In vivo recovery of the injured anal sphincter after repair and injection of myogenic stem cells: an experimental model.

OBJECTIVE: This study aims to evaluate in vivo function of the external anal sphincter after transection and repair augmented with myogenic stem cells, and to establish normative electromyography parameters of the rodent external anal sphincter. DESIGN AND SETTING: Thirty-three Sprague-Dawley rodents underwent baseline needle electromyography of the external anal sphincter. Motor unit action potentials were obtained and normative parameters established. Animals were randomly assigned to a myogenic stem cell group (n = 24) or control group (n = 9). All underwent proctoepisiotomy. The control group underwent layered repair with phosphate-buffered saline injection to the external anal sphincter. The treatment group underwent identical repair with injection of myogenic stem cells 5.0 × 10. Baseline anal pressure recordings were collected and repeated 2 weeks postintervention, and electromyography was repeated at 2 and 4 weeks. Groups were compared across 3 time points with the use of repeated measures ANOVA. MAIN OUTCOME MEASURES: The primary outcomes measured were the functional recovery of rat anal sphincters after stem cell transplantation as assessed by objective electromyography and anal pressure measures. RESULTS: A mean of 17 motor unit action potentials were sampled per animal. At 2 weeks postrepair, there was a significant difference between control and transplant groups with respect to amplitude, duration, turns, and phases (p < 0.01 for each). No significant electromyography differences were seen at 4 weeks. Resting and peak anal pressures declined significantly at 2 weeks postinjury in the control but not in the stem cell group. LIMITATIONS: Use of a murine animal population limited the subjective feedback and wider applicability. CONCLUSIONS: In vivo functional studies show recovery of anal sphincter pressures and electromyography to preinjury levels by day 14 in the myogenic stem cell group but not controls. At 4 weeks, all electromyography parameters returned to baseline irrespective of group. Restoration of function may be accelerated by the transplantation of myogenic stem cells and associated trophic factors.

Functional analysis of the KCS-like element of the interferon-inducible RNA-specific adenosine deaminase ADAR1 promoter.

The ADAR1 gene encodes an RNA-specific adenosine deaminase that alters the functional activity of both viral and cellular RNAs by posttranscriptional adenosine-to-inosine RNA editing. The interferon (IFN) responsive PI promoter of the ADAR1 gene possesses an IFN-stimulated response element (ISRE) responsible for IFN-inducibility, as well as an adjacent upstream sequence, designated kinase conserved sequence-like (KCS-l) element. The KCS-l element is similar to the 15-bp KCS element so far unique to the human and mouse RNA-dependent PKR kinase gene promoters. The KCS element of the PKR kinase (PKR) promoter is essential for both basal and IFN-inducible PKR promoter activity. We have now examined the functional properties of the KCS-l element of the ADAR1 PI promoter. Electrophoretic mobility shift assays (EMSAs) detected constitutively expressed nuclear proteins that bound selectively to the ADAR1 KCS-l element. Competition EMSA and antibody supershift assays indicated that ADAR1 KCS-l-binding proteins shared some properties with PKR KCS-binding proteins. However, transient transfection analyses performed with ADAR1 PI promoter constructs possessing deletion and substitution mutant forms of the KCS-l element revealed that the ADAR1 KCS-l element was not essential for either basal or IFN-inducible promoter activity. Substitution of the ADAR1 KCS-l element with the PKR KCS element increased both basal and inducible ADAR1 PI promoter activity. These results suggest that the KCS-l element of the ADAR1 PI promoter is not functionally equivalent to the KCS element of the PKR promoter.

The promoter-proximal KCS element of the PKR kinase gene enhances transcription irrespective of orientation and position relative to the ISRE element and is functionally distinct from the KCS-like element of the ADAR deaminase Promoter.

