Prevention of postpartum stress incontinence in primigravidae with increased bladder neck mobility: a randomised controlled trial of antenatal pelvic floor exercises.

OBJECTIVE: To test whether supervised pelvic floor exercises antenatally will reduce the incidence of postpartum stress incontinence in at-risk primigravidae with bladder neck mobility, ultrasonically proven. DESIGN: Single blind, randomised controlled trial. SETTING: Antenatal clinic in a UK NHS Trust Hospital. SAMPLE: Two hundred and sixty-eight primigravidae attending an antenatal clinic at approximately 20 weeks of gestation with bladder neck mobility, on standardised valsalva, of 5 mm or more linear movement. The median age was 28, ranging from 16 to 47 years. INTERVENTION: Patients randomised to supervised pelvic floor exercises (n = 139) attended a physiotherapist at monthly intervals from 20 weeks until delivery. The exercises comprised three repetitions of eight contractions each held for six seconds, with two minutes rest between repetitions. These were repeated twice daily. At 34 weeks of gestation the number of contractions per repetition was increased to 12. Both the untreated control group and the study group received verbal advice on pelvic floor exercises from their midwives antenatally. MAIN OUTCOME MEASURES: Subjective reporting of stress incontinence at three months postpartum. Pelvic floor strength, using perineometry, and bladder neck mobility measured by perineal ultrasound. RESULTS: Of the 268 women enrolled, information on the main outcome variable was available for 110 in the control group and 120 in the study group. Fewer women in the supervised pelvic floor exercise group reported postpartum stress incontinence, 19.2% compared with 32.7% in the control group (RR 0.59 [0.37-0.92]). There was no change in bladder neck mobility and no difference in pelvic floor strength between groups after exercise, although all those developing postpartum stress incontinence had significantly poorer perineometry scores than those who were continent. CONCLUSIONS: The findings suggest that antenatal supervised pelvic floor exercises are effective in reducing the risk of postpartum stress incontinence in primigravidae with bladder neck mobility.

Is anatomical failure following anterior vaginal repair associated with weak native vaginal tissues? A biomechanical and immunohistochemical study.

INTRODUCTION AND HYPOTHESIS: This study was performed to determine whether anatomical recurrence of cystocoele 1 year after anterior vaginal repair is related to biomechanical properties and/or the content of collagen in the vaginal wall and epithelial tissues. METHODS: In this prospective, observational study in a UK teaching hospital, we assessed women undergoing surgery for symptomatic anterior compartment prolapse. Outcome measures were anatomical recurrence, biomechanical strength and collagen content in vaginal tissues. In part one of the study, 42 women underwent biomechanical testing of full-thickness anterior vaginal wall tissue samples to determine the elastic moduli and yield stress. In part two, 59 women underwent immunohistochemical testing of anterior vaginal wall tissue samples to determine tissue content of procollagen I; collagen types I, III, V; and matrix metalloproteinases 1 and 2 (MMP-1 and 2). Results were then compared with anatomical outcome at 1 year postsurgery. RESULTS: Differences in yield strain in all outcome groups (optimal, satisfactory and unsatisfactory) were not statistically significant. Considerable variation was found in collagen type I in both satisfactory and unsatisfactory groups. There was no difference or correlation with procollagen, collagen types III and V, and MMP-1 and recurrence of pelvic organ prolapse (POP) between groups. There was a weak correlation between collagen type I and higher yield stress in both groups. CONCLUSIONS: Anatomical failure of anterior repair does not appear to be related to the biomechanical strength or collagen content of the anterior vaginal wall.

A randomised controlled trial of abdominal versus laparoscopic sacrocolpopexy for the treatment of post-hysterectomy vaginal vault prolapse: LAS study.

