Early stages of salmon calcitonin aggregation: effect induced by ageing and oxidation processes in water and in the presence of model membranes

The natural ageing- and hydrogen peroxide-induced aggregation of salmon calcitonin were studied in water and in the presence of dipalmitoylphosphatidylcholine (DPPC) liposomes. The early stages of the aggregation process at low protein concentration were investigated by means of Circular Dichroism spectroscopy (CD) and conventional and immunogold labelling Transmission Electron Microscopy (TEM). In buffered water solution, salmon calcitonin showed a two-stage conformational variation related to fibril formation and phase-separation of larger aggregates. A first stage, characterised by small conformational changes but a decrease in dichroic band intensity, was followed by a second stage, 6 days after, leading to higher conformational variations and aggregations. Salmon calcitonin showed a distinct modification in the secondary structure and aggregate morphology in the presence of hydrogen peroxide with respect to natural ageing, indicating that the two aggregation processes (natural and chemical-induced) followed a distinct mechanism. The oxidised forms of the peptide were separated by liquid chromatography. The same study was performed in the presence of DPPC liposomes. The results obtained by conventional and immunogold labelling TEM evidenced that salmon calcitonin in buffered water solution essentially does not enter the liposomes but forms around them a fibril network characterised by the same conformational changes after 6 days. The oxidised sample in the presence of liposomes showed a "fibrils hank", separated from liposomes. The presence of liposomes did not affect either the aggregation or the conformational modifications yet observed by TEM and CD in water solution.     

Identification of Risk Pathways and Functional Modules for Coronary Artery Disease Based on Genome-wide SNP Data

Coronary artery disease(CAD) is a complex human disease, involving multiple genes and their nonlinear interactions, which often act in a modular fashion. Genome-wide single nucleotide polymorphism(SNP) profiling provides an effective technique to unravel these underlying genetic interplays or their functional involvements for CAD. This study aimed to identify the susceptible pathways and modules for CAD based on SNP omics. First, the Wellcome Trust Case Control Consortium(WTCCC) SNP datasets of CAD and control samples were used to assess the jointeffect of multiple genetic variants at the pathway level, using logistic kernel machine regression model. Then, an expanded genetic network was constructed by integrating statistical gene–gene interactions involved in these susceptible pathways with their protein–protein interaction(PPI)knowledge. Finally, risk functional modules were identified by decomposition of the network. Of 276 KEGG pathways analyzed, 6 pathways were found to have a significant effect on CAD. Other than glycerolipid metabolism, glycosaminoglycan biosynthesis, and cardiac muscle contraction pathways, three pathways related to other diseases were also revealed, including Alzheimer's disease, non-alcoholic fatty liver disease, and Huntington's disease. A genetic epistatic network of 95 genes was further constructed using the abovementioned integrative approach. Of 10 functional modules derived from the network, 6 have been annotated to phospholipase C activity and cell adhesion molecule binding, which also have known functional involvement in Alzheimer's disease.These findings indicate an overlap of the underlying molecular mechanisms between CAD and Alzheimer's disease, thus providing new insights into the molecular basis for CAD and its molecular relationships with other diseases.     

A simple and efficient expression and purification system using two newly constructed vectors

Structural biology places a high demand on proteins both in terms of quality and quantity. Although many protein expression and purification systems have been developed, an efficient and simple system which can be easily adapted is desirable. Here, we report a new system which combines improved expression, solubility screening and purification efficiency. The system is based on two newly constructed vectors, pEHISTEV and pEHISGFPTEV derived from a pET vector. Both vectors generate a construct with an amino-terminal hexahistidine tag (His-tag). In addition, pEHISGFPTEV expresses a protein with an N-terminal His-tagged green fluorescent protein (GFP) fusion to allow rapid quantitation of soluble protein. Both vectors have a tobacco etch virus (TEV) protease cleavage site that allows for production of protein with only two additional N-terminal residues and have the same multiple cloning site which enables parallel cloning. Protein purification is a simple two-stage nickel affinity chromatography based on the His tag removal. A total of seven genes were tested using this system. Expression was optimised using pEHISGFPTEV constructs by monitoring the GFP fluorescence and the soluble target proteins were quantified using spectrophotometric analysis. All the tested proteins were purified with sufficient quantity and quality to attempt structure determination. This system has been proven to be simple and effective for structural biology. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable.     

