Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine

NAD-dependent ADP-ribosylation is one of the posttranslational protein modifications. On mammalian cells, glycosylphosphatidylinositol-anchored cell surface ADP-ribosyltransferases (ARTs) ADP-ribosylate other cell surface proteins and thereby affect important cellular functions. Here we describe convenient flow-cytometric and immunoblot assays for monitoring ADP-ribosylation of cell surface proteins on living cells by exploiting the capacity of ARTs to utilize etheno-NAD as substrate. Etheno-ADP-ribosylation of cell surface proteins can be detected by flow cytometry with 1G4, a monoclonal antibody specific for ethenoadenosine. Labeling of cells with 1G4 is dependent on the expression of cell surface ARTs and occurs only after incubation of ART-expressing cells with etheno-NAD and not with etheno-ADP-ribose. Dose-response analyses show efficient 1G4 staining of ART-expressing cells at micromolar etheno-NAD concentrations. Half-maximal staining is obtained with 1-2 micro M etheno-NAD, saturation is reached at 5-20 micro M etheno-NAD. Immunoblot analyses confirm that ART-expressing cells incorporate ethenoadenosine covalently (i.e., SDS resistant) into several cell surface proteins. The flow-cytometric 1G4 staining assay can be used to identify subpopulations of cells expressing cell surface ART activity and to select ART(hi) cell variants. The immunoblot 1G4 staining assay can also be used to identify etheno-ADP-ribosylated target proteins. These new assays hold promise for many interesting applications in biochemistry and cell biology.     

The family of toxin-related ecto-ADP-ribosyltransferases in humans and the mouse

ADP-ribosyltransferases including toxins secreted by Vibrio cholera, Pseudomonas aerurginosa, and other pathogenic bacteria inactivate the function of human target proteins by attaching ADP-ribose onto a critical amino acid residue. Cross-species polymerase chain reaction (PCR) and database mining identified the orthologs of these ADP-ribosylating toxins in humans and the mouse. The human genome contains four functional toxin-related ADP-ribosyltransferase genes (ARTs) and two related intron-containing pseudogenes; the mouse has six functional orthologs. The human and mouse ART genes map to chromosomal regions with conserved linkage synteny. The individual ART genes reveal highly restricted expression patterns, which are largely conserved in humans and the mouse. We confirmed the predicted extracellular location of the ART proteins by expressing recombinant ARTs in insect cells. Two human and four mouse ARTs contain the active site motif (R-S-EXE) typical of arginine-specific ADP-ribosyltransferases and exhibit the predicted enzyme activities. Two other human ARTs and their murine orthologues deviate in the active site motif and lack detectable enzyme activity. Conceivably, these ARTs may have acquired a new specificity or function. The position-sensitive iterative database search program PSI-BLAST connected the mammalian ARTs with most known bacterial ADP-ribosylating toxins. In contrast, no related open reading frames occur in the four completed genomes of lower eucaryotes (yeast, worm, fly, and mustard weed). Interestingly, these organisms also lack genes for ADP-ribosylhydrolases, the enzymes that reverse protein ADP-ribosylation. This suggests that the two enzyme families that catalyze reversible mono-ADP-ribosylation either were lost from the genomes of these nonchordata eucaryotes or were subject to horizontal gene transfer between kingdoms.     

Dendritic polyglycerol: a new versatile biocompatible-material

Polyglycerol represents the first hyperbranched polymer that can be prepared in a controlled synthesis. It is characterized by the combination of a stable, biocompatible polyether scaffold, high-end group functionality and a compact, well-defined dendrimer-like architecture. These characteristics can be used to generate new materials properties and for biomedical applications to molecularly amplify or multiply effects or to create extremely high local concentrations of drugs, molecular labels, or probe moieties. Therefore, dendritic polyglycerols are expected to lead to new strategies for 'molecular medicine'. In this brief summary, the current state of the art in polyglycerol research is given, focusing on applications in life sciences.     

Cutting edge: a natural P451L mutation in the cytoplasmic domain impairs the function of the mouse P2X7 receptor

The P2X7 receptor (P2X(7)R) is an ATP-gated channel that mediates apoptosis of cells of the immune system. The capacity of P2X(7)R to form large pores depends on its large cytoplasmic tail, which harbors a putative TNFR-related death domain. Previous transfection studies indicated that mouse P2X(7)R forms pores much less efficiently than its counterparts from humans and rats. In this study, we demonstrate that an allelic mutation (P451L) in the predicted death domain of P2X(7)R confers a drastically reduced sensitivity to ATP-induced pore formation in cells from some commonly used strains of mice, i.e., C57BL/6 and DBA/2. In contrast, most other strains of mice, including strains derived from wild mice, carry P451 at this position as do rats and humans. The effects of the P451L mutation resemble those of the E496A mutation in human P2X(7)R. These P2X(7)R mutants may provide useful tools to decipher the molecular mechanisms leading to pore formation.     

Cellular organization of Bacillus subtilis: sodium dodecyl sulfate-induced cell partitioning into zebra structures.

