Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coil and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His6FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund`s adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity. (c) 2010 Elsevier Ltd. All rights reserved.

Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel alpha-amylase inhibitory properties

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1). Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein`s biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1), showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of alpha-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian alpha-amylase activity in vitro.

Effects of Extrusion on the Emulsifying Properties of Rumen and Soy Protein

Defatted rumen protein and soy protein concentrate were extruded in a 15.5:1 L/D single-screw extruder at the optimum conditions for their expansion (150A degrees C and 35% moisture, and 130A degrees C and 35% moisture, respectively). Emulsions were produced with these proteins and studied by rheology and time domain low-resolution (1)H nuclear magnetic resonance (TD-NMR). Extrusion increased storage modulus of rumen protein emulsions. The opposite was observed for soy protein. Mechanical relaxation showed the existence of three relaxing components in the emulsions whose relative contributions were changed by extrusion. Likewise, spin-spin relaxation time constants (T (2)) measured by TD-NMR also showed three major distinct populations of protons in respect to their mobility that were also altered by extrusion. Extrusion increased surface hydrophobicity of both rumen and soy protein. Solubility of rumen protein increased with extrusion whereas soy protein had its solubility decreased after processing. Extrusion promoted molecular reorganization of protein, increasing its superficial hydrophobicity, affecting its interfacial properties and improving its emulsifying behavior. The results show that extrusion can promote the use of rumen, a by-product waste from the meat industry, in human nutrition by replacing soy protein in food emulsions.

Effects of milk supplementation with skim milk powder, whey protein concentrate and sodium caseinate on acidification kinetics, rheological properties and structure of nonfat stirred yogurt

Milk supplementation with milk proteins in four different levels was used to investigate the effect on acidification and textural properties of yogurt. Commercial skim milk powder was diluted in distilled water, and the supplements were added to give different enriched-milk bases; these were heat treated at 90 degrees C for 5 min. These mixtures were incubated with the bacterial cultures for fermentation in a water bath, at 42 degrees C, until pH 4.50 was reached. Chemical changes during fermentation were followed by measuring the pH. Protein concentration measurements, microbial counts of Lactobacillus bulgaricus and Streptococcus thermophilus, and textural properties (G`, G ``, yield stress and firmness) were determined after 24 h of storage at 4 degrees C. Yogurt made with milk supplemented with sodium caseinate resulted in significant properties changes, which were decrease in fermentation time, and increase in yield stress, storage modulus, and firmness, with increases in supplement level. Microstructure also differed from that of yogurt produced with milk supplemented with skim milk powder or sodium caseinate. (C) 2009 Elsevier Ltd. All rights reserved.

Thermal study and physico-chemical characterization of some functional properties of guava seeds protein isolate (Psidium guajava)

The guava seed protein isolate ( PI) was obtained from the protein precipitation belonging to the class of the gluteline (Ip 4.5). The conditions for the preparation of the PI were determined by both the solubility curve and simultaneous thermogravimetry-differential thermal analysis (TG-DTA): pH 11.5, absence of NaCl and whiteners and T=( 25 +/- 3) degrees C. Under these conditions a yield of 77.0 +/- 0.4%, protein content of 94.2 +/- 0.3, ashes 0.50 +/- 0.05% and thermal stability, T= 200 degrees C, were obtained. The TG-DTA curves and the PI emulsification capacity study showed the presence of hydrophobic microdomains at pH 11.5 and 3.0 suggesting a random coil protein conformation and, to pH 10.0, an open protein conformation. The capacity of emulsification (CE), in the absence of NaCl, was verified for: 1 - pH 3.0 and 8.5, using the IP extracted at pH 10.0 and 11.5, CE >= 343 +/- 5 g of emulsified oil/g of protein; 2 - pH 6.60 just for the PI obtained at pH 11.5, CE >= 140 +/- 8 g of emulsified oil/g of protein.

