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Abstract Background The H-NS protein is a global regulator of gene expression in bacteria and can also bind transposition complexes (transpososomes). In Tn5 transposition H-NS promotes transpososome assembly in vitro and disruption of the hns gene causes a modest decrease in Tn5 transposition (three- to five-fold). This is consistent with H-NS acting as a positive regulator of Tn5 transposition. Molecular determinants for H-NS binding to the Tn5 transpososome have not been determined, nor has the strength of the interaction been established. There is also uncertainty as to whether H-NS regulates Tn5 transposition in vivo through an interaction with the transposition machinery as disruption of the hns gene has pleiotropic effects on Escherichia coli, the organism used in this study. Results In the current work we have further examined determinants for H-NS binding to the Tn5 transpososome through both mutational studies on Tn5 termini (or 'transposon ends') and protein-protein cross-linking analysis. We identify mutations in two different segments of the transposon ends that abrogate H-NS binding and characterize the affinity of H-NS for wild type transposon ends in the context of the transpososome. We also show that H-NS forms cross-links with the Tn5 transposase protein specifically in the transpososome, an observation consistent with the two proteins occupying overlapping binding sites in the transposon ends. Finally, we make use of the end mutations to test the idea that H-NS exerts its impact on Tn5 transposition in vivo by binding directly to the transpososome. Consistent with this possibility, we show that two different end mutations reduce the sensitivity of the Tn5 system to H-NS regulation. Conclusions H-NS typically regulates cellular functions through its potent transcriptional repressor function. Work presented here provides support for an alternative mechanism of H-NS-based regulation, and adds to our understanding of how bacterial transposition can be regulated.

The histone-like nucleoid structuring protein (H-NS) is an important regulator of stress response and virulence genes in gram-negative bacteria. In addition to binding regulatory regions of genes in a structure-specific manner, H-NS also binds in a structure-specific manner to sites in the Tn10 transpososome, allowing it to act as a positive regulator of Tn10 transposition. This is the only example to date of H-NS regulating a transposition system by interacting directly with the transposition machinery. In general, transposition complexes tend to include segments of deformed DNA and given the capacity of H-NS to bind such structures, and the results from the Tn10 system, we asked if H-NS might regulate another transposition system (Tn5) by directly binding the transposition machinery. We show in the current work that H-NS does bind Tn5 transposition complexes and use hydroxyl radical footprinting to characterize the H-NS interaction with the Tn5 transpososome. We also show that H-NS can promote Tn5 transpososome formation in vitro, which correlates with the Tn5 system showing a dependence on H-NS for transposition in vivo. Taken together the results suggest that H-NS might play an important role in the regulation of many different bacterial transposition systems and thereby contribute directly to lateral gene transfer.

DNA transposition reactions typically involve a strand transfer step wherein the transposon ends are covalently joined by the transposase protein to a short target site. There is very little known about the transposase-DNA interactions that direct this process, and thus our overall understanding of the dynamics of DNA transposition reactions is limited. Tn5 presents an attractive system for defining such interactions because it has been possible to solve the structure of at least one Tn5 transposition intermediate: a transpososome formed with pre-cleaved ends. However, insertion specificity in the Tn5 system is low and this has hampered progress in generating target-containing transpososomes that are homogeneous in structure (i.e. where a single target site is engaged) and therefore suitable for biochemical and structural analysis. We have developed a system where the Tn5 transpososome integrates almost exclusively into a single target site within a short DNA fragment. The key to establishing this high degree of insertion specificity was to use a target DNA with tandem repeats of a previously characterized Tn5 insertion hotspot. The target DNA requirements to form this strand transfer complex are evaluated. In addition, we show that target DNAs missing single phosphate groups at specific positions are better substrates for strand transfer complex formation relative to the corresponding unmodified DNA fragments. Moreover, utilization of missing phosphate substrates can increase the degree of target site selection. A method for concentrating and partially purifying the Tn5 strand transfer complex is described.

