Interaction of the C-Terminal Peptide of Pulmonary Surfactant Protein B (SP-B) with a Bicellar Lipid Mixture Containing Anionic Lipid

The hydrophobic lung surfactant SP-B is essential for respiration. SP-B promotes spreading and adsorption of surfactant at the alveolar air-water interface and may facilitate connections between the surface layer and underlying lamellar reservoirs of surfactant material. SP-B_63–78 is a cationic and amphipathic helical peptide containing the C-terminal helix of SP-B. ²H NMR has been used to examine the effect of SP-B_63–78 on the phase behavior and dynamics of bicellar lipid dispersions containing the longer chain phospholipids DMPC-d ₅₄ and DMPG and the shorter chain lipid DHPC mixed with a 3∶1∶1 molar ratio. Below the gel-to-liquid crystal phase transition temperature of the longer chain components, bicellar mixtures form small, rapidly reorienting disk-like particles with shorter chain lipid components predominantly found around the highly curved particle edges. With increasing temperature, the particles coalesce into larger magnetically-oriented structures and then into more extended lamellar phases. The susceptibility of bicellar particles to coalescence and large scale reorganization makes them an interesting platform in which to study peptide-induced interactions between lipid assemblies. SP-B_63–78 is found to lower the temperature at which the orientable phase transforms to the more extended lamellar phase. The peptide also changes the spectrum of motions contributing to quadrupole echo decay in the lamellar phase. The way in which the peptide alters interactions between bilayered micelle structures may provide some insight into some aspects of the role of full-length SP-B in maintaining a functional surfactant layer in lungs.     

Statistical Determinants of Selective Ionic Complexation: Ions in Solvent, Transport Proteins, and Other “Hosts”

To provide utility in understanding the molecular evolution of ion-selective biomembrane channels/transporters, globular proteins, and ionophoric compounds, as well as in guiding their modification and design, we present a statistical mechanical basis for deconstructing the impact of the coordination structure and chemistry of selective multidentate ionic complexes. The deconstruction augments familiar ideas in liquid structure theory to realize the ionic complex as an open ion-ligated system acting under the influence of an “external field” provided by the host (or surrounding medium). Using considerations derived from this basis, we show that selective complexation arises from exploitation of a particular ion's coordination preferences. These preferences derive from a balance of interactions much like that which dictates the Hofmeister effect. By analyzing the coordination-state space of small family IA and VIIA ions in simulated fluid media, we derive domains of coordinated states that confer selectivity for a given ion upon isolating and constraining particular attributes (order parameters) of a complex comprised of a given type of ligand. We demonstrate that such domains may be used to rationalize the ion-coordinated environments provided by selective ionophores and biological ion channels/transporters of known structure, and that they can serve as a means toward deriving rational design principles for ion-selective hosts.     

Vaccinia Virus F13L Protein with a Conserved Phospholipase Catalytic Motif Induces Colocalization of the B5R Envelope Glycoprotein in Post-Golgi Vesicles

The wrapping of intracellular mature vaccinia virions by modified trans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.     

Tandem Cell‐Free Protein Synthesis as a Tool for Rapid Screening of Optimal Molecular Chaperones

A simple and flexible method is developed for rapid screening of molecular chaperones that enhance the functional expression of recombinant proteins. A panel of molecular chaperones are transiently expressed in a reaction mixture of cell‐free protein synthesis and then a target protein is subsequently expressed in the presence of these presynthesized molecular chaperones. The biological activity of the cell‐free synthesized target protein is compared to identify the effective molecular chaperones. This strategy successfully identifies individual and combinations of bacterial molecular chaperones that markedly improved the functional expression of horseradish peroxidase. The authors believe that the presented strategy provides a versatile platform for the optimal production of functional proteins, and can also be extended to studies of other interacting proteins.     

