Highly immunogenic and totally synthetic lipopeptides as self-adjuvanting immunocontraceptive vaccines

In this study, we describe the synthesis of various lipopeptides based on the sequence of luteinizing hormone-releasing hormone (LHRH) and report on their abilities to induce Abs against this "self" hormone when inoculated into mice in the absence of additional adjuvant. The peptides consisted of a colinear CD4(+) T helper cell epitope from the L chain of influenza virus hemagglutinin and LHRH, which has B cell epitopes but no T cell epitopes present in its sequence. Lipids were attached either at the N terminus or between the T cell epitope and LHRH, in the approximate center of the peptide. The lipopeptide constructs displayed different solubilities and immunological properties that depended not only on the lipid content but also on the position of attachment of the lipids. Some of these constructs were highly immunogenic, inducing high titers of Ab, which were capable of efficiently sterilizing female mice when administered in saline by s.c. or intranasal routes. The most effective vaccines were highly soluble, contained the dipalmitoyl-S-glyceryl cysteine moiety, and had this lipid attached at the center of the molecule. The relative ability of the lipopeptides to induce an Ab response in the absence of external adjuvant was reflected by their ability to up-regulate the surface expression of MHC class II molecules on immature dendritic cells. These results demonstrate that the composition and position within peptide vaccines of self-adjuvanting lipid groups can influence the ability to induce the maturation of dendritic cells and, in turn, the magnitude of the resulting Ab response.     

Biodegradable polymeric nanospheres formed by temperature-induced phase transition in a mixture of poly(lactide-co-glycolide) and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer

The mixture of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer(F-127) and PLGA (poly(lactide-co-gycolide)) forms a liquid state above their phase transition temperatures, and the phase-separated state is induced by decreasing the temperature below the phase transition temperature. On the basis of the temperature-induced phase transition behavior in the mixture of F-127 and PLGA, a novel method for the preparation of drug-loaded PLGA nanospheres was designed and characterized by measuring the loading amount, the encapsulation efficiency, and the drug release pattern. Paclitaxel, used as a potent anticancer drug, was selected as a model drug.     

Deamidation, but not truncation, decreases the urea stability of a lens structural protein, betaB1-crystallin

Crystallins, the major structural proteins in the lens of the eye, are maintained with little turnover throughout the lifetime of the host. With time, lens crystallins undergo post-translational modifications that may play an important role in loss of vision during aging and cataract formation. Specific modifications include deamidation and truncation. Urea-induced denaturation was studied for recombinantly expressed wild-type betaB1 (WT), the deamidated mutant (Q204E), an N-terminally truncated mutant (betaB1(DeltaN41)), and other truncated versions of these proteins generated by calpain II digestion. Tryptophan fluorescence was used to monitor loss of global tertiary structure. Loss of secondary structure was followed by circular dichroism, and electron paramagnetic resonance site-directed spin labeling was used to monitor loss of tertiary structure selectively in the N-terminal domain. Our results indicated that the deamidated mutant was significantly destabilized relative to WT. Q204E showed a two-step denaturation curve with transitions at 4.1 and 7.2 M urea, whereas denaturation of WT occurred in a cooperative single step with a transition midpoint of 5.9 M urea. Unfolding of WT was completely reversible, whereas Q204E failed to fully refold. Prolonged incubation under denaturing conditions led to aggregation, which was also more pronounced for Q204E dimers than for WT. Truncation of 41 residues from the N-terminus or 47 and 5 residues from the N- and C-termini did not affect stability. These studies indicated that a single-site deamidation could significantly diminish the stability of lens betaB1-crystallin, supporting the idea that such modifications may play an important role in age-related cataract formation.     

PROTEOME-3D: an interactive bioinformatics tool for large-scale data exploration and knowledge discovery

Comprehensive understanding of biological systems requires efficient and systematic assimilation of high-throughput datasets in the context of the existing knowledge base. A major limitation in the field of proteomics is the lack of an appropriate software platform that can synthesize a large number of experimental datasets in the context of the existing knowledge base. Here, we describe a software platform, termed PROTEOME-3D, that utilizes three essential features for systematic analysis of proteomics data: creation of a scalable, queryable, customized database for identified proteins from published literature; graphical tools for displaying proteome landscapes and trends from multiple large-scale experiments; and interactive data analysis that facilitates identification of crucial networks and pathways. Thus, PROTEOME-3D offers a standardized platform to analyze high-throughput experimental datasets for the identification of crucial players in co-regulated pathways and cellular processes.     