多肽、蛋白类药物脂质体的研究进展

脂质体做为多肽、蛋白类药物一种新型给药载体有控制药物释放、降低药物的毒性、提高药物的靶向性等突出优点,具有广阔的应用前景。本文通过查阅近10年来多肽和蛋白类药物脂质体研究的相关资料,总结论述了脂质体作为多肽、蛋白类药物载体在新的制备方法、新型脂质体、给药途径、产业化进展四个方面的最新研究动向,指出了多肽、蛋白类药物脂质体在研究应用中存在的不足,并展望了多肽、蛋白类药物脂质体未来发展的方向。     

Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes

The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24 h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine™ RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications.     

Evaluation of quantitative parameters of the interaction of antibody-bearing liposomes with target antigens

The model system for the analysis of targeted liposomes is proposed--the layer of protein antigen adsorbed on polystyrene wells. Antibodies were treated with palmitoyl chloride and liposomes were produced by the cholate dialysis method in the presence of the modified protein (7 X 10(-4) mol protein/mol lipid). Affinity of antibody-bearing liposomes to the antigen on the surface of Multiwell plates was studied, and apparent dissociation constant value was estimated: KD was in the range 1.5 to 5 X 10(-9) M liposomes. Sequential transfers of liposomes in antigen-coated plates revealed that the high-affinity fraction of liposomes is adsorbed first. The bound fraction has 1.7-times-higher protein content. For effective in vivo targeting it would be necessary to have high-affinity liposomes and a high concentration of the target antigen.     

Exomer: A coat complex for transport of select membrane proteins from the trans-Golgi network to the plasma membrane in yeast

A yeast plasma membrane protein, Chs3p, transits to the mother-bud neck from a reservoir comprising the trans-Golgi network (TGN) and endosomal system. Two TGN/endosomal peripheral proteins, Chs5p and Chs6p, and three Chs6p paralogues form a complex that is required for the TGN to cell surface transport of Chs3p. The role of these peripheral proteins has not been clear, and we now provide evidence that they create a coat complex required for the capture of membrane proteins en route to the cell surface. Sec7p, a Golgi protein required for general membrane traffic and functioning as a nucleotide exchange factor for the guanosine triphosphate (GTP)-binding protein Arf1p, is required to recruit Chs5p to the TGN surface in vivo. Recombinant forms of Chs5p, Chs6p, and the Chs6p paralogues expressed in baculovirus form a complex of approximately 1 MD that binds synthetic liposomes in a reaction requiring acidic phospholipids, Arf1p, and the nonhydrolyzable GTPgammaS. The complex remains bound to liposomes centrifuged on a sucrose density gradient. Thin section electron microscopy reveals a spiky coat structure on liposomes incubated with the full complex, Arf1p, and GTPgammaS. We termed the novel coat exomer for its role in exocytosis from the TGN to the cell surface. Unlike other coats (e.g., coat protein complex I, II, and clathrin/adaptor protein complex), the exomer does not form buds or vesicles on liposomes.     

The use of liposomes to study COPII- and COPI-coated vesicle formation and membrane protein sorting

We have established systems that reconstitute the biogenesis of coated transport vesicles with liposomes made of pure lipids and purified coat proteins. Optimization of the lipid composition in the liposomes allowed the efficient binding of both coat protein I and coat protein II (COPII) coat subunits. Coated vesicles of approximately the size generated from biomembranes were detected and characterized by centrifugation analysis and electron microscopy. A variation of this budding reaction allowed us to measure the sorting of v-SNARE proteins into synthetic COPII vesicles. We developed a novel system to tether glutathione S-transferase (GST)-hybrid proteins to the surface of liposomes formulated with a glutathione-derivatized phospholipid. This system allowed us to detect the positive role of cytoplasmic domains of two v-SNARE proteins that are packaged into COPII vesicles. Therefore, both generation of coated vesicles and protein sorting into the vesicles can be reproduced with liposomes and purified proteins.     

Use of a simple mathematical model to predict the behavior of Escherichia coli overproducing beta-lactamase within continuous single- and two-stage reactor systems

A simple mathematical model is developed to help explain the complex population dynamics of an Escherichia coli host-plasmid expression/excretion system for beta-lactamase within single- and two-stage reactors. The model successfully integrates the individual regulatory (tac promoter induction), genetic (runaway plasmid replication), and population dynamics (culture instability) aspects of the system. The model predicts, and experiment confirms, that high-level beta-lactamase production and excretion cannot be easily maintained in single-stage reactors using the current plasmid construction. Stable target protein production and excretion is mathematically predicted, and experimentally confirmed, within two-stage reactors. The model is used to provide insight into engineering a more stable host-vector expression/excretion system for use in single-stage reactors. (c) 1993 John Wiley & Sons, Inc.     

