The hU3-55K protein requires 15.5K binding to the box B/C motif as well as flanking RNA elements for its association with the U3 small nucleolar RNA in Vitro

The 15.5K protein directly binds to the 5' stem-loop of the U4 small nuclear RNA, the small nucleolar (sno) RNA box C/D motif, and the U3 snoRNA-specific box B/C motif. The box B/C motif has also been shown to be essential for the association of the U3 small nucleolar ribonucleoprotein-specific protein hU3-55K. We therefore set out to determine how 15.5K and hU3-55K recognize the box B/C motif. By using an in vitro assembly assay, we show that hU3-55K effectively binds a sub-fragment of the U3 snoRNA surrounding the B/C motif that we have named the U3BC RNA. The association of hU3-55K with the U3BC RNA is dependent on the binding of 15.5K to the box B/C motif. The association of hU3-55K with the U3BC RNA was found to be also dependent on a conserved RNA structure that flanks the box B/C motif. Furthermore, we show that hU3-55K, a WD 40 repeat containing protein, directly cross-links to the U3BC RNA. Our data support a new structural model of the box B/C region of the U3 snoRNA in which the box B/C motif is base-paired to form a structure highly similar to that of both the U4 5' stem-loop and the box C/D motif.     

Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages

Surfactant protein-A (SP-A) plays multiple roles in pulmonary host defense, including stimulating bacterial phagocytosis by innate immune cells. Previously, SP-A was shown to interact with complement protein C1q. Our goal was to further characterize this interaction and elucidate its functional consequences. Radiolabeled SP-A bound solid-phase C1q but not other complement proteins tested. The lectin activity of SP-A was not required for binding to C1q. Because C1q is involved in bacterial clearance but alone does not efficiently enhance the phagocytosis of most bacteria, we hypothesize that SP-A enhances phagocytosis of C1q-coated antigens. SP-A enhanced by sixfold the percentage of rat alveolar macrophages in suspension that phagocytosed C1q-coated fluorescent beads. Furthermore, uptake of C1q-coated beads was enhanced when either beads or alveolar macrophages were preincubated with SP-A. In contrast, SP-A had no significant effect on the uptake of C1q-coated beads by alveolar macrophages adhered to plastic slides. We conclude that SP-A may serve a protective role in the lung by interacting with C1q to enhance the clearance of foreign particles.     

In vitro manipulations of vitamin C and vitamin E concentrations alter intracellular O2- production of hybrid striped bass (Morone chrysops x Morone saxatilis) head-kidney cells

To examine the mechanism by which vitamins C and E alter phagocyte function, a series of in vitro manipulations were conducted with cells isolated from the head-kidney of hybrid striped bass (average weight 680 g) fed a diet supplemented with minimum requirement levels of vitamins C and E for 2 weeks. Head-kidney phagocytes were cultured in media containing physiologically deficient (23 microM, adequate (45 microM) or excessive (182 microM) concentrations of vitamin C, and physiologically deficient (5 microM), adequate (9 microM) or excessive (32 microM) concentrations of vitamin E for 18 h. Following culture and stimulation, levels of reactive oxygen intermediates and hydrogen peroxide were determined. There were no effects of vitamin C or vitamin E concentrations on hydrogen peroxide or extracellular O2- generation. Intracellular O2- production, however, was significantly (P < or = 0.05) affected. When vitamin C was supplied at deficient levels to the medium, vitamin E elevated O2- production to levels not different from those of cells incubated with requirement levels of both vitamins. Similarly, when vitamin E was deficient in the media, vitamin C supplementation at requirement levels normalised intracellular O2- production. This data provides support for the presence of a vitamin C and vitamin E sparing mechanism in phagocytic head-kidney cells of hybrid striped bass and yield some insight into the mechanisms by which vitamin C and vitamin E function in immunomodulation.     

