R-body-producing bacteria.

Until 10 years ago, R bodies were known only as diagnostic features by which endosymbionts of paramecia were identified as kappa particles. They were thought to be limited to the cytoplasm of two species in the Paramecium aurelia species complex. Now, R bodies have been found in free-living bacteria and other Paramecium species. The organisms now known to form R bodies include the cytoplasmic kappa endosymbionts of P. biaurelia and P. tetraurelia, the macronuclear kappa endosymbionts of P. caudatum, Pseudomonas avenae (a free-living plant pathogen), Pseudomonas taeniospiralis (a hydrogen-oxidizing soil microorganism), Rhodospirillum centenum (a photosynthetic bacterium), and a soil bacterium, EPS-5028, which is probably a pseudomonad. R bodies themselves fall into five distinct groups, distinguished by size, the morphology of the R-body ribbons, and the unrolling behavior of wound R bodies. In recent years, the inherent difficulties in studying the organization and assembly of R bodies by the obligate endosymbiont kappa, have been alleviated by cloning and expressing genetic determinants for these R bodies (type 51) in Escherichia coli. Type 51 R-body synthesis requires three low-molecular-mass polypeptides. One of these is modified posttranslationally, giving rise to 12 polypeptide species, which are the major structural subunits of the R body. R bodies are encoded in kappa species by extrachromosomal elements. Type 51 R bodies, produced in Caedibacter taeniospiralis, are encoded by a plasmid, whereas bacteriophage genomes probably control R-body synthesis in other kappa species. However, there is no evidence that either bacteriophages or plasmids are present in P. avenae or P. taeniospiralis. No sequence homology was detected between type 51 R-body-encoding DNA and DNA from any R-body-producing species, except C. varicaedens 1038. The evolutionary relatedness of different types of R bodies remains unknown.     

Viral genetic determinants of T-cell killing and immunodeficiency disease induction by the feline leukemia virus FeLV-FAIDS.

Within the fatal immunodeficiency disease-inducing strain of feline leukemia virus, FeLV-FAIDS, are viruses which range in pathogenicity from minimally (clone 61E is the prototype) to acutely pathogenic, most of the latter of which are also replication defective (clone 61C is the prototype). Mixtures of 61E and 61C virus and chimeras generated between them, but not 61E alone, killed feline T cells. T-cell killing depended on changes within a 7-amino-acid region near the C terminus of the gp70 env gene or was achieved independently by changes within a 109-amino-acid region encompassing the N terminus of gp70. The carboxy-terminal change was also sufficient for induction of fatal immunodeficiency disease in cats. Other changes within the 61C gp70 gene enhanced T-cell killing, as did changes in the long terminal repeat, the latter of which also enhanced virus replication. T-cell killing correlated with high levels of intracellular unintegrated and proviral DNA, all of which were blocked by treatment of infected cells with sera from 61C-immune cats or with a neutralizing monoclonal antibody. These findings indicate that T-cell killing is a consequence of superinfection and that the mutations in env critical to pathogenicity of the immunosuppressive variant result in a failure to establish superinfection interference in infected cells.     

Characterization of Caedibacter endonucleobionts from the macronucleus of Paramecium caudatum and the identification of a mutant with blocked R-body synthesis

Cytology, DNA and host-symbiont relationships of x-like endosymbionts from Paramecium caudatum are described. The symbionts (Caedibacter caryophila, sp. nov.) live in the macronuclei of their hosts. They confer the killer trait upon their hosts and appear well adapted to their endonucleobiotic way of life. R bodies (proteinaceous ribbons associated with killing) are produced, but differ significantly from any of the four R-body classes previously described. C. caryophila and their R bodies were isolated. DNA was extracted from purified symbionts and used to demonstrate that one P. caudatum line harbors a natural mutant which is deficient in R-body production. Melting studies indicate a GC content of 34.6%. No sequence homology between the C. caryophila DNA and the coding sequence for type 51 R-body production was observed. C. caryophila is parasitic, causing the death of its hosts in starving cultures.     