Glutathione disulfide liposomes – A research tool for the study of glutathione disulfide associated functions and dysfunctions

Glutathione disulfide (GSSG) is the oxidized form of glutathione (GSH). GSH is a tripeptide present in the biological system in mM concentration and is the major antioxidant in the body. An increase in GSSG reflects an increase in intracellular oxidative stress and is associated with disease sates. The increase has also been demonstrated to lead to an increase in protein S-glutathionylation that can affect the structure and function of proteins. Protein S-glutathionylation serves as a regulatory mechanism during cellular oxidative stress. Though GSSG is commercially available, its roles in various GSSG-associated normal/abnormal physiological functions have not been fully delineated due to the reason that GSSG is not cell membrane permeable and a lack of method to specifically increase GSSG in cells. We have developed cationic liposomes that can effectively deliver GSSG into cells. Various concentrations of GSSG liposomes can be conveniently prepared. At 1 mg/mL, the GSSG liposomes effectively increased intracellular GSSG by 27.1±6.9 folds (n=3) in 4 h and led to a significant increase in protein S-glutathionylation confirming that the increased GSSG is functionally effective. The Trypan blue assay demonstrated that GSSG liposomes were not cytotoxic; the cell viability was greater than 95% after cells were treated with the GSSG liposomes for 4 h. A stability study showed that the dry form of the GSSG liposomes were stable for at least 70 days when stored at ?80 °C. Our data demonstrate that the GSSG liposomes can be a valuable tool in studying GSSG-associated physiological/pathological functions.     

New pulp biobleaching system involving manganese peroxidase immobilized in a silica support with controlled pore sizes

Attempts have been made to use manganese peroxidase (MnP) for chlorine-free pulp biobleaching, but they have not been commercially viable because of the enzyme's low stability. We developed a new pulp biobleaching method involving mesoporous material-immobilized manganese peroxidase from Phanerochaete chrysosporium. MnP immobilized in FSM-16, a folded-sheet mesoporous material whose pore size is nearly the same as the diameter of the enzyme, had the highest thermal stability and tolerance to H(2)O(2). MnP immobilized in FSM-16 retained more than 80% of its initial activity even after 10 days of continuous reaction. We constructed a thermally discontinuous two-stage reactor system, in which the enzyme (39 degrees C) and pulp-bleaching (70 degrees C) reactions were performed separately. When the treatment of pulp with MnP by means of the two-stage reactor system and alkaline extraction was repeated seven times, the brightness of the pulp increased to about 88% within 7 h after completion of the last treatment.     

Association of blood proteins with large unilamellar liposomes in vivo. Relation to circulation lifetimes.

The proteins associated with liposomes in the circulation of mice were analyzed in order to determine whether bound proteins significantly influence the fate of liposomes in vivo. Liposomes were administered intravenously via the dorsal tail vein of CD1 mice and were isolated from blood after 2 min in the absence of coagulation inhibitors using a rapid "spin column" procedure. Various negatively charged liposomes exhibiting markedly different clearance properties were studied; notably, these included liposomes containing 10 mol % ganglioside GM1 which has been previously shown to effectively limit liposomal uptake by the fixed macrophages of the reticuloendothelial system. The protein binding ability (PB; g of protein/mol of lipid) of the liposomes was quantitated and related to the circulation half-life (tau 1/2) of the liposomes. Liposomes having similar membrane surface charge imparted by different anionic phospholipids were found to exhibit markedly different protein binding potentials. Furthermore, PB values determined from the in vivo experiments were found to be inversely related to circulation half-lives. PB values in excess of 50 g of protein/mol of lipid were observed for rapidly cleared liposomes such as those containing cardiolipin or phosphatidic acid (tau 1/2 less than 2 min). PB values for ganglioside GM1-containing liposomes (tau 1/2 greater than 2 h) were significantly less (PB less than 15 g of total protein/mol of total lipid). PB values were also determined for liposomes recovered from in vitro incubations with isolated human serum; relative PB values obtained from these in vitro experiments were in agreement with relative PB values measured from in vivo experiments. PB values, therefore, could be a useful parameter for predicting the clearance behavior of liposomes in the circulation. Liposomes exhibiting increased PB values in vivo were shown by immunoblot analysis to bind more immune opsonins, leading to a higher probability of phagocytic uptake. Finally, based on results obtained using the in vitro system, it is suggested that the mechanism by which ganglioside GM1 prolongs the murine circulation half-life of liposomes is by reducing the total amount of blood protein bound to the liposomes in a relatively nonspecific manner.     