Measurement of the pre-ejection period (PEP), a non-invasive index of cardiac function, was made in a human fetus with a disturbance of cardiac rhythm manifested by: (i) intermittent mechanically ineffective extrasystoles (ventricular premature beats) and (ii) pauses. The mean interval between the sinus beats before and following extrasystoles was virtually the same as that before and following pauses. The PEP was shortened at the first sinus beat following both extrasystoles and pauses, but to a greater extent following extrasystoles. The PEP was still shortened at the second sinus beat following extrasystoles, but prolonged at the same cardiac cycle following pauses. These data demonstrate that fetal cardiac post-extrasystolic potentiation was taking place and that this was due in part to inotropic potentiation of the post-extrasystolic beats. Inotropic potentiation of post-extrasystolic beats is due to a fundamental property of cardiac muscle relating frequency of stimulation to strength of contraction (the so-called 'force-interval' relationship). Our data present the first evidence, to our knowledge, that the inotropic state of the human fetal heart in vivo is modulated by this mechanism.

Serial ovarian ultrasound and daily assessments of plasma concentrations of pituitary and ovarian hormones were used to investigate ovarian function in 175 women with unexplained infertility. Their endocrine and ultrasound profiles were compared with similarly derived data from 43 normal volunteers. Fifty-one (29.1%) of the study group showed subnormal luteal phase rises in progesterone concentrations, described as poor progesterone surge (PPS) cycles. Within this group, 23 women (45.1%) demonstrated luteal cyst formation, a pattern not seen in any of the control cycles. High concentrations of follicle stimulating hormone (FSH) and reduced concentrations of oestradiol (E2) were observed in the follicular phases of the PPS cycles suggesting that the phenomenon is a product of abnormal follicular metabolism. An association of PPS with infertility exists, perhaps related to a combination of disturbances in the follicular micro-environment compromising oocyte quality, a failure of oocyte release, and impaired endometrial receptivity.

A prospective, controlled study of ovarian function using ovarian ultrasound and daily plasma hormone estimations (estradiol, progesterone [P], follicle-stimulating hormone [FSH], luteinizing hormone [LH]) was carried out on 175 spontaneously cycling patients with unexplained infertility. Forty-one (23.4%) demonstrated luteal phase cyst formation. In 21 cycles the dominant follicle reduced in size after the LH peak (cystic corpus luteum cycles), and in 20 no shrinkage was seen (luteinized unruptured follicles). Progesterone concentrations in the early luteal phase were significantly reduced in the luteinized unruptured follicle cycles. Elevation in plasma FSH was seen in the early follicular and luteal phases of both cyst forming groups and may be due to disturbances in ovarian metabolism. Follicular rupture is important for efficient P release by the corpus luteum.

Cardiac electromechanical intervals were measured during labour in 103 unselected human fetuses. Mean values (+/- SD) for the three intervals measured were 76.01 +/- 9.93 ms for the pre-ejection period, 41.56 +/- 8.51 ms for the isovolumetric contraction time and 156.23 +/- 16.58 ms for the left ventricular ejection time. These are consistent with previously reported data from our own and other centres. the intervals studied did not alter significantly as labour proceeded or during uterine contractions. There was no consistent correlation between them and fetal scalp blood PO2, PCO2 or pH, although the pre-ejection period was shortened considerably in three very acidaemic or hypoxic fetuses, and it was prolonged in association with tight nuchal cord entanglement and variable decelerations in fetal heart rate. These findings raise the possibility of using recorded cardiac electromechanical intervals to determine the clinical significance of different fetal heart-rate patterns that suggest the possibility of fetal distress.

To find an effective routine screening method for small-for-dates fetuses 474 women with singleton pregnancies participated in a two-stage ultrasonic examination schedule. At the first-stage examination, which was conducted in early pregnancy, fetal crown-rump length or biparietal diameter was measured for an accurate assessment of gestational age, which was essential for interpreting the results of the second-stage examination. The second-stage examination was performed at 34-36 weeks and entailed measuring seven fetal variables, the results of which were assessed singly and in combination after delivery to identify the best indicator of small-for-dates fetuses. Fetal head measurements proved to be the least sensitive indicators of growth retardation, correctly identifying only 56-59% of cases. Measurements of trunk area and circumference, however, correctly identified 81% and 83% of cases respectively, but the most effective screening index was the product of crown-rump length and trunk area: with this index 34 out of 36 small-for-dates fetuses (94%) were correctly identified. Calculating the product of crown-rump length and trunk area from ultrasonic displays is quick and simple, and combined with the first-stage examination is a highly reliable screening method for small-for-dates fetuses.

