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The onset of preeclampsia at or near to term is associated with low maternal and neonatal morbidity and mortality. In contrast, those patients (1%) who suffer early onset preeclampsia engender significant maternal and perinatal morbidity and mortality. Therefore, because of the lack of proven prophylaxis for preeclampsia, prediction of risk or identification of subclinical disease is desirable to identify patients for more intensive observation. There are certain at-risk groups of patients such as those with chronic hypertension, pregestational diabetes, multifetal gestation, and previous preeclampsia. These patients account for the majority of cases of preeclampsia in multiparas, yet only account for 14% of preeclampsia in nulliparous women. Thus, the majority of cases of preeclampsia arises from nulliparous women without medical complications at low risk. Differences in the time of onset, severity, and organ system involvement suggest there may be different underlying etiologies that ultimately lead to preeclampsia manifested as the triad of maternal hypertension, proteinuria, and edema. Distinct markers therefore may identify subgroups of at-risk patients with separate underlying causes. These markers ultimately could be used for diagnosis of disease before the clinical appearance of maternal disease (hypertension, proteinuria, and edema). Based on data from patients with established disease, with the involvement of various organ systems, potential candidate markers would include renal function (kallikrein-creatinine); coagulation and fibrinolytic systems and platelet activation (platelet volume); markers of vascular function (fibronectin, prostacyclin, thromboxane) and oxidant stress (lipid peroxides, 8-isoprostane, antioxidants, anticardiolipin antibodies, hemoglobin, iron, transferrin, homocysteine, hypertriglyceridemia, albumin isoforms); placental peptide hormones (CRH, CRHbp, activin, inhibin, hCG); vascular resistance (uteroplacental flow velocity waveforms); genetic markers; insulin resistance; and glucose intolerance. Although cross-sectional studies have identified these potential markers, they need to be evaluated in prospective longitudinal studies with rigorous definition of outcome to determine if they are useful in predicting preeclampsia and whether they can identify different subgroups of patients.

Two sensitive and selective quantitative methods for 6-oxo-PGF1a, the stable hydrolysis product of prostacyclin are described and compared. Prostaglandins were extracted from biological fluids with organic solvents. Samples for gas chromatographic mass spectrometric analysis required additional thin-layer chromatographic separation prior to conversion to the O-methyloxime, methyl ester, tri-trimethylsilyl ether. Ion fragments at m/z418 and 508 (protium) and m/z422 and 512 (deuterium) were monitored to prepare 6-oxo-PGF1astandard curves. The gas chromatographic mass spectrometric detection limit was 500 pg injected on column with a method coefficient of variation of 11.6% at this level. At 590 pg ml-1the coefficient of variation for reproducibility of measurement was 1.77%. Antisera raised against a 6-oxo-PGF1a-bovine serum albumin conjugate in sheep had a higher titre and greater selectivity than those raised in rabbits. Cross-reaction of sheep antisera with all prostaglandins and fatty acids tested was less than 0.5%. The radioimmunoassay limit of detection was 60 pg ml-1(6 pg per tube) with a coefficient of variation of 10.4% (intra-assay) and 10.75% (inter-assay). The double blind comparison of gas chromatographic mass spectrometric and radioimmunoassay quantitation of the same samples gave a correlation coefficient of 0.97. Both methods offer sensitivity, selectivity and reproducibility. Gas chromatography mass spectrometry is necessary to validate the radioimmunoassay method which offers advantages of time, sample capacity and volume, expenditure and sensitivity.

The Tokamak Physics Experiment is designed to develop the scientific basis for a compact and continuously operating tokamak fusion reactor. It is based on an emerging class of tokamak operating modes, characterized by beta limits well in excess of the Troyon limit, confinement scaling well in excess of H-mode, and bootstrap current fractions approaching unity. Such modes are attainable through the use of advanced, steady state plasma controls including strong shaping, current profile control, and active particle recycling control. Key design features of the TPX are superconducting toroidal and poloidal field coils; actively-cooled plasma-facing components; a flexible heating and current drive system; and a spacious divertor for flexibility. Substantial deuterium plasma operation is made possible with an in-vessel remote maintenance system, a lowactivation titanium vacuum vessel, and shielding of ex-vessel components. The facility will be constructed as a national project with substantial participation by U.S. industry. Operation will begin with first plasma in the year 2000.

