Positions of multiple insertions in SSU rDNA of lichen-forming fungi

Lichen-forming fungi, in symbiotic associations with algae, frequently have nuclear small subunit ribosomal DNA (SSU rDNA) longer than the 1,800 nucleotides typical for eukaryotes. The lichen-forming ascomycetous fungus Lecanora dispersa contains insertions at eight distinct positions of its SSU rDNA; the lichen-forming fungi Calicium tricolor and Porpidia crustulata each contain one insertion. Insertions are not limited to fungi that form lichens; the lichen ally Mycocalicium albonigrum also contains two insertions. Of the 11 insertion positions now reported for lichen-forming fungi and this ally, 6 positions are known only from lichen-forming fungi. Including the 4 newly reported in this study, insertions are now known from at least 17 positions among all reported SSU rDNA sequences. Insertions, most of which are Group I introns, are reported in fungal and protistan lineages and occur at corresponding positions in genomes as phylogenetically distant as the nuclei of fungi, green algae, and red algae. Many of these positions are exposed in the mature rRNA tertiary structure and may be subject to independent insertion of introns. Insertion of introns, accompanied by their sporadic loss, accounts for the scattered distribution of insertions observed within the SSU rDNA of these diverse organisms.      

Partial reconstruction of the microvillus core bundle: characterization of villin as a Ca(++)-dependent, actin-bundling/depolymerizing protein

The brush border, isolated from chicken intestine epithelial cells, contains the 95,000 relative molecular mass (M(r)) polypeptide, villin. This report describes the purification and characterization of villin as a Ca(++)-dependent, actin bundling/depolymerizing protein. Then 100,000 g supernatant from a Ca(++) extract of isolated brush borders is composed of three polypeptides of 95,000 (villin), 68,000 (fimbrin), and 42,000 M(r) (actin). Villin, following purification from this extract by differential ammonium sulfate precipitation and ion-exchange chromatography, was mixed with skeletal muscle F-actin. Electron microscopy of negatively stained preparations of these villin-actin mixtures showed that filament bundles were present. This viscosity, sedimentability, and ultrastructural morphology of filament bundles are dependent on the villin:actin molar ratio, the pH, and the free Ca(++) concentration in solution. At low free Ca(++) (less than 10(-6) M), the amount of protein in bundles, when measured by sedimentation, increased as the villin: actin molar ratio increased and reached a plateau at approximately a 4:10 ratio. This behavior correlates with the conversion of single actin filaments into filament bundles as detected in the electron microscope. At high free Ca(++) (more than 10(-6) M), there was a decrease in the apparent viscosity in the villin-actin mixtures to a level measured for the buffer. Furthermore, these Ca(++) effects were correlated with the loss of protein sedimented, the disappearance of filament bundles, and the appearance of short fragments of filaments. Bundle formation is also pH-sensitive, being favored at mildly acidic pH. A decrease in the pH from 7.6 to 6.6 results in an increase in sedimentable protein and also a transformation of loosly associated actin filaments into compact actin bundles. These results are consistent with the suggestions that villin is a bundling protein in the microvillus and is responsible for the Ca(++)-sensitive disassembly of the microvillar cytoskeleton. Thus villin may function in the cytoplasm as a major cytoskeletal element regulating microvillar shape.     

Synthesis and biophysical characterization of engineered topographic immunogenic determinants with alpha alpha topology.

