Novel bacterial endosymbionts of Acanthamoeba spp. related to the Paramecium caudatum symbiont Caedibacter caryophilus

Acanthamoebae are increasingly being recognized as hosts for obligate bacterial endosymbionts, most of which are presently uncharacterized. In this study, the phylogeny of three Gram-negative, rod-shaped endosymbionts and their Acanthamoeba host cells was analysed by the rRNA approach. Comparative analyses of 16S rDNA sequences retrieved from amoebic cell lysates revealed that the endosymbionts of Acanthamoeba polyphaga HN-3, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39 are related to the Paramecium caudatum endosymbionts Caedibacter caryophilus, Holospora elegans a n d Holospora obtusa . With overall 16S rRNA sequence similarities to their closest relative, C. caryophilus , of between 87% and 93%, these endosymbionts represent three distinct new species. In situ hybridization with fluorescently labelled endosymbiont-specific 16S rRNA-targeted probes demonstrated that the retrieved 16S rDNA sequences originated from the endosymbionts and confirmed their intracellular localization. We propose to classify provisionally the endosymbiont of Acanthamoeba polyphaga HN-3 as ' Candidatus Caedibacter acanthamoebae', the endosymbiont of Acanthamoeba sp. strain UWC9 as ' Candidatus Paracaedibacter acanthamoebae' and the endosymbiont of Acanthamoeba sp. strain UWE39 as ' Candidatus Paracaedibacter symbiosus'. The phylogeny of the Acanthamoeba host cells was analysed by comparative sequence analyses of their 18S rRNA. Although Acanthamoeba polyphaga HN-3 clearly groups together with most of the known Acanthamoeba isolates (18S rRNA sequence type 4), Acanthamoeba sp. UWC9 and UWE39 exhibit < 92% 18S rRNA sequence similarity to each other and to other Acanthamoeba isolates. Therefore, we propose two new sequence types (T13 and T14) within the genus Acanthamoeba containing, respectively, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39.     

The genes encoding bovine SP-A, SP-D, MBL-A, conglutinin, CL-43 and CL-46 form a distinct collectin locus on Bos taurus chromosome 28 (BTA28) at position q.1.8-1.9

Collectins are a group of C-type lectins involved in the innate immune system, where they mediate and modulate clearance of pathogens. The health status of cattle is of major economical and ethical concern; therefore, the study of bovine collectins is of importance. The collectins conglutinin, CL-43 and CL-46 are only present in Bovidae and the characterization of their genes indicates that they are structural descendants of another collectin, lung surfactant protein D (SP-D). In this study, we assembled BAC clones into a contig spanning 330-1150 kb, which includes the bovine genes encoding the collectins SP-A (SFTPA), SP-D (SFTPD), mannan-binding lectin A (MBL1), CL-43 (COLEC9), CL-46 (COLEC13) and conglutinin (COLEC8). In the same contig, we also identified a gene that potentially encodes a novel conglutinin-like collectin (COLEC14). The arrangement of STFPA, SFTPD and MBL1 is homologous to the organization found in humans and mice, whereas the Bovidae-specific collectin genes, COLEC8, COLEC9 and COLEC13, extend from SFTPD. Proximal to the collectin locus at BTA28q1.8-1.9, and included in the contig, we found the microsatellite IDVGA8, which may be a valuable marker for tracking polymorphisms in the linked collectin genes.     

Discovery of the novel candidate phylum "Poribacteria" in marine sponges

Marine sponges (Porifera) harbor large amounts of commensal microbial communities within the sponge mesohyl. We employed 16S rRNA gene library construction using specific PCR primers to provide insights into the phylogenetic identity of an abundant sponge-associated bacterium that is morphologically characterized by the presence of a membrane-bound nucleoid. In this study, we report the presence of a previously unrecognized evolutionary lineage branching deeply in the domain Bacteria that is moderately related to the Planctomycetes, Verrucomicrobia, and Chlamydia lines of decent. Because members of this lineage showed <75% 16S rRNA gene sequence similarity to known bacterial phyla, we suggest the status of a new candidate phylum, named "Poribacteria", to acknowledge the affiliation of the new bacterium with sponges. The affiliation of the morphologically conspicuous sponge bacterium with the novel phylogenetic lineage was confirmed by fluorescence in situ hybridization with newly designed probes targeting different sites of the poribacterial 16S rRNA. Consistent with electron microscopic observations of cell compartmentalization, the fluorescence signals appeared in a ring-shaped manner. PCR screening with "Poribacteria"-specific primers gave positive results for several other sponge species, while samples taken from the environment (seawater, sediments, and a filter-feeding tunicate) were PCR negative. In addition to a report for Planctomycetes, this is the second report of cell compartmentalization, a feature that was considered exclusive to the eukaryotic domain, in prokaryotes.     

Involution of seminiferous tubules in aged hamsters: an ultrastructural, immunohistochemical and quantitative morphological study

In this study, we examined the age-related changes on morphometric parameters and ultrastructure of seminiferous tubules, and on the expression of extracellular matrix proteins in lamina propria of Syrian hamsters. A significant decrease in the percentage of normal tubules and an increase in the percentage of hypospermatogenic and arrested maturation tubules was observed with aging. Aged animals showed a decrease in tubular diameter, tubular lumen, seminiferous epithelium volume and total tubular volume. However, the total length of seminiferous tubules was significantly increased with aging. The most important ultrastructural changes with aging were the thickening of the lamina propria, the presence of diverse abnormalities in the spermiogenesis process, degeneration of germ cells, and vacuolization and flattening of Sertoli cells showing abundant lipofucsin droplets and residual bodies. Laminin immunoreactivity was found along the lamina propria of seminiferous tubules both in young and aged animals. Fibronectin immunoreactivity was found along the lamina propria and blood vessels. Both laminin and fibronectin total volume of immunostaining per testis was increased in aged hamsters. In conclusion, the age-related changes in seminiferous tubules of hamster include: a decrease in tubular width and an increase in tubular length; widening of the lamina propria caused by a more extensive connective matrix between the peritubular cells and the basal membrane; and a strong disarrangement of the seminiferous epithelium, including germ cell degeneration and important alterations in both spermiogenesis and Sertoli cell structure.     

