Identification of T cell epitopes on the 33-kDa fragment of Plasmodium yoelii merozoite surface protein 1 and their antibody-independent protective role in immunity to blood stage malarial

Merozoite surface protein 1 (MSP1) of malaria parasites undergoes proteolytic processing at least twice before invasion into a new RBC. The 42-kDa fragment, a product of primary processing, is cleaved by proteolytic enzymes giving rise to MSP1(33), which is shed from the merozoite surface, and MSP1(19), which is the only fragment carried into a new RBC. In this study, we have identified T cell epitopes on MSP1(33) of Plasmodium yoelii and have examined their function in immunity to blood stage malaria. Peptides 20 aa in length, spanning the length of MSP1(33) and overlapping each other by 10 aa, were analyzed for their ability to induce T cell proliferation in immunized BALB/c and C57BL/6 mice. Multiple epitopes were recognized by these two strains of mice. Effector functions of the dominant epitopes were then investigated. Peptides Cm15 and Cm21 were of particular interest as they were able to induce effector T cells capable of delaying growth of lethal P. yoelii YM following adoptive transfer into immuno-deficient mice without inducing detectable Ab responses. Homologs of these epitopes could be candidates for inclusion in a subunit vaccine.

Linearization of a naturally occurring circular protein maintains structure but eliminates hemolytic activity

Cyclotides are a recently discovered family of disulfide rich proteins from plants that contain a circular protein backbone. They are exceptionally stable, as exemplified by their use in native medicine of the prototypic cyclotide kalata B1. The peptide retains uterotonic activity after the plant from which it is derived is boiled to make a medicinal tea. The circular backbone is thought to be in part responsible for the stability of the cyclotides, and to investigate its role in determining structure and biological activity, an acyclic derivative, des-(24-28)-kalata B1, was chemically synthesized and purified. This derivative has five residues removed from the 29-amino acid circular backbone of kalata B1 in a loop region corresponding to a processing site in the biosynthetic precursor protein. Two-dimensional NMR spectra of the peptide were recorded, assigned, and used to identify a series of distance, angle, and hydrogen bonding restraints. These were in turn used to determine a representative family of solution structures. Of particular interest was a determination of the structural similarities and differences between des-(2428)-kalata B1 and native kalata B1. Although the overall three-dimensional fold remains very similar to that of the native circular protein, removal of residues 24-28 of kalata B1 causes disruption of some structural features that are important to the overall stability. Furthermore, loss of hemolytic activity is associated with backbone truncation and linearization.

Localization of the Epstein-Barr virus protein LMP 1 to exosomes

The Epstein-Barr virus latent membrane protein (LMP 1) functions as a constitutively active signalling molecule and associates in lipid rafts clustered with other signalling molecules. Using immunofluorescent confocal microscopy, LMP 1 was shown to have an heterogeneous distribution among individual cells which was not related to the cell cycle stage. LMP 1 was shown to localize to intracellular compartments in cells other than the plasma membrane, Co-labelling of cells with both an LIMP 1 antibody and an antibody to the Golgi protein GS15 revealed that the intracellular LMP 1 partly co-localized with the Golgi apparatus. Further confirmation of intracellular LMP 1 localization was obtained by immunoelectron microscopy with rabbit polyclonal LIMP 1 antibodies and cryosectioning. As well as being present in intracellular foci, LMP 1 co-localized in part with MHC-II and was present on exosomes derived from a lymphoblastoid cell line. Preparations of LMP 1 containing exosomes were shown to inhibit the proliferation of peripheral blood mononuclear cells, suggesting that LIMP 1 could be involved in immune regulation. This may be of particular relevance in EBV-associated tumours such as nasopharyngeal carcinoma and Hodgkin's disease, as LMP 1-containing exosomes may be taken up by infiltrating T-lymphocytes, where LMP 1 could exert an anti-proliferative effect, allowing the tumour cells to evade the immune system.

The trans-membrane protein p25 forms highly specialized domains that regulate membrane composition and dynamics