Model peptides with predetermined secondary, tertiary, and quaternary conformation have been successfully designed, synthesized, and characterized in an attempt to mimic the three-dimensional structure of an antigenic determinant. This work is a continuing effort to map the antigenic structure of the protein antigen lactate dehydrogenase C4 (LDH-C4) to develop a contraceptive vaccine. A putative topographic determinant with alpha alpha topology which associates into four-helix bundles was designed on the basis of the framework model of protein folding. An idealized amphiphilic 18-residue sequence (alpha 1) and a 40-residue alpha alpha fold (alpha 3) have been shown to form stable 4-helix structures in solution with a free energy of association on the order of -20.8 kcal/mol (tetramerization of alpha 1) and -7.8 kcal/mol (dimerization of alpha 3). Both alpha 1 and alpha 3 form stable monolayers at the air-water interface. The CD spectra of Langmuir-Blodgett monolayers are characteristically alpha-helical. Both CD and FTIR spectroscopic studies reval a high degree of secondary structure. The SAXS data strongly suggest that the helices are arranged in a four-helix bundle since the radius of gyration of 17.2 A and the vector distribution function are indicative of a prolate ellipsoid of axial dimensions and molecular weight appropriate for the four-helix bundle. The major contribution to the formation and stabilization of alpha 1 and alpha 3 is believed to be hydrophobic interaction between the amphiphilic alpha-helices. The displayed heptad repeat, helix dipole, ion pairs, and the loop sequence may have also contributed to the overall stability and antiparallel packing of the helices. A detailed structural analysis of a relevant topographic immunogenic determinant will elucidate the nature of antigen-antibody interactions as well as provide insight into protein folding intermediates.     

On the inhibition of muscle membrane chloride conductance by aromatic carboxylic acids

25 aromatic carboxylic acids which are analogs of benzoic acid were tested in the rat diaphragm preparation for effects on chloride conductance (G(Cl)). Of the 25, 19 were shown to reduce membrane G(Cl) with little effect on other membrane parameters, although their apparent K(i) varied widely. This inhibition was reversible if exposure times were not prolonged. The most effective analog studied was anthracene-9-COOH (9-AC; K(i) = 1.1 x 10(-5) M). Active analogs produced concentration-dependent inhibition of a type consistent with interaction at a single site or group of sites having similar binding affinities, although a correlation could also be shown between lipophilicity and K(i). Structure-activity analysis indicated that hydrophobic ring substitution usually increased inhibitory activity while para polar substitutions reduced effectiveness.

These compounds do not appear to inhibit G(Cl) by altering membrane surface charge and the inhibition produced is not voltage dependent. Qualitative characteristics of the I-V relationship for Cl(-) current are not altered. Conductance to all anions is not uniformly altered by these acids as would be expected from steric occlusion of a common channel. Concentrations of 9-AC reducing G(Cl) by more than 90 percent resulted in slight augmentation of G(I). The complete conductance sequence obtained at high levels of 9-AC was the reverse of that obtained under control conditions. Permeability sequences underwent progressive changes with increasing 9-AC concentration and ultimately inverted at high levels of the analog. Aromatic carboxylic acids appear to inhibit G(Cl) by binding to a specific intramembrane site and altering the selectivity sequence of the membrane anion channel.

     

Protein glycosylation mutants of procyclic Trypanosoma brucei: defects in the asparagine-glycosylation pathway

We employed a genetic approach to study protein glycosylation in the procyclic form of the parasite Trypanosoma brucei. Two different mutant parasites, ConA 1-1 and ConA 4-1, were isolated from mutagenized cultures by selecting cells which resisted killing or agglutination by concanavalin A. Both mutant cells show reduced concanavalin A binding. However, the mutants have different phenotypes, as indicated by the fact that ConA 1-1 binds to wheat germ agglutinin but ConA 4-1 and wild type do not. A blot probed with concanavalin A revealed that many proteins in both mutants lost the ability to bind this lectin, and the blots resembled one of wild type membrane proteins treated with PNGase F. This finding suggested that the mutants had altered asparagine- linked glycosylation. This conclusion was confirmed by studies on a flagellar protein (Fla1) and procyclic acidic repetitive protein (PARP). Structural analysis indicated that the N- glycan of wild type PARP is exclusively Man5GlcNAc2 whereas that in both mutants is predominantly a hybrid type with a terminal N- acetyllactosamine. The occupancy of the PARP glycosylation site in ConA 4-1 was much lower than that in ConA 1-1. These mutants will be useful for studying trypanosome glycosylation mechanisms and function.      