INTRODUCTION AND HYPOTHESIS: This prospective multi-centre true two-sided equivalence trial was designed to test the clinical equivalence of open (ASCP) and laparoscopic (LSCP) sacrocolpopexy using objective and subjective outcomes METHODS: The study was carried out in three urogynaecology units in England, UK and the patient population consisted of women referred with symptomatic and bothersome post-hysterectomy vaginal vault prolapse at least 1 cm above or beyond the hymeneal remnants. The interventions were either abdominal or laparoscopic sacrocolpopexy following randomisation to one of the types of surgery. RESULTS: For the primary outcome (point C on the POP-Q) the results at 1 year were -6.63 cm for the open ASCP and -6.67 cm for the LSCP respectively. Subjective outcomes at 1 year showed that 90% of the ASCP group and 80% of the LSCP group were "much better". There were improvements with regard to blood loss, haemoglobin and shorter length of stay in the LSCP group compared with the ASCP group. CONCLUSION: This fully powered randomised controlled trial comparing open and laparoscopic sacrocolpopexy has shown clinical equivalence.

Signalling through phosphoinositide 3-kinases: the lipids take centre stage.

Phosphoinositide 3-kinases (PI3Ks) phosphorylate inositol lipids at the 3' position of the inositol ring to generate the 3-phosphoinositides PI(3)P, PI(3,4) P2 and PI(3,4,5) P3. Recent research has shown that one way in which these lipids function in signal transduction and membrane trafficking is by interacting with 3-phosphoinositide-binding modules in a broad variety of proteins. Specifically, certain FYVE domains bind PI(3)P whereas certain pleckstrin homology domains bind PI(3,4) P2 and/or PI(3,4,5) P3. Also in 1998, PTEN - a major tumour suppressor in human cancer - was also shown to antagonise PI3K signalling by removing the 3-phosphate from 3-phosphoinositides.

An idiotypic cross-reaction between allotype a3 and allotype a negative rabbit antibodies to streptococcal carbohydrate.

Two antibodies to Group C streptococcal carbohydrate isolated from an individual rabbit had similar relative binding affinities for a Group C immuno-adsorbent column. Their light chains were similar, if not identical, as were the constant regions of their heavy chains. Differences in the variable regions of the H chains of the two antibodies were detected by chemical analysis. The two antibodies had serologically identical idiotypic determinants although one antibody possessed the a3 allotype and the other had no detectable group a marker. The occurrence of such antibodies indicates the absence of obligatory associations between group a allotypes and idiotypic specificities, despite the fact that both determinants have antigenic components in the V(H) region of the H chain.

Recognition of viral glycoproteins by influenza A-specific cross-reactive cytolytic T lymphocytes.

Two populations of cytolytic T lymphocytes (CTL) generated after influenza A virus infection can be distinguished into one with specificity for the sensitizing hemagglutinin type and a second with cross-reactivity for antigens induced by other type-A influenza viruses. The molecules carrying the antigenic determinants recognized by the cross-reactive CTL were studied. In L-929 cells abortively infected with fowl plague virus, matrix (M) protein synthesis is specifically inhibited, whereas the envelope glycoproteins, hemagglutinin and neuraminidase, are synthesized and incorporated into the plasma membrane. These target cells were lysed by cross-reactive CTL. The envelope proteins of type A/Victoria virus were separated from the other virion components and reconstituted into lipid vesicles that lacked M protein that subsequently were used to prepare artificial target cells. Target-cell formation with vesicles was achieved by addition of fusion-active Sendai virus. These artificial target cells were also susceptible to lysis by cross-reactive CTL. In contrast to previous observations that suggested that the M protein of influenza viruses is recognized by these effector cells, we present evidence that the antigencic determinants induced by the viral glycoproteins are recognized.

Phosphatidyl-inositol 3-kinase: a key enzyme in diverse signalling processes.

Phosphatidylinositol (PI) 3-kinase associates in signal-transducing complexes with activated growth factor receptors and other protein tyrosine kinases. The enzyme may also act downstream of receptors that are not tyrosine kinases in terminally differentiated cells. The recent cloning of the catalytic subunit of PI 3-kinase has revealed a structural similarity to a yeast protein important in vacuolar protein sorting. This finding provides some interesting clues to the function of PI3-kinase in diverse cellular responses.

Sequence and expression of human estrogen receptor complementary DNA.

