Generation of Mouse Small Intestinal Epithelial Cell Lines That Allow the Analysis of Specific Innate Immune Functions

Cell lines derived from the small intestine that reflect authentic properties of the originating intestinal epithelium are of high value for studies on mucosal immunology and host microbial homeostasis. A novel immortalization procedure was applied to generate continuously proliferating cell lines from murine E19 embryonic small intestinal tissue. The obtained cell lines form a tight and polarized epithelial cell layer, display characteristic tight junction, microvilli and surface protein expression and generate increasing transepithelial electrical resistance during in vitro culture. Significant up-regulation of Cxcl2 and Cxcl5 chemokine expression upon exposure to defined microbial innate immune stimuli and endogenous cytokines is observed. Cell lines were also generated from a transgenic interferon reporter (Mx2-Luciferase) mouse, allowing reporter technology-based quantification of the cellular response to type I and III interferon. Thus, the newly created cell lines mimic properties of the natural epithelium and can be used for diverse studies including testing of the absorption of drug candidates. The reproducibility of the method to create such cell lines from wild type and transgenic mice provides a new tool to study molecular and cellular processes of the epithelial barrier.     

Presence and Analysis of Plasmids in Human and Animal Associated Arcobacter Species

In this study, we report the screening of four Arcobacter species for the presence of small and large plasmids. Plasmids were present in 9.9% of the 273 examined strains. One Arcobacter cryaerophilus and four Arcobacter butzleri plasmids were selected for further sequencing. The size of three small plasmids isolated from A. butzleri and the one from A. cryaerophilus strains ranged between 4.8 and 5.1 kb, and the size of the large plasmid, isolated from A. butzleri, was 27.4 kbp. The G+C content of all plasmids ranged between 25.4% and 26.2%. A total of 95% of the large plasmid sequence represents coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried 35 coding sequences, including seven genes in a contiguous region of 11.6 kbp that encodes an orthologous type IV secretion system found in the Wolinella succinogenes genome, Helicobacter pylori and Campylobacter jejuni plasmids, which makes this plasmid interesting for further exploration.     

The 12 kDa protein of potato virus M displays properties of a nucleic acid-binding regulatory protein

The 3' terminal 1.4 kb segment of potato virus M (PVM) genomic RNA was cloned and sequenced. This part of the viral genome encodes the capsid protein CP as well as a 12 kDa protein of as yet unknown function. Both proteins were expressed in bacteria and their nucleic acid-binding properties studied. The 12 kDa protein (pr12), but not the capsid protein bound single- and double-stranded nucleic acids. This property of pr12 in conjunction with a zinc finger motif located adjacent to a basic region of the 12 kDa protein suggests that it may act as a regulatory factor during virus replication.     

Infraspecific variation in Cerastium alpinum s. lat. (Caryophyllaceae) in Central Norway

Within Cerastium alpinum s. lat., several taxa have been circumscribed. Three infraspecific taxa are currently recognized in Nordic literature, but the opinions have varied about their taxonomic levels and relations to each other. Populations of these three taxa — alpinum, glabratum and lanatum — were investigated in Central Norway to elucidate taxonomic relationships on a regional scale. Morphometric analyses (PCA ordination) based on two data sets; one with 61 characters, and one without the 35 indumentum characters, gave different results. The former partly support the use of a higher rank for glabratum , but this analysis may put too much weight on indumentum characters. The reduced data set gave a more continuous transition between the three taxa, with no special status for glabratum. Crossing experiments between the taxa indicated full cross compatibility as far as relative seed set was concerned. Finally, vegetation analysis (DCA ordination) indicated that the three taxa have somewhat different ecological demands. This suggests that they, in spite of hybridization abilities, normally are maintained as separate entities due to habitat differences. A delimitation of three infraspecific taxa at the same level within C. alpinum s. lat. is thus supported by this study. Whether these taxa should be treated as subspecies or varieties strongly depends upon the definitions of these categories. Viewed in a wide geographical context, however, the arguments for subspecific rank are strengthened.     

The effect of grass maturing and root decay on N2O production in soil

The N₂O flux from the surface of grass-covered pots was only significant following grass maturing. Removal of the above-ground plant material resulted in an immediate and long-lasting increase in N₂O production in the soil. The results suggest that easily available organic matter from the roots stimulates the denitrification when the plants are damaged. Grass cutting might therefore result in a marked nitrogen loss through denitrification. The quantitative effect was equal in soil with and without succinate added. The size of the anaerobic zone around the roots is therefore sufficient to allow for denitrification activity mediated by increased organic matter availability because of plant cutting.     

