfaces of the surface and internal membranes; the plasma membrane, the tonoplast, and the membranes of cytoplasmic organelles (e.g., chloroplasts, granules, etc) primarily contribute to the β₁-, β₂-, and β₃-dispersion, respectively. The whole dielectric dispersion curve was satisfactorily interpreted in terms of a spherical cell model taking a large vacuole and cytoplasmic organelles into account. Using this model the capacitances of the plasma membranes and the tonoplasts were estimated to be 0.6-0.7 μF/cm² and 0.9-1.0 μF/cm², respectively.     

Nitrophenol Chemi-Sensor and Active Solar Photocatalyst Based on Spinel Hetaerolite Nanoparticles

In this contribution, a significant catalyst based on spinel ZnMn₂O₄ composite nanoparticles has been developed for electro-catalysis of nitrophenol and photo-catalysis of brilliant cresyl blue. ZnMn₂O₄ composite (hetaerolite) nanoparticles were prepared by easy low temperature hydrothermal procedure and structurally characterized by X-ray powder diffraction (XRD), field emission scanning electron microscopy (FESEM), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR) and UV-visible spectroscopy which illustrate that the prepared material is optical active and composed of well crystalline body-centered tetragonal nanoparticles with average size of ∼38±10 nm. Hetaerolite nanoparticles were applied for the advancement of a nitrophenol sensor which exhibited high sensitivity (1.500 µAcm⁻² mM⁻¹), stability, repeatability and lower limit of detection (20.0 µM) in short response time (10 sec). Moreover, hetaerolite nanoparticles executed high solar photo-catalytic degradation when applied to brilliant cresyl blue under visible light.     

Design and In Vitro Evaluation of Finasteride-Loaded Liquid Crystalline Nanoparticles for Topical Delivery

In this study, liquid crystalline nanoparticles (LCN) have been proposed as new carrier for topical delivery of finasteride (FNS) in the treatment of androgenetic alopecia. To evaluate the potential of this nanocarrier, FNS-loaded LCN was prepared by ultrasonication method and characterized for size, shape, in vitro release, and skin permeation–retention properties. The particle size ranged from 153.8 to 170.2 nm with a cubical shape and exhibited controlled release profile with less than 20% of the drug released in the first 24 h. The release profile was significantly altered with addition of different additives. Formulation with lower monoolein exhibited higher skin permeation with a flux rate of 0.061 ± 0.005 μg cm⁻² h⁻¹ in 24 h. The permeation however, significantly increased with glycerol, propylene glycol, and polyethylene glycol 400, while it declined for the addition of oleic acid. A similar trend was observed with skin retention study. In conclusion, FNS-loaded LCN could be advocated as a viable alternative for oral administration of the drug.Key words: androgenetic alopecia, finasteride, liquid crystalline nanoparticles, release, skin permeation–retention     

Estrogen induces two distinct cholesterol crystallization pathways by activating ERα and GPR30 in female mice

To distinguish the lithogenic effect of the classical estrogen receptor α (ERα) from that of the G protein-coupled receptor 30 (GPR30), a new estrogen receptor, on estrogen-induced gallstones, we investigated the entire spectrum of cholesterol crystallization pathways and sequences during the early stage of gallstone formation in gallbladder bile of ovariectomized female wild-type, GPR30(^−/−), ERα(^−/−), and GPR30(^−/−)/ERα(^−/−) mice treated with 17β-estradiol (E₂) at 6 µg/day and fed a lithogenic diet for 12 days. E₂ disrupted biliary cholesterol and bile salt metabolism through ERα and GPR30, leading to supersaturated bile and predisposing to the precipitation of cholesterol monohydrate crystals. In GPR30(^−/−) mice, arc-like and tubular crystals formed first, followed by classical parallelogram-shaped cholesterol monohydrate crystals. In ERα(^−/−) mice, precipitation of lamellar liquid crystals, typified by birefringent multilamellar vesicles, appeared earlier than cholesterol monohydrate crystals. Both crystallization pathways were accelerated in wild-type mice with the activation of GPR30 and ERα by E₂. However, cholesterol crystallization was drastically retarded in GPR30(^−/−)/ERα(^−/−) mice. We concluded that E₂ activates GPR30 and ERα to produce liquid crystalline versus anhydrous crystalline metastable intermediates evolving to cholesterol monohydrate crystals from supersaturated bile. GPR30 produces a synergistic lithogenic action with ERα to enhance E₂-induced gallstone formation.     

