Stc1: a critical link between RNAi and chromatin modification required for heterochromatin integrity.

In fission yeast, RNAi directs heterochromatin formation at centromeres, telomeres, and the mating type locus. Noncoding RNAs transcribed from repeat elements generate siRNAs that are incorporated into the Argonaute-containing RITS complex and direct it to nascent homologous transcripts. This leads to recruitment of the CLRC complex, including the histone methyltransferase Clr4, promoting H3K9 methylation and heterochromatin formation. A key question is what mediates the recruitment of Clr4/CLRC to transcript-bound RITS. We have identified a LIM domain protein, Stc1, that is required for centromeric heterochromatin integrity. Our analyses show that Stc1 is specifically required to establish H3K9 methylation via RNAi, and interacts both with the RNAi effector Ago1, and with the chromatin-modifying CLRC complex. Moreover, tethering Stc1 to a euchromatic locus is sufficient to induce silencing and heterochromatin formation independently of RNAi. We conclude that Stc1 associates with RITS on centromeric transcripts and recruits CLRC, thereby coupling RNAi to chromatin modification.

The potential role of unregulated autonomous bladder micromotions in urinary storage and voiding dysfunction; overactive bladder and detrusor underactivity.

The isolated bladder shows autonomous micromotions, which increase with bladder distension, generate sensory nerve activity, and are altered in models of urinary dysfunction. Intravesical pressure resulting from autonomous activity putatively reflects three key variables; the extent of micromotion initiation, distances over which micromotions propagate, and overall bladder tone. In vivo, these variables are subordinate to the efferent drive of the central nervous system. In the micturition cycle storage phase, efferent inhibition keeps autonomous activity generally at a low level, where it may signal 'state of fullness', whilst maintaining compliance. In the voiding phase, mass efferent excitation elicits generalised contraction (global motility initiation). In lower urinary tract dysfunction, efferent control of the bladder can be impaired, for example due to peripheral 'patchy' denervation. In this case, loss of efferent inhibition may enable unregulated micromotility, and afferent stimulation, predisposing to urinary urgency. If denervation is relatively slight, the detrimental impact on voiding may be low, as the adjacent innervated areas may be able to initiate micromotility synchronous with the efferent nerve drive, so that even denervated areas can contribute to the voiding contraction. This would become increasingly inefficient the more severe the denervation, such that ability of triggered micromotility to propagate sufficiently to engage the denervated areas in voiding declines, so the voiding contraction increasingly develops the characteristics of underactivity. In summary, reduced peripheral coverage by the dual efferent innervation (inhibitory and excitatory) impairs regulation of micromotility initiation and propagation, potentially allowing emergence of overactive bladder and, with progression, detrusor underactivity.

Modulation of Bladder Wall Micromotions Alters Intravesical Pressure Activity in the Isolated Bladder.

Micromotions are phasic contractions of the bladder wall. During urine storage, such phasic activity has little effect on intravesical pressure, however, changed motile activity may underlie urodynamic observations such as detrusor overactivity. The potential for bladder motility to affect pressure reflects a summation of the overall movements, comprising the initiation, propagation, and dissipation components of micromotions. In this study, the influence of initiation of micromotions was investigated using calcium activated chloride channel blocker niflumic acid, and the effect of propagation using blockers of gap junctions. The overall bladder tone was modulated using isoprenaline. Isolated tissue strips and whole bladder preparations from juvenile rats were used. 18ß-glycyrrhetinic acid was used to block gap junctions, reducing the amplitude and frequency of micromotions in in vitro and ex vivo preparations. Niflumic acid reduced the frequency of micromotions but had no effect on the amplitude of pressure fluctuations. Isoprenaline resulted in a reduction in pressure fluctuations and a decrease in pressure baseline. Using visual video data analysis, bladder movement was visible, irrespective of lack of pressure changes, which persisted during bladder relaxation. However, micromotions propagated over shorter distances and the overall bladder tone was reduced. All these results suggest that phasic activity of the bladder can be characterised by a combination of initiation and propagation of movement, and overall bladder tone. At any given moment, intravesical pressure recordings are an integration of these parameters. This synthesis gives insight into the limitations of clinical urodynamics, where intravesical pressure is the key indicator of detrusor activity.

Anoctamin-1 in the juvenile rat urinary bladder.

PURPOSE: To investigate presence, location and functional role of calcium-activated chloride channel (CaCC) Anoctamin-1 (Ano1) in rat urinary bladder. MATERIALS AND METHODS: Bladders from 3 week old Wistar rats were studied. End-point PCR on total mRNA was used to assess the expression of Ano1. Immunofluorescent labelling of whole mount bladder tissue imaged with confocal microscope allowed localization of Ano1 and vimentin immunopositive cells. The effects of CaCC blockers: niflumic acid (NFA) (3,10,30 µM) and 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (10, 30 µM) on spontaneous phasic contractile activity of intact (with mucosa) and denuded (without mucosa) detrusor strips were measured under isometric tension in organ baths (n = 141, N = 60). RESULTS: Ano1 expression was found at mRNA level in mucosa and detrusor layers. Confocal microscopy revealed presence of Ano1 immunopositive cells in mucosa and in detrusor layers; a subpopulation of vimentin positive cells expressed Ano1. Both chloride channel blockers reduced the amplitude and frequency of phasic contractions in denuded and intact strips. CONCLUSIONS: Ano1 is expressed in rat urinary bladder and is present in cells sharing markers with interstitial cells. CaCC blockers reduced phasic activity of the bladder tissue. Ano1 is expressed in the bladder and plays a role in its spontaneous phasic contractile activity.

