Pore Formation in Lipid Bilayer Membranes made of Phosphatidylcholine and Cholesterol Followed by Means of Constant Current

This paper describes the application of chronopotentiometry to lipid bilayer research. The experiments were performed on bilayer lipid membranes composed of phosphatidylcholine and cholesterol and formed using the painting technique. Chronopotentiometric (U = f(t)) measurements were used to determine the membrane capacitance, resistance, and breakdown voltage as well as pore conductance and diameter.     

Kinetic Theory Model for Ion Movement through Biological Membranes: I. Field-Dependent Conductances in the Presence of Solution Symmetry

A model for ion movement through specialized sites in the plasma membrane is presented and analyzed using techniques from nonequilibrium kinetic theory. It is assumed that ions traversing these specialized regions interact with membrane molecules through central conservative forces. The membrane molecules are approximated as massive spherical scattering centers so that ionic fractional energy losses per collision are much less than one. Equations for steady-state membrane ionic currents and conductances as functions of externally applied electric field strength are derived and numerically analyzed, under the restriction of identical solutions on each size of the membrane and constant electric fields within the membrane. The analysis is carried through for a number of idealized ion-membrane molecule central force interactions. For any interaction leading to a velocity-dependent ion-membrane molecule collision frequency, the membrane chord conductance is a function of the externally applied electric field. Interactions leading to a collision frequency that is an increasing (decreasing) function of ionic velocity are characterized by chord conductances that are decreasing (increasing) functions of field strength. For ion-neutral molecule interactions, the conductance is such a rapidly decreasing function of field strength that the slope conductance becomes negative for all field strengths above a certain value.     

Pleckstrin Associates with Plasma Membranes and Induces the Formation of Membrane Projections: Requirements for Phosphorylation and the NH2-terminal PH Domain

Pleckstrin homology (PH) domains are sequences of ~100 amino acids that form “modules” that have been proposed to facilitate protein/protein or protein/lipid interactions. Pleckstrin, first described as a substrate for protein kinase C in platelets and leukocytes, is composed of two PH domains, one at each end of the molecule, flanking an intervening sequence of 147 residues. Evidence is accumulating to support the hypothesis that PH domains are structural motifs that target molecules to membranes, perhaps through interactions with Gβγ or phosphatidylinositol 4,5-bisphosphate (PIP₂), two putative PH domain ligands. In the present studies, we show that pleckstrin associates with membranes in human platelets. We further demonstrate that, in transfected Cos-1 cells, pleckstrin associates with peripheral membrane ruffles and dorsal membrane projections. This association depends on phosphorylation of pleckstrin and requires the presence of its NH₂-terminal, but not its COOH-terminal, PH domain. Moreover, PH domains from other molecules cannot effectively substitute for pleckstrin's NH₂terminal PH domain in directing membrane localization. Lastly, we show that wild-type pleckstrin actually promotes the formation of membrane projections from the dorsal surface of transfected cells, and that this morphologic change is similarly PH domain dependent. Since we have shown previously that pleckstrin-mediated inhibition of PIP₂ metabolism by phospholipase C or phosphatidylinositol 3-kinase also requires pleckstrin phosphorylation and an intact NH₂-terminal PH domain, these results suggest that: (a) pleckstrin's NH₂terminal PH domain may regulate pleckstrin's activity by targeting it to specific areas within the cell membrane; and (b) pleckstrin may affect membrane structure, perhaps via interactions with PIP₂ and/or other membrane-bound ligands.     

Photoelectron Quantum Yields of Hemin, Hemoglobin, and Apohemoglobin: Possible Applications to Photoelectron Microscopy of Heme Proteins in Biological Membranes

Hemoglobin is examined as a model system for intrinsic photoelectron labeling experiments. The absolute photoelectron quantum yields of hemin, hemoglobin, and apohemoglobin thin films were measured in the 180-230 nm wavelength region. Hemin exhibits a quantum yield of approximately 6 × 10^-4 electrons per incident photon at 180 nm, 9 × 10^-5 electrons per incident photon at 210 nm, and 2 × 10^-6 electrons per incident photon at 230 nm. At all wavelengths the hemin curve lies approximately a factor of 20 above that of hemoglobin and two orders of magnitude above that of apohemoglobin. High image contrast is observed between hemin and apohemoglobin in low magnification photoelectron micrographs, suggesting the feasibility of intrinsic labeling studies involving heme proteins. The quantum yield of hemoglobin is discussed in terms of linear contributions from heme groups and protein weighted by their relative surface areas. The fractional surface areas based on the known structure of hemoglobin are consistent with values derived from the quantum yields of hemin and apohemoglobin.     