A study of lipid bilayer membrane stability using precise measurements of specific capacitance

A method is described for measuring the specific capacitance (C_m) of lipid bilayer membranes with an estimated experimental error of only 1%. The gross capacitance was measured with an AC Wheatstone bridge and a photographic technique was used to determine the area of thin membrane. The results of measurements on oxidized cholesterol-decane membranes formed in 1 × 10^-2 M KCl show that C_m depends upon temperature, voltage, time, and the age of the bulk membrane solutions. For a freshly thinned membrane (from 5 week old solution), C_m increases exponentially from an initial value of 0.432 ±0.021 (SD) μF/cm² with a time constant of ~15 min. A 100 mv potential applied across the membrane for 10-20 min prior to making measurements eliminated this time dependence and produced final-state membranes. C_m of final-state membranes depends upon applied voltage (V_a) and obeys the equation C_m = C₀ + βV_a² where V_a V_DC + V^rms_AC. C₀ and β depend upon temperature; C₀ decreases linearly with temperature while β increases linearly. At 20°C, C₀ = 0.559 ±0.01 (SD) μF/cm² and β = 0.0123 ±0.0036 (SD) (μF/cm²)/(mv²) and at 34°C, C₀ = 0.472 ±0.01 and β = 0.0382 ±0.0039. These variations in C_m are interpreted as resulting from thickness changes. The possibility that they result from diffuse layer and/or membrane dielectric phenomena is discussed and found to be unlikely. The results are discussed in terms of membrane stability by constructing hypothetical potential energy vs. thickness curves.     

Oscillatory Flow-induced Proliferation of Osteoblast-like Cells Is Mediated by ��v��3 and ��1 Integrins through Synergistic Interactions of Focal Adhesion Kinase and Shc with Phosphatidylinositol 3-Kinase and the Akt/mTOR/p70S6K Pathway

Interstitial flow in and around bone tissue is oscillatory in nature and affects the mechanical microenvironment for bone cell growth and formation. We investigated the role of oscillatory shear stress (OSS) in modulating the proliferation of human osteoblast-like MG63 cells and its underlying mechanisms. Application of OSS (0.5 ± 4 dynes/cm²) to MG63 cells induced sustained activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR/p70S6K (p70S6 kinase) signaling cascades and hence cell proliferation, which was accompanied by increased expression of cyclins A and D1, cyclin-dependent protein kinases-2, -4, and -6, and bone formation-related genes (c-fos, Egr-1, and Cox-2) and decreased expression of p21^CIP1 and p27^KIP1. OSS-induced activation of PI3K/Akt/mTOR/p70S6K and cell proliferation were inhibited by specific antibodies or small interference RNAs of α_vβ₃ and β₁ integrins and by dominant-negative mutants of Shc (Shc-SH2) and focal adhesion kinase (FAK) (FAK(F397Y)). Co-immunoprecipitation assay showed that OSS induces sustained increases in association of Shc and FAK with α_vβ₃ and β₁ integrins and PI3K subunit p85, which were abolished by transfecting the cells with FAK(F397Y) or Shc-SH2. OSS also induced sustained activation of ERK, which was inhibited by the specific PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and was required for OSS-induced activation of mTOR/p70S6K and proliferation in MG63 cells. Our findings provide insights into the mechanisms by which OSS induces osteoblast-like cell proliferation through activation of α_vβ₃ and β₁ integrins and synergistic interactions of FAK and Shc with PI3K, leading to the modulation of downstream ERK and Akt/mTOR/p70S6K pathways.     

Enhanced Topical Delivery of Finasteride Using Glyceryl Monooleate-Based Liquid Crystalline Nanoparticles Stabilized by Cremophor Surfactants

