A novel CD4-CD8alpha+CD205+CD11b- murine spleen dendritic cell line: establishment, characterization and functional analysis in a model of vaccination to toxoplasmosis

Dendritic cells (DCs) play an essential role in the induction of immune responses to pathogen infections. Native DCs are difficult to obtain in large numbers and consequently the vast majority of DCs employed in all experiments are derived from bone marrow progenitors. In an attempt to solve this problem, we have established a novel CD8alpha(+) DC line (H-2(k)) from spleen, which we have named SRDC line, and which is easy to culture in vitro. These cells display similar morphology, phenotype and activity to CD4(-)CD8alpha(+)CD205(+)CD11b(-) DCs purified ex vivo. Toxoplasma gondii antigen was shown to be taken up by these cells and to increase class I and class II major histocompatibility complex (MHC), CD40, CD80 and CD86 surface expression. We report that vaccination with T. gondii antigen-pulsed SRDCs, which synthesize large amounts of interleukin-12, induced protective immune responses against this intracellular pathogen in syngeneic CBA/J mice. This protection was associated with strong cellular and humoral immune responses at systemic and intestinal levels. Spleen and mesenteric lymph node cell proliferations were correlated with a Th1/Th2-type response and a specific SRDC homing to spleen and intestine was observed. The SRDC or CD4(-)CD8alpha(+)CD205(+)CD11b(-) DC line can be expected to be a very useful tool for immunobiology studies of DC.     

Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach

Identification and characterization of multi-protein complexes is an important step toward an integrative view of protein-protein interaction networks that determine protein function and cell behavior. The limiting factor for identifying protein complexes is the method for their separation. Blue native PAGE (BN-PAGE) permits a high-resolution separation of multi-protein complexes under native conditions. To date, BN-PAGE has only been applicable to purified material. Here, we show that dialysis permits the analysis of multi-protein complexes of whole cellular lysates by BN-PAGE. We visualized different multi-protein complexes by immunoblotting including forms of the eukaryotic proteasome. Complex dynamics after gamma interferon stimulation of cells was studied, and an antibody shift assay was used to detect protein-protein interactions in BN-PAGE. Furthermore, we identified defined protein complexes of various proteins including the tumor suppressor p53 and c-Myc. Finally, we identified multi-protein complexes via mass spectrometry, showing that the method has a wide potential for functional proteomics.     

Enlightenment of old ideas from new investigations: more questions regarding the evolution of bacteriogenic light organs in squids

Bioluminescence is widespread among many different types of marine organisms. Metazoans contain two types of luminescence production, bacteriogenic (symbiotic with bacteria) or autogenic, via the production of a luminous secretion or the intrinsic properties of luminous cells. Several species in two families of squids, the Loliginidae and the Sepiolidae (Mollusca: Cephalopoda) harbor bacteriogenic light organs that are found central in the mantle cavity. These light organs are exceptional in function, that is, the morphology and the complexity suggests that the organ has evolved to enhance and direct light emission from bacteria that are harbored inside. Although light organs are widespread among taxa within the Sepiolidae, the origin and development of this important feature is not well studied. We compared light organ morphology from several closely related taxa within the Sepiolidae and combined molecular phylogenetic data using four loci (nuclear ribosomal 28S rRNA and the mitochondrial cytochrome c oxidase subunit I and 12S and 16S rRNA) to determine whether this character was an ancestral trait repeatedly lost among both families or whether it evolved independently as an adaptation to the pelagic and benthic lifestyles. By comparing other closely related extant taxa that do not contain symbiotic light organs, we hypothesized that the ancestral state of sepiolid light organs most likely evolved from part of a separate accessory gland open to the environment that allowed colonization of bacteria to occur and further specialize in the eventual development of the modern light organ.     