Effect of magnetic therapy on selected physical performances

The purpose of this study was to investigate the effects of magnetic therapy in the form of shoe insoles on vertical jump, bench squat, 40-yd dash, and a soccer-specific fitness test performance. Subjects were 14 collegiate male soccer players who were pretested, retested 3 weeks later, and then placed into a double-blind control or treatment group using a matching procedure. The control group received magnetic shoe insoles with a rating of 125 gauss, and the treatment group received insoles with a rating of 600 gauss. Subjects wore the insoles during practice and games for 7 weeks and were then retested. Results indicated significant differences among test scores during the 3 time periods but not between the treatment and control groups. There was a decline in 40-yd dash performance from the initial evaluation (5.10 seconds) to the final evaluation (5.08 seconds). There were no other significant differences. Within the limitations of the study, magnetic therapy did not improve physical performance.     

Identification of H-specific determinants in flagellin of four Escherichia coli strains

The H serogroup of Escherichia coli is determined by the flagellar antigen, flagellin. Sequence analysis of the flagellin gene, fliC, reveals a central variable region and the highly conserved N- and C-termini. This variable region has been shown to encode both H-specific and cross-reactive epitopes. Using polyclonal antibodies, we mapped the linear H-specific determinants in flagellin from four E. coli serotypes O157:H10, 0138:H14, O157:H42 and O157:H43. The specificity of all potential fragments was verified with 52 ECRC (Escherichia coli Reference Center) H-specific antisera. Our results indicated that: (a) a specific determinant of H10 flagellin (1263 bp long) maps to the region covering amino acid residues 305-331; (b) a specific determinant of H14 flagellin (1653 bp long) maps to the region covering amino acid residues 430-461; (c) a specific determinant of H42 flagellin (1281 bp long) maps to a region covering amino acid residues 171-201; and (d) a specific determinant of H43 flagellin (1506 bp long) maps to a region covering amino acid residues 200-260.     

Ltx1, a mouse locus that influences the susceptibility of macrophages to cytolysis caused by intoxication with Bacillus anthracis lethal factor, maps to chromosome 11

The lethal factor (LF) toxin that is produced by Bacillus anthracis plays an important role in the pathogenesis of anthrax. LF has mononuclear phagocyte-specific intoxicating effects that are not well understood. We have identified genetic differences in inbred mouse strains that determine whether their cultured macrophages are susceptible to the cytolytic effect of LF intoxication. Our identification of resistant and susceptible mouse strains enabled us to analyse crosses between these strains and to map a single responsible gene (called Ltx1 ) to chromosome 11. Ltx1 probably influences intoxication events that occur after the delivery of LF to the cytosol, as all mouse macrophages are killed by polypeptides containing the catalytic domain of Diphtheria toxin fused to the domain of LF required for cytosolic transport. Furthermore, the susceptibility phenotype is dominant to resistance, suggesting that resistance is caused by an absence of or polymorphism in a molecule that acts jointly with, or downstream of, the activity of LF. Our mapping of Ltx1 is a crucial first step in its positional cloning, which will provide more information about the mechanism of LF intoxication.     

Characterizing Tropical Tree Species Growth Strategies: Learning from Inter-Individual Variability and Scale Invariance

Understanding how tropical tree species differ in their growth strategies is critical to predict forest dynamics and assess species coexistence. Although tree growth is highly variable in tropical forests, species maximum growth is often considered as a major axis synthesizing species strategies, with fast-growing pioneer and slow-growing shade tolerant species as emblematic representatives. We used a hierarchical linear mixed model and 21-years long tree diameter increment series in a monsoon forest of the Western Ghats, India, to characterize species growth strategies and question whether maximum growth summarizes these strategies. We quantified both species responses to biotic and abiotic factors and individual tree effects unexplained by these factors. Growth responses to competition and tree size appeared highly variable among species which led to reversals in performance ranking along those two gradients. However, species-specific responses largely overlapped due to large unexplained variability resulting mostly from inter-individual growth differences consistent over time. On average one-third of the variability captured by our model was explained by covariates. This emphasizes the high dimensionality of the tree growth process, i.e. the fact that trees differ in many dimensions (genetics, life history) influencing their growth response to environmental gradients, some being unmeasured or unmeasurable. In addition, intraspecific variability increased as a power function of species maximum growth partly as a result of higher absolute responses of fast-growing species to competition and tree size. However, covariates explained on average the same proportion of intraspecific variability for slow- and fast-growing species, which showed the same range of relative responses to competition and tree size. These results reflect a scale invariance of the growth process, underlining that slow- and fast-growing species exhibit the same range of growth strategies.