Local re-utilization of thymidine in normal mouse tissues as measured with iododeoxyuridine

Thymidine (TdR) and its analogue, iododeoxyuridine (IdUdr), were used to quantitate nucleoside re-utilisation in vivo. Significantly different results obtained, however, depending upon what form of isotopically labelled iododeoxyuridine is used. No measurable local thymidine re-utilization was found in mouse thymus, spleen or bone marrow when the retention of [3H]IdUdR was compared with [14C]TdR. On the other hand, significant differences were found between the retention of [125I]IdUdR and [3H]IdUdR, which is attributed to de-iodination of iododeoxyuridine. Some thymidine re-utilization was found in duodenum using both [3H]IdUdR and [125I]IdUdR. Information on the in vivo distribution of TdR and the contention that a large degree of TdR re-utilization in the thymus is evidence of extensive cell death must be re-interpreted in the light of these results. In addition, evidence for little or no local re-utilization in some tissues will greatly simplify the use of [11C]TdR as an imaging agent for measuring tissue proliferation in vivo with positron emission tomography (PET).     

Inhibition of Na+/Ca2+ exchanger activity in cardiac and skeletal muscle sarcolemmal vesicles by monoclonal antibody 44D7

Monoclonal antibodies 44D7 and 4F2 inhibited specifically the Na+-dependent Ca2+ fluxes characteristic of the Na+/Ca2+ exchanger in cardiac and skeletal muscle sarcolemmal vesicles. Preincubation of membrane vesicles with monoclonal antibody 44D7 inhibited 90% of the Na+-dependent Ca2+ uptake measured in the first 10 s of the reaction and 50% of that measured after 60 s. Ca2+/calmodulin-dependent ATPase activity and ATP-dependent Ca2+ uptake by sarcolemmal vesicles were not affected by monoclonal antibody 44D7 whereas the Na+-dependent release of accumulated Ca2+ was inhibited. In the presence of the 44D7 antigen isolated from human kidney, monoclonal antibody 44D7 could no longer inhibit Na+-dependent Ca2+ fluxes. The distribution of 4F2 antigenic activity in the isolated muscle membrane fractions correlated with that of Na+/Ca2+ exchanger activity; cardiac and skeletal muscle sarcolemmal vesicles expressed higher levels of the antigen than skeletal muscle transverse tubule membrane, while no antigen could be detected in sarcoplasmic reticulum membranes. Our results suggest that monoclonal antibodies 44D7 and 4F2 interact either directly with the Na+/Ca2+ exchanger molecules or with some other protein(s) responsible for the regulation of this activity in the heart and skeletal muscle.     

Phylogenetic relationships of bacterial endosymbionts of Paramecium aurelia: polynucleotide sequence relationships of 51 kappa and its mutants.

Hydroxyapatite chromatographic procedures were used to investigate the deoxyribonucleic acid (DNA) sequence relationships of kappa of Paramecium tetraurelia stock 51 and the organisms that have been designated as mutants of 51 kappa. Of the "mutants" studied, only 51m43 kappa possessed a high percentage (89%) of DNA sequences homologous to those of 51 kappa. All other "mutant" strains possessed less than 25% polynucleotide sequence homology to 51 kappa DNA. The three strains of pi endosymbionts (51m1 pi, 51m43 pi, and 139 pi) share greater than 75% DNA sequence homology with each other and approximately 50% DNA sequence homology with 138 mu, the mate-killer endosymbiont found in P. octaurelia. Only 23% of the 51 kappa DNA sequences were found to be homologous with those of 51m1 kappa. The data indicate that of the "mutants" studied, only 51m43 kappa could be a mutant of 51 kappa. The pi endosymbionts comprise a closely related group of organisms that are also related to 138 mu but not to any of the kappas tested. The group of organisms designated as kappa appears to be comprised of at least two distinct phylogenetic groups.     

Orthologous gene-expression profiling in multi-species models: search for candidate genes

Microarray-driven gene-expression profiles are generally produced and analyzed for a single specific experimental model. We have assessed an analytical approach that simultaneously evaluates multi-species experimental models within a particular biological condition using orthologous genes as linkers for the various Affymetrix microarray platforms on multi-species models of ventilator-associated lung injury. The results suggest that this approach may be a useful tool in the evaluation of biological processes of interest and selection of process-related candidate genes.     

RESOURCERER: a database for annotating and linking microarray resources within and across species

Microarray expression analysis is providing unprecedented data on gene expression in humans and mammalian model systems. Although such studies provide a tremendous resource for understanding human disease states, one of the significant challenges is cross-referencing the data derived from different species, across diverse expression analysis platforms, in order to properly derive inferences regarding gene expression and disease state. To address this problem, we have developed RESOURCERER, a microarray-resource annotation and cross-reference database built using the analysis of expressed sequence tags (ESTs) and gene sequences provided by the TIGR Gene Index (TGI) and TIGR Orthologous Gene Alignment (TOGA) databases [now called Eukaryotic Gene Orthologs (EGO)].