Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry

The septins are a conserved family of GTP-binding proteins that, in the baker''s yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified.Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen.     

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.     

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.     

Oat mesophyll protoplasts: their response to various feeder cultures

Oat (Avena sativa L.) mesophyll protoplasts were recently demonstrated to be capable of dedifferentiation, repeated divisions, and colony formation. Since the development of oat mesophyll protoplasts is decisively influenced by the nature of the used feeder culture (species, variety and concentration), we conducted a systematic study of this parameter. Generally, graminaceous feeders promoted protoplast proliferation, while dicot species repressed protoplast divisions. The beneficial effect of those feeders that promote divisions was counterbalanced by a factor that causes necrosis. The correct balance between promotion of divisions or necrosis depended on the nature of the feeder and its plating density.     

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.     

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.     

Midbrain auditory sensitivity in the spring peeper (Pseudacris crucifer): correlations with behavioral studies

1. We derived audiograms from recordings of multiunit activity in the torus semicircularis of 10 males and 6 females of the spring peeper from central Missouri, USA. We used free-field stimulation with tone bursts that had temporal properties similar to typical advertisement calls and that ranged in frequency from 500-6000 Hz. 2. Audiograms from different electrode positions in the same animal had the same general shape. There was no evidence of tonotopy. 3. Audiograms showed two regions of maximal sensitivity: a low-frequency region (500-700 Hz); and a high-frequency region (2000-4000 Hz). Absolute thresholds and frequencies of maximum sensitivity varied considerably from individual to individual. 4. Audiograms derived from all individuals of each sex indicated that in the high-frequency region, corresponding to the frequency range of advertisement calls, males were more broadly tuned than females. However, tuning in both sexes was relatively weak, and the data predict relatively little selectivity in behavioral responses over the entire range of variation in frequency of the advertisement call in local populations. 5. The results are discussed in terms of behavioral experiments with both males and females from the same populations in central Missouri. We show that merely summarizing the audiograms based on estimates of minimum thresholds of a population or species may mask significant individual differences in tuning. Moreover, most behavioral studies are conducted at playback levels considerably above threshold. For these reasons, behavioral selectivity is not always accurately predicted by inspection of "average" audiograms.