The mechanism by which the estrogen receptor and other steroid hormone receptors regulate gene expression in eukaryotic cells is not well understood. In this study, a complementary DNA clone containing the entire translated portion of the messenger RNA for the estrogen receptor from MCF-7 human breast cancer cells was sequenced and then expressed in Chinese hamster ovary (CHO-K1) cells to give a functional protein. An open reading frame of 1785 nucleotides in the complementary DNA corresponded to a polypeptide of 595 amino acids and a molecular weight of 66,200, which is in good agreement with published molecular weight values of 65,000 to 70,000 for the estrogen receptor. Homogenates of transformed Chinese hamster ovary cells containing a protein that bound [3H]estradiol and sedimented as a 4S complex in salt-containing sucrose gradients and as an 8 to 9S complex in the absence of salt. Interaction of this receptor-[3H]estradiol complex with a monoclonal antibody that is specific for primate ER confirms the identity of the expressed complementary DNA as human estrogen receptor. Amino acid sequence comparisons revealed significant regional homology among the human estrogen receptor, the human glucocorticoid receptor, and the putative v-erbA oncogene product. This suggests that steroid receptor genes and the avian erythroblastosis viral oncogene are derived from a common primordial gene. The homologous region, which is rich in cysteine, lysine, and arginine, may represent the DNA-binding domain of these proteins.

Expression of a platelet-derived growth factor-like protein in simian sarcoma virus transformed cells.

The near identity of the partial amino acid sequence of human platelet-derived growth factor (PDGF) and that predicted for p28sis, the putative transforming protein of the simian sarcoma virus (SSV), suggests expression of a growth factor activity may be central for transformation by SSV. It is now reported that SSV-transformed cells but not control cells contain a growth factor activity that is identical to PDGF in immunoassay, in mitogenic dose response, and in specific mitogenic activity. The protein immunoprecipitated by antiserum to human PDGF has an apparent molecular weight of 20,000, identical to that of p20sis, the putative intracellular degradation product of p28sis. The results support the concept that expression of a PDGF-like molecule, which appears to be the product of the viral-sis gene, is responsible for the abnormal regulation of growth is SSV-transformed cells.

The complete primary structure of protein kinase C--the major phorbol ester receptor.

Protein kinase C, the major phorbol ester receptor, was purified from bovine brain and through the use of oligonucleotide probes based on partial amino acid sequence, complementary DNA clones were derived from bovine brain complementary DNA libraries. Thus, the complete amino acid sequence of bovine protein kinase C was determined, revealing a domain structure. At the amino terminal is a cysteine-rich domain with an internal duplication; a putative calcium-binding domain follows, and there is at the carboxyl terminal a domain that shows substantial homology, but not identity, to sequences of other protein kinase.

Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways.

A new family of protein kinase C-related genes has been identified in bovine, human, and rat genomes. The alpha-, beta-, and gamma-type protein kinase sequences are highly homologous, include a kinase domain, and potential calcium-binding sites, and they contain interspersed variable regions. The corresponding genes are located on distinct human chromosomes; the possibility of even greater genetic complexity of this gene family is suggested by Northern and Southern hybridization analyses.

Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting.

The VPS34 gene product (Vps34p) is required for protein sorting to the lysosome-like vacuole of the yeast Saccharomyces cerevisiae. Vps34p shares significant sequence similarity with the catalytic subunit of bovine phosphatidylinositol (PI) 3-kinase [the 110-kilodalton (p110) subunit of PI 3-kinase], which is known to interact with activated cell surface receptor tyrosine kinases. Yeast strains deleted for the VPS34 gene or carrying vps34 point mutations lacked detectable PI 3-kinase activity and exhibited severe defects in vacuolar protein sorting. Overexpression of Vps34p resulted in an increase in PI 3-kinase activity, and this activity was specifically precipitated with antisera to Vps34p. VPS34 encodes a yeast PI 3-kinase, and this enzyme appears to regulate intracellular protein trafficking decisions.

Phosphoinositide 3-kinases: a conserved family of signal transducers.

Phosphoinositide 3-kinases (PI3Ks) generate lipids that are implicated in receptor-stimulated signalling and in the regulation of membrane traffic. Several distinct classes of PI3Ks have now been identified that have been conserved throughout eukaryotic evolution. Potential signalling pathways downstream of PI3Ks have been elucidated and PI3K function is now being characterised in several model organisms.

The long-term effectiveness of antenatal pelvic floor muscle training: eight-year follow up of a randomised controlled trial.