Interaction analysis of a ladder-shaped polycyclic ether and model transmembrane peptides in lipid bilayers by using Förster resonance energy transfer and polarized attenuated total reflection infrared spectroscopy

Ladder-shaped polycyclic ethers (LSPs) are predicted to interact with membrane proteins; however, the underlying mechanism has not been satisfactorily elucidated. It has been hypothesized that LSPs possess non-specific affinity to α-helical segments of transmembrane proteins. To verify this hypothesis, we constructed a model LSP interaction system in a lipid bilayer. We prepared 5 types of α-helical peptides and reconstituted them in liposomes. The reconstitution and orientation of these peptides in the liposomes were examined using polarized attenuated total reflection infrared (ATR-IR) spectroscopy and gel filtration. The results revealed that 4 peptides were retained in liposomes, and 3 of them formed stable transmembrane structures. The interaction between the LSP and the peptides was investigated using Förster resonance energy transfer (FRET). In the lipid bilayer, the LSP strongly recognized the peptides that possessed aligned hydrogen donating groups with leucine caps. We propose that this leucine-capped 16-amino acid sequence is a potential LPS binding motif.     

Adapter protein for site-specific conjugation of payloads for targeted drug delivery

High-affinity interactions of two fragments of human RNase I (1-15-aa Hu-tag and 21-125-aa HuS adapter protein) can be used for assembly of targeting drug delivery complexes. In this approach, a targeting protein is expressed as a fusion protein with a 15-aa Hu-tag, while HuS is conjugated to a drug (or a drug carrier) creating a "payload" module, which is then bound noncovalently to the Hu-tag of the targeting protein. Although this approach eliminates chemical modifications of targeting proteins, the payload modules are still constructed by random cross-linking of drugs or drug carriers to an adapter protein that might lead to functional heterogeneity of the complexes. To avoid this problem, we engineered an adapter protein HuS(N88C) with an unpaired cysteine in position 88 that can be directly modified without interference with activity of assembled targeting complexes. HuS(N88C) binds Hu-tagged annexin V with K(D) of 50 +/- 6 nM, which is comparable to that of wild-type HuS. To demonstrate the utility of HuS(N88C) for developing uniform payload modules, we constructed a HuS(N88C)-lipid conjugate and inserted it into preformed liposomes loaded with a fluorescent dye. Targeting proteins, Hu-tagged vascular endothelial growth factor or Hu-tagged annexin V, were docked to liposomes decorated with HuS, and the assembled complexes delivered liposomes selectively to target cells.     

Construction of a modular yeast two-hybrid cDNA library from human EST clones for the human genome protein linkage map

Identification of all human protein–protein interactions will lead to a global human protein linkage map that will provide important information for functional genomics studies. The yeast two-hybrid system is a powerful molecular genetic approach for studying protein–protein interactions. To apply this technology to generate a human protein linkage map, the first step is to construct two-hybrid cDNA libraries that cover the entire human genome. With a homologous recombination-mediated approach, we have constructed a modular human EST-derived yeast two-hybrid library in the Gal4 activation domain-based vector, pACT2. Quality analysis of this library indicated that the approach of constructing two-hybrid cDNA libraries from individually arrayed human EST clones is feasible, and such a two-hybrid library is suitable for detecting protein–protein interactions. This is also the first time that a comprehensive two-hybrid system cDNA library has been constructed from a collection of individually arrayed EST clones.     

The characterisation of liposomes with covalently attached proteins

The problem of characterising liposomes with covalently attached proteins has been analysed theoretically in terms of a normal weight distribution of liposome diameters. The polydispersity of protein conjugation is considered in terms of the width (standard deviation) of the liposome size distribution. It is shown that the weight-average number of proteins per liposome is a convenient parameter to use to define the protein content of proteoliposomes. Two types of proteoliposome have been prepared (small unilamellar vesicles and reverse phase evaporation vesicles) in which wheat germ agglutinin is covalently coupled to the liposomal surface. The liposomes cover a range of weight average diameter from 65 to 240 nm and of polydispersity (weight to number average diameter (dw/dn) from 2.6 to 11.4. The liposomes have been characterised by chemical analysis and photon correlation spectroscopy and the results are discussed in terms of the theoretical consequences of an equivalent normal weight distribution of diameters.     

