Thick filament mechano-sensing is a calcium-independent regulatory mechanism in skeletal muscle.

Recent X-ray diffraction studies on actively contracting fibres from skeletal muscle showed that the number of myosin motors available to interact with actin-containing thin filaments is controlled by the stress in the myosin-containing thick filaments. Those results suggested that thick filament mechano-sensing might constitute a novel regulatory mechanism in striated muscles that acts independently of the well-known thin filament-mediated calcium signalling pathway. Here we test that hypothesis using probes attached to the myosin regulatory light chain in demembranated muscle fibres. We show that both the extent and kinetics of thick filament activation depend on thick filament stress but are independent of intracellular calcium concentration in the physiological range. These results establish direct control of myosin motors by thick filament mechano-sensing as a general regulatory mechanism in skeletal muscle that is independent of the canonical calcium signalling pathway.

Mechanics of myosin function in white muscle fibres of the dogfish, Scyliorhinus canicula.

The contractile properties of muscle fibres have been extensively investigated by fast perturbation in sarcomere length to define the mechanical characteristics of myofilaments and myosin heads that underpin refined models of the acto-myosin cycle. Comparison of published data from intact fast-twitch fibres of frog muscle and demembranated fibres from fast muscle of rabbit shows that stiffness of the rabbit myosin head is only ∼62% of that in frog. To clarify if and how much the mechanical characteristics of the filaments and myosin heads vary in muscles of different animals we apply the same high resolution mechanical methods, in combination with X-ray diffraction, to fast-twitch fibres from the dogfish (Scyliorhinus canicula). The values of equivalent filament compliance (C(f)) measured by X-ray diffraction and in mechanical experiments are not significantly different; the best estimate from combining these values is 17.1 ± 1.0 nm MPa(−1). This value is larger than Cf in frog, 13.0 ± 0.4 nm MPa(−1). The longer thin filaments in dogfish account for only part of this difference. The average isometric force exerted by each attached myosin head at 5°C, 4.5 pN, and the maximum sliding distance accounted for by the myosin working stroke, 11 nm, are similar to those in frog, while the average myosin head stiffness of dogfish (1.98 ± 0.31 pN nm(−1)) is smaller than that of frog (2.78 ± 0.30 pN nm(−1)). Taken together these results indicate that the working stroke responsible for the generation of isometric force is a larger fraction of the total myosin head working stroke in the dogfish than in the frog.

Probing myosin structural conformation in vivo by second-harmonic generation microscopy.

Understanding of complex biological processes requires knowledge of molecular structures and measurement of their dynamics in vivo. The collective chemomechanical action of myosin molecules (the molecular motors) in the muscle sarcomere represents a paradigmatic example in this respect. Here, we describe a label-free imaging method sensitive to protein conformation in vivo. We employed the order-based contrast enhancement by second-harmonic generation (SHG) for the functional imaging of muscle cells. We found that SHG polarization anisotropy (SPA) measurements report on the structural state of the actomyosin motors, with significant sensitivity to the conformation of myosin. In fact, each physiological/biochemical state we probed (relaxed, rigor, isometric contraction) produced a distinct value of polarization anisotropy. Employing a full reconstruction of the contributing elementary SHG emitters in the actomyosin motor array at atomic scale, we provide a molecular interpretation of the SPA measurements in terms of myosin conformations. We applied this method to the discrimination between attached and detached myosin heads in an isometrically contracting intact fiber. Our observations indicate that isometrically contracting muscle sustains its tetanic force by steady-state commitment of 30% of myosin heads. Applying SPA and molecular structure modeling to the imaging of unstained living tissues provides the basis for a generation of imaging and diagnostic tools capable of probing molecular structures and dynamics in vivo.

Structural changes in myosin motors and filaments during relaxation of skeletal muscle.