Intraguild Predation and Feeding Preferences in Three Species of Phytoseiid Mite Used for Biological Control

The ecological impact of introduced biological control agents on native species of arthropods is a matter of considerable debate. This study investigated the ability of the non-native predatory mite Neoseiulus californicus to feed on the native Typhlodromus pyri and vice versa, as both species now co-occur in UK orchards. Typhlodromips montdorensis is a candidate for introduction into the UK as a glasshouse biological control agent. The ability of T. montdorensis to feed on the widely used N. californicus was investigated to identify possible intraguild predation, which might influence the effectiveness of either or both species as predators of Tetranychus urticae. Both N. californicus and T. pyri consumed larval stages of each other, but in choice experiments both showed a preference for T. urticae. Both N. californicus and T. montdorensis also fed on each other, but whereas N. californicus again showed a preference for T. urticae, T. montdorensis fed equally on T. urticae and N. californicus. Interactions between N. californicus and T. pyri and N. californicus and T. montdorensis are discussed in relation to their effectiveness as biological control agents in the glasshouse and the natural control of spider mite in the field.     

Peroxidase and Tyrosinase Are Present in Secondary Lysosomes That Degrade Photosensory Membranes of the Crayfish Photoreceptor: Possible Role in Pigment Granule Formation

Three enzymes (acid phosphatase, peroxidase, and tyrosinase) were localized by electron microscopy within the retina of crayfish Orconectes limosus. Peroxidase activity was observed only in lamellar bodies, which are secondary lysosomes and degrade photosensory membrane. After H₂O₂ was omitted from the reaction medium, peroxidase activity in lamellar bodies was partly inhibited but was not missing completely. After addition of sodium pyruvate, which inhibits endogenous generation of H₂O₂, staining of lamellar bodies was absent. Tyrosinase activity was found in lamellar bodies and in small vesicles within the rhabdoms similar to those found positive for acid phosphatase. Granules (500–700 nm in diameter) with an electron opaque matrix and mature screening pigment granules showed tyrosinase activity. Moreover, lamellar structures within membrane-bound organelles that additionally contained screening pigment-like granules were electron dense because of tyrosinase activity. After addition of phenylthiourea (PTU) to the incubation medium, lamellar bodies did not generally contain electron dense deposits, although weak staining of single membranes still was sometimes observed. After addition of sodium pyruvate in combination with PTU, no staining was detected. The possible role of tyrosinase in ommochrome synthesis within secondary lysosomes that degrade photosensory membrane is discussed.     

Double Domain Swapping in Bovine Seminal RNase: Formation of Distinct N- and C-swapped Tetramers and Multimers with Increasing Biological Activities

Bovine seminal (BS) RNase, the unique natively dimeric member of the RNase super-family, represents a special case not only for its additional biological actions but also for the singular features of 3D domain swapping. The native enzyme is indeed a mixture of two isoforms: M = M, a dimer held together by two inter-subunit disulfide bonds, and MxM, 70% of the total, which, besides the two mentioned disulfides, is additionally stabilized by the swapping of its N-termini.When lyophilized from 40% acetic acid, BS-RNase oligomerizes as the super-family proto-type RNase A does. In this paper, we induced BS-RNase self-association and analyzed the multimers by size-exclusion chromatography, cross-linking, electrophoresis, mutagenesis, dynamic light scattering, molecular modelling. Finally, we evaluated their enzymatic and cytotoxic activities.Several BS-RNase domain-swapped oligomers were detected, including two tetramers, one exchanging only the N-termini, the other being either N- or C-swapped. The C-swapping event, confirmed by results on a BS-K113N mutant, has been firstly seen in BS-RNase here, and probably stabilizes also multimers larger than tetramers.Interestingly, all BS-RNase oligomers are more enzymatically active than the native dimer and, above all, they display a cytotoxic activity that definitely increases with the molecular weight of the multimers. This latter feature, to date unknown for BS-RNase, suggests again that the self-association of RNases strongly modulates their biological and potentially therapeutic properties.     