Vitamin A in liposomes. Inhibition of complement binding and alteration of membrane structure.

Incorporation of vitamin A aldehyde (retinal) into liposomes had an inhibitory effect on the amount of human complement protein bound in the presence of specific antiserum. The total membrane-bound protein was directly measured on liposomes which were washed after incubation in antiserum and fresh human serum (complement). At every concentration of complement, decreased protein binding was found with liposomes which contained retinal. Binding of the third component of complement (C3) was also measured directly on washed liposomes and was found to be decreased in the presence of retinal. The diminution in protein binding due to retinal was not caused by differences in the amount of antibody bound and this was shown by two experiments. First, specific antibody protein binding to liposomes was directly measured and was essentially unaffected by retinal. Second, liposomes were prepared from lipid extracts of sheep erythrocytes. These liposomes were used as as immunoadsorbants to remove antisheep erythrocyte antibodies. The immunoadsorbant capacity was the same in both the presence and the absence of retinal. A further conclusion from these experiments was that retinal did not change the number of liposomal glycolipid antigen molecules available for antibody binding and thus presumably did not change the total number of lipid molecules present on the outer surface of the liposomes. Retinal did have an effect on the geometric structure of the liposomes. Size distribution measurements were performed in the diameter range of 1-6.35 mum by using an electronic particle size analyzer (Coulter Counter). Liposomes containing retinal were shifted toward smaller sizes and had less total surface area and volume. It was suggested that retinal-containing liposomes may have had a tighter packing of the molecules in the phospholipid bilayer. This effect of retinal on liposomal structure may have been responsible for the observed decreased binding of C3 and total complement protein.     

Interaction of diphtheria toxin fragments A, B and protein crm 45 with liposomes

Diphtheria toxin, its fragments A, B and the protein serologically related to toxin, crm 45, have been studied for their hydrophobicity using the method of charge shift electrophoresis. These molecules were then assayed for liposome interaction. The results have shown that the diphtheria toxin B fragment behaves as an amphiphatic protein because it contains a hydrophobic domain located in that portion of the B chain which remains in protein crm 45. Toxin fragment A is hydrophilic. Incubation of protein crm 45 or toxin fragment B with preformed liposomes leads to association of these proteins with lipid vesicles. Fragment A does not interact with liposomes. Binding of protein crm 45 with lipid vesicles is dependent on time and temperature. Protein crm 45 is unidirectionally associated with liposomes, its enzymic fragment A directed outside the liposome. Fragment B or protein crm 45, upon binding with liposomes, does not affect the permeability of the vesicles.     

Designing Lipid Nanostructures for Local Delivery of Biologically Active Macromolecules

This study focuses on the possible therapeutic utility of liposomes in the local treatment of inflammatory disorders, specifically rheumatoid arthritis (RA). Our purpose was to design a depot delivery system of an anti-inflammatory glycoprotein, lactoferrin (Lf), using positive multivesicular liposomes and to investigate its in vivo efficiency. Lactoferrin (Lf) has previously been shown to have therapeutic potential in mice with collagen-induced arthritis (CIA) after intra-articular (i.a.) injection. In order to protect Lf from enzymatic degradation and to maintain an adequate concentration in the joint, liposomes have been used as carriers for controlled drug delivery. Based on our previous findings we compared the ability of free Lf and Lf encapsulated in liposomes to suppress established joint inflammation and to modulate the cytokine response of lymph node (LN) T lymphocytes in DBA/1 mice with CIA. The anti-inflammatory effect of Lf formulated in positive liposomes was more pronounced compared with the free protein. After a single i.a. injection of liposomal Lf the arthritic score significantly decreased continuously for 2 weeks while in the case of free Lf for only 3–4 days. The cytokine levels produced by LN T cells showed decreased pro-inflammatory cytokines (TNF-α and IFN-γ) accompanied by increased anti-inflammatory cytokines (IL-5 and especcialy IL-10) in encapsulated compared with free Lf. When compared with free Lf, liposomal Lf decreased the expression of costimulatory molecules on DCs, reduced pro-inflammatory (TNF) and increased anti-inflammatory (IL-10) cytokine production. Using CIA model we have studied the liposome trafficking following i.a. administration and we have identified DCs as a target for liposomes in the draining LN. Our results suggest that the entrapment of Lf in liposomes may modify its pharmacodynamic profile and could have great potential as controlled delivery system in the treatment of RA and other local inflammatory conditions.