Structural changes in myosin motors and filaments during relaxation from short tetanic contractions of intact single fibres of frog tibialis anterior muscles at sarcomere length 2.14 mum, 4 degrees C were investigated by X-ray diffraction. Force declined at a steady rate for several hundred milliseconds after the last stimulus, while sarcomere lengths remained almost constant. During this isometric phase of relaxation the intensities of the equatorial and meridional M3 X-ray reflections associated with the radial and axial distributions of myosin motors also recovered at a steady rate towards their resting values, consistent with progressive net detachment of myosin motors from actin filaments. Stiffness measurements confirmed that the fraction of motors attached to actin declined at a constant rate, but also revealed a progressive increase in force per motor. The interference fine structure of the M3 reflection suggested that actin-attached myosin motors are displaced towards the start of their working stroke during isometric relaxation. There was negligible recovery of the intensities of the meridional and layer-line reflections associated with the quasi-helical distribution of myosin motors in resting muscle during isometric relaxation, and the 1.5% increase in the axial periodicity of the myosin filament associated with muscle activation was not reversed. When force had decreased to roughly half its tetanus plateau value, the isometric phase of relaxation abruptly ended, and the ensuing chaotic relaxation had an exponential half-time of ca 60 ms. Recovery of the equatorial X-ray intensities was largely complete during chaotic relaxation, but the other X-ray signals recovered more slowly than force.

Inhibition of steroid sulfatase activity in endometriotic implants by STX64 (667Coumate): a potential new therapy.

Endometriosis is a common and debilitating condition of women in their reproductive age that is associated with pain and infertility. Current medical treatments are only partially effective and associated with wide-ranging side effects. New understanding of local estrogen production by endometriotic tissue and the availability of powerful suppressing drugs may herald a new era in the treatment of this condition.

Mechanism and functional consequences of loss of FOXO1 expression in endometrioid endometrial cancer cells.

The forkhead transcription factor FOXO1, a downstream target of phosphatidylinositol-3-kinase/Akt signalling pathway, regulates cyclic differentiation and apoptosis in normal endometrium, but its role in endometrial carcinogenesis is unknown. Screening of endometrial cancer cell lines demonstrated that FOXO1 is expressed in HEC-1B cells, but not in Ishikawa cells, which in turn highly express the FOXO1 targeting E3-ubiquitin ligase Skp2. FOXO1 transcript levels were also lower in Ishikawa cells and treatment with the proteasomal inhibitor was insufficient to restore expression. Lack of FOXO1 expression in Ishikawa cells was not accounted for by differential promoter methylation or activity, but correlated with increased messenger RNA (mRNA) turnover. Comparative analysis demonstrated that HEC-1B cells proliferate slower, but are more resistant to paclitaxel-mediated cell death than Ishikawa cells, which were partially reversed upon silencing of FOXO1 in HEC-1B cells or its re-expression in Ishikawa cells. We further show that FOXO1 is required for the expression of the growth arrest- and DNA-damage-inducible gene GADD45alpha. Analysis of biopsy samples demonstrated a marked loss of FOXO1 and GADD45alpha mRNA and protein expression in endometrioid endometrial cancer compared to normal endometrium. Together, these observations suggest that loss of FOXO1 perturbs endometrial homeostasis, promotes uncontrolled cell proliferation and increases susceptibility to genotoxic insults.

Inhibition of steroid sulphatase activity in endometriotic implants by 667 COUMATE: a potential new therapy.

BACKGROUND: Local biosynthesis of estrogens is thought to be important for the maintenance and growth of endometriotic implants. In addition to the formation of estrogen via the aromatase pathway, steroid sulphatase (STS), which is responsible for the hydrolysis of estrogen sulphates, may be an important source of estrogens in endometriosis. METHODS: Eutopic and ectopic endometrial samples from 14 women with minimal or mild (MM) endometriosis and from 13 women with moderate to severe (MS) endometriosis were analysed for aromatase and STS activities. RESULTS: Aromatase and STS activity were detected in all samples. STS enzyme activity in both eutopic and ectopic endometrium was considerably higher and less variable than aromatase activity. Moreover, STS, but not aromatase, activity in endometriotic implants correlated with the severity of the disease (mean +/- SEM: 203 +/- 38 nmol/4 h/g wet weight tissue in MM disease versus 423 +/- 44 nmol/4 h/g wet weight tissue in MS endometriosis, P < 0.001). The STS inhibitor 667 COUMATE almost completely blocked STS activity (>99%) in both eutopic and ectopic tissues. CONCLUSIONS: The high levels of STS activity detected in ectopic endometrium and the correlation with severity of disease suggest that STS inhibitors could be useful for the treatment of endometriosis.

Magnetic resonance imaging in preeclampsia and eclampsia complicated by visual disturbance and other neurological abnormalities.

