Solution structure of the motile major sperm protein (MSP) of Ascaris suum - evidence for two manganese binding sites and the possible role of divalent cations in filament formation

The major sperm protein (MSP) of Ascaris suum mediates amoeboid motility by forming an extensive intermeshed system of cytoskeletal filaments analogous to that formed by actin in many other amoeboid cells. MSP is a dimeric molecule that polymerizes to form non-polar filaments constructed from two helical subfilaments that wind round one another. Moreover, MSP filaments can interact with one another to form higher-order assemblies without requiring the range of accessory proteins usually employed in actin-based systems. A knowledge of how MSP polymerizes and forms the hierarchical series of helical MSP macromolecular assemblies is fundamental to understanding locomotion in these cells. Here we describe the solution structure of MSP dimers determined by NMR spectroscopy under conditions where MSP does not polymerize to form filaments. The solution structure is indistinguishable from that observed in putative MSP subfilament helices by X-ray crystallography, indicating that MSP polymerization is not accompanied by a major conformational change. We also show that the rate of MSP polymerization associated with movement of vesicles in an in vitro motility assay is enhanced by the presence of magnesium and manganese ions and use NMR to show that the primary residues that bind these ions are 24-25 and 83-86. These residues are distant from the interface formed between MSP dimers in subfilament helices, and so are probably not involved in this level of polymerization. Instead the manganese and magnesium ion binding appears to be associated with the assembly of subfilaments into filaments and their subsequent aggregation into bundles.     

Screening and chemoprophylaxis for tuberculosis infection in college populations

Lyme borreliosis is the most frequent tick-borne disease in the Northern hemisphere. Here we describe the first isolation of Borrelia burgdorferi sensu lato in Bulgaria: the midguts of 47 Ixodes ricinus obtained by flagging from the Central park in Sofia, Bulgaria were cultivated for borreliae in BSK medium. The eight isolates obtained from the ticks and one skin isolate from a Bulgarian patient with erythema migrans were subjected to phenotypic [outer surface protein A (OspA) serotyping] and genotypic analysis (pulsed-field gel electrophoresis typing followed by large restriction fragment pattern analysis after MluI digestion, polymerase chain reaction with 16S rRNA-directed oligonucleotide probes, and restriction fragment length polymorphism analysis of rrf-rrl intergenic spacer amplicons). The skin isolate was B. burgdorferi sensu stricto, as were four of the tick isolates; the other four tick isolates were B. garinii representing three different OspA serotypes (types 3, 5 and 7). These findings confirm the wide geographic distribution of the different B. garinii-associated OspA serotypes in Europe (shown here for the first time for the Southeastern part of Europe) and of B. burgdorferi sensu stricto in the Western hemisphere. These findings have implications for development of diagnostic tests and a borrelia vaccine in Southeastern Europe.     

Specific immune induction following DNA-based immunization through in vivo transfection and activation of macrophages/antigen-presenting cells

The initiation of an adaptive immune response requires Ag presentation in combination with the appropriate activation signals. Classically, Ag presentation and immune activation occur in the lymph node and spleen, where a favorable organ architecture and rich cellular help can enhance the process. Recently, several investigators have reported the use of DNA expression cassettes to elicit cellular and humoral immunity against diverse pathogens. Although the immune mechanisms involved are still poorly understood, plasmid inoculation represents a model system for studying immune function in response to invading pathogens. In this report, we demonstrate the presence of activated macrophages or dendritic cells in the blood lymphocyte pool and peripheral tissues of animals inoculated with DNA expression cassettes. These cells are directly transfected in vivo, present Ag, and display the surface proteins CD80 and CD86. Our studies indicate that these cells function as APC and can activate naive T lymphocytes. They may represent an important first step APC in genetic immunization and natural infection.     