The Conserved Proline Residue in the LOB Domain of LBD18 Is Critical for DNA-Binding and Biological Function

Dear Editor, The LATERAL ORGAN BOUNDARIES DOMAIN （LBD）/ ASYMMETRICLEAVES2-LIKE （ASL） genes （hereafter referred to as LBD） encode proteins containing a conserved plant-specific LOB domain and play roles in lateral organ development （Iwakawa et al., 2002; Shuai et al., 2002; Majer and Hochholdinger, 2011）. The LOB domain is approximately 100 amino acids in length and contains a conserved four-Cys motif （CX2CX6CX3C）, the Gly-Ala-Ser block （GAS）, and the leucine-zipper-like coiled-coil motif （LX6LX3LX6L） （Shuai et al., 2002）. The leucine-zipper-like coiled-coil motif in the LOB domain is predicted to function in protein dimerization. LOB, AS2, and LBD4 preferentially bind unique DNA sequences in electrophoretic mobility shift assays （EMSAs） （Husbands et al., 2007）. The LOB domain of AS2 cannot be functionally replaced by those of other members of the LOB family,     

Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0–2.2 M) than the formation of the native state (0–1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7–2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.     

Theoretical Study of Possible Reaction Mechanisms for the Formation of Carbodiimide in the Interstellar Medium (ISM) and Polarizabilities of Carbodiimide

Origins of Life and Evolution of Biospheres - The Structure of carbodiimide has been studied by using quantum chemical methods. Carbodiimide (HNCNH) has been detected towards Sagittarius B2 (N) in...     

Nonreciprocal Packaging of Human Immunodeficiency Virus Type 1 and Type 2 RNA: a Possible Role for the p2 Domain of Gag in RNA Encapsidation

The ability of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) to cross-package each other’s RNA was investigated by cotransfecting helper virus constructs with vectors derived from both viruses from which the gag and pol sequences had been removed. HIV-1 was able to package both HIV-1 and HIV-2 vector RNA. The unspliced HIV-1 vector RNA was packaged preferentially over spliced RNA; however, unspliced and spliced HIV-2 vector RNA were packaged in proportion to their cytoplasmic concentrations. The HIV-2 helper virus was unable to package the HIV-1 vector RNA, indicating a nonreciprocal RNA packaging relationship between these two lentiviruses. Chimeric proviruses based on HIV-2 were constructed to identify the regions of the HIV-1 Gag protein conferring RNA-packaging specificity for the HIV-1 packaging signal. Two chimeric viruses were constructed in which domains within the HIV-2 gag gene were replaced by the corresponding domains in HIV-1, and the ability of the chimeric proviruses to encapsidate an HIV-1-based vector was studied. Wild-type HIV-2 was unable to package the HIV-1-based vector; however, replacement of the HIV-2 nucleocapsid by that of HIV-1 generated a virus with normal protein processing which could package the HIV-1-based vector. The chimeric viruses retained the ability to package HIV-2 genomic RNA, providing further evidence for a lack of reciprocity in RNA-packaging ability between the HIV-1 and HIV-2 nucleocapsid proteins. Inclusion of the p2 domain of HIV-1 Gag in the chimera significantly enhanced packaging.     

The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex

BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response.     

In Situ Solvent Formation Microextraction in the Presence of Ionic Liquid for Preconcentration and Speciation of Arsenic in Saline Samples and Total Arsenic in Biological Samples by Electrothermal Atomic Absorption Spectrometry

In this modality, the extraction phase is formed in situ while simultaneously extracting analytes. First, a water-miscible ionic liquid (IL) ([Hmim][BF₄]), capable of complete dissolving in the aqueous solution, was added to the sample. Then, an ion-exchange reagent (NaPF₆) was added to obtain the hydrophobic IL ([Hmim][PF₆]) that acted as the analyte extractant to form the cloudy homogeneous solution for the preconcentration and speciation of trace amounts of As (III) and As (V) with electrothermal atomic absorption spectrometry (ETAAS) detection. In situ solvent formation microextraction is a simple and rapid method for extraction and preconcentration of metal ions from sample solutions containing high concentration of salt. Some effective factors that influence the microextraction efficiency were investigated and optimized. Under the optimum experimental conditions, the limit of detection (3 σ) and the enrichment factor were 6 ng L^-1 and 198, respectively. The obtained relative standard deviation was 4.78%. The proposed method was successfully applied for the determination of As (III) and As (V) in water samples, food salts, and total As in biological samples.