This study describes magnetic resonance imaging findings in women presenting with neurological complications associated with preeclampsia and eclampsia. One eclamptic and two preeclamptic women were studied after presenting with postpartum neurological events. In two women the brain increased in size on the initial follow-up images, following the same pattern seen in normal pregnancy. In the other woman, the brain was decreased in size at 13 days postpartum but increased in size at six weeks postpartum. This initial reduction in brain size may reflect the resolution of cerebral oedema resulting from underlying pathological processes.

Association of maternal endothelial dysfunction with preeclampsia.

CONTEXT: Preeclampsia is believed to result from release of placental factors that damage maternal vascular endothelium. However, because most studies have been conducted during pregnancy, it has not been possible to separate maternal from placental mechanisms underlying endothelial dysfunction in preeclampsia. OBJECTIVE: To determine whether endothelial function is impaired in nonpregnant women with previous preeclampsia and whether endothelial dysfunction is mediated by oxidative stress. DESIGN AND SETTING: Case-control study conducted at 3 hospital maternity units in London, England, between July 1997 and June 2000. PARTICIPANTS: A total of 113 women with previous preeclampsia (n = 35 with recurrent episodes; n = 78 with a single episode) and 48 women with previous uncomplicated pregnancies, all of whom were at least 3 months (median, 3 years) postpartum. MAIN OUTCOME MEASURES: Brachial artery flow-mediated (endothelium-dependent) and glyceryl trinitrate-induced (endothelium-independent) dilatation were compared between previously preeclamptic women and controls. To investigate oxidative stress, these measurements were repeated after administration of ascorbic acid, 1 g intravenously, in 15 cases and 15 controls. RESULTS: Mean (SD) flow-mediated dilatation was lower in women with previous preeclampsia compared with controls (recurrent group, 0.9% [4.1%]; single-episode group, 2.7% [3.5%]; and control group, 4.7% [4.3%]; P<.001). In contrast, glyceryl trinitrate-induced dilatation was similar in the 3 groups (recurrent, 19.5% [5.9%]; single-episode, 21.0% [8.0%]; and control, 21.0% [8.3%]; P =.65). Impaired flow-mediated dilatation in previously preeclamptic women was not accounted for by recognized vascular risk factors. Ascorbic acid administration increased flow-mediated dilatation in previously preeclamptic women (baseline, 2.6% [3.3%]; after administration, 5.6% [3.0%]; P =.001) but not in controls (baseline, 6.2% [3.3%]; after administration, 6.7% [5.0%]; P =.72). CONCLUSIONS: Our results indicate that endothelial function is impaired in women with previous preeclampsia and is not explained by established maternal risk factors but is reversed by antioxidant ascorbic acid administration.

Correlation between umbilical arterial flow and placental morphology.

Umbilical artery blood velocity waveforms were compared with the morphological aspects of the vascular bed of 30 placentas using quantitative methods. An abnormal pulsatility index was found to be highly correlated with a lower number of arteries in the tertiary stem villi as well as a lower placental weight and birth weight. The reduction in the number of arteries seems to be due to an early developmental arrest of placental angiogenesis and is partly compensated for by increased formation of capillaries in the terminal villi, since the number of capillaries is independent of the number of arteries.

Exencephaly in autosomal dominant brachydactyly syndrome.

Exencephaly was diagnosed at 17 weeks in a 27-year-old primigravida with abnormalities of the hands and a family history suggestive of autosomal dominant brachydactyly and clinodactyly. In this family there was also a history of 'anencephaly'. To our knowledge, this is the first report on the association of exencephaly and autosomal dominant brachydactyly. As the relationship between hand and cranial anomalies is well established, we suggest that this association in our case could be due to a defect in the same gene.

Acute twin-twin transfusion: a possible mechanism for brain-damaged survivors after intrauterine death of a monochorionic twin.

Intrauterine death of one twin in monochorionic pregnancies is associated with increased mortality and morbidity for the survivor. This has been attributed to the consequences of intrauterine disseminated intravascular coagulation (DIC) initiated by the dead twin. We describe a case in which the fetal cerebral and renal lesions typically found in survivors occurred without any derangement in coagulation. Instead, acute twin-twin transfusion was suggested by the presence of severe anemia in the surviving fetus at delivery. We suggest that the lesions frequently found in the survivors are often due to acute hemodynamic and ischemic changes resulting from acute twin-twin transfusion at the time of intrauterine death, rather than to late-onset DIC. This hypothesis has an important implication for future management: Intervention must occur before intrauterine death if neurologic sequelae in the survivor are to be prevented.