Unusual organic osmolytes in deep-sea animals: adaptations to hydrostatic pressure and other perturbants

Shallow-living marine invertebrates use free amino acids as cellular osmolytes, while most teleosts use almost no organic osmolytes. Recently we found unusual osmolyte compositions in deep-sea animals. Trimethylamine N-oxide (TMAO) increases with depth in muscles of some teleosts, skates, and crustaceans (up to 300 mmol/kg at 2900 m). Other deep-sea animals had high levels of (1). scyllo-inositol in echinoderms, gastropods, and polychaetes, (2). that polyol plus beta-alanine and betaine in octopods, (3). hypotaurine, N-methyltaurine, and unidentified methylamines in vestimentiferans from hydrothermal vents and cold seeps, and (4). a depth-correlated serine-phosphate osmolyte in vesicomyid clams from trench seeps. We hypothesize that some of these solutes counteract effects of hydrostatic pressure. With lactate dehydrogenase, actin, and pyruvate kinase, 250 mM TMAO (but not glycine) protected both ligand binding and protein stability against pressure. To test TMAO in living cells, we grew yeast under pressure. After 1 h at 71 MPa, 3.5 h at 71 MPa, and 17 h at 30 MPa, 150 mM TMAO generally doubled the number of cells that formed colonies. Sulfur-based osmolytes which are not correlated with depth, such as hypotaurine and thiotaurine, are probably involved in sulfide metabolism and detoxification. Thus deep-sea osmolytes may have at least two other roles beyond acting as simple compatible osmotica.     

Body zinc distribution profile during N-methyl-N-nitrosourea-induced mammary tumorigenesis in rats at various levels of dietary zinc intake

Zinc distribution is apparently altered in breast cancer patients. It is unclear if this apparent zinc redistribution is a consequence of altered zinc nutrition or tissue-specific response to breast cancer. Our objectives were to assess effects of N-methyl-N-nitrosourea-treatment and N-methyl-N-nitrosourea-induced mammary tumorigenesis on body zinc-distribution profile in rats and to assess effects of dietary zinc intake on the body zinc-distribution profile during N-methyl-N-nitrosourea treatment and N-methyl-N-nitrosourea-induced mammary tumorigenesis in rats. Female Sprague-Dawley rats were assigned to zinc-deficient (3 mg/kg diet) or zinc-adequate (31 mg/kg diet) ad libitum or pair-fed group. Rats were sham treated or N-methyl-N-nitrosourea treated (50 mg/kg body weight; Experiment 1 or 40 mg/kg body weight; Experiment 2) (n=6). In both experiments, the zinc concentration was significantly higher (6–19 times) in mammary tumor than in mammary gland. Tissue zinc concentration was essentially unaffected by N-methyl-N-nitrosourea treatment and tumor bearing, but was reduced by zinc deficiency in the bone, kidney, and liver. Overall, higher mammary tumor zinc concentration and absence of zinc redistribution during N-methyl-N-nitrosourea treatment and N-methyl-N-nitrosourea-induced mammary tumorigenesis, regardless of zinc intakes, indicates zinc accumulation in mammary tumors. Because zinc is essential for growth and cancer is characterized by uncontrolled growth, this zinc accumulation suggests an involvement of zinc in N-methyl-N-nitrosourea-induced rat mammary tumorigenesis.     

Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells

Interactions between microtubules (MTs) and filamentous actin (f-actin) are involved in directed cell locomotion, but are poorly understood. To test the hypothesis that MTs and f-actin associate with one another and affect each other's organization and dynamics, we performed time-lapse dual-wavelength spinning-disk confocal fluorescent speckle microscopy (FSM) of MTs and f-actin in migrating newt lung epithelial cells. F-actin exhibited four zones of dynamic behavior: rapid retrograde flow in the lamellipodium, slow retrograde flow in the lamellum, anterograde flow in the cell body, and no movement in the convergence zone between the lamellum and cell body. Speckle analysis showed that MTs moved at the same trajectory and velocity as f-actin in the cell body and lamellum, but not in the lamellipodium or convergence zone. MTs grew along f-actin bundles, and quiescent MT ends moved in association with f-actin bundles. These results show that the movement and organization of f-actin has a profound effect on the dynamic organization of MTs in migrating cells, and suggest that MTs and f-actin bind to one another in vivo.     