Oxidant Injury in PC12 Cells—A Possible Model of Calcium "Dysregulation" in Aging: II. Interactions with Membrane Lipids

Abstract: In a model recently developed to study the parameters altering vulnerability to oxidative stress, it was shown via image analysis that H₂O₂-exposed PC12 cells exhibited increased levels of intracellular Ca²⁺ (baseline), decreases in K⁺-stimulated Ca²⁺ levels (peak), and decreased poststimulation Ca²⁺ clearance (recovery). The present experiments were performed to determine if the response patterns in these parameters to oxidative stress would be altered after modification of membrane lipid composition induced by incubating the PC12 cells with 660 µ M cholesterol (CHL) in the presence or absence of 500 µ M sphingomyelin (SPH) before low (5 µ M ) or high (300 µ M ) H₂O₂ exposure. Neither CHL nor SPH had synergistic effects with high concentrations of H₂O₂ on baseline. However, CHL in the presence or absence of SPH reversed the effect of low concentrations of H₂O₂ on baseline. SPH decreased significantly the cell's ability to clear excess Ca²⁺ in the presence or absence of H₂O₂ and increased significantly the level of conjugated dienes (CDs). It is surprising that in the cells pretreated with CHL, the CD levels were not significantly different from controls. However, in the presence of SPH, the effects of CHL on CDs were altered. These results suggest that the ratios of membrane lipids could be of critical importance in determining the vulnerability to oxidative stress and Ca²⁺ translocation in membranes. This may be of critical importance in aging where there is increased membrane SPH and significant loss of calcium homeostasis.     

Changes in Intracellular Free Calcium Concentration during Illumination of Invertebrate Photoreceptors : Detection with Aequorin

Aequorin, which luminesces in the presence of calcium, was injected into photoreceptor cells of Limulus ventral eye. A bright light stimulus elicited a large increase in aequorin luminescence, the aequorin response, indicating a rise of intracellular calcium ion concentration, Ca_i. The aequorin response reached a maximum after the peak of the electrical response of the photoreceptor, decayed during a prolonged stimulus, and returned to an undetectable level in the dark. Reduction of Ca_o reduced the amplitude of the aequorin response by a factor no greater than 3. Raising Ca_o increased the amplitude of the aequorin response. The aequorin response became smaller when membrane voltage was clamped to successively more positive values. These results indicate that the stimulus-induced rise of Ca_i may be due in part to a light-induced influx of Ca and in part to release of Ca from an intracellular store. Our findings are consistent with the hypothesis that a rise in Ca_i is a step in the sequence of events underlying light-adaptation in Limulus ventral photoreceptors. Aequorin was also injected into photoreceptors of Balanus. The aequorin responses were similar to those recorded from Limulus cells in all but two ways: (a) A large sustained aequorin luminescence was measured during a prolonged stimulus, and (b) removal of extracellular calcium reduced the aequorin response to an undetectable level.     

Desert geophytes under dew and fog: The “curly-whirlies” of Namaqualand (South Africa)

In the semidesert of Namaqualand and adjacent regions of the former Cape Province, South Africa, there occurs an assemblage of geophytes belonging to eight monocot families and some Oxalis species that exhibit special morphological adaptations of their aerial parts to harvest and absorb water from dew and fog, the main source of moisture in this region. Most of them pass their vegetative phase in winter. Leaves and in some cases axes, display a circinate, helical, tortuose, or serpentine shape and/or their margins are undulate or crispate, or (and) are provided with a ciliate or fimbriate pilosity of uncommon appearance. These morphomes, rare elsewhere among monocotyledons, promote an increased deposit of dew and fog by enlargement of surfaces and edges, keeping at the same time the overall size of the leaves restricted. They improve the water budget of these plants in three ways: (1) remnant water on the aerial parts retards the transpiration stress at day-time; (2) although special organs for direct absorption seem to be absent, field and laboratory tests show, that considerable uptake of water occurs but in quantities not exceeding that capacity found in many non-desert plants; (3) the water harvest of the leaves dripping to the soil and reaching the root zone, where it is stored in tubers, bulbs, corms and rhizomes, appears to be the main contribution. Experiments using artificial, directional fog and metal models imitating the natural profiles demonstrate that a surplus of water in efficiency rates of 0.1-66% is collected by the various surface types compared to a standard model with a non-sculptured (plain) frontal surface of the same size. The higher rates are sufficient to moisten the underlying soil down to the rhizosphere. The circumference of roots and storage organs probably does not exceed the area shadowed by the foliage. Namaqualand geophytes with such morphologies apparently form a novel desert biotype: as mesophytes they represent a distinguished strategy besides that of succulents and other xerophytes.     