[The clinical use of levosulpiride in retinal fluorangiography]. / Considerazione sull'uso clinico della levo-sulpiride nella fluorangiografia retinica.

The value of administering 25 mg of levosulpiride per os approximately one hour before the sodium fluorescein bolus used in fluorangiography is assessed in order to avoid to the onset of nausea and/or vomiting during and after the test. The study was performed in 35 patients. No nausea and/or vomiting was observed in over 90% of cases treated.

Limited role of fetal blood sampling in prediction of outcome in intrauterine growth retardation.

Fetal acid-base status was evaluated on 66 blood samples taken for rapid karyotyping from 58 growth-retarded fetuses. Before blood sampling, doppler blood flow studies of the umbilical artery showed end-diastolic frequencies to be absent in 32 fetuses (group 1) and present in 26 (group 2). Fetuses with chromosomal (n = 4) or structural (n = 8) abnormalities were excluded from subsequent analysis. Gestational age at blood sampling (27.8 [95% CI 26.5-29.1] vs 32.2 [30.4-34.1] weeks) and time from sampling to delivery (median 2 (range 0-35] vs 14 [0-77] days) were significantly lower in group 1 than group 2. There were no perinatal deaths in group 2 whereas mortality in group 1 was 65.4%. There were significant differences between the groups at blood sampling in pH, pO2, pCO2, base equivalents, and nucleated-red-cell count, but within group 1 these measurements were similar in surviving fetuses and those who died perinatally. Since acid-base determination does not predict perinatal outcome in growth-retarded fetuses, fetal blood sampling has a limited role in monitoring fetal wellbeing.

Twin pregnancy complicated by single intrauterine death. Problems and outcome with conservative management.

During an 11-year period we encountered 16 pregnancies in which one twin died in utero and the pregnancy continued. Eight of these twin pregnancies were monochorionic. None of the women developed severe disseminated intravascular coagulation. The fetal outcome indicates that the prognosis for a surviving dichorionic twin is relatively good, with immaturity the main hazard. By contrast the surviving monochorionic twin has a poor prognosis with a high frequency of neurological damage. This damage is not related to intrapartum or neonatal problems and at present cannot be diagnosed before birth. There is no evidence that birth of the surviving twin by caesarean section will improve the prognosis. Early diagnosis of monochorionic twins and subsequent ultrasound follow up should identify fetal growth discrepancy and possible twin to twin transfusion requiring early delivery.

When does death occur in an acardiac twin? Ultrasound diagnostic difficulties.

Fetal acardia is a rare abnormality of multiple pregnancies, which is lethal for the affected fetus and can cause death in 50% of normal co-twins. Antenatal recognition with early ultrasound is essential to institute a prospective management to improve the outcome. Our communication outline the difficulties which may be encountered in ultrasound diagnosis. In particular the problem of distinguishing a fetal heart from large pulsating mediastinal vessels, which can be present in these fetuses, and the difficulty of diagnosing death in an acardiac fetus. Our report confirms that the co-twin remains at increased risk of sudden death, even without ultrasound evidence of cardiac failure or biochemical compromise. The finding in this fetus of intravascular fibrin deposits suggests the possibility of acute disseminated intravascular coagulation, not previously reported in association with an acardiac twin.

Maternal pyrexia associated with the use of epidural analgesia in labour.

To establish the effect of pain relief on maternal temperature during labour forty patients who went into spontaneous labour with a single fetus, had a normal temperature (less than 37.5 degrees C), and had no clinical evidence of infection were investigated prospectively. They were divided into two comparable groups--one receiving pethidine and the other epidural analgesia. Both groups had much the same temperatures at the beginning of labour and before any analgesic administration. The mean temperature in the pethidine group remained constant during labour, whereas in the epidural analgesia group it showed a significant rise after only 6 hours of labour. This rise was not related to any clinical evidence of infection. Patients receiving epidural analgesia during labour are at increased risk of developing pyrexia. This pyrexia may be the result of vascular and thermoregulatory modifications induced by epidural analgesia.