Trans-membrane proteins of the p24 family are abundant, oligomeric proteins predominantly found in cis-Golgi membranes. They are not easily studied in vivo and their functions are controversial. We found that p25 can be targeted to the plasma membrane after inactivation of its canonical KKXX motif (KK to SS, p25SS), and that p25SS causes the co-transport of other p24 proteins beyond the Golgi complex, indicating that wild-type p25 plays a crucial role in retaining p24 proteins in cis-Golgi membranes. We then made use of these observations to study the intrinsic properties of these proteins, when present in a different membrane context. At the cell surface, the p25SS mutant segregates away from both the transferrin receptor and markers of lipid rafts, which are enriched in cholesterol and glycosphingolipids. This suggests that p25SS localizes to, or contributes to form, specialized membrane domains, presumably corresponding to oligomers of p25SS and other p24 proteins. Once at the cell surface, p25SS is endocytosed, together with other p24 proteins, and eventually accumulates in late endosomes, where it remains confined to well-defined membrane regions visible by electron microscopy. We find that this p25SS accumulation causes a concomitant accumulation of cholesterol in late endosomes, and an inhibition of their motility - two processes that are functionally linked. Yet, the p25SS-rich regions themselves seem to-exclude not only Lamp1 but also accumulated cholesterol. One may envision that p25SS accumulation, by excluding cholesterol from oligomers, eventually overloads neighboring late endosomal membranes with cholesterol beyond their capacity (see Discussion). In any case, our data show that p25 and presumably other p24 proteins are endowed with the intrinsic capacity to form highly specialized domains that control membrane composition and dynamics. We propose that p25 and other p24 proteins control the fidelity of membrane transport by maintaining cholesterol-poor membranes in the Golgi complex.

Dietary interactions influence the effects of bovine folate-binding protein on the bioavailability of tetrahydrofolates in rats

The newborns of mammals have a high folate demand, yet obtain adequate folate nutrition solely from their mothers' milk despite its low folate content. Milk folate is entirely bound by an excess of folate-binding protein (FBP), prompting speculation that FBP may affect the bioavailability of the limited folate supply. Previous research has shown that FBP-bound folic acid is more gradually absorbed, thereby reducing the peak plasma folate concentration and preventing loss into the urine. Natural folates are reduced derivatives of folic acid, with milk predominantly containing 5-methyltetrahydrofolate, yet little research has been carried out to determine the role of FBP in the bioavailability of reduced folates. We studied the effect of FBP on folate nutrition of rats in both single-dose and 4-wk feeding experiments. The effect of FBP was influenced by the presence of other milk components. FBP increased bioavailability of dietary folate when it was consumed with other whey proteins or with soluble casein. However, in the presence of acid-precipitated casein or a whey preparation enriched in lipids, bioavailability was decreased. These results highlight the difficulties of extrapolating from experimental results obtained using purified diets alone and of studying interactions among dietary components. They suggest that the addition of FBP-rich foods to folate-rich foods could enhance the bioavailability of natural folates, but that the outcome of such a combination would depend on interactions with other components of the diet.

Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily

The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant, and fungal kingdoms and is implicated in human brain tumor growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterization, cloning and structural homology modeling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to >95% purity by activity-guided fractionation using a para-nitroanilide substrate based on the putative cleavage site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins that have a cysteine-rich C-terminal tail. A homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.

Inhibition of 19-kDa C-terminal region of merozoite surface protein-1-specific antibody responses in neonatal pups by maternally derived 19-kDa C-terminal region of merozoite surface protein-1-specific antibodies but not whole parasite-specific antibodies

Immunizing pregnant women with a malaria vaccine is one approach to protecting the mother and her offspring from malaria infection. However, specific maternal Abs generated in response to vaccination and transferred to the fetus may interfere with the infant's ability to respond to the same vaccine. Using a murine model of malaria, we examined the effect of maternal 19-kDa C-terminal region of merozoite surface protein-1 (MSP1(19)) and Plasmodium yoelii Abs on the pups' ability to respond to immunization with MSP1(19). Maternal MSPI,g-specific Abs but not A yoelii-specific Abs inhibited Ab production following MSP1(19) immunization in 2-wk-old pups. This inhibition was correlated with the amount of maternal MSP1(19) Ab present in the pup at the time of immunization and was due to fewer specific B cells. Passively acquired Ab most likely inhibited the development of an Ab response by blocking access to critical B cell epitopes. If a neonate's ability to respond to MSP1(19) vaccination depends on the level of maternal Abs present at the time of vaccination, it may be necessary to delay immunization until Abs specific for the vaccinating Ag have decreased.