The aim of this study was to investigate the capability of two surfactants, Cremophor RH 40 (RH) and Cremophor EL (EL), to prepare liquid crystalline nanoparticles (LCN) and to study its influence on the topical delivery of finasteride (FNS). FNS-loaded LCN was formulated with the two surfactants and characterized for size distribution, morphology, entrapment efficiency, in vitro drug release, and skin permeation/retention. Influence of FNS-loaded LCN on the conformational changes on porcine skin was also studied using attenuated total reflectance Fourier-transform infrared spectroscopy. Transmission electron microscopical image confirmed the formation of LCN. The average particle size of formulations was in the range of 165.1–208.6 and 153.7–243.0 nm, respectively. The formulations prepared with higher surfactant concentrations showed faster release and significantly increased skin permeation. Specifically, LCN prepared with RH 2.5% presented higher permeation flux (0.100 ± 0.005 μgcm⁻²h⁻¹) compared with lower concentration (0.029 ± 0.007 μgcm⁻²h⁻¹). Typical spectral bands of lipid matrix of porcine skin were shifted to higher wavenumber, indicating increased degree of disorder of the lipid acyl chains which might cause fluidity increase of stratum corneum. Taken together, Cremophor surfactants exhibited a promising potential to stabilize the LCN and significantly augmented the skin permeation of FNS.KEY WORDS: Cremophor, finasteride, liquid crystalline nanoparticles, skin permeation–retention     

Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β? Interface

In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABA_ARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABA_AR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343–19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABA_AR responses. In the α1β3γ2 GABA_AR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ⁺-β⁻ subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β⁺-α⁻ subunit interfaces. GABA inhibits S-[³H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2–15′) in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[³H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ⁺-β⁻ site, based upon the distance in GABA_AR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABA_AR function and provide a first demonstration that an intersubunit-binding site in the GABA_AR transmembrane domain binds negative and positive allosteric modulators.     

Structure of collagen receptor integrin α(1)I domain carrying the activating mutation E317A

We have analyzed the structure and function of the integrin α₁I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α₁I C139S/E317A was a higher avidity collagen binder than α₁I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α₁I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α₂I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α₂I structure, has not changed its position in the activated α₁I variant. During the integrin activation, Glu³³⁵ on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the β₁ subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu³³⁵. This indicates that the stabilization of helix 7 into its downward position is not required if the α₁ MIDAS is already open. To conclude, the activated α₁I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg²⁸⁷-Glu³¹⁷ ion pair has just broken during the integrin activation.     

Cerium oxide and iron oxide nanoparticles abolish the antibacterial activity of ciprofloxacin against gram positive and gram negative biofilm bacteria

Metal oxide nanoparticles have been suggested as good candidates for the development of antibacterial agents. Cerium oxide (CeO₂) and iron oxide (Fe₂O₃) nanoparticles have been utilized in a number of biomedical applications. Here, the antibacterial activity of CeO₂ and Fe₂O₃ nanoparticles were evaluated on a panel of gram positive and gram negative bacteria in both the planktonic and biofilm cultures. Additionally, the effect of combining CeO₂ and Fe₂O₃ nanoparticles with the broad spectrum antibiotic ciprofloxacin on tested bacteria was investigated. Thus, minimum inhibitory concentrations (MICs) of CeO₂ and Fe₂O₃ nanoparticles that are required to inhibit bacterial planktonic growth and bacterial biofilm, were evaluated, and were compared to the MICs of the broad spectrum antibiotic ciprofloxacin alone or in the presence of CeO₂ and Fe₂O₃ nanoparticles. Results of this study show that both CeO₂ and Fe₂O₃ nanoparticles fail to inhibit bacterial growth and biofilm biomass for all the bacterial strains tested. Moreover, adding CeO₂ or Fe₂O₃ nanoparticles to the broad spectrum antibiotic ciprofloxacin almost abolished its antibacterial activity. Results of this study suggest that CeO₂ and Fe₂O₃ nanoparticles are not good candidates as antibacterial agents, and they could interfere with the activity of important antibiotics.     

Fate and Phytotoxicity of CeO2 Nanoparticles on Lettuce Cultured in the Potting Soil Environment

Cerium oxide nanoparticles (CeO₂ NPs) have been shown to have significant interactions in plants. Previous study reported the specific-species phytotoxicity of CeO₂ NPs by lettuce (Lactuca sativa), but their physiological impacts and vivo biotransformation are not yet well understood, especially in relative realistic environment. Butterhead lettuce were germinated and grown in potting soil for 30 days cultivation with treatments of 0, 50, 100, 1000 mg CeO₂ NPs per kg soil. Results showed that lettuce in 100 mg·kg^-1 treated groups grew significantly faster than others, but significantly increased nitrate content. The lower concentrations treatment had no impact on plant growth, compared with the control. However, the higher concentration treatment significantly deterred plant growth and biomass production. The stress response of lettuce plants, such as Superoxide dismutase (SOD), Peroxidase (POD), Malondialdehyde(MDA) activity was disrupted by 1000 mg·kg^-1 CeO₂ NPs treatment. In addition, the presence of Ce (III) in the roots of butterhead lettuce explained the reason of CeO₂ NPs phytotoxicity. These findings demonstrate CeO₂ NPs modification of nutritional quality, antioxidant defense system, the possible transfer into the food chain and biotransformation in vivo.     