Presence of the Novel Pituitary Protein „7B2” in Bovine Chromaffin Granules: Possible Co-Release of 7B2 and Catecholamine as Induced by Nicotine

We observed the presence of the novel pituitary protein "7B2" and its release in the bovine adrenal medulla. The 7B2 concentration (mean +/- SEM) in extracts of the bovine adrenal medulla was 952 +/- 155 pg/mg tissue (n = 6). 7B2 was distributed in the chromaffin granule fraction prepared from the bovine adrenal medulla and was released by high K+ and/or nicotine from cultured cells of the bovine adrenal medulla. Co-release of 7B2 with catecholamine induced by nicotine from the cultured bovine chromaffin cells was also observed. In an analysis of the bovine adrenal medulla chromaffin granule fraction on gel permeation chromatography, there was a major peak with an apparent molecular weight of 45,000, whereas a major peak with an apparent molecular weight of 20,000 was found in that on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On reverse-phase HPLC, a major peak with a retention time of 35 min was observed in the bovine chromaffin granule fraction and in the bovine anterior pituitary extract. These findings indicate that 7B2 is a secretory protein in the bovine adrenal medulla. The possibility that 7B2 might be released with catecholamine, possibly in response to stress, warrants investigation.     

Surfactant Protein SP-B Strongly Modifies Surface Collapse of Phospholipid Vesicles: Insights from a Quartz Crystal Microbalance with Dissipation

Pulmonary surfactant protein B (SP-B) facilitates the rapid transfer of phospholipids from bilayer stores into air-liquid interfacial films along the breathing cycle, and contributes to the formation of a surface-associated multilayer reservoir of surfactant to optimize the stability of the respiratory interface. To obtain more insights into the mechanisms underlying this transfer and multilayer formation, we established a simple model system that captures different features of SP-B action. We monitored the formation of supported planar bilayers from the collapse of intact phospholipid vesicles on a silica surface using a technique called quartz crystal microbalance with dissipation, which provides information on changes in membrane thickness and viscosity. At physiologically relevant concentrations, SP-B dramatically alters vesicle collapse. This manifests itself as a reduced buildup of intact vesicles on the surface before collapse, and allows the stepwise buildup of multilayered deposits. Accumulation of lipids in these multilayer deposits requires the presence of SP-B in both the receptor and the arriving membranes, surrounded by a comparable phospholipid charge. Thus, the quartz crystal microbalance with dissipation system provides a useful, simplified way to mimic the effect of surfactant protein on vesicle dynamics and permits a detailed characterization of the parameters governing reorganization of surfactant layers.     

Partially Unfolded Forms of the Prion Protein Populated under Misfolding-promoting Conditions: CHARACTERIZATION BY HYDROGEN EXCHANGE MASS SPECTROMETRY AND NMR*

The susceptibility of the cellular prion protein (PrP^C) to convert to an alternative misfolded conformation (PrP^Sc), which is the key event in the pathogenesis of prion diseases, is indicative of a conformationally flexible native (N) state. In the present study, hydrogen-deuterium exchange (HDX) in conjunction with mass spectrometry and nuclear magnetic resonance spectroscopy were used for the structural and energetic characterization of the N state of the full-length mouse prion protein, moPrP(23–231), under conditions that favor misfolding. The kinetics of HDX of 34 backbone amide hydrogens in the N state were determined at pH 4. In contrast to the results of previous HDX studies on the human and Syrian hamster prion proteins at a higher pH, various segments of moPrP were found to undergo different extents of subglobal unfolding events at pH 4, a pH at which the protein is known to be primed to misfold to a β-rich conformation. No residual structure around the disulfide bond was observed for the unfolded state at pH 4. The N state of the prion protein was observed to be at equilibrium with at least two partially unfolded forms (PUFs). These PUFs, which are accessed by stochastic fluctuations of the N state, have altered surface area exposure relative to the N state. One of these PUFs resembles a conformation previously implicated to be an initial intermediate in the conversion of monomeric protein into misfolded oligomer at pH 4.     