The Origin of Nonmonotonic Complex Behavior and the Effects of Nonnative Interactions on the Diffusive Properties of Protein Folding

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Organic acids and protein compounds causing the photoluminescence properties of natural rubber membranes and the quenching phenomena from Au nanoparticle incorporation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Generation and Biological Properties of a Recombinant Dodecahedron Containing the Short Fiber Protein of the Human Adenovirus 41

Objective: In order to gain further insight into the function of the enteric adenovirus short fiber (SF), we have constructed a recombinant dodecahedron containing the SF protein of HAdV-41 and the HAdV-3 penton base. Methods: Recombinant baculoviruses expressing the HAdV-41 SF protein and HAdV-3 penton base were cloned and amplified in Sf9 insect cells. Recombinant dodecahedra were expressed by coinfection of High Five (TM) cells with both baculoviruses, 72 h post-infection. Cell lysate was centrifuged on sucrose density gradient and the purified recombinant dodecahedra were recovered. Results: Analysis by negative staining electron microscopy demonstrated that chimeric dodecahedra made of the HAdV-3 penton base and decorated with the HAdV-41 SF were successfully generated. Next, recombinant dodecahedra were digested with pepsin and analyzed by Western blot. A 'site-specific' proteolysis of the HAdV-41 SF was observed, while the HAdV-3 penton base core was completely digested. Conclusion: These results show that, in vitro, the HAdV-41 SF likely undergoes proteolysis in the gastrointestinal tract, its natural environment, which may facilitate the recognition of receptors in intestinal cells. The results obtained in the present study may be the basis for the development of gene therapy vectors towards the intestinal epithelium, as well as orally administered vaccine vectors, but also for the HAdV-41 SF partner identification. Copyright (C) 2011 S. Karger AG, Basel

Biomimetic coating of organic polymers with a protein-functionalized layer of calcium phosphate: the surface properties of the carrier influence neither the coating characteristics nor the incorporation mechanism or release kinetics of the protein

Polymers that are used in clinical practice as bone-defect-filling materials possess many essential qualities, such as moldability, mechanical strength and biodegradability, but they are neither osteoconductive nor osteoinductive. Osteoconductivity can be conferred by coating the material with a layer of calcium phosphate, which can be rendered osteoinductive by functionalizing it with an osteogenic agent. We wished to ascertain whether the morphological and physicochemical characteristics of unfunctionalized and bovine-serum-albumin (BSA)-functionalized calcium-phosphate coatings were influenced by the surface properties of polymeric carriers. The release kinetics of the protein were also investigated. Two sponge-like materials (Helistat® and Polyactive®) and two fibrous ones (Ethisorb and poly[lactic-co-glycolic acid]) were tested. The coating characteristics were evaluated using state-of-the-art methodologies. The release kinetics of BSA were monitored spectrophotometrically. The characteristics of the amorphous and the crystalline phases of the coatings were not influenced by either the surface chemistry or the surface geometry of the underlying polymer. The mechanism whereby BSA was incorporated into the crystalline layer and the rate of release of the truly incorporated depot were likewise unaffected by the nature of the polymeric carrier. Our biomimetic coating technique could be applied to either spongy or fibrous bone-defect-filling organic polymers, with a view to rendering them osteoconductive and osteoinductive.

BIOCHEMICAL PROPERTIES OF GABA (B) RECEPTORS (BACLOFEN, ADENYLATE CYCLASE, CYCLIC-AMP, PHOSPHOLIPASE, PROTEIN KINASE C)

(gamma)-Aminobutyric acid (GABA), a neurotransmitter in the mammalian central nervous system, influences neuronal activity by interacting with at least two pharmacologically and functionally distinct receptors. GABA(,A) receptors are sensitive to blockade by bicuculline, are associated with benzodiazepine and barbiturate binding sites, and mediate chloride flux. The biochemical and pharmacolocal properties of GABA(,B) receptors, which are stereoselectively activated by (beta)-p-chlorophenyl GABA (baclofen), are less well understood. The aim of this study was to define these features of GABA(,B) receptors, with particular emphasis on their possible relationship to the adenylate cyclase system in brain.^ By themselves, GABA agonists have no effect on cAMP accumulation in rat brain slices. However, some GABA agonists markedly enhance the cAMP accumulation that results from exposure to norepinephrine, adenosine, VIP, and cholera toxin. Evidence that this response is mediated by the GABA(,B) system is provided by the finding that it is bicuculline-insensitive, and by the fact that only those agents that interact with GABA(,B) binding sites are active in this regard. GABA(,B) agonists are able to enhance neurotransmitter-stimulated cAMP accumulation in only certain brain regions, and the response is not influenced by phosphodiesterase inhibitors, although is totally dependent on the availability of extracellular calcium. Furthermore, data suggest that inhibition of phospholipase A(,2), a calcium-dependent enzyme, decreases the augmenting response to baclofen, although inhibitors of arachidonic acid metabolism are without effect. These findings indicate that either arachidonic acid or lysophospholipid, products of PLA(,2)-mediated degradation of phospholipids, mediates the augmentation. Moreover, phorbol esters, compounds which directly activate protein kinase C, were also found to enhance neurotransmitter-stimulated cAMP accumulation in rat brain slices. Since this enzyme is known to be stimulated by unsaturated fatty acids such as arachidonate, it is proposed that GABA(,B) agonists enhance cAMP accumulation by fostering the production of arachidonic acid which stimulates protein kinase C, leading to the phosphorylation of some component of the adenylate cyclase system. Thus, GABA, through an interaction with GABA(,B) receptors, modulates neurotransmitter receptor responsiveness in brain. The pharmocological manipulation of this response could lead to the development of therapeutic agents having a more subtle influence than current drugs on central nervous system function. ^