What the Distribution of Cell Lengths in the Root Meristem Does and Does Not Reveal About Cell Division

Roots have long been realized to be useful material for studies of cell division. Despite this long history of use, the behavior of cells in the meristem is often misinterpreted. A common error is to argue that differences in cell length reflect differences in cell division rate. In this article we explain the fallacy behind this argument and show how the analysis of cell length distribution can lead to insight about the root meristem. These observations support a model for the root meristem where cells of various tissues grow at the same relative growth rate and divide at the same frequency, indicating that these growth parameters are built into the cells at a fundamental level. The differences in cell length between various tissues appear to arise at their formation, first at the tissue initials and ultimately in the seed. Length differences among mature cells may be enhanced by differences in the location within the meristem where division ceases. Discovering mechanisms regulating the length of initial cells and the position where cells cease division requires a realistic understanding of how growth constrains the division behavior of dividing cells.     

Synthesis of the alpha-L-Araf-(1-->2)-beta-D-Galp-(1-->6)-beta-D-Galp-(1-->6)-[alpha-L-Araf-(1-->2)]-beta-D-Galp-(1-->6)-D-Gal hexasaccharide as a possible repeating unit of the cell-cultured exudates of Echinacea purpurea arabinogalactan.

For the characterization of the supposed epitope of an arabinogalactan, isolated from the extract of the cell-cultured Echinacea purpurea, the title hexasaccharide was synthesized. The whole synthetic route was based on the 6-O-(methoxydimethyl)methyl ether (MIP) protecting group strategy. 2-O-Benzyl-3,4-O-isopropylidene-6-O-(methoxydimethyl)methyl-beta-D-galactopyranosyl-(1-->6)-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranose was used to prepare the desired glycosyl donor and glycosyl acceptor both carrying a persistent O-benzyl group at position 2'. Reaction of the digalactose donor and the digalactose acceptor resulted in a beta-(1-->6)-linked galactose-containing tetrasaccharide in which OH-2' and OH-2"' were substituted with benzyl groups. Hydrogenolytic removal of the benzyl groups of the tetragalactose compound gave the diol aglycon which was diarabinosylated in one step to furnish the protected target compound, whose deprotection led to the title hexasaccharide. All of the synthesized compounds were characterized by 1H and 13C NMR spectra, as well as by MALDI-TOF mass-spectrometry measurements.     

Redox properties of human manganese superoxide dismutase and active-site mutants

The redox potential of human manganese superoxide dismutase (MnSOD) has been difficult to determine because of the problem of finding suitable electron mediators. We have found that ferricyanide and pentacyanoaminoferrate can be used as electron mediators, although equilibration is very slow with a half-time near 6 h. Values of the midpoint potential were determined both by allowing enzyme and mediators to equilibrate up to 38 h and by reductive titration adding dithionite to enzyme and mediator. An overall value of the midpoint potential was found to be 393 +/- 29 mV. To elucidate the role of His30 and Tyr34 in the active site of human MnSOD, we have also measured the redox properties of the site-specific mutants His30Asn (H30N) and Tyr34Phe (Y34F) and compared them with the wild-type enzyme. Crystal structures have shown that each mutation interrupts a hydrogen bond network in the active site, and each causes a 10-fold decrease in the maximal velocity of catalysis of superoxide dismutation as compared with wild type. The present study shows that H30N and Y34F human MnSOD have very little effect, within experimental uncertainty, on the redox potential of the active-site metal. The redox potentials determined electrochemically were 365 +/- 28 mV for H30N and 435 +/- 30 mV for Y34F MnSOD. These results suggest that the role of His30 and Tyr34 is more in support of catalysis, probably proton transport, and not in the tuning of the redox potential.     

Electrolyte changes in plasma and urine of athletes during acute and rigorous bed rest and ambulatory conditions