OBJECTIVE: To determine the long-term effectiveness of antenatal pelvic floor muscle training (PFMT) on stress urinary incontinence (SUI). DESIGN: Eight-year follow up of a randomised controlled trial (RCT). SETTING: Acute NHS Teaching Trust. POPULATION: Participants in an RCT of antenatal PFMT 8 years previously. METHOD: Participants were asked about the presence of SUI, impact on quality of life, frequency of performance of PFMT and details of subsequent deliveries. MAIN OUTCOME MEASURE: The prevalence of SUI at 8 years. RESULTS: One hundred and sixty-four (71%) of the original 230 women responded. The significant improvement in postnatal SUI originally shown in the PFMT group compared with controls (19.2 versus 32.7%, P = 0.02) at 3 months was not evident 8 years later (35.4 versus 38.8%, P = 0.7). On direct questioning, 68.4% of the study group claimed that they still performed PFMT as taught during the study, with 38.0% of them performing this twice or more per week. There was no difference in outcome between those who performed PFMT twice or more per week compared with those performing PFMT less frequently. There were no differences in quality-of-life domains between the study and the control groups at 8 years. CONCLUSION: The initially beneficial effect of supervised antenatal PFMT on SUI did not continue for a long term despite the majority claiming to still perform PFMT. These findings are in keeping with those of other studies and raise concerns about the long-term efficacy of PFMT. Strategies to improve compliance with PFMT are required.

Receptor signalling: to sevenless, a daughter.

Genetic studies with Drosophila identified what appeared to be a linear signalling cassette connecting extracellular signals to nuclear responses. But the discovery of a substrate for the Sevenless receptor indicates that the concept of a single, linear pathway may be an oversimplification.

A family of phosphoinositide 3-kinases in Drosophila identifies a new mediator of signal transduction.

BACKGROUND: Mammalian phosphoinositide 3-kinases (PI 3-kinases) are involved in receptor-mediated signal transduction and have been implicated in processes such as transformation and mitogenesis through their role in elevating cellular phosphatidylinositol (3,4,5)-trisphosphate. Additionally, a PI 3-kinase activity which generates phosphatidylinositol 3-phosphate has been shown to be required for protein trafficking in yeast. RESULTS: We have identified a family of three distinct PI 3-kinases in Drosophila, using an approach based on the polymerase chain reaction to amplify a region corresponding to the conserved catalytic domain of PI 3-kinases. One of these family members, PI3K_92D, is closely related to the prototypical PI 3-kinase, p110 alpha; PI3K_59F is homologous to Vps34p, whereas the third, PI3K_68D, is a novel PI 3-kinase which is widely expressed throughout the Drosophila life cycle. The PI3K_68D cDNA encodes a protein of 210 kDa, which lacks sequences implicated in linking p110 PI 3-kinases to p85 adaptor proteins, but contains an amino-terminal proline-rich sequence, which could bind to SH3 domains, and a carboxy-terminal C2 domain. Biochemical analyses demonstrate that PI3K_68D has a novel substrate specificity in vitro, restricted to phosphatidylinositol and phosphatidylinositol 4-phosphate, and is unable to phosphorylate phosphatidylinositol (4,5)-bisphosphate, the implied in vivo substrate for p110. CONCLUSIONS: A family of PI 3-kinases in Drosophila, including a novel class represented by PI3K_68D, is described. PI3K_68D has the potential to bind to signalling molecules containing SH3 domains, lacks p85-adaptor-binding sequences, has a Ca(2+)-independent phospholipid-binding domain and displays a restricted in vitro substrate specificity, so it could define a novel signal transduction pathway.

Regulation of imaginal disc cell size, cell number and organ size by Drosophila class I(A) phosphoinositide 3-kinase and its adaptor.