HER-2/neu Cancer Vaccines: Present Status and Future Prospects

Immunotherapeutic approaches to cancer should focus on novel undertakings that modulate immune responses by synergistic enhancement of anti-tumor immunological parameters. Cancer vaccines should preferably be composed of multiple defined tumor antigen specific B- and T-cell epitopes. The main focus of this article is to briefly review the present status of Her-2/neu vaccine strategies and to describe the innovative strategies developed in my laboratory for a vaccine against HER-2/neu (ErbB-2) with emphasis on the humoral arm of the immune response. Elucidating the underlining mechanisms of anti-tumor effects elicited by peptide vaccines against a self-protein is a requirement for developing an immunotherapeutic strategy that might be effective in human cancer vaccines. Our approach entails the identification of biologically relevant epitopes, establishing relevant in vitro assays for monitoring vaccine efficacy, devising strategies to engineer conformationally dependent sequences, developing highly immunogenic vaccines for an outbred population and delivering the immunogen/vaccine in a safe and efficacious vehicle, utilizing transgenic animal models for assessing tumor development, and developing challenge models using transplantable tumors to study efficacy of vaccine constructs. We have developed a multi-HER-2/neu B-cell epitope approach and shown in preclinical studies that immunization with a combination of two B-cell epitope was more effective in preventing mammary tumors than a single epitope. We have translated that work to the clinic (OSU 0105) in an FDA approved, NCI sponsored “Phase 1 Active Immunotherapy trial with Chimeric and Multi-epitope based peptide vaccine targeting HER-2 oncoprotein and nor-MDP adjuvant in patients with metastatic and/or recurrent solid tumors” at the James Cancer Hospital at the Ohio State University. The correlation between overexpression of HER-2/neu and up-regulation of VEGF has been demonstrated in breast cancer patients. Thus, blocking angiogenesis is an attractive strategy to inhibit tumor growth, invasion, and metastasis. The hypothesis that combination of anti-angiogenic therapy and tumor immunotherapy of cancer may be synergistic is an important future goal. In this review, I will discuss insights into our preclinical studies that might aid in the design of the next generation of cancer vaccines and become an integrated component of prophylactic/preventive and therapeutic approach.     

Engineered conformation-dependent VEGF peptide mimics are effective in inhibiting VEGF signaling pathways

Angiogenesis, or formation of new blood vessels, is crucial to cancer tumor growth. Tumor growth, progression, and metastasis are critically influenced by the production of the pro-angiogenic vascular endothelial growth factor (VEGF). Promising anti-angiogenic drugs are currently available; however, their susceptibilities to drug resistance and long term toxicity are serious impediments to their use, thus requiring the development of new therapeutic approaches for safe and effective angiogenic inhibitors. In this work, peptides were designed to mimic the VEGF-binding site to its receptor VEGFR-2. The VEGF conformational peptide mimic, VEGF-P3(CYC), included two artificial cysteine residues, which upon cyclization constrained the peptide in a loop native-like conformation to better mimic the anti-parallel structure of VEGF. The engineered cyclic VEGF mimic peptide demonstrated the highest affinity to VEGFR-2 by surface plasmon resonance assay. The VEGF peptide mimics were evaluated as inhibitors in several in vitro assays in which VEGF-dependent signaling pathways were observed. All VEGF mimics inhibited VEGFR-2 phosphorylation with VEGF-P3(CYC) showing the highest inhibitory effects when compared with unstructured peptides. Additionally, we show in several angiogenic in vitro assays that all the VEGF mimics inhibited endothelial cell proliferation, migration, and network formation with the conformational VEGF-P3 (CYC) being the best. The VEGF-P3(CYC) also caused a significant delay in tumor development in a transgenic model of VEGF(+/-)Neu2-5(+/-). These results indicate that the structure-based design is important for the development of this peptidomimetic and for its anti-angiogenic effects.     