Rigorous bed rest (RBR) induces significant electrolyte changes, but little it is not known about the effect of acute bed rest (ABR) (i.e., abrupt confinement to a RBR). The aim of this study was to measure urinary and plasma electrolyte changes during ABR and RBR conditions. The studies were done during 3 d of a pre-bed-rest (BR) period and during 7 d of an ABR and RBR period. Thirty male trained athletes aged, 24.4 ± 6.6 yr were chosen as subjects. They were divided equally into three groups: unrestricted ambulatory control subjects (UACS), acute-bed-rested subjects (ABRS), and rigorous-bed-rested subjects (RBRS). The UACS group experienced no changes in professional training and daily activities. The ABRS were submitted abruptly to a RBR regimen and without having any prior knowledge of the exact date and time when they would be subjected to an RBR regimen. The RBRS were subjected to an RBR regime on a predetermined date and time known to them from the beginning of the study. Sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), and phosphate (P) in plasma and urine, plasma renin activity (PRA) and plasma aldosterone (PA), physical characteristics, peak oxygen uptake, and food and water intakes were measured. Urinary Na, K, Ca, Mg, and P excretion and plasma Na, K, Mg, Ca, and P concentration, PRA, and PA concentration increased significantly (p ≤ 0.01), whereas body weight, peak oxygen uptake, and food and water intakes decreased significantly in the ABRS and RBRS groups when compared with the UACS group. However, urinary and plasma Na, K, Mg, P, and Ca, PRA, and PA values increased much faster and were much greater in the ABRS group than in the RBRS group. Plasma and urinary Na, K, Ca, Mg, and P, PRA and PA levels, food and water intakes, body weight, and peak oxygen uptake did not change significantly in the UACS group when compared with its baseline control values. It was shown that RBR and ABR conditions induce significant increases in urinary and plasma electrolytes; however, urinary and plasma electrolyte changes appeared much faster and were much greater in the ABRS group than the RBRS group. It was concluded that the more abruptly motor activity is ended, the faster and the greater the urinary and plasma electrolyte change.     

Composition and distribution of the pelagic and sympagic algal assemblages in the Laptev Sea during autumnal freeze-up

The phytoplankton and ice algal assemblages in the SiberianLaptev Sea during the autumnal freeze-up period of 1995 aredescribed. The spatial distribution of algal taxa (diatoms,dinoflagellates, chrysophytes, chlorophytes) in the newly formedice and waters at the surface and at 5 m depth differed considerablybetween regions. This was also true for algal biomass measuredby in situ fluorescence, chlorophyll (Chl) a and taxon-specificcarbon content. Highest in situ fluorescence and Chl a concentrations(ranging from 0.1 to 3.2 µg l^–1) occurred in surfacewaters with maxima in Buor Khaya Bay east of Lena Delta. Thealgal standing stock on the shelf consisted mainly of diatoms,dinoflagellates, chrysophytes and chlorophytes with a totalabundance (excluding unidentified flagellates <10 µm)in surface waters of 351–33 660 cells l^–1. Highestalgal abundance occurred close to the Lena Delta. Phytoplanktonbiomass (phytoplankton carbon; PPC) ranged from 0.1 to 5.3 µgC l^–1 in surface waters and from 0.3 to 2.1 µg Cl^–1 at 5 m depth, and followed the distribution patternof abundances. However, the distribution of Chl a differed considerablyfrom the distribution pattern shown by PPC. The algal assemblagein the sea ice, which could not be quantified due to high sedimentload, was dominated by diatom species, accompanied by dinoflagellates.Thus, already during the early stage of autumnal freeze-up,incorporation processes, selective enrichment and subsequentgrowth lead to differences between surface water and sea icealgal assemblages.     

Variability for resistance to Fusarium solani culture filtrate and fusaric acid among somaclones in pea

Pea (Pisum sativum L.) somaclones of cultivars Adept, Komet and Bohatýr were obtained after selection in vitro with Fusarium solani filtrate and fusaric acid (FA). R2 regenerants were analysed by random amplification of polymorphic DNA (RAPD; OPAB4, P-14, UBC-556) and inter-retrotransposon amplification polymorphism (IRAP; Ogre) markers. Marker UBC-556 showed different banding patterns for each cultivar, but without specific bands for selected and control plants. Markers OPAB4, P14 and Ogre were useful for clear discrimination between selected and non-selected variants of all three cultivars. Flow cytometry analysis proved the same genome size of selected and non-selected pea lines. Therefore in vitro selection by pathogen derived agents could be the efficient method for obtaining of pea somaclones with increased resistance to F. solani.