Rat granulosa cell cytosol contains a second oestrogen-binding species (SOB) distinguished from the classical oestrogen receptor by its lower dissociation constant (approx. 45 nmol/l) and the ability to bind oestrogens, antioestrogens, androgens and progesterone but not diethylstilboestrol. The SOB and the oestrogen receptor can be further distinguished by their differential adsorption to spheroidal hydroxylapatite and Concanavalin A–Sepharose. Addition of chaotropic salts or molybdate to granulosa cell cytosol did not alter the concentration of SOB or oestrogen receptor measured, indicating that there are no 'masked' binding sites in the two species caused by aggregation phenomena. The association rate of oestradiol with SOB at 4°C (1·72 ± 0·27(s.e.m.) × 108mol/h) and 25°C (4·50 ± 0·36 × 108mol/h) was faster than with the oestrogen receptor (7·20 ± 0·15 × 107mol/h and 1·23 ± 0·15 × 108mol/h respectively). The biphasic dissociation kinetics of [3H]oestradiol from the oestrogen receptor at 25°C (rate constants k−1= 0·30±0·07/min and k−2= 3·73±0·57 × 10−3/min) were similar to those reported in other target tissues but the dissociation of [3H]oestradiol from SOB appeared to be much more rapid and could not be measured by the Sephadex LH-20 separation method employed for determining receptor kinetics. Using sucrose density-gradient (SDG) analysis and Sephacryl S-200 gel chromatography the oestrogen receptor fractionated in an aggregated form (10·3S, Stokes radius >5·2 nm) in low ionic strength buffers and as a small species (4·4S, Stokes radius 3·5 nm) in buffers containing 0·4 m-KCl. However, the SOB fractionated as 2–3S, Stokes radius 3·7–4·0 nm at low ionic strength and as 5·8S, Stokes radius 3·5 nm in 0·4 m-KCl. In contrast to the receptor from other target tissues the granulosa cell oestrogen receptor did not bind to the artificial acceptor matrix oligo(dT)-cellulose and heat activation did not promote a 4S to 5S conversion when analysed on SDG. The salt-extracted form of nuclear receptor sedimented at 4·6S, mol. wt 69–72 000 on SDG.J. Endocr.(1984) 102, 93–102

The increased fetal-placental vascular resistance seen in preeclampsia and/or IUGR has been empirically ascribed to deficiencies in synthesis of vasodilator autocoids in this vascular bed. However, abnormal development of the vascular tree and thus increased resistance to flow could lead to alterations in production of autocoids as a compensatory response, particularly as physical factors such as flow/shear stress over endothelial cells are potent stimuli to autocoid synthesis. The nitric oxide radical (NO) is a potent vasodilator of the human fetal-placental vasculature. Alterations in NO synthesis have been reported in the placenta in preeclampsia. The bioactivity of NO is regulated by its interaction with and inactivation by the superoxide anion. However, the product of this interaction, the strong-lived oxidant peroxynitrite, while acting acutely as a vasodilator, acts in a chronic manner to exert deleterious cytotoxic effects and causes vascular dysfunction. Production and action of peroxynitrite can be identified by the presence of nitrotyrosine residues. We have determined the immunohistochemical localization and quantification of the endothelial nitric oxide synthase isoform (eNOS) and of nitrotyrosine residues (NT) in placental villous tissue from normotensive pregnancies in comparison with those complicated by preeclampsia and/or IUGR. Whereas, there was no difference in quantity of eNOS immunostaining in syncytiotrophoblast between the four groups of tissues, more intense eNOS staining was seen in the villous vascular endothelium of preeclamptic and/or IUGR placentae. This parallels our previous observation of increased concentrations of nitrite, a breakdown product of NO, in the umbilical circulation of these pregnancies. No NT immunostaining was seen in trophoblast cells of any placentae or in the vascular endothelium of normotensive placentae. However, significantly more intense NT immunostaining was apparent in the villous vascular endothelium of the pathologic placentae and diffusing into vascular smooth muscle and stroma. Therefore, the increased vascular resistance and abnormal umbilical blood flow velocity waveforms seen in preeclampsia and/or IUGR cannot simply be ascribed to reduced synthesis and action of vasodilator autocoids in fetal placental vasculature. Rather, increased synthesis of some of these vasodilator autocoids e.g., NO may be occurring in the first instance as an adaptive or compensatory response to increased resistance or to a primary anatomic defect found in this vasculature. The effect of this increased autocoid expression will be to try to vasodilate this vasculature to increase blood flow to the placenta and increase oxygen and nutrient extraction. However, in common with many other conditions of oxidative stress where endothelial dysfunction is seen, concomitant increased synthesis of nitric oxide and superoxide may lead to the formation of the deleterious compound peroxynitrite, which results in vascular damage. The relative rates and sites of synthesis and the opportunity for interaction between these radicals may be key to determining whether potentially beneficial or deleterious effects are seen.

Peripheral plasma levels of immunoreactive 6-oxo-PGF1 alpha, the stable hydrolysis product of prostacyclin, were significantly higher in female patients with tumours of the genital tract than in normal controls. In the groups with malignant tumours, these high levels declined after operation and/or radiotherapy if the tumour responded to treatment. In patients who did not respond to treatment or with tumour recurrence, levels of plasma 6-oxo-PGF1 alpha remained high or even rose further. Benign gynaecological tumours were also associated with significantly raised plasma 6-oxo-PGF1 alpha levels, and these fell to normal levels immediately on surgical removal of the tumour. Possible reasons for these alterations are described. Further investigations are warranted to see whether serial measurements of plasma 6-oxo-PGF1 alpha could be used as a prognostic index for the clinical status of patients with gynaecological tumours.