Interfacial Kinetic and Binding Properties of Mammalian Group IVB Phospholipase A2 (cPLA2β) and Comparison with the Other cPLA2 Isoforms

The cytosolic (group IV) phospholipase A₂ (cPLA₂s) family contains six members. We have prepared recombinant proteins for human α, mouse β, human γ, human δ, human ϵ, and mouse ζ cPLA₂s and have studied their interfacial kinetic and binding properties in vitro. Mouse cPLA₂β action on phosphatidylcholine vesicles is activated by anionic phosphoinositides and cardiolipin but displays a requirement for Ca²⁺ only in the presence of cardiolipin. This activation pattern is explained by the effects of anionic phospholipids and Ca²⁺ on the interfacial binding of mouse cPLA₂β and its C2 domain to vesicles. Ca²⁺-dependent binding of mouse cPLA₂β to cardiolipin-containing vesicles requires a patch of basic residues near the Ca²⁺-binding surface loops of the C2 domain, but binding to phosphoinositide-containing vesicles does not depend on any specific cluster of basic residues. Human cPLA₂δ also displays Ca²⁺- and cardiolipin-enhanced interfacial binding and activity. The lysophospholipase, phospholipase A₁, and phospholipase A₂ activities of the full set of mammalian cPLA₂s were quantified. The relative level of these activities is very different among the isoforms, and human cPLA₂δ stands out as having relatively high phospholipase A₁ activity. We also tested the susceptibility of all cPLA₂ family members to a panel of previously reported inhibitors of human cPLA₂α and analogs of these compounds. This led to the discovery of a potent and selective inhibitor of mouse cPLA₂β. These in vitro studies help determine the regulation and function of the cPLA₂ family members.     

Characteristics of an R-Phycoerythrin with Two γ Subunits Prepared from Red Macroalga Polysiphonia urceolata

An R-phycoerythrin (R-PE) was isolated by gel filtrations on Sepharose CL-4B and Sephadex G-150 from the phycobiliprotein extract of the marine red macroalga Polysiphonia urceolata Grev and further purified by ion exchange chromatography on DEAE-Sepharose Fast Flow. The purified R-PE showed three absorption peaks at 498 nm, 538 nm, 566 nm and one fluorescent emission maximum at 577 nm. Although the R-PE showed a single band on the examination by native PAGE, it exhibited two very close bands at pH about 4.7 in native isoelectric focusing (IEF). Polypeptide analysis of the R-PE demonstrated that it contained four chromophore-carrying subunits, α^18.2, β^20.6, γ^31.6 (γ''), γ^34.6 (γ), and no colorless polypeptide; its subunit composition was 6α^18.2:6β^20.6:1 γ^31.6:2γ^34.6. The α and β subunits were distributed within a acidic pH range from 5.0 to 6.0 in denaturing IEF and the γ subunits were in a basic pH range from 7.6 to 8.1. These results reveal that the prepared R-PE may exist in two hexamers of γ (αβ)₃ γ (αβ)₃γ'' and γ (αβ)₃ γ''(αβ)₃ γ and that the R-PE participate in the rod domain assembly of P. urceolata phycobilisomes by stacking each of its trimer (αβ)₃ face-to-face with the aid of one γ subunit (γ or γ'').     

Stable Seamless Interfaces and Rapid Ionic Conductivity of Ca–CeO2/LiTFSI/PEO Composite Electrolyte for High‐Rate and High‐Voltage All‐Solid‐State Battery