Voiding dysfunction due to detrusor underactivity: an overview.

Detrusor underactivity (DUA) is defined as a voiding contraction of reduced strength and/or duration, which prolongs urination and/or prevents complete emptying of the bladder within a 'normal' period of time. This issue is associated with voiding and postmicturition urinary symptoms, and can predispose to urinary infections and acute urinary retention. The aetiology of DUA is influenced by multiple factors, including ageing, bladder outlet obstruction, neurological disease, and autonomic denervation. The true prevalence of this condition remains unknown, as most data come from referral populations. Urodynamic testing is used to diagnose the condition, either by assessing the relationship between bladder pressures and urinary flow, or by interrupting voiding to measure detrusor pressure change under isovolumetric conditions. Current treatments for DUA have poor efficacy and tolerability, and often fail to improve quality of life; muscarinic receptor agonists, in particular, have limited efficacy and frequent adverse effects. Bladder emptying might be achieved through Valsalva straining, and intermittent or indwelling catheterization, although sacral nerve stimulation can reduce dependency on catheterization. Novel stem-cell-based therapies have been attempted; however, new drugs that increase contractility are currently largely conceptual, and the complex pathophysiology of DUA, difficulty achieving organ specificity of treatment, the limited availability of animal models, and the subjective nature of current outcome measures must be addressed to facilitate the development of such agents.

A systematic genetic screen identifies new factors influencing centromeric heterochromatin integrity in fission yeast.

BACKGROUND: Heterochromatin plays important roles in the regulation and stability of eukaryotic genomes. Both heterochromatin components and pathways that promote heterochromatin assembly, including RNA interference, RNAi, are broadly conserved between the fission yeast Schizosaccharomyces pombe and humans. As a result, fission yeast has emerged as an important model system for dissecting mechanisms governing heterochromatin integrity. Thus far, over 50 proteins have been found to contribute to heterochromatin assembly at fission yeast centromeres. However, previous studies have not been exhaustive, and it is therefore likely that further factors remain to be identified. RESULTS: To gain a more complete understanding of heterochromatin assembly pathways, we have performed a systematic genetic screen for factors required for centromeric heterochromatin integrity. In addition to known RNAi and chromatin modification components, we identified several proteins with previously undescribed roles in heterochromatin regulation. These included both known and newly characterised splicing-associated proteins,which are required for proper processing of centromeric transcripts by the RNAi pathway, and COP9 signalosome components Csn1 and Csn2, whose role in heterochromatin assembly can be explained at least in part by a role in the Ddb1-dependent degradation of the heterochromatin regulator Epe1. CONCLUSIONS: This work has revealed new factors involved in RNAi-directed heterochromatin assembly in fission yeast. Our findings support and extend previous observations that implicate components of the splicing machinery as a platform for RNAi, and demonstrate a novel role for the COP9 signalosome in heterochromatin regulation.

Phasic contractions in urinary bladder from juvenile versus adult pigs.

AIMS: Alterations in properties of the bladder with maturation are relevant physiologically and pathophysiologically. The aim of this study was to investigate alterations in bladder properties with maturation in juvenile vs. adult pig, focussing on differences between layers of the bladder wall (mucosa vs. detrusor) and the presence and functional contribution of interstitial cells (ICs). METHODS: Basal and cholinergic-induced phasic contractions (PCs) in mucosal and denuded-detrusor strips from juvenile and adult pigs were assessed. Expression of c-kit, a marker of ICs, was investigated in the mucosa and the detrusor layers of the pig bladder. The functional role of ICs in mediating PCs was examined using imatinib. RESULTS: Mucosal strips from juvenile and adult pig bladders demonstrated basal PCs whilst denuded-detrusor strips did not. PCs of mucosal strips from juvenile pigs were significantly greater than those from adult bladders. Immunoreactivity for c-kit was detected in mucosa and detrusor layers of pig bladder. Histological studies demonstrated a distinct layer of smooth muscle between the urothelium and bladder detrusor, termed the muscularis mucosa. Imatinib was only effective in inhibiting PCs in mucosal strips from juvenile pigs. Imatinib inhibited the carbachol-induced PCs of both juvenile and adult denuded-detrusor strips, although strips from juvenile bladders demonstrated a trend towards being more sensitive to this inhibition. CONCLUSIONS: We confirm the presence of c-kit positive ICs in pig urinary bladder. The enhanced PCs of mucosal strips from juvenile animals could be due to altered properties of ICs or the muscularis mucosa in the bladders of these animals.