Mouse deformed epidermal autoregulatory factor 1 recruits a LIM domain factor, LMO-4, and CLIM coregulators

Nuclear LIM domains interact with a family of coregulators referred to as Clim/Ldb/Nli. Although one family member, Clim-2/Ldb-1/Nli, is highly expressed in epidermal keratinocytes, no nuclear LIM domain factor is known to be expressed in epidermis. Therefore, we used the conserved LIM-interaction domain of Clim coregulators to screen for LIM domain factors in adult and embryonic mouse skin expression libraries and isolated a factor that is highly homologous to the previously described LIM-only proteins LMO-1, -2, and -3. This factor, referred to as LMO-4, is expressed in overlapping manner with Clim-2 in epidermis and in several other regions, including epithelial cells of the gastrointestinal, respiratory and genitourinary tracts, developing cartilage, pituitary gland, and discrete regions of the central and peripheral nervous system. Like LMO-2, LMO-4 interacts strongly with Clim factors via its LIM domain. Because LMO/Clim complexes are thought to regulate gene expression by associating with DNA-binding proteins, we used LMO-4 as a bait to screen for such DNA-binding proteins in epidermis and isolated the mouse homologue of Drosophila Deformed epidermal autoregulatory factor 1 (DEAF-1), a DNA-binding protein that interacts with regulatory sequences first described in the Deformed epidermal autoregulatory element. The interaction between LMO-4 and mouse DEAF-1 maps to a proline-rich C-terminal domain of mouse DEAF-1, distinct from the helix-loop-helix and GATA domains previously shown to interact with LMOs, thus defining an additional LIM-interacting domain.     

Arrangement of subunits in 20 S particles consisting of NSF, SNAPs, and SNARE complexes

The present study attempts to identify the antigen-presenting cells in the retina, utilizing bone marrow-transplanted chimeric rats. Two types of chimeras were used: one produced by transplanting bone marrow cells from F1 hybrids of Lewis and Brown Norway (BN) into sublethally irradiated Brown Norway rats (LBN/F1-->BN), followed by adoptive transfer of S-antigen-specific T cells obtained from Lewis rats; the second produced by transplanting bone marrow cells from BN rats into sublethally irradiated F1 hybrids (BN-->LBN/F1), followed by adoptive transfer of S-antigen-specific T cells obtained from F1 hybrids. As controls, Lewis, F1 hybrids and BN rats also received adoptive transfer of syngeneic uveitogenic T cell lines. All animals were killed on the seventh day after adoptive transfer and their eyes and pineal glands were analysed immunohistochemically, utilizing antibody directed against Lewis specific MHC class II molecules(OX-3). The analyses revealed the development of uveoretinitis and pinealitis in both types of chimeras and in the Lewis and F1 hybrid rats. BN rats did not develop uveoretinitis. OX-3-positive cells were found in the retina and the pineal glands of both types of chimeras, and in the Lewis and F1 hybrid rats but not in the BN rats. These cells in the retina expressed dendritic morphology and perivascular distribution. Retinal pigment epithelia, Müller cells and the vascular endothelia of both chimeras, the two strains, and the F1 hybrid rats did not demonstrate OX-3-positive staining. These results suggest that the bone marrow-derived cells in the retina and pineal gland may present S-antigen to T cells, initiating the cascade of uveoretinitis and pinealitis.     

In vivo high resolution MRI of the calcaneus: differences in trabecular structure in osteoporosis patients

The purpose of this study was to use high resolution (HR) magnetic resonance (MR) images of the calcaneus to investigate the trabecular structure of patients with and without osteoporotic hip fractures and to compare these techniques with bone mineral density (BMD) in differentiating fracture and nonfracture patients. Axial and sagittal HR MR images of the calcaneus were obtained in 50 female (23 postmenopausal patients with osteoporotic hip fractures and 27 postmenopausal controls). A three-dimensional gradient-echo sequence was used with a slice thickness of 500 micron and in plane resolution of 195 x 195 micron. Texture analysis was performed using morphological features, analogous to standard histomorphometry and fractal dimension. Additionally, BMd measurements of the hip (dual-energy X-ray absorptiometry) were obtained in all patients. Significant differences between both patient groups were obtained using morphological parameters and fractal dimension as well as hip BMD (p < 0.05). Odds ratios for the texture parameters apparent (app.) bone volume/total volume and app. trabecular separation were higher than for hip BMD. Receiver operator characteristic values of texture measures and hip BMD were comparable. In conclusion, trabecular structure measures derived from HR MR images of the calcaneus can differentiate between postmenopausal women with and without osteoporotic hip fractures.