Experimental evaluation of the relationship between lethal or non-lethal virulence and transmission success in malaria parasite infections

Background  

Evolutionary theory suggests that the selection pressure on parasites to maximize their transmission determines their optimal host exploitation strategies and thus their virulence. Establishing the adaptive basis to parasite life history traits has important consequences for predicting parasite responses to public health interventions. In this study we examine the extent to which malaria parasites conform to the predicted adaptive trade-off between transmission and virulence, as defined by mortality. The majority of natural infections, however, result in sub-lethal virulent effects (e.g. anaemia) and are often composed of many strains. Both sub-lethal effects and pathogen population structure have been theoretically shown to have important consequences for virulence evolution. Thus, we additionally examine the relationship between anaemia and transmission in single and mixed clone infections.     

Peptide vaccines of the HER-2/neu dimerization loop are effective in inhibiting mammary tumor growth in vivo

Human epidermal growth factor receptor-2 (HER-2)/neu (ErbB2), a member of the epidermal growth factor family of receptors, is overexpressed in 20-30% of breast cancers. It is an attractive target for receptor-directed antitumor therapy using mAbs. Unlike other epidermal growth factor receptor family members, HER-2/neu does not bind a high-affinity ligand, but rather functions as the preferred dimerization partner. Pertuzumab (Omnitarg) is a humanized mAb directed against the HER-2/neu dimerization domain that inhibits receptor signaling. The recent definition of the crystal structure of the HER-2/neu-pertuzumab complex demonstrated that the receptor dimerization region encompassed residues 266-333. Based on the three-dimensional structure of the complex, we have designed three conformational peptide constructs (sequences 266-296, 298-333, and 315-333) to mimic regions of the dimerization loop of the receptor and to characterize their in vitro and in vivo antitumor efficacy. All the constructs elicited high-affinity peptide Abs that inhibited multiple signaling pathways including HER-2/neu-specific inhibition of cellular proliferation and cytoplasmic receptor domain phosphorylation. All the peptide Abs showed Ab-dependent cellular cytotoxicity to varying degrees with the 266-296 constructs being equally effective as compared with Herceptin. The 266-296 peptide vaccine had statistically reduced tumor onset in both transplantable tumor models (FVB/n and BALB/c) and significant reduction in tumor development in two transgenic mouse tumor models (BALB-neuT and VEGF(+/-)Neu2-5(+/-)). The 266-296 construct represents the most promising candidate for antitumor vaccination and could also be used to treat a variety of cancers with either normal or elevated expression of HER-2 including breast, lung, ovarian, and prostate.     

Peptide-based inhibition of NF-κB rescues diaphragm muscle contractile dysfunction in a murine model of Duchenne muscular dystrophy

Deterioration of diaphragm function is one of the prominent factors that contributes to the susceptibility of serious respiratory infections and development of respiratory failure in patients with Duchenne Muscular Dystrophy (DMD). The NF-κB signaling pathway has been implicated as a contributing factor of dystrophic pathology, making it a potential therapeutic target. Previously, we demonstrated that pharmacological inhibition of NF-κB via a small NEMO Binding Domain (NBD) peptide was beneficial for reducing pathological features of mdx mice. Now, we stringently test the effectiveness and clinical potential of NBD by treating mdx mice with various formulations of NBD and use diaphragm function as our primary outcome criteria. We found that administering DMSO-soluble NBD rescued 78% of the contractile deficit between mdx and wild-type (WT) diaphragm. Interestingly, synthesis of a GLP NBD peptide as an acetate salt permitted its solubility in water, but as a negative consequence, also greatly attenuated functional efficacy. However, replacing the acetic acid counterion of the NBD peptide with trifluoroacetic acid retained the peptide's water solubility and significantly restored mdx diaphragm contractile function and improved histopathological indices of disease in both diaphragm and limb muscle. Together, these results support the feasibility of using a mass-produced, water-soluble NBD peptide for clinical use.