Epigenetic silencing of monoallelically methylated miRNA loci in precancerous colorectal lesions.

Epigenetic silencing of protein-encoding genes is common in early-stage colorectal tumorigenesis. Less is known about the methylation-mediated silencing of genes encoding microRNAs (miRNAs), which are also important epigenetic modulators of gene expression. Using quantitative PCR, we identified 56 miRNAs that were expressed in normal colorectal mucosa and in HT29 colorectal cancer cells treated with demethylating agents but not in untreated HT29 cells, suggesting that they probably undergo methylation-induced silencing during colorectal tumorigenesis. One of these, miR-195, had recently been reported to be underexpressed in colorectal cancers and to exert tumor-suppressor effects in colorectal cancer cells. We identified the transcription start site (TSS) for primary miRNA (pri-miR)-497/195, the primary precursor that yields miR-195 and another candidate on our list, miR-497, and a single CpG island upstream to the TSS, which controls expression of both miRNAs. Combined bisulfite restriction analysis and bisulfite genomic sequencing studies revealed monoallelic methylation of this island in normal colorectal mucosa (50/50 samples) and full methylation in most colorectal adenomas (38/50; 76%). The hypermethylated precancerous lesions displayed significantly downregulated expression of both miRNAs. Similar methylation patterns were observed at two known imprinted genes, MEG3 and GNAS-AS1, which encode several of the 56 miRNAs on our list. Imprinting at these loci was lost in over half the adenomas (62% at MEG3 and 52% at GNAS-AS1). Copy-number alterations at MEG3, GNAS-AS1 and pri-miR-497/195, which are frequent in colorectal cancers, were less common in adenomas and confined to tumors displaying differential methylation at the involved locus. Our data show that somatically acquired, epigenetic changes at monoallelically methylated regions encoding miRNAs are relatively frequent in sporadic colorectal adenomas and might contribute to the onset and progression of these tumors.

Lemur tyrosine kinase-2 signalling regulates kinesin-1 light chain-2 phosphorylation and binding of Smad2 cargo.

A recent genome-wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9, but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3ß (GSK3ß). KLC2 phosphorylation by GSK3ß induces the release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3ß on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-ß (TGFß) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFß-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling.

Perceptions of clients and veterinarians on what attributes constitute 'a good vet'.

The perceptions of veterinarians and small animal (SA) clients on what attributes constitute 'a good veterinarian' were examined by a questionnaire survey. The respondents were asked to record how important they considered 20 attributes for a veterinary surgeon to have on a five-point scale from 'not at all important' to 'very important'. In addition, they were asked to list which attributes they considered to be the three most important attributes in a veterinary surgeon; finally, they were asked whether there were any additional attributes that they considered to be highly desirable in a veterinary surgeon. In total, 407 SA clients, 243 SA veterinarians and 61 non-SA veterinarians completed the questionnaire. There were significant differences in the proportion of clients who considered an attribute to be 'very important' compared with SA veterinarians for 12 of the 20 attributes (P<0.005). A larger proportion of clients considered 'confidence', 'knowledge about veterinary medicine and surgery', 'cleanliness', 'good at explaining technical terms', 'patience', 'clear about cost of treatment', 'ability to work in a team', 'honesty', 'politeness', 'decisiveness', 'good with animals' and 'good practical skills' to be 'very important' attributes than the SA veterinarians; a larger proportion of SA veterinarians considered 'good communication skills' to be a 'very important' attribute than the clients.

Latrophilin, neurexin, and their signaling-deficient mutants facilitate alpha -latrotoxin insertion into membranes but are not involved in pore formation.

Pure alpha-latrotoxin is very inefficient at forming channels/pores in artificial lipid bilayers or in the plasma membrane of non-secretory cells. However, the toxin induces pores efficiently in COS-7 cells transfected with the heptahelical receptor latrophilin or the monotopic receptor neurexin. Signaling-deficient (truncated) mutants of latrophilin and latrophilin-neurexin hybrids also facilitate pore induction, which correlates with toxin binding irrespective of receptor structure. This rules out the involvement of signaling in pore formation. With any receptor, the alpha-latrotoxin pores are permeable to Ca(2+) and small molecules including fluorescein isothiocyanate and norepinephrine. Bound alpha-latrotoxin remains on the cell surface without penetrating completely into the cytosol. Higher temperatures facilitate insertion of the toxin into the plasma membrane, where it co-localizes with latrophilin (under all conditions) and with neurexin (in the presence of Ca(2+)). Interestingly, on subsequent removal of Ca(2+), alpha-latrotoxin dissociates from neurexin but remains in the membrane and continues to form pores. These receptor-independent pores are inhibited by anti-alpha-latrotoxin antibodies. Our results indicate that (i) alpha-latrotoxin is a pore-forming toxin, (ii) receptors that bind alpha-latrotoxin facilitate its insertion into the membrane, (iii) the receptors are not physically involved in the pore structure, (iv) alpha-latrotoxin pores may be independent of the receptors, and (v) pore formation does not require alpha-latrotoxin interaction with other neuronal proteins.

Tetramerisation of alpha-latrotoxin by divalent cations is responsible for toxin-induced non-vesicular release and contributes to the Ca(2+)-dependent vesicular exocytosis from synaptosomes.