NH2-terminal Deletion of β-Catenin Results in Stable Colocalization of Mutant β-Catenin with Adenomatous Polyposis Coli Protein and Altered MDCK Cell Adhesion

β-Catenin is essential for the function of cadherins, a family of Ca²⁺-dependent cell–cell adhesion molecules, by linking them to α-catenin and the actin cytoskeleton. β-Catenin also binds to adenomatous polyposis coli (APC) protein, a cytosolic protein that is the product of a tumor suppressor gene mutated in colorectal adenomas. We have expressed mutant β-catenins in MDCK epithelial cells to gain insights into the regulation of β-catenin distribution between cadherin and APC protein complexes and the functions of these complexes. Full-length β-catenin, β-catenin mutant proteins with NH₂-terminal deletions before (ΔN90) or after (ΔN131, ΔN151) the α-catenin binding site, or a mutant β-catenin with a COOH-terminal deletion (ΔC) were expressed in MDCK cells under the control of the tetracycline-repressible transactivator. All β-catenin mutant proteins form complexes and colocalize with E-cadherin at cell–cell contacts; ΔN90, but neither ΔN131 nor ΔN151, bind α-catenin. However, β-catenin mutant proteins containing NH₂-terminal deletions also colocalize prominently with APC protein in clusters at the tips of plasma membrane protrusions; in contrast, full-length and COOH-terminal– deleted β-catenin poorly colocalize with APC protein. NH₂-terminal deletions result in increased stability of β-catenin bound to APC protein and E-cadherin, compared with full-length β-catenin. At low density, MDCK cells expressing NH₂-terminal–deleted β-catenin mutants are dispersed, more fibroblastic in morphology, and less efficient in forming colonies than parental MDCK cells. These results show that the NH₂ terminus, but not the COOH terminus of β-catenin, regulates the dynamics of β-catenin binding to APC protein and E-cadherin. Changes in β-catenin binding to cadherin or APC protein, and the ensuing effects on cell morphology and adhesion, are independent of β-catenin binding to α-catenin. These results demonstrate that regulation of β-catenin binding to E-cadherin and APC protein is important in controlling epithelial cell adhesion.     

Monophosphoryl Lipid A‐Adjuvanted Virosomes with Ni‐Chelating Lipids for Attachment of Conserved Viral Proteins as Cross‐Protective Influenza Vaccine

Induction of CD8⁺ cytotoxic T cells (CTLs) to conserved internal influenza antigens, such as nucleoprotein (NP), is a promising strategy for the development of cross‐protective influenza vaccines. However, influenza NP protein alone cannot induce CTL immunity due to its low capacity to activate antigen‐presenting cells (APCs) and get access to the MHC class I antigen processing pathway. To facilitate the generation of NP‐specific CTL immunity the authors develop a novel influenza vaccine consisting of virosomes with the Toll‐like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) and the metal‐ion‐chelating lipid DOGS‐NTA‐Ni incorporated in the membrane. In vitro, virosomes with incorporated MPLA induce stronger activation of APCs than unadjuvanted virosomes. Virosomes modified with DOGS‐NTA‐Ni show high conjugation efficacy for his‐tagged proteins and facilitate efficient uptake of conjugated proteins by APCs. Immunization of mice with MPLA‐adjuvanted virosomes with attached NP results in priming of NP‐specific CTLs while MPLA‐adjuvanted virosomes with admixed NP are inefficient in priming CTLs. Both vaccines induce equally high titers of NP‐specific antibodies. When challenged with heterosubtypic influenza virus, mice immunized with virosomes with attached or admixed NP are protected from severe weight loss. Yet, unexpectedly, they show more weight loss and more severe disease symptoms than mice immunized with MPLA‐virosomes without NP. Taken together, these results indicate that virosomes with conjugated antigen and adjuvant incorporated in the membrane are effective in priming of CTLs and eliciting antigen‐specific antibody responses in vivo. However, for protection from influenza infection NP‐specific immunity appears not to be advantageous.     

Increased Immunogenicity to LipL32 of <Emphasis Type="Italic">Leptospira interrogans</Emphasis> when Expressed as a Fusion Protein with the Cholera Toxin B Subunit

Leptospirosis is one of the most widespread zoonosis in the world. The development of a recombinant leptospira vaccine remains a challenge. In this study, we cloned the Leptospira interrogans open reading frame (ORF) coding the external membrane protein LipL32, an immunodominant antigen found in all pathogenic leptospira, downstream of the highly immunogenic cholera toxin B subunit (CTB) ORF. Expression and assembly of the CTB-LipL32 fusion protein into oligomeric structures of pentameric size were observed in soluble fractions by Western blot analysis. The CTB-LipL32 protein demonstrated strong affinity for monosialotetrahexosylgaglioside (GM1-ganglioside) in an enzyme-linked immunosorbent assay (ELISA), suggesting that the antigenic sites for binding and proper folding of the pentameric CTB structure were conserved. Furthermore, antisera against LipL32 also recognized the CTB-LipL32 fusion protein, suggesting that LipL32 also conserved its antigenic sites, a fact confirmed by an ELISA assay showing soluble CTB-LipL32 recognition by sera from convalescent patients. In addition, soluble CTB-LipL32 generated higher specific titers in mice immunized without external adjuvant than co-administration of CTB with LipL32. The data presented here provide support for CTB-LipL32 as a promising antigen for use in the control and study of leptospirosis.     