Post-integration stabilization of a transposon vector by terminal sequence deletion in Drosophila melanogaster

Germline transformation systems for nearly 20 insect species have been derived from transposable elements, allowing the development of transgenic insects for basic and applied studies. These systems use a defective nonautonomous vector that results in stable vector integrations after the disappearance of transiently provided transposase helper plasmid, which is essential to maintain true breeding lines and consistent transgene expression that would otherwise be lost after vector remobilization. The risk of remobilization by an unintended transposase source has so far not been a concern for laboratory studies, but the prospective use of millions of transgenic insects in biocontrol programs will likely increase the risk, therefore making this a critical issue for the ecological safety of field release programs. Here we describe an efficient method that deletes a terminal repeat sequence of a transposon vector after genomic integration. This procedure prevents transposase-mediated remobilization of the other terminal sequence and associated genes, ensuring their genomic stability.     

The crystal structure of a high affinity RNA stem-loop complexed with the bacteriophage MS2 capsid: further challenges in the modeling of ligand-RNA interactions

We have determined the structure to 2.8 A of an RNA aptamer (F5), containing 2'-deoxy-2-aminopurine (2AP) at the -10 position, complexed with MS2 coat protein by soaking the RNA into precrystallised MS2 capsids. The -10 position of the RNA is an important determinant of binding affinity for coat protein. Adenine at this position in other RNA stem-loops makes three hydrogen bonds to protein functional groups. Substituting 2AP for the -10 adenine in the F5 aptamer yields an RNA with the highest yet reported affinity for coat protein. The refined X-ray structure shows that the 2AP base makes an additional hydrogen bond to the protein compared to adenine that is presumably the principal origin of the increased affinity. There are also slight changes in phosphate backbone positions compared to unmodified F5 that probably also contribute to affinity. Such phosphate movements are common in structures of RNAs bound to the MS2 T = 3 protein shell and highlight problems for de novo design of RNA binding ligands.     

PML regulates p53 stability by sequestering Mdm2 to the nucleolus

The promyelocytic leukaemia (PML) tumour-suppressor protein potentiates p53 function by regulating post-translational modifications, such as CBP-dependent acetylation and Chk2-dependent phosphorylation, in the PML-Nuclear Body (NB). PML was recently shown to interact with the p53 ubiquitin-ligase Mdm2 (refs 4-6); however, the mechanism by which PML regulates Mdm2 remains unclear. Here, we show that PML enhances p53 stability by sequestering Mdm2 to the nucleolus. We found that after DNA damage, PML and Mdm2 accumulate in the nucleolus in an Arf-independent manner. In addition, we found that the nucleolar localization of PML is dependent on ATR activation and phosphorylation of PML by ATR. Notably, in Pml(-/-) cells, sequestration of Mdm2 to the nucleolus was impaired, as well as p53 stabilization and the induction of apoptosis. Furthermore, we demonstrate that PML physically associates with the nucleolar protein L11, and that L11 knockdown impairs the ability of PML to localize to nucleoli after DNA damage. These findings demonstrate an unexpected role of PML in the nucleolar network for tumour suppression.     

Behavioural structuring of relatedness in the spotted hyena (Crocuta crocuta) suggests direct fitness benefits of clan-level cooperation

Spotted hyenas (Crocuta crocuta) are gregarious carnivores that live in multigenerational social groups, called clans, containing one to several matrilines. Members of multiple matrilines within a clan cooperate during dangerous interactions with inter- and intraspecific competitors. The evolution of cooperation may be influenced by relatedness between individuals, which in turn is influenced by reproductive skew and mate choice, dispersal and territorial behaviours. Behavioural data exist for spotted hyenas, but corresponding data on patterns of relatedness are unavailable; this lack of data makes it difficult to assess the relative importance of selection pressures favouring cooperative behaviour within and among groups. Therefore we conducted a longitudinal analysis of relatedness within a single large clan of spotted hyenas, as well as a cross-sectional analysis of relatedness among hyenas from multiple clans. Within a clan, patterns of relatedness reflected known pedigree relationships, and relatedness was higher within than among matrilines, even across generations. Although mean within-matriline relatedness varied among matrilines, it did not decline with matriline rank. On average, clan members were not related closely, due to high levels of male-mediated gene flow among clans, and relatedness declined very slightly across clan borders. Low mean relatedness within clans suggests that spotted hyenas cooperate with unrelated clan-mates against close paternal kin in other clans. Our data also suggest that spotted hyenas must derive large net direct fitness benefits from group living and cooperation.     

NRMD: Nuclear Receptor Mutation Database

The NRMD is a database for nuclear receptor mutation information. It includes mutation information from SWISS-PROT/TrEMBL, several web-based mutation data resources, and data extracted from the literature in a fully automatic manner. Because it is also possible to add mutations manually, a hundred mutations were added for completeness. At present, the NRMD contains information about 893 mutations in 54 nuclear receptors. A common numbering scheme for all nuclear receptors eases the use of the information for many kinds of studies. The NRMD is freely available to academia and industry as a stand-alone version at: www.receptors.org/NR/.