Stable and seamless interfaces among solid components in all‐solid‐state batteries (ASSBs) are crucial for high ionic conductivity and high rate performance. This can be achieved by the combination of functional inorganic material and flexible polymer solid electrolyte. In this work, a flexible all‐solid‐state composite electrolyte is synthesized based on oxygen‐vacancy‐rich Ca‐doped CeO₂ (Ca–CeO₂) nanotube, lithium bis(trifluoromethanesulfonyl)imide (LiTFSI), and poly(ethylene oxide) (PEO), namely Ca–CeO₂/LiTFSI/PEO. Ca–CeO₂ nanotubes play a key role in enhancing the ionic conductivity and mechanical strength while the PEO offers flexibility and assures the stable seamless contact between the solid electrolyte and the electrodes in ASSBs. The as‐prepared electrolyte exhibits high ionic conductivity of 1.3 × 10^?4 S cm^?1 at 60 °C, a high lithium ion transference number of 0.453, and high‐voltage stability. More importantly, various electrochemical characterizations and density functional theory (DFT) calculations reveal that Ca–CeO₂ helps dissociate LiTFSI, produce free Li ions, and therefore enhance ionic conductivity. The ASSBs based on the as‐prepared Ca–CeO₂/LiTFSI/PEO composite electrolyte deliver high‐rate capability and high‐voltage stability.     

Integration of Fourier Transform Infrared Spectroscopy,Fluorescence Spectroscopy,Steady-state Kinetics and Molecular Dynamics Simulations of Gαi1 Distinguishes between the GTP Hydrolysis and GDP Release Mechanism

Gα subunits are central molecular switches in cells. They are activated by G protein-coupled receptors that exchange GDP for GTP, similar to small GTPase activation mechanisms. Gα subunits are turned off by GTP hydrolysis. For the first time we employed time-resolved FTIR difference spectroscopy to investigate the molecular reaction mechanisms of Gα_i1. FTIR spectroscopy is a powerful tool that monitors reactions label free with high spatio-temporal resolution. In contrast to common multiple turnover assays, FTIR spectroscopy depicts the single turnover GTPase reaction without nucleotide exchange/Mg²⁺ binding bias. Global fit analysis resulted in one apparent rate constant of 0.02 s⁻¹ at 15 °C. Isotopic labeling was applied to assign the individual phosphate vibrations for α-, β-, and γ-GTP (1243, 1224, and 1156 cm⁻¹, respectively), α- and β-GDP (1214 and 1134/1103 cm⁻¹, respectively), and free phosphate (1078/991 cm⁻¹). In contrast to Ras·GAP catalysis, the bond breakage of the β-γ-phosphate but not the P_i release is rate-limiting in the GTPase reaction. Complementary common GTPase assays were used. Reversed phase HPLC provided multiple turnover rates and tryptophan fluorescence provided nucleotide exchange rates. Experiments were complemented by molecular dynamics simulations. This broad approach provided detailed insights at atomic resolution and allows now to identify key residues of Gα_i1 in GTP hydrolysis and nucleotide exchange. Mutants of the intrinsic arginine finger (Gα_i1-R178S) affected exclusively the hydrolysis reaction. The effect of nucleotide binding (Gα_i1-D272N) and Ras-like/all-α interface coordination (Gα_i1-D229N/Gα_i1-D231N) on the nucleotide exchange reaction was furthermore elucidated.     

Phosphatidylinositol-4,5-Bisphosphate Enhances Anionic Lipid Demixing by the C2 Domain of PKCα

The C2 domain of PKCα (C2α) induces fluorescence self-quenching of NBD-PS in the presence of Ca²⁺, which is interpreted as the demixing of phosphatidylserine from a mixture of this phospholipid with phosphatidylcholine. Self-quenching of NBD-PS was considerably increased when phosphatidylinositol-4,5-bisphosphate (PIP₂) was present in the membrane. When PIP₂ was the labeled phospholipid, in the form of TopFluor-PIP₂, fluorescence self-quenching induced by the C2 domain was also observed, but this was dependent on the presence of phosphatidylserine. An independent indication of the phospholipid demixing effect given by the C2α domain was obtained by using ²H-NMR, since a shift of the transition temperature of deuterated phosphatidylcholine was observed as a consequence of the addition of the C2α domain, but only in the presence of PIP₂. The demixing induced by the C2α domain may have a physiological significance since it means that the binding of PKCα to membranes is accompanied by the formation of domains enriched in activating lipids, like phosphatidylserine and PIP₂. The formation of these domains may enhance the activation of the enzyme when it binds to membranes containing phosphatidylserine and PIP₂.     