In the hope of reducing perinatal risks associated with retardation of intrauterine growth a previously described two stage ultrasound screening schedule was evaluated by a controlled trial in 877 women with low risk single pregnancies. The two stages of ultrasound examination were an assessment of gestational age during early pregnancy followed by measurement of length from crown to rump and area of trunk at between 34 and 36 weeks' gestation. The product of crown to rump length and trunk area was calculated. The sensitivity of this schedule in identifying in advance 94% of babies who were small for dates at birth, with 90% specificity, and the speed and simplicity of measurement confirmed the accuracy and feasibility of two stage ultrasonography as a screening procedure. The controlled trial did not, however, show any benefit from its routine application in these low risk pregnancies.

The maternal serum alpha-fetoprotein concentration was measured between 16 and 20 weeks in 145 twin pregnancies in which neither fetus had a neural tube defect. When the maternal serum alpha-fetoprotein concentration was less than two multiples of the singleton median, pregnancy outcome was good; the extended perinatal mortality rate was 32.6/1000, mean birth weights for the first and second twins were 2507 and 2443 gm, respectively, and mean gestation at delivery was 36 weeks, 6 days. When the maternal serum alpha-fetoprotein concentration was greater than four multiples of the singleton median, the outcome was poor; the extended perinatal mortality was 400/1000, mean birth weights were 1963 and 1523 gm, and mean gestation at delivery was 32 weeks, 4 days. The negative correlations of maternal serum alpha-fetoprotein concentration with birth weight and gestation at delivery were highly significant. Maternal serum alpha-fetoprotein concentration in midpregnancy is a useful predictor of outcome in twin pregnancy, independent of the occurrence of neural tube defect, and it appears to be related to the timing of delivery rather than fetal growth.

Fetal systolic time intervals (pre-ejection period, iso-volumetric contraction time and ventricular ejection time) can be measured using Doppler ultrasound to detect valve movements and the fetal ECG to indicate the onset of ventricular depolarisation. Continuous recordings of FSTI, fetal heart rate and uterine activity depict changes reflecting progressive cord compression and have the potential for providing improved differentiation of late and variable decelerations. However, the continual adjustment of the ultrasound transducer required to maintain a good signal limits the technique. Recent improvements in phono indicate that this may provide a useful alternative, provided that suitable signal-processing is developed.

A new method of estimating fetal weight from the circumference of the fetal trunk, measured by ultrasound, is described. Comparison of weights estimated in this way with the actual birth weights of 50 fetuses, delivered not more than 48 hours later, shows a mean error in prediction of only 75 g, and in 94 per cent the error was less than 145 g. With such accuracy, the method is of clinical value, particularly in anticipating difficult delivery and in monitoring growth of the fetus at risk.

Is amniocentesis safe? Does it still have a place in prenatal diagnosis? These questions continue to be asked, but it remains the cornerstone of diagnosis while other methods are being evaluated.

Calculation of the product of crown-rump length and trunk area (CRL x TA), as measured by ultrasound between 34 and 36 weeks and combined with accurate assessment of gestational age in early pregnancy, was previously shown to be highly effective in detecting the small-for-dates fetus in a largely unselected series of patients. To assess the value of this two-stage schedule in high-risk pregnancies, 202 patients with singleton pregnancies at risk of fetal growth retardation were studied. Of the 53 babies that were small-for-dates at birth, 49 (92%) were identified in advance by CRL x TA measurement. In contrast to previous findings, measurement of trunk area (TA) alone was similarly effective, identifying in advance 48 (91%) of these small-for-dates babies.