A panel of candidate tumor antigens in colorectal cancer revealed by the serological selection of a phage displayed cDNA expression library

In the last few years it has been shown that the humoral immune response in cancer patients is a rich source of putative cancer vaccine candidates. To fully explore the complex information present within the Ab repertoire of cancer patients, we have applied a method, serological Ag selection, to molecularly define tumor Ags recognized by the humoral immune response in colorectal cancer (CRC). First, we built a cDNA display library by cloning a cDNA library from CRC cell line HT-29 for expression as a fusion protein with a filamentous phage minor coat protein, pVI. This cDNA display library was then enriched on pooled sera from CRC patients who had undergone active specific immunization with autologous tumor. We identified a panel of 19 clones reactive with the serum pool. Seventeen of 19 (89%) clones showed reactivity with one or more of the eight Ag-reactive sera, conversely six of eight (75%) sera were reactive with at least one of the 19 clones. Sequencing revealed that these 19 clones represented 13 different Ags. A detailed serological analysis of the 13 different Ags showed preferential reactivity to sera of cancer patients for six different Ags. Four of these Ags displayed increased serum reactivity after the active specific immunization procedure. Furthermore, one of the six Ags, a novel Ag homologous to HSPC218, showed restricted expression in normal testis, suggesting that it belongs to the cancer-testis Ag family. Some of the Ags we have identified may be candidates for tumor vaccination, for sero-diagnosis of cancer, as prognostic markers, or as probes for monitoring tumor cell-based vaccination trials.     

Genomic analysis of Hox clusters in the sea lamprey Petromyzon marinus

The sea lamprey Petromyzon marinus is among the most primitive of extant vertebrates. We are interested in the organization of its Hox gene clusters, because, as a close relative of the gnathostomes, this information would help to infer Hox cluster organization at the base of the gnathostome radiation. We have partially mapped the P. marinus Hox clusters using phage, cosmid, and P1 artificial chromosome libraries. Complete homeobox sequences were obtained for the 22 Hox genes recovered in the genomic library screens and analyzed for cognate group identity. We estimate that the clusters are somewhat larger than those of mammals (roughly 140 kbp vs. 105 kbp) but much smaller than the single Hox cluster of the cephalochordate amphioxus (at more than 260 kb). We never obtained more than three genes from any single cognate group from the genomic library screens, although it is unlikely that our screen was exhaustive, and therefore conclude that P. marinus has a total of either three or four Hox clusters. We also identify four highly conserved non-coding sequence motifs shared with higher vertebrates in a genomic comparison of Hox 10 genes.     

DNA-encoded fetal liver tyrosine kinase 3 ligand and granulocyte macrophage-colony-stimulating factor increase dendritic cell recruitment to the inoculation site and enhance antigen-specific CD4+ T cell responses induced by DNA vaccination of outbred animals

DNA-based immunization is a contemporary strategy for developing vaccines to prevent infectious diseases in animals and humans. Translating the efficacy of DNA immunization demonstrated in murine models to the animal species that represent the actual populations to be protected remains a significant challenge. We tested two hypotheses directed at enhancing DNA vaccine efficacy in outbred animals. The first hypothesis, that DNA-encoding fetal liver tyrosine kinase 3 ligand (Flt3L) and GM-CSF increases dendritic cell (DC) recruitment to the immunization site, was tested by intradermal inoculation of calves with plasmid DNA encoding Flt3L and GM-CSF followed by quantitation of CD1(+) DC. Peak DC recruitment was detected at 10-15 days postinoculation and was significantly greater (p < 0.05) in calves in the treatment group as compared with control calves inoculated identically, but without Flt3L and GM-CSF. The second hypothesis, that DNA encoding Flt3L and GM-CSF enhances immunity to a DNA vector-expressed Ag, was tested by analyzing the CD4(+) T lymphocyte response to Anaplasma marginale major surface protein 1a (MSP1a). Calves immunized with DNA-expressing MSP1a developed strong CD4(+) T cell responses against A. marginale, MSP1a, and specific MHC class II DR-restricted MSP1a epitopes. Administration of DNA-encoding Flt3L and GM-CSF before MSP1a DNA vaccination significantly increased the population of Ag-specific effector/memory cells in PBMC and significantly enhanced MSP1a-specific CD4(+) T cell proliferation and IFN-gamma secretion as compared with MHC class II DR-matched calves vaccinated identically but without Flt3L and GM-CSF. These results support use of these growth factors in DNA vaccination and specifically indicate their applicability for vaccine testing in outbred animals.