A novel procedure of alpha-latrotoxin (alpha LTX) purification has been developed. Pure alpha LTX has been demonstrated to exist as a very stable homodimer. Such dimers further assemble into tetramers, and Ca(2+), Mg(2+) or higher toxin concentrations facilitate this process. However, when the venom is treated with EDTA, purified alpha LTX loses the ability to tetramerise spontaneously; the addition of Mg(2+) or Ca(2+) restores this ability. This suggests that alphaLTX has some intrinsically bound divalent cation(s) that normally support its tetramerisation. Single-particle cryoelectron microscopy and statistical image analysis have shown that: 1) the toxin has a non-compact, branching structure; 2) the alpha LTX dimers are asymmetric; and 3) the tetramers are symmetric and have a 25 A-diameter channel in the centre. Both alpha LTX oligomers bind to the same receptors in synaptosomes and rat brain sections. To study the effects of the dimers and tetramers on norepinephrine release from rat cerebrocortical synaptosomes, we used the EDTA-treated and untreated toxin preparations. The number of tetramers present in a preparation correlates with alpha LTX pore formation, suggesting that the tetramers are the pore-forming species of alpha LTX. The toxin actions mediated by the pore include: 1) Ca(2+) entry from the extracellular milieu; and 2) passive efflux of neurotransmitters via the pore that occurs independently of Ca(2+). The Ca(2+)-dependent alpha LTX-stimulated secretion conforms to all criteria of vesicular exocytosis but also depends upon intact intracellular Ca(2+) stores and functional phospholipase C (PLC). The Ca(2+)-dependent effect of the toxin is stronger when dimeric alpha LTX is used, indicating that higher receptor occupancy leads to its stronger activation, which contributes to stimulation of neuroexocytosis. In contrast, the Ca(2+)-independent release measured biochemically represents leakage of neurotransmitters through the toxin pore. These results are discussed in relation to the previously published observations.

Structure of alpha-latrotoxin oligomers reveals that divalent cation-dependent tetramers form membrane pores.

We report here the first three-dimensional structure of alpha-latrotoxin, a black widow spider neurotoxin, which forms membrane pores and stimulates secretion in the presence of divalent cations. We discovered that alpha-latrotoxin exists in two oligomeric forms: it is dimeric in EDTA but forms tetramers in the presence of Ca2+ or Mg2+. The dimer and tetramer structures were determined independently at 18 A and 14 A resolution, respectively, using cryo-electron microscopy and angular reconstitution. The alpha-latrotoxin monomer consists of three domains. The N- and C-terminal domains have been identified using antibodies and atomic fitting. The C4-symmetric tetramers represent the active form of alpha-latrotoxin; they have an axial channel and can insert into lipid bilayers with their hydrophobic base, providing the first model of alpha-latrotoxin pore formation.

Investigations into the preferences of laboratory rats for nest-boxes and nesting materials.

Nest-boxes and nesting materials were considered potentially useful items with which to enhance the environment of rats housed in standard laboratory cages. This study was carried out to determine whether such items are actually used by rats, and if so, what features are important in their design. Laboratory rats were allowed to choose between four commercially available nest-boxes. Nest-boxes were preferred to other parts of the cage but the nest-box most frequently selected was not suitable for routine laboratory use. Accordingly a new nest-box was designed, incorporating features apparently attractive to the animals. This was a simple structure of opaque perspex, consisting of a roof and three walls. Similarly, rats were exposed to six commercially available nesting materials and those consisting of long paper strips were most preferred.

Operant studies to determine the strength of preference in laboratory rats for nest-boxes and nesting materials.

Previous work has shown that laboratory rats preferred to use nest-boxes and nesting materials rather than empty parts of the cage. In preference tests, they chose opaque or semi-opaque nest-boxes and long strips of soft paper nesting material. Choice tests to demonstrate a preference between nest-boxes and nesting material were not possible because nesting materials were carried into the nest-boxes. Furthermore, preference tests did not show how important these items were to the animals. Accordingly, operant tests were conducted, in which the rats had to lift a weighted door in order to gain access to an empty cage, or one containing a nest-box, nesting material or both items. By progressively increasing the weight of the door in subsequent trials, it was shown that the rats would carry out more work to reach a nest-box, with or without nesting material, than to reach an empty cage.

The use of a novel operant test to determine the strength of preference for flooring in laboratory rats.

A previous study showed that laboratory rats preferred to dwell on a solid floor rather than a grid one, particularly when resting (Manser et al. 1995). The strengths of this preference was investigated in an operant trial using a novel test apparatus, which consisted of a grid-floored cage and a solid-floored cage, joined via a central box containing a barrier whose weight was adjustable. Trials in which rats had to lift the barrier in order to explore the whole apparatus were alternated with those in which the rats were confined on the grid floor and then had to lift the barrier in order to reach the solid floor. The latter trials were carried out at the beginning of the light period when the rats were seeking a resting place. In both trials, the weight of the barrier was progressively increased for each rat, until a maximum weight was found which it would lift either to explore its environment (weight A) or to reach the solid floor (weight B). No significant differences were found between weights A and B, showing that rats would work as hard to reach a solid floor to rest on as they would to explore a novel environment. The apparatus used could with some modifications, be appropriate for use in other operant studies in laboratory rats.

An investigation into the effects of solid or grid cage flooring on the welfare of laboratory rats.

The welfare of laboratory rats housed on either solid or grid floors was investigated in several ways. No differences were found in body weight gain, food consumption or water consumption amongst rats housed in either condition. When handling was standardized between the 2 groups, there was no correlation between flooring and docility. Preference testing revealed that rats chose to dwell on solid floors rather than grids, regardless of previous housing experience. This preference for solid floors was particularly marked (88%) when the animals were resting and much less marked during activity (55.4%). Since the rats were observed to spend 70 to 75% of their time resting, it was concluded that their welfare was likely to be improved by housing them on solid floors.