A new rapid test of fetal lung maturity (the AmnioStat-FLM) was compared with two currently available rapid tests and with 2-dimensional thin-layer chromatography (2-d TLC). Forty-nine specimens of liquor obtained within 72 h of delivery were assessed, 27 of which were contaminated with blood or meconium. The AmnioStat-FLM compared well with spectrophotometry, the shake test and with 2-d TLC. Only the Amniostat-FLM and the 2-d TLC were reliable in the presence of contamination. The results suggest that the AmnioStat-FLM is a reliable, rapid test and unaffected by the presence of contamination.

In a study of 766 amniotic fluids, collected from pregnancies between 26 weeks and term, phosphatidylglycerol (PG) was identified in a greater proportion than was a mature lecithin/sphingomyelin (L/S) ratio at all gestational ages between 28 and 38 weeks regardless of the underlying pregnancy complication. The early appearance of PG was particularly striking in amniotic fluids obtained after preterm rupture of membranes. Since PG has been previously shown to be a useful indicator of the risk of neonatal respiratory distress syndrome, its appearance before a mature L/S ratio suggests that its detection offers a considerable advantage in the management of high-risk obstetric problems in which the earliest possible indication that the fetal lungs are mature is required.

Because respiratory distress syndrome (RDS) may occur in one twin but not the other it may be misleading to assess fetal lung maturity using amniotic fluid from only one sac. We compared the amniotic fluid lecithin/sphingomyelin (L/S), phosphatidyl glycerol/sphingomyelin (PG/S) and phosphatidyl inositol/sphingomyelin (PI/S) ratios between co-twins and co-triplets in 32 sets of twins and three set of triplets. In the twin pregnancies we found a weak correlation for L/S ratio but a much improved one for PG/S and PI/S. The concordance between sacs for all three ratios was better in monozygotic than in dizygotic twins. The efficacy of amniotic fluid PG in the determination of fetal lung maturity was demonstrated and the discrepancies between the sacs was much less for PG than for the L/S ratios. Employing the L/S ratio combined with the presence or absence of PG should reduce false results to a minimum.

Based on the analysis of 561 amniotic fluid samples obtained within 72 h of delivery, including 288 samples collected from the vagina, the detection of phosphatidylglycerol (PG) in the fluid together with the determination of the lecithin/sphingomyelin (L/S) ratio gave an accurate prediction of the risk of the newborn infant developing respiratory distress syndrome (RDS). In the presence of PG, regardless of the L/S ratio, only 0.6% of the babies developed RDS, while absent PG was associated with an 82.8% incidence of RDS. The predictive ability was improved by knowing the L/S ratio since in the presence of both PG and a mature L/S ratio (greater than 2.0), no baby developed RDS whereas 3.4% of them did when the ratio was immature despite the presence of PG. The test appears to be a useful determinant of the risk of RDS in babies born to diabetic mothers and it seems particularly effective in assessing amniotic fluid collected vaginally. It is recommended that laboratories dealing with amniotic fluid from high-risk pregnancies should detect PG as well as measuring the L/S ratio.

Because of both the advantage of speed compared with thin layer chromatography (TLC) and the dearth of high pressure liquid chromatography (HPLC) methods for phospholipid separation, it was decided to investigate the use of HPLC with a differential refractometer as detector for the separation and quantitation of amniotic fluid phospholipids required for the prediction of fetal lung maturity. A method was devised which gave results which compared well with those from TLC both in terms of quantitation and predictive value. Despite this, the method was found to lack sufficient reliability for application to the routine clinical assessment of fetal lung maturity. The method does, however, offer a good alternative to two dimensional TLC with phosphate analysis in research work involving quantitation of phosphatidyl inositol, phosphatidyl glycerol and particularly lecithin.