Cells of Bacillus subtilis heated in high concentrations of sodium dodecyl sulfate (5%) and then washed free of detergent with a hot salt solution (80 C) become structurally reorganized into regions of densely compacted cytoplasm (termed zebras) and regions of sparsely filled material (termed spaces). Size distribution studies of zebras indicate that division-suppressed mutants and wild-type cells both yield zebras of comparable length. Similarly the lengths of zebras found in populations emerging from spores are uniform in one-, two-, three-, and four-zebra-containing cells. In contrast, the length of spaces is slightly larger than that of zebras and is unusually large in two-zebra-containing cells. The locations of zebras and spaces along cell length have been studied in spore out-growth populations. A statistical procedure developed previously in genome location investigations was used to analyze the location of zebras along cell length. The data indicate that as cells elongate, new sites arise where the cell contents are strongly bound to the cell surface. Within filament populations produced by division-suppressed mutants there is a linear relationship of mean filament length and zebra number per filament. These data indicate that cytoplasm in filaments with no obvious structural compartmentalizations may be organized into units associated with particular regions of cell surface. The attachment of cell contents to the cell surface may involve deoxyribonucleic acid. Zebra-containing cells digested with proteolytic enzyme and ribonuclease are converted to cells that contain a crystalline-like granule fixed at the location of each zebra. Exposure to deoxyribonuclease mobilizes these granules within the cell wall.     

A general model of reaction kinetics in biological systems

Dynamic mathematical models in biotechnology require, besides the information about the stoichiometry of the biological reaction system, knowledge about the reaction kinetics. Modulation phenomena like limitation, inhibition and activation occur in different forms of competition with the key enzymes responsible for the respective metabolic reaction steps. The identification of a priori unknown reaction kinetics is often a critical task due to the non-linearity and (over-) parameterization of the model equations introduced to account for all the possible modulation phenomena. The contribution of this paper is to propose a general formulation of reaction kinetics, as an extension of the Michaelis-Menten kinetics, which allows limitation/activation and inhibition effects to be described with a reduced number of parameters. The versatility of the new model structure is demonstrated with application examples.     

Parasite-mediated selection in experimental metapopulations of Daphnia magna

In metapopulations, only a fraction of all local host populations may be infected with a given parasite species, and limited dispersal of parasites suggests that colonization of host populations by parasites may involve only a small number of parasite strains. Using hosts and parasites obtained from a natural metapopulation, we studied the evolutionary consequences of invasion by single strains of parasites in experimental populations of the cyclical parthenogen Daphnia magna. In two experiments, each spanning approximately one season, we monitored clone frequency changes in outdoor container populations consisting of 13 and 19 D. magna clones, respectively. The populations were either infected with single strains of the microsporidian parasites Octosporea bayeri or Ordospora colligata or left unparasitized. In both experiments, infection changed the representation of clones over time significantly, indicating parasite-mediated evolution in the experimental populations. Furthermore, the two parasite species changed clone frequencies differently, suggesting that the interaction between infection and competitive ability of the hosts was specific to the parasite species. Taken together, our results suggest that parasite strains that invade local host populations can lead to evolutionary changes in the genetic composition of the host population and that this change is parasite-species specific.     

Tumor necrosis factor alpha induction of NF-kappaB requires the novel coactivator SIMPL

A myriad of stimuli including proinflammatory cytokines, viruses, and chemical and mechanical insults activate a kinase complex composed of IkappaB kinase beta (IKK-beta), IKK-alpha, and IKK-gamma/N, leading to changes in NF-kappaB-dependent gene expression. However, it is not clear how the NF-kappaB response is tailored to specific cellular insults. Signaling molecule that interacts with mouse pelle-like kinase (SIMPL) is a signaling component required for tumor necrosis factor alpha (TNF-alpha)-dependent but not interleukin-1-dependent NF-kappaB activation. Herein we demonstrate that nuclear localization of SIMPL is required for type I TNF receptor-induced NF-kappaB activity. SIMPL interacts with nuclear p65 in a TNF-alpha-dependent manner to promote endogenous NF-kappaB-dependent gene expression. The interaction between SIMPL and p65 enhances p65 transactivation activity. These data support a model in which TNF-alpha activation of NF-kappaB dependent-gene expression requires nuclear relocalization of p65 as well as nuclear relocalization of SIMPL, generating a TNF-alpha-specific induction of gene expression.     

Livestock trade history, geography, and parasite strains: the mitochondrial genetic structure of Echinococcus granulosus in Argentina

A sample of 114 isolates of Echinococcus granulosus (Cestoda: Taeniidae) collected from different host species and sites in Argentina has been sequenced for 391 bp from the mitochondrial cytochrome c oxidase subunit I gene to analyze genetic variability and population structure. Nine different haplotypes were identified, 5 of which correspond to already characterized strains. Analysis of molecular variance and nested clade analysis of the distribution of haplotypes among localities within 3 main geographic regions indicate that geographic differentiation accounts for the overall pattern of genetic variability in E. granulosus populations. Significant geographic differentiation is also present when the sheep strain alone is considered. Our results suggest that geographic patterns are not due to actual restricted gene flow between regions but are rather a consequence of past history, probably related to the time and origin of livestock introduction in Argentina.