Numbers of granulosa cells obtained from follicles of immature rats increased from 1·6 × 105cells/ovary on day 8 to 7·1 × 106cells/ovary on day 40 of age, the day of vaginal opening and first pro-oestrus. Very high levels of cytosol oestrogen receptor were found on day 8 (175 000 sites/cell) but by day 19 20 000 sites/cell were found. Nuclear receptor concentrations were highest on day 12(5400 ± 1470 (s.d.) sites/cell) and again on day 21 (5400 ± 2300 sites/cell). After day 21 both cytosol and nuclear oestrogen receptor concentrations fell and remained low until nuclear concentrations rose at day 40. Two consecutive daily injections of FSH/LH (5 i.u.) increased cell number over control in animals killed on day 22, gave no significant alteration in animals killed on day 26 or 28 but decreased numbers in animals aged 32 and 35 days. Only on day 22 was the increase in cell number associated with an increase in nuclear oestrogen receptor concentrations. Indeed on days 32 and 35 increased nuclear receptor concentrations were associated with a decreased cell number.J. Endocr.(1987) 112,333–338

The dissociation constants (Kd) and steroid specificities of oestrogen-binding species in rat granulosa cell cytosol and nuclei have been studied. Preliminary work, where diethylstilboestrol was employed as competitor in binding assays, identified the oestrogen receptor in whole ovarian tissue nuclei (Kd0·35 ±0.09 nmol/l) and cytosol (Kd0·39 ± 0·03 nmol/l). Isolation of granulosa cells revealed that the majority of this receptor (75–96%) was present in these cells. Specificity studies on the binding of [3H]oestradiol in granulosa cell cytosol indicated the presence of an additional class of oestrogen-binding sites which were, however, not present in nuclei. Saturation analysis over an extended range of [3H]oestradiol concentrations and using unlabelled oestradiol as competitor revealed a binding species of Kd45·8± 6·9 nmol/l (capacity 16·7 pmol/mg cytosol protein) for oestradiol in addition to the cytosol oestrogen receptor of Kd0·58 ± nmol/l (capacity 2·8 pmol/mg cytosol protein). The low affinity of this novel species implies that the dextran-coated charcoal techniques used in previous studies on ovarian oestrogen-binding species would cause dissociation of ligand and not allow it to be measured.The second oestrogen-binding species displayed affinity for oestradiol-17β, oestriol, oestrone, testosterone, 5α-dihydrotestosterone, methyltrienolone, progesterone and the antioestrogens tamoxifen, nafoxidine and clomiphene citrate. The species, however, did not bind diethylstilboestrol, a characteristic shared with other low affinity cytosol oestrogen-binding species which have been reported in dog prostate, chick oviduct and male rat liver but not shared with uterine type II oestrogen receptors. It can be further distinguished from the oestrogen receptor by differential ammonium sulphate precipitation and the stability of its ligand binding at temperatures above 55 °C where the oestrogen receptor–ligand interaction is rapidly lost.Concentrations of nuclear oestrogen receptor in granulosa cells (2200 sites/cell) were similar to those found in other target tissues but a high proportion of this receptor (70%) was 'unoccupied' or available for binding at 4 °C and the majority (75%) was resistant to extraction with 0·4 m-KCl. As the second oestrogen-binding species could not be detected in granulosa cell nuclei it is unlikely to be involved directly in eliciting genomic responses to hormonal stimulation. It is more probable that it regulates the level of the free intracellular steroid to which the oestrogen receptor of the granulosa cell (the predominant site of oestrogen biosynthesis) is exposed.J. Endocr.(1984) 102, 83–91

The interaction of rat uterine cytosol oestrogen-receptor complexes with the synthetic acceptor oligo(dT)-cellulose was studied. Differences in the stability of receptor complexes and their ability to bind to oligo(dT)-cellulose on storage at 4 degrees C or when exposed to increased temperatures indicated heterogeneity of steroid- and oligonucleotide-binding sites. Dilution, dialysis and (NH4)2SO4 precipitation increased the interaction of receptor complexes with oligo(dT)-cellulose (a step termed activation). This increase may be the result of the removal of low-molecular-weight cytosol components which inhibit receptor activation, dimerization to the 5 S form, which binds to oligo(dT)-cellulose, or interaction of 5 S receptor with the oligonucleotide. Cytosol oestradiol-receptor complexes exhibited biphasic dissociation kinetics. All these manipulations resulted in an increase in the proportion of the slow-dissociating component equivalent to the increase in receptor binding to oligo(dT)-cellulose. In contrast, addition of 10mM-sodium molybdate to cytosol decreased both oligo(dT)-cellulose binding and the proportion of receptor with slow dissociation kinetics. The inclusion of proteinase inhibitors did not affect interactions of receptor with oligo(dT)-cellulose nor the dissociation kinetics. These results suggest that oligo(dT)-cellulose binding may serve to quantify the proportion of cytosol receptor in an active form capable of nuclear interaction and to help to ascertain whether a receptor system is fully functional. This binding procedure could prove useful in the evaluation of oestrogen responsivity under normal and pathological conditions.