Enrichment, isolation and characterisation of ruminal bacteria that degrade non-protein amino acids from the tropical legume Acacia angustissima

Acacia angustissima has been proposed as a protein supplement in countries where low quality forages predominate. A number of non-protein amino acids have been identified in the leaves of A. angustissima and these have been linked to toxicity in ruminants. The non-protein amino acid 4-n-acetyl-2,4-diaminobutyric acid (ADAB) has been shown to be the major amino acid in the leaves of A. angustissima. The current study aimed to identify micro-organisms from the rumen environment capable of degrading ADAB by using a defined rumen-simulating media with an amino acid extract from A. angustissima. A mixed enrichment culture was obtained that exhibited substantial ADAB-degrading ability. Attempts to isolate an ADAB-degrading micro-organism were carried out, however no isolates were able to degrade ADAB in pure culture. This enrichment culture was also able to degrade the non-protein amino acids diaminobutyric acid (DABA) and diaminopropionic acid (DAPA) which have structural similarities to ADAB. Two isolates were obtained which could degrade DAPA. One isolate is a novel Grain-positive rod (strain LPLR3) which belongs to the Firmicutes and is not closely related to any previously isolated bacterium. The other isolate is strain LPSR1 which belongs to the Gammaproteobacteria and is closely related (99.93% similar) to Klebsiella pneumoniae subsp. ozaenae. The studies demonstrate that the rumen is a potential rich source of undiscovered micro-organisms which have novel capacities to degrade plant secondary compounds. (c) 2005 Elsevier B.V. All rights reserved.

The human papillomavirus type 16 E7 protein binds human interferon regulatory factor-9 via a novel PEST domain required for transformation

It is critical that viruses are able to avoid the antiviral activities of interferon (IFN). We have shown previously that the human papillomavirus (HPV) is able to avoid IFN-alpha via interaction of the HPV-16 E7 protein with IFN regulatory factor-9 (IRF-9). Here, we investigated the details of the interaction using HPV-16 E7 peptide mapping to show that IRF-9 binds HPV-16 E7 in a domain encompassing amino acids 25-36. A closer examination of this region indicates this is a novel proline, glutamate, serine, and threonine-rich (PEST) domain, with a PEST score of 8.74. We have also mapped the region of interaction within IRF-9 and found that amino acids 354-393 play an important role in binding to HPV-16 E7. This region of IRF-9 encompasses the IRF association domain (IAD), a region important for protein-protein interaction central to IRF function. Finally, we used alanine-scanning mutagenesis to determine if E7-IRF-9 interaction was important for E7-mediated cellular transformation and found that the HPV-16 E7 mutants Y25A, E26A, S31A, S32A, and E35A, but not L28A and N29A, caused loss of transformation ability. Preliminary data suggest loss of IRF-9 interaction with E7 mutants correlated with transformation. Our work suggests E7- IRF- 9 interaction is important for the transforming ability of HPV-16 E7 and that HPV-16 E7 may interact with other IRF proteins that have IAD domains.

Cytokine secretion via cholesterol-rich lipid raft-associated SNAREs at the phagocytic cup

Lipopolysaccharide-activated macrophages rapidly synthesize and secrete tumor necrosis factor alpha(TNF alpha) to prime the immune system. Surface delivery of membrane carrying newly synthesized TNF alpha is controlled and limited by the level of soluble N-ethylmaleimide-sensitive factor attachment protein receptor ( SNARE) proteins syntaxin 4 and SNAP-23. Many functions in immune cells are coordinated from lipid rafts in the plasma membrane, and we investigated a possible role for lipid rafts in TNF alpha trafficking and secretion. TNF alpha surface delivery and secretion were found to be cholesterol-dependent. Upon macrophage activation, syntaxin 4 was recruited to cholesterol-dependent lipid rafts, whereas its regulatory protein, Munc18c, was excluded from the rafts. Syntaxin 4 in activated macrophages localized to discrete cholesterol-dependent puncta on the plasma membrane, particularly on filopodia. Imaging the early stages of TNF alpha surface distribution revealed these puncta to be the initial points of TNF alpha delivery. During the early stages of phagocytosis, syntaxin 4 was recruited to the phagocytic cup in a cholesterol-dependent manner. Insertion of VAMP3-positive recycling endosome membrane is required for efficient ingestion of a pathogen. Without this recruitment of syntaxin 4, it is not incorporated into the plasma membrane, and phagocytosis is greatly reduced. Thus, relocation of syntaxin 4 into lipid rafts in macrophages is a critical and rate-limiting step in initiating an effective immune response.

The matricellular protein SPARC is internalized in Sertoli, Leydig, and germ cells during testis differentiation

The gene encoding the matricellular protein secreted protein, acidic and rich in cysteine (SPARC) was identified in a screen for genes expressed sex-specifically during mouse gonad development, as being strongly upregulated in the male gonad from very early in testis development. We present here a detailed analysis of SPARC gene and protein expression during testis development, from 11.5 to 15.5 days post coitum (dpc). Section in situ hybridization analysis revealed that SPARC mRNA is expressed by the Sertoli cells in the testis cords and the fetal Leydig cells, found within the interstitial space between the testis cords. Immunodetection with anti-SPARC antibody showed that the protein was located inside the testis cords, within the cytoplasm of Sertoli and germ cells. In the interstitium, SPARC was present intracellularly within the Leydig cells. The internalization of SPARC in Sertoli, Leydig, and germ cells suggests that it plays an intracellular regulatory role in these cell types during fetal testis development.

