Slow photo-cross-linking kinetics of benzophenone-labeled voltage sensors of ion channels

Voltage-gated ion channels have voltage sensors that move in response to changes in membrane potential. This movement regulates the gates that control access of ions to the permeation pathway. To study the coupling between voltage sensors and gates, we immobilize the voltage sensors, using a bifunctional photo-cross-linking reagent that can be attached to an introduced cysteine, and observe the consequences for gate movement [Horn, R., Ding, S., and Gruber, H. J. (2000) J. Gen. Physiol. 116, 461-475]. UV irradiation of the benzophenone adduct attached to the cysteine residue immobilizes the voltage sensors, S4 segments, of both Na(+) and Shaker K(+) channels. Here we examine the kinetics of S4 immobilization after a brief UV flash. Immobilization has an exponential time course with time constants of >200 ms for Shaker and 17 ms for Na(+) channels, whereas the triplet excited state lifetime of the benzophenone adduct is <1 ms. This result suggests that H-atom abstraction by benzophenone is rapid and that the rate-limiting step in immobilization is the recombination of alkyl and ketyl free radicals generated by H-abstraction. H-Abstraction is also 2.7-fold more efficient at a hyperpolarized voltage than at a depolarized membrane potential in Shaker S4 segments. S4 immobilization after a UV flash can be prevented by depolarization of Shaker channels, suggesting that movement in the activation pathway is capable of separating the ketyl and alkyl free radicals. Exploiting the unique charge movement and gating properties of the L382V mutant of Shaker, we show that free radical separation follows S4 movement itself and is relatively independent of the movement of activation gates.     

Genotoxicity assessment in aquatic environments under the influence of heavy metals and organic contaminants

The genotoxicity of river water and sediment including interstitial water was evaluated by microscreen phage-induction and Salmonella/microsome assays. Different processes used to fractionate the sediment sample were compared using solvents with different polarities. The results obtained for mutagenic activity using the Salmonella/microsome test were negative in the water and interstitial water samples analysed using the direct concentration method. The responses in the microscreen phage-induction assay showed the presence of genotoxic or indicative genotoxic activity for at least one water sample of each site analysed using the same concentration method. Similar results were obtained for interstitial water samples, i.e. absence of mutagenic activity in the Salmonella/microsome test and presence of genotoxic activity in the microscreen phage-induction assay. Metal contamination, as evidenced by the concentrations in stream sediments, may also help explain some of these genotoxic results. Stream sediment organic extracts showed frameshift mutagenic activity in the ether extract detected by Salmonella/microsome assay. The concentrates evaluated by microscreen phage-induction assay identified the action of organic compounds in the non-polar, medium polar and polar fractions. Thus, the microscreen phage-induction assay has proven to be a more appropriate methodology than the Salmonella/microsome test to analyse multiple pollutants in this ecosystem where both organic compounds and heavy metals are present.     

Evaluation of an in vitro coculture model for the blood-brain barrier: Comparison of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources

Summary Cocultures of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources, American Type Culture Collection and European Collection of Animal Cell Cultures, were evaluated as an in vitro model for the blood-brain barrier. Monolayers of endothelial cells grown in the presence or absence of glial cells were examined for transendothelial electrical resistance, sucrose permeability, morphology, multidrug resistance-associated protein expression, and P-glycoprotein expression and function. Coculture of glial cells with endothelial cells increased electrical resistance and decreased sucrose permeability across European endothelial cell monolayers, but had no effect on American endothelial cells. Coculture of European glial cells with endothelial cells caused cell flattening and decreased cell stacking with both European and American endothelial cells. No P-glycoprotein or multidrug resistance-associated protein was immunodetected in endothelial cells grown in glial cell-conditioned medium. Functional P-glycoprotein was demonstrated in American endothelial cells selected in vinblastine-containing medium over eight passages, but these cells did not form a tight endothelium. In conclusion, while European glial cells confer blood-brain barrier-like morphology and barrier integrity to European endothelial cells in coculture, the European endothelial-glial cell coculture model does not express P-glycoprotein, normally found at the blood-brain barrier. Further, the response of endothelial cells to glial factors was dependent on cell source, implying heterogeneity among cell populations. On the basis of these observations, the umbilical vein endothelial cell-glial cell coculture model does not appear to be a viable model for predicting blood-brain barrier penetration of drug molecules.     

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.