Ultrastructure of flame cells and protonephridial capillaries of Craspedella and Didymorchis (Plathelminthes,Rhabdocoela)

Summary The ultrastructure of the flame cells and protonephridial capillaries of the Rhabdocoela Craspedella sp. and Didymorchis sp., ectocommensals on the freshwater crayfish Cherax destructor in eastern Australia is described. The flame cells of both species have variable numbers of cilia without distinct rootlets and with decreasing numbers of axonemal tubules towards the ciliary tips. Bundles of microtubules extend from the cytoplasm adjacent to the ciliary rootlets through the ribs of the weir apparatus into the distal cytoplasmic tube, where the numbers of microtubules gradually decrease. The weir apparatus is formed by a single row of longitudinal ribs connected by a membrane. In Craspedella, but not in Didymorchis, the ribs have external branched leptotriches. Mitochondria are common in the wall of the flame cell of both species. The protonephridial capillary just above the end of the ciliary tuft narrows in both species and bends sharply in Craspedella. The lumen of the flame cell and the capillary is lined by a dark layer of cytoplasm; there is no enlargement of the surface area by microvilli or lamellae. Centrioles were seen in the capillary wall of Craspedella, and in Didymorchis the cytoplasm around the capillaries has a very loose and light appearance. The ultrastructure of the flame cells and capillaries of both species corresponds closely to that of Temnocephala sp.Abbreviations in the figures BB basal body - CE centriole - L leptotrich - M microtubules - ME membrane of weir apparatus - MI mitochondrion - PC protonephridial capillary - R rib (rod) of weir apparatus     

Gill monogenea of Rastrelliger spp. (Scombridae)

The following gill monogeneans are described, based on a survey of 240 Rastrelliger kanagurta, 12 R. faughni and 185 R. brachysoma (Scombridae) from many geographical areas: Eyelavera typica from R. kanagurta, R. faughni and R. brachysoma, Indomazocraes jagannath from R. kanagurta and R. faughni, Kuhnia sprostonae from R. kanagurta and R. brachysoma, and Kuhnia scombercolias from R. kanagurta and R. brachysoma. Eyelavera parukhini Lebedev, 1980 is synonymised with E. typica, Scomberocotyle eyela Unnithan, 1964 with Indomazocraes jagannath, Kuhnia microlepidotusi Gupta & Krishna, 1977 and K. kanagurta Mamaev & Parukhin, 1986 with K. sprostonae, K. arabica Mamaev & Parukhin, 1986 with K. scombercolias Nasir & Fuentes Zambrano, 1983. It is emphasized that populations of Monogenea from the same host species or genus in different geographical areas are likely to be conspecific, and should not be described as different species, if they differ only slightly from each other. Monogenea that differ from insufficiently described species in minor detail should not be described as new species unless material of the original species has been examined.     

Light- and electron-microscopic immunocytochemistry of peptidergic neurons innervating thoracico-abdominal neurohaemal areas in the blowfly

Summary Ventral thoracic neurosecretory cells (VTNCs) of the blowflies, Calliphora erythrocephala and C. vomitoria, innervating thoracic neuropil and the dorsal neural sheath of the thoracico-abdominal ganglion have been shown to be immunoreactive to a variety of mammalian peptide antisera. In the neural sheath the VTNC terminals form an extensive neurohaemal network that is especially dense over the abdominal ganglia. The same areas are invaded by separate, ut overlapping serotonin-immunoreactive (5-HT-IR) projections derived from neuronal cell bodies in the suboesophageal ganglion. Immunocytochemical studies with different antisera, applied to adjacent sections at the lightmicroscopic level, combined with extensive cross-absorption tests, suggest that the perikarya of the VTNCs contain co-localized peptides related to gastrin/cholecystokinin (CCK), bovine pancreatic polypeptide (PP), Met- and Leuenkephalin and Met-enk-Arg⁶-Phe⁷ (Met-enk-RF). Electron-microscopic immunogold-labeling shows that some of the terminals in the dorsal sheath react with several of the individual peptide antisera, whilst others with similar cytology are non-immunoreactive. In the same region, separate terminals with different cytological characteristics contain 5-HT-IR. Both 5-HT-IR and peptidergic terminals are localized outside the cellular perineurium beneath the acellular permeable sheath adjacent to the haemocoel. Hence, we propose that various bioactive substances may be released from thoracic neurosecretory neurons into the circulating haemolymph to act on peripheral targets. The same neurons may also interact by synaptic or modulatory action in the CNS in different neuropil regions of the thoracic ganglion.     

Diminished oestrogen sensitivity and increased ovulation rate in adult female rats after prepubertal treatment with oestrogen or pimozide

Immature female rats were implanted with oestradiol benzoate or cholesterol in the medial preoptic area at different ages, and the inhibition of the ovariectomy-induced increase of LH secretion by s.c. injected oestradiol was investigated. Medial preoptic oestrogen implants reduced the inhibition of LH secretion in 4-week-old rats, but not in younger animals. Elevation of the circulating oestrogen concentration or suppression of the central nervous dopamine activity by daily injections of oestradiol and pimozide, respectively, from Day 26 to the day of vaginal opening, i.e. during the time when the mechanism of the oestrogen-induced desensitization of the negative oestrogen feedback matures, resulted in considerable diminution of the LH-inhibiting effect of oestradiol in ovariectomized adult females. In intact cyclic rats, both prepubertal treatments led to a significant increase of the average number of eggs per ovulation that was mainly caused by reduction of the number of animals with a low ovulation rate.     

Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.