Particle formation during soy protein precipitation

The current success of soy foods is driving soy ingredient manufacturers to develop innovative products for food manufacturers. One such innovation is separating the soy proteins glycinin and beta-conglycinin to take advantage of their individual functional and nutritional properties. Precipitation by acidification is a low-cost method for separating these two proteins. Separation is achieved by preferentially precipitating glycinin at pH ~ 6 while beta-conglycinin remains in solution. Understanding the particle formation during protein precipitation is important as it can influence the efficiency of the precipitation process as well as subsequent downstream processes such as the particle-liquid separation step, usually achieved by centrifugation. Most of the previous soy protein precipitation studies are limited to precipitation at pH 4 as this is the pH range most commonly used in the commercial manufacturing of soy protein isolates. To date, there have been no studies on the particle formation during precipitation at pH > 5.Precipitation of soy protein is generally thought to occur by the rapid formation of primary particles in the size range of 0.1 - 0.3 microns followed by aggregation of these particles via collision to aggregates of size about 1 - 50 microns. The formation of the primary particles occurs on a time scale much shorter than that of the overall precipitation process (Nelson and Glatz, 1985). This study shows that precipitation of soy protein is indeed rapid. At high pH levels, binary liquid-liquid separation occurs forming a protein-rich heavy phase. The protein-rich phase appears as droplets which can be coalesced to form a uniform bulk layer under centrifugation forces. Upon lowering the pH level by the addition of acid, further protein is precipitated as amorphous material which binds the droplets together to form aggregates of amorphous precipitates. Liquid-liquid separation has been observed in many protein solutions but this phenomenon has only scarcely been reported in the literature for soy proteins. It presents an exciting opportunity for an innovative product. Features of the liquid-phase protein such as protein yield and purity will be characterized.

Cross-sectional and prospective relationships of interleukin-6 and C-reactive protein with physical performance in elderly persons: MacArthur Studies of Successful aging

Although IL-6 has been shown to predict onset of disability in older persons and both IL-6 and CRP are associated with motality risk, these markers of inflammation have only limited associations with physical performance, except for walking measures and grip strength at baseline, and do not predict change in performance 7 years later in a high-functioning subset of older adults.

Rich task implementation: modernism meets postmodernism

School renewal', 'productive pedagogies', 'rich tasks', 'New Basics', 'key learning areas'--these are some of the discourses of change in selected Queensland schools. This paper will report on teaching as an insider/outsider in a school's Health and Physical Education department during a time of intense pressure for structural, curriculum and pedagogical shifts. As a teacher/researcher, I spent ten weeks in a government secondary school attempting to implement rich tasks as well as collect data using formal and informal interviews, field note, and document analyses, with a focus upon teachers', students' and administrators' sense of change processes and outcomes. It is suggested that the processes of, and barriers to, curriculum change in this context are best explained in terms of tensions between modernist and postmodernist phenomena.

Geographic variation and positive selection on M7 lysin, an acrosomal sperm protein in mussels (Mytilus spp.)

Successful fertilization in free-spawning marine organisms depends on the interactions between genes expressed on the surfaces of eggs and sperm. Positive selection frequently characterizes the molecular evolution of such genes, raising the possibility that some common deterministic process drives the evolution of gamete recognition genes and may even be important for understanding the evolution of prezygotic isolation and speciation in the marine realm. One hypothesis is that gamete recognition genes are subject to selection for prezygotic isolation, namely reinforcement. In a previous study, positive selection on the gene coding for the acrosomal sperm protein M7 lysin was demonstrated among allopatric populations of mussels in the Mytilus edulis species group (M. edulis, M. galloprovincialis, and M. trossulus). Here, we expand sampling to include M7 lysin haplotypes from populations where mussel species are sympatric and hybridize to determine whether there is a pattern of reproductive character displacement, which would be consistent with reinforcement driving selection on this gene. We do not detect a strong pattern of reproductive character displacement; there are no unique haplotypes in sympatry nor is there consistently greater population structure in comparisons involving sympatric populations. One distinct group of haplotypes, however, is strongly affected by natural selection and this group of haplotypes is found within M. galloprovincialis populations throughout the Northern Hemisphere concurrent with haplotypes common to M. galloprovincialis and M. edulis. We suggest that balancing selection, perhaps resulting from sexual conflicts between sperm and eggs, maintains old allelic diversity within M. galloprovincialis.