The RNA-dependent protein kinase PKR promoter is interferon (IFN) inducible and possesses a novel 15-base pair (bp) constitutive activator element, designated kinase conserved sequence (KCS), in addition to an IFN-stimulated response element (ISRE). Deletion of the KCS element or point mutations within the KCS element greatly reduce both basal and IFN-inducible PKR promoter activity. The IFN-inducible RNA-specific adenosine deaminase ADAR1 promoter possesses a KCS-like (KCS-l) element. The sequences of the KCS and KCS-l elements and their positions relative to the cognate ISRE element are similar between the PKR and ADAR1 promoters. However, substitution of the ADAR1 KCS-l element for the KCS element of the PKR promoter resulted in significantly reduced basal and IFN-inducible promoter activities comparable to either point mutation or entire deletion of the PKR KCS element. The PKR KCS element selectively bound nuclear proteins more efficiently than did the ADAR1 KCS-l element. Reversing the positions of the KCS and ISRE elements of the PKR promoter relative to one another or reversing the orientation of either element while conserving the naturally occurring 4-bp spacing between the two elements did not significantly reduce basal or IFN-inducible promoter activity. Taken together, these results are consistent with the notion that the KCS and ISRE elements of the PKR promoter function as a unit.

Patient characteristics associated with a successful pessary fitting.

OBJECTIVE: : The objective of the study was to assess if patient characteristics, including Pelvic Organ Prolapse Quantification measurements, are predictive of successful pessary fitting in women with pelvic organ prolapse (POP) and/or incontinence. METHODS: : This was a retrospective chart review of patients who underwent a pessary fitting for POP and/or incontinence. Multiple demographic parameters, pessary fitting data, and Pelvic Organ Prolapse Quantification measurements were examined. RESULTS: : Complete data were available on 158 patients, and 59% were successfully fit. Shorter total vaginal length (TVL), less than 8 cm, was associated with an unsuccessful pessary fitting (odds ratio [OR], 0.1; 95% confidence interval [CI], 0.02-0.46). A genital hiatus (GH)/TVL ratio of less than 0.9 was predictive of successful fitting (OR, 12.5; 95% CI, 1.5-102). Patients with a prior hysterectomy were more likely to have an unsuccessful pessary fitting (OR, 0.41; 95% CI, 0.22-0.8). The GH/TVL ratio and TVL were predictors only in patients with a previous hysterectomy. CONCLUSIONS: : Patients with a previous hysterectomy and a TVL of less than 8 cm or a GH/TVL ratio of 0.9 or greater can be counseled that successful pessary fitting is unlikely.

DNA damage-binding proteins and heterogeneous nuclear ribonucleoprotein A1 function as constitutive KCS element components of the interferon-inducible RNA-dependent protein kinase promoter.

Protein kinase regulated by RNA (PKR) plays important roles in many cellular processes including virus multiplication and cell growth, differentiation, and apoptosis. The promoter of the PKR gene possesses a novel 15-bp element designated KCS, positioned upstream of a consensus interferon (IFN)-stimulated response element, that is required for both basal and interferon-inducible transcription. Protein binding to the KCS element is not dependent upon IFN treatment and correlates with transcriptional activity of the PKR promoter. The identity of KCS-binding proteins (KBP) that selectively bind at the KCS element is largely unknown, except for the transcription factor Sp1. We now have purified KBP from HeLa cell nuclear extracts by ion-exchange and DNA-affinity chromatography steps and then identified four constituent proteins of the KBP complex by mass spectrometry and immunochemistry: KBP120 and KBP45 are the damaged DNA-binding protein subunits, p127 DDB1 and p48 DDB2, respectively; KBP100 is the transcription factor Sp1; and KBP35 is the heterogeneous nuclear ribonucleoprotein A1. The steady-state levels of these four KCS-binding proteins in human cells are not altered by IFN treatment. Components of the KBP complex bind selectively and constitutively to the KCS element in the absence of IFN treatment, both in vitro as measured by competition electrophoretic mobility shift assay (EMSA) and DNA pull-down assays and in vivo as measured by chromatin immunoprecipitation assays. Depletion of DDB2 by antisense strategy reduces KBP complex formation by EMSA. These results provide new insight into the biochemical identity and activity of proteins involved in PKR promoter function.