BACKGROUND: Class I(A) phosphoinositide 3-kinases (PI 3-kinases) have been implicated in the regulation of several cellular processes including cell division, cell survival and protein synthesis. The size of Drosophila imaginal discs (epithelial structures that give rise to adult organs) is maintained by factors that can compensate for experimentally induced changes in these PI 3-kinase-regulated processes. Overexpression of the gene encoding the Drosophila class I(A) PI 3-kinase, Dp110, in imaginal discs, however, results in enlarged adult organs. These observations have led us to investigate the role of Dp100 and its adaptor, p60, in the control of imaginal disc cell size, cell number and organ size. RESULTS: Null mutations in Dp110 and p60 were generated and used to demonstrate that they are essential genes that are autonomously required for imaginal disc cells to achieve their normal adult size. In addition, modulating Dp110 activity increases or reduces cell size in the developing imaginal disc, and does so throughout the cell cycle. The inhibition of Dp110 activity reduces the rate of increase in cell number in the imaginal discs, suggesting that Dp110 normally promotes cell division and/or cell survival. Unlike direct manipulation of cell-cycle progression, manipulation of Dp110 activity in one compartment of the disc influences the size of that compartment and the size of the disc as a whole. CONCLUSIONS: We conclude that during imaginal disc development, Dp110 and p60 regulate cell size, cell number and organ size. Our results indicate that Dp110 and p60 signalling can affect growth in multiple ways, which has important implications for the function of signalling through class I(A) PI 3-kinases.

Three-dimensional solution structure of the pleckstrin homology domain from dynamin.

BACKGROUND: The pleckstrin homology (PH) domain is a region of approximately 100 amino acids, defined by sequence similarity, that has been found in about 60 proteins, many of which are involved in signal transduction downstream of cell surface receptors; the function of PH domains is unknown. The only clue to the function of PH domains is the circumstantial evidence that they may link beta gamma subunits of G proteins to second messenger systems. Knowledge of the three-dimensional structures of PH domains should help to elucidate the roles they play in the proteins that contain them. RESULTS: Using homonuclear and heteronuclear magnetic resonance spectroscopy, we have determined the solution structure of the PH domain of the GTPase dynamin, one of a number of proteins that have PH domains and interact with GTP. The fold of the dynamin PH domain is composed of two antiparallel beta-sheets, which pack face-to-face at an angle of approximately 60 degrees. The first beta-sheet comprises four strands (residues 13-58) from the amino-terminal half of the protein sequence; the second beta-sheet contains three strands (residues 63-99). A single alpha-helix (residues 102-116) flanks one edge of the interface between the two sheets, parallel in orientation to the second sheet, in an alpha/beta roll motif similar to that of the B oligomer of verotoxin-1 from Escherichia coli. CONCLUSIONS: The structure of the dynamin PH domain is very similar to the recently reported structures of the pleckstrin and spectrin PH domains. This shows that, despite the low level of sequence similarity between different PH domains, they do have a characteristic polypeptide fold. On the basis of our structure, the suggestion that PH domains engage in coiled-coil interactions with G protein beta gamma subunits seems unlikely and should be re-evaluated.

Wiskott-Aldrich syndrome protein (WASp) is a binding partner for c-Src family protein-tyrosine kinases.

BACKGROUND: Receptor-mediated signal transduction requires the assembly of multimeric complexes of signalling proteins, and a number of conserved protein domains, such as the SH2, SH3 and PH domains, are involved in mediating protein-protein interactions in such complexes. The identification of binding partners for these domains has added considerably to our understanding of signal-transduction pathways, and the purpose of this work was to identify SH3-binding proteins in haematopoietic cells. RESULTS: We performed affinity-chromatography experiments with a panel of GST-SH3 fusion proteins (composed of glutathione-S-transferase appended to various SH3 domains) to search for SH3-binding proteins in a human megakaryocytic cell line. Protein microsequencing identified one of the SH3-binding proteins as WASp, the protein that is defective in Wiskott-Aldrich syndrome (WAS) and isolated X-linked thrombocytopenia. WASp bound preferentially in vitro to SH3 domains from c-Src family kinases, and analysis of proteins expressed in insect cells using a baculovirus vector demonstrated a specific interaction between WASp and the Fyn protein-tyrosine kinase. Finally, in vivo experiments showed that WASp and Fyn physically associate in human haematopoietic cells. CONCLUSIONS: Haematopoietic cells from individuals with WAS exhibit defects in cell morphology and signal transduction, including reduced proliferation and tyrosine phosphorylation in response to stimulatory factors. Members of the c Src family of protein-tyrosine kinases, including Fyn, are involved in a range of signalling pathways - such as those regulating cytoskeletal structure - in both haematopoietic and non-haematopoietic cells. Our data suggest that binding of Fyn to WASp may be a critical event in such signalling pathways in haematopoietic cells.