Mitogenic and oncogenic properties of the small G protein Rap1b

It has been widely reported that the small GTP-binding protein Rap1 has an anti-Ras and anti-mitogenic activity. Thus, it is generally accepted that a normal physiological role of Rap1 proteins is to antagonize Ras mitogenic signals, presumably by forming nonproductive complexes with proteins that are typically effectors or modulators of Ras. Rap1 is activated by signals that raise intracellular levels of cAMP, a molecule that has long been known to exert both inhibitory and stimulatory effects on cell growth. We have now tested the intriguing hypothesis that Rap1 could have mitogenic effects in systems in which cAMP stimulates cell proliferation. The result of experiments addressing this possibility revealed that Rap1 has full oncogenic potential. Expression of Rap1 in these cells results in a decreased doubling time, an increased saturation density, and an unusual anchorage-dependent morphological transformation. Most significantly, however, Rap1-expressing cells formed tumors when injected into nude mice. Thus, we propose that the view that holds Rap1 as an antimitogenic protein should be restricted and conclude that Rap1 is a conditional oncoprotein.

In vitro properties of the first ORF protein from mouse LINE-1 support its role in ribonucleoprotein particle formation during retrotransposition

LINEs are transposable elements, widely distributed among eukaryotes, that move via reverse transcription of an RNA intermediate. Mammalian LINEs have two ORFs (ORF1 and ORF2). The proteins encoded by these ORFs play important roles in the retrotransposition process. Although the predicted amino acid sequence of ORF1 is not closely related to any known proteins, it is highly basic; thus, it has long been hypothesized that ORF1 protein functions to bind LINE-1 (L1) RNA during retrotransposition. Cofractionation of ORF1 protein and L1 RNA in extracts from both mouse and human embryonal carcinoma cells indicated that ORF1 protein binds L1 RNA, forming a ribonucleoprotein particle. Based on UV crosslinking and electrophoretic mobility-shift assays using purified components, we demonstrate here that the ORF1 protein encoded by mouse L1 binds nucleic acids with a strong preference for RNA and other single-stranded nucleic acids. Furthermore, multiple copies of ORF1 protein appear to bind single-stranded nucleic acid in a manner suggesting positive cooperativity; such binding characteristics are likely to be facilitated by the protein–protein interactions detected among molecules of ORF1 polypeptide by coimmunoprecipitation. These observations are consistent with the formation of ribonucleoprotein particles containing L1 RNA and ORF1 protein and provide additional evidence for the role of ORF1 protein during retrotransposition of L1.

Direct molecular level measurements of the electrostatic properties of a protein surface

In this work, we used direct measurements with the surface force apparatus to determine the pH-dependent electrostatic charge density of a single binding face of streptavidin. Mean field calculations have been used with considerable success to model electrostatic potential fields near protein surfaces, but these models and their inherent assumptions have not been tested directly at the molecular level. Using the force apparatus and immobilized, oriented monolayers of streptavidin, we measured a pI of 5–5.5 for the biotin-binding face of the protein. This differs from the pI of 6.3 for the soluble protein and confirms that we probed the local electrostatic features of the macromolecule. With finite difference solutions of the linearized Poisson–Boltzmann equation, we then calculated the pH-dependent charge densities adjacent to the same face of the protein. These calculated values agreed quantitatively with those obtained by direct force measurements. Although our study focuses on the pH-dependence of surface electrostatics, this direct approach to probing the electrostatic features of proteins is applicable to investigations of any perturbations that alter the charge distribution of the surfaces of immobilized molecules.