Incorporation of Counter Ions in Organic Molecules: New Strategy in Developing Dopant‐Free Hole Transport Materials for Efficient Mixed‐Ion Perovskite Solar Cells

Hole transport matertial (HTM) as charge selective layer in perovskite solar cells (PSCs) plays an important role in achieving high power conversion efficiency (PCE). It is known that the dopants and additives are necessary in the HTM in order to improve the hole conductivity of the HTM as well as to obtain high efficiency in PSCs, but the additives can potentially induce device instability and poor device reproducibility. In this work a new strategy to design dopant‐free HTMs has been presented by modifying the HTM to include charged moieties which are accompanied with counter ions. The device based on this ionic HTM X44 dos not need any additional doping and the device shows an impressive PCE of 16.2%. Detailed characterization suggests that the incorporated counter ions in X44 can significantly affect the hole conductivity and the homogeneity of the formed HTM thin film. The superior photovoltaic performance for X44 is attributed to both efficient hole transport and effective interfacial hole transfer in the solar cell device. This work provides important insights as regards the future design of new and efficient dopant free HTMs for photovotaics or other optoelectronic applications.     

霍乱毒素B亚基融合蛋白表达载体的构建

采用PCR技术将霍乱弧菌CTB基因的终止密码子定点突变并引入一个EcoR Ⅰ位点,然后与人工合成的Linker连接,构建成了新颖的融合蛋白表达载体。在CTB 3′端的Linker上有四个单一人酶切位点,在任何限制性位点融合外源基因序列后,都可在3′端形成转译终止密码子。     

Comparison of Plantago Species from Nutrient-rich and Nutrient-poor Conditions: Growth Response, ATPases and Lipids of the Roots, as Affected by the Level of Mineral Nutrition

Two groups of Plantago species, one of relatively nutrient-rich habitats (Plantago major L., P. major L. ssp. pleiosperma Pilger and P. martrima L.) and the other of relatively nutrient poor habitats (P. lanceolata L., P. media L. and P. coronopus L.), were compared with respect to the response of the plants to alterations of the level of mineral nutrition. The comparison deals with growth response and root cell membrane functions (Ca²⁺ and Mg²⁺ stimulated ATPases and lipids). As far as these parameters are concerned species from relatively nutrientpoor habitats are not much affected by bringing the plants to high-salt conditions, while the reverse was true for species from relatively nutrient-rich habitats when transferred to low salt medium.     

Identification of cyk, a Cyclin B2 Kinase, as a Novel Calcium/Calmodulin-Dependent Protein Kinase II and Its Role during Xenopus laevis Oocyte Maturation

Recently, we have partially purified and characterized a specific cell cycle-regulated cyclin B2 kinase (cyk) from prophase oocytes of Xenopus laevis after an ATP-gamma-S activation step (R. Derua, I. Stevens, E. Waelkens, A. Fernandez, N. Lamb, W. Merlevede, and J. Goris, 1997, Exp. Cell Res. 230, 310-324). In the present paper we describe its purification to homogeneity. We could identify the kinase as a special form of calcium/calmodulin-dependent protein kinase II (CaMKII), consisting of five isoforms with molecular masses ranging from 52 to 83 kDa. At least three of them could be considered as novel. Using an in vivo assay with a synthetic peptide (cyktide), an activation of the kinase was shown at about 50% maturation. Further evidence for this observation came from the injection of the calcium chelator BAPTA and the specific cyk/CaMKII inhibitor AIP. A delay of oocyte maturation of at least 1 h was observed. Besides serine 53, a second cyk phosphorylation site in cyclin B2 was identified as threonine 41. Site-directed mutagenesis of these sites indicated that phosphorylation of these sites in Xenopus cyclin B2 was not required for the hallmark functions of cyclin B2.