A phospholipid profile, the main features of which were the lecithin/sphingomyelin (L/S) ratio and the presence or absence of phosphatidylglycerol (PG), was determined in amniotic fluid from 188 patients. There was a mature profile (L/S ratio of at least 2 . 0 and detectable PG) in 145 patients, including seven insulin-dependent diabetics, and noe of their babies developed respiratory distress syndrome (RDS). The L/S ratio was less than 2 . 0 and PG absent in 12 patients, nine of whose babies developed RDS, whereas only three small babies (delivered between 28 and 35 weeks because of fulminant pre-eclampsia or severe abruptio placentae) out of 31 developed RDS when the L/S ratio was less than 2 . 0 but PG was present. When amniotic fluid was collected from the vagina only one out of 69 babies developed RDS when PG was present (regardless of the L/S ratio), while all of seven babies developed RDS when PG was absent. It is concluded that the amniotic fluid phospholipid profile, particularly the presence or absence of PG, gives an accurate assessment of fetal lung maturation. The profile may prove a useful adjunct to the management of high-risk pregnancies, especially after premature membrane rupture and perhaps also when the mother is diabetic.

Intravenous salbutamol, a beta-adrenoceptor stimulant, given to nine patients in normal labour, with continous monitoring of uterine activity and of the maternal and fetal cardiovascular systems, was shown to decrease uterine activity significantly; maternal and fetal heart rates were significantly increased, and maternal systolic and diastolic arterial pressures were significantly decreased during the infusion, although no treatment had to be discontinued because of these effects. Apart from worsening of low back pain during the infusion in one patients, subjective side-effects were trival. With the salbutamol infusion continued at an effective maintenance rate, the carioselective beta-adrenoceptor blocking drug, practolol, given intravenously, reduced the maternal heart rate (although not significantly) but it did not alter the fetal heart rate; it also appeared to interfere transiently with the inhibiting action of salbutamol on uterine activity, but cerevical dilatation was arrested until the salbutamol infusion was discontinued. At least in five patients, labour remained suppressed until oxytoxin was infused intravenously.

Adapting Sir Dugald Baird's concept of primary obstetric causes of perinatal mortality, a revised clinico-pathological classification has been evolved to take account of new knowledge and developments, and to direct attention to potentially avoidable deaths and to where intensified efforts and investigation are needed. Categories highlighting the importance of intrauterine growth retardation, unexplained intrauterine death and spontaneous premature labour have been introduced, intrapartum hypoxia is separated from birth trauma, and infection again has its own category. Regular perinatal audit at one obstetric hospital, since 1979, has shown that the new system provides a workable and useful means for classifying not only perinatal deaths, but also late abortions, late neonatal deaths and perinatally related infant deaths. The rate of total perinatally related wastage, defined in this way, was almost twice that for perinatal mortality (22.8 compared with 11.9 per 1000 births). The former is advocated as a more realistic index for the audit of perinatal care. The revised and extended system is put forward as a contribution to the current debate on classifying and reporting such wastage, in the hope that it may be tested as a model for regional as well as hospital surveys.

One hundred eighty-six pregnancies with elevated maternal serum alpha-fetoprotein (AFP) between 16 and 20 weeks' gestation, but with normally formed single fetuses, were analyzed retrospectively. In comparison with matched control subjects, there was an increased incidence of low birth weight, preterm delivery, intrauterine growth retardation, and other clinical complications, especially when maternal serum AFP was abnormally high on more than one occasion. These findings could not be explained by the occurrence of threatened abortion or the performance of amniocentesis. It is suggested that where maternal serum AFP screening for fetal neural tube defects is already established as a cost-effective routine procedure, the additional recognition of some pregnancies at very high risk of other, perinatal complications is of practical value. However, maternal serum AFP testing in the second trimester cannot be recommended as a cost-effective screening method for detecting low birth weight infants, having a sensitivity in this series of only 11%. Many (33%) of the low birth weight infants detected in this way were very small (birth weights less than 1.5 kg); 73% of the predicted preterm births were very premature (before 34 weeks of gestation), and 72% of the identified growth-retarded infants were severely effected (weighing less than the fifth percentile for gestational age).

The normal range and trends of the ratio between lecithin and sphingomyelin in the amniotic fluid, estimated on thin-layer chromatograms, have been established for the last trimester. The accuracy of the ratio as an index of fetal lung development and of the risk of neonatal respiratory distress has been confirmed. The usual increase in the ratio towards term was not found in some pregnancies complicated by diabetes or severe rhesus incompatibility. Determination of the ratio may prove a valuable guide in the management of pregnancies with these complications.