Further studies on the isolation and properties of alpha-chain sub-units of haemoglobin

1. α^A-Chain sub-units have been isolated from neutral haemolysates containing certain unstable β-chain haemoglobin variants. They are identical, in electrophoretic properties, tryptic peptide analyses and their ability to recombine with Hb-β^A₄ at neutral pH, with the α^A-chain sub-units isolated from Hb-A at acid pH. Abnormal α^G-chain sub-units have been prepared from haemolysates containing the α-chain variant Hb-G. 2. α^A-Chain sub-units prepared by either method are eluted from Sephadex at low haemoglobin concentration as monomer sub-units. The elution volume, sedimentation coefficient and apparent molecular weight are, however, concentration-dependent, suggesting an equilibrium between monomers and higher polymers with the former as the predominant species. Elevated temperatures cause the α^A-chain sub-unit to aggregate and denature. 3. The product of the reaction between the α^A-chain sub-unit and Hb-β^A₄ has been characterized as Hb-A. Only Hb-A and Hb-G can be detected as the products when a mixture of α^A- and α^G-chain sub-units reacts with Hb-β^A₄. The absence of the species α^Aα^Gβ^A₂ can be explained in terms of the rapid equilibrium between whole and half haemoglobin molecules and the reassortment of αβ sub-units during the separation process.     

Essential Role of the EF-hand Domain in Targeting Sperm Phospholipase Cζ to Membrane Phosphatidylinositol 4,5-Bisphosphate (PIP2)

Sperm-specific phospholipase C-ζ (PLCζ) is widely considered to be the physiological stimulus that triggers intracellular Ca²⁺ oscillations and egg activation during mammalian fertilization. Although PLCζ is structurally similar to PLCδ1, it lacks a pleckstrin homology domain, and it remains unclear how PLCζ targets its phosphatidylinositol 4,5-bisphosphate (PIP₂) membrane substrate. Recently, the PLCδ1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes. Those critical cationic EF-hand residues in PLCδ1 are notably conserved in PLCζ. We investigated the potential role of these conserved cationic residues in PLCζ by generating a series of mutants that sequentially neutralized three positively charged residues (Lys-49, Lys-53, and Arg-57) within the mouse PLCζ EF-hand domain. Microinjection of the PLCζ EF-hand mutants into mouse eggs enabled their Ca²⁺ oscillation inducing activities to be compared with wild-type PLCζ. Furthermore, the mutant proteins were purified, and the in vitro PIP₂ hydrolysis and binding properties were monitored. Our analysis suggests that PLCζ binds significantly to PIP₂, but not to phosphatidic acid or phosphatidylserine, and that sequential reduction of the net positive charge within the first EF-hand domain of PLCζ significantly alters in vivo Ca²⁺ oscillation inducing activity and in vitro interaction with PIP₂ without affecting its Ca²⁺ sensitivity. Our findings are consistent with theoretical predictions provided by a mathematical model that links oocyte Ca²⁺ frequency and the binding ability of different PLCζ mutants to PIP₂. Moreover, a PLCζ mutant with mutations in the cationic residues within the first EF-hand domain and the XY linker region dramatically reduces the binding of PLCζ to PIP₂, leading to complete abolishment of its Ca²⁺ oscillation inducing activity.     

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.     

Variability in Bioreactivity Linked to Changes in Size and Zeta Potential of Diesel Exhaust Particles in Human Immune Cells

Acting as fuel combustion catalysts to increase fuel economy, cerium dioxide (ceria, CeO₂) nanoparticles have been used in Europe as diesel fuel additives (Envirox™). We attempted to examine the effects of particles emitted from a diesel engine burning either diesel (diesel exhaust particles, DEP) or diesel doped with various concentrations of CeO₂ (DEP-Env) on innate immune responses in THP-1 and primary human peripheral blood mononuclear cells (PBMC). Batches of DEP and DEP-Env were obtained on three separate occasions using identical collection and extraction protocols with the aim of determining the reproducibility of particles generated at different times. However, we observed significant differences in size and surface charge (zeta potential) of the DEP and DEP-Env across the three batches. We also observed that exposure of THP-1 cells and PBMC to identical concentrations of DEP and DEP-Env from the three batches resulted in statistically significant differences in bioreactivity as determined by IL-1β, TNF-α, IL-6, IFN-γ, and IL-12p40 mRNA (by qRT-PCR) and protein expression (by ELISPOT assays). Importantly, bioreactivity was noted in very tight ranges of DEP size (60 to 120 nm) and zeta potential (−37 to −41 mV). Thus, these physical properties of DEP and DEP-Env were found to be the primary determinants of the bioreactivity measured in this study. Our findings also point to the potential risk of over- or under- estimation of expected bioreactivity effects (and by inference of public health risks) from bulk DEP use without taking into account potential batch-to-batch variations in physical (and possibly chemical) properties.