Image analysis of protein profiles from paired microvillous and basal syncytiotrophoblast plasma membranes from term human placenta and characterization of IgG binding to membrane vesicles.

Image analysis of SDS-PAGE profiles of highly purified, paired maternal-facing (microvillous; MVM) and fetal-facing (basal; BM) plasmalemma membrane vesicles from six term human placentae showed that, while individual MVM or BM profiles were extremely reproducible, the two membrane populations were substantially different--although all of the seven major bands of molecular mass, 98.4, 79.4, 71.1, 45.1, 40.9, 39.5 and 34.5 kDa found in MVM were present, albeit in differing amounts, in BM. BM were characterized by the presence of five low molecular weight bands which were not present in MVM. Despite this consistency of the membrane preparations, binding of 125I-IgG or its fragments showed marked variability in both MVM and BM. At pH 7.4, both MVM and BM bound similar amounts of 125I-IgG with Kd values of 5.2 +/- 1.9 x 10(-6) M (s.e., n = 8) and 2.9 +/- 0.4 x 10(-6) M respectively, (P > 0.05). There were 1.2-1.6 x 10(15) binding sites/mg protein. Affinity constants for Fc fragment binding to MVM and BM were similar to those for IgG, although the Bmax value for BM Fc binding was greatly reduced compared to that for IgG (P > 0.001). Fab binding to MVM and BM was also saturable but substantially lower than that of Fc, whereas binding of F(ab')2 was low and linear. Both MVM and BM bound marginally more IgG at pH 6.0 than at pH 7.4. These data provide further evidence for receptor-mediated transcytosis of maternal IgG across the placenta and confirm that the placental IgG transporter differs from classical Fc gamma receptors.

Sequential preparation of highly purified microvillous and basal syncytiotrophoblast membranes in substantial yield from a single term human placenta: inhibition of microvillous alkaline phosphatase activity by EDTA.

The human placental syncytiotrophoblast is a highly polarised epithelial layer responsible for regulating materno-fetal exchange. We here describe a novel procedure for isolating paired fractions of the maternal-facing and fetal-facing plasma membranes from this syncytium, from a single placenta, without the need for homogenisation procedures. This reduces the potential for contamination of these membrane fractions by intracellular membranes, or from plasma membranes from other cell types within the placenta. Microvillous membrane vesicles (MVM) were obtained by gentle stirring of dispersed villous tissue. The tissue sedimented at the end of this procedure was subjected to sequential ultrasonication to release the basal membrane (BM). Crude MVM was subsequently purified on a discontinuous sucrose gradient. Crude BM was further purified using either discontinuous Ficoll or sucrose gradients. The Ficoll procedure, while producing a BM fraction extremely enriched in marker enzyme, resulted in unacceptably low protein recoveries and hence the sucrose gradient procedure was also adopted for BM. Yields for MVM and BM produced on sucrose density gradients approached 30 mg/100 g tissue. The MVM fraction was composed of vesicles of 232 +/- 9 (S.E.) nm diameter of which nearly 90% were 'right side out'. These membranes were 37-fold enriched in the marker enzyme alkaline phosphatase. Purified BM vesicles were 317 +/- 14 nm in diameter, also approximately 90% 'right side out' and over 40-fold enriched in dihydroalprenolol binding. Cross-contamination or contamination from intracellular membranes was negligible. MVM alkaline phosphatase activity was shown to be inhibitable in a dose- and time-dependent manner by EDTA present in the storage buffer.

Sleepwalking, night terrors, and consciousness.

OBJECTIVE: To determine some personality and psychoneurotic characteristics of adults who have the sleepwalking-night terrors syndrome. DESIGN: Prospective assessment of two groups of consecutive patients with a firm diagnosis of either of two specific sleep disorders as established clinically and by polysomnography. SETTING: Outpatient sleep disorders clinic and sleep laboratory in a tertiary referral centre. PATIENTS: 12 Patients referred consecutively to the clinic in whom a diagnosis of sleepwalking (six) or night terrors (six) was confirmed. MAIN OUTCOME MEASURES: Psychological characteristics as measured at the time of clinical assessment by means of the Eysenck personality questionnaire, the hostility and direction of hostility questionnaire, and the Crown-Crisp experiential index. RESULTS: Both groups scored exceptionally highly on the hysteria scale of the Crown-Crisp experiential index and the night terrors group also scored highly on the anxiety scale. The patients with sleepwalking also scored highly on a measure of externally directed hostility. CONCLUSIONS: The physiological and psychological features identified in these patients, possibly reflecting different expressions of a constitutional cerebral characteristic, may be explored in terms of hysterical dissociation. The findings contribute to the debate concerning the nature of sleepwalking, in particular with and without the forensic aspects.

Uptake of alpha 2-macroglobulin-trypsin complex by human placenta is mediated by a microvillous membrane receptor.

The fate of native alpha 2-macroglobulin (alpha 2M) or its trypsin complex (alpha 2M-T) was studied in the isolated dually-perfused lobule of term human placenta. [125I]-alpha 2M added to the maternal circuit was unchanged during the course of the perfusion with minimal activity becoming associated with the placental tissue. Transfer of radioactivity into the fetal circulation accounted for only 0.07 per cent of the initial dose after 2 h. In contrast, [125I]-alpha 2M-T was rapidly taken up into the placental tissue (nearly 28 per cent of the initial dose during the 2-h perfusion) and breakdown products were released into both maternal and fetal circulations. At the end of 2 h, radioactivity levels on the fetal side were 13 times higher than those found with the native protein. These indications of a classical receptor-mediated uptake and breakdown pathway were confirmed in experiments in which the acidotrophic agent chloroquine was added to the maternal circuit prior to the alpha 2M-T. In the presence of chloroquine, tissue uptake was inhibited and the subsequent release of radioactive degradation products into the fetal circuit was similar to the levels seen with alpha 2M. Incubation of term trophoblast cells at 37 degrees C with [125I]-alpha 2M-T revealed over three-fold greater cell-associated activity than was found with the native protein. In another series of experiments, a purified microvillous membrane fraction was prepared from term placentae using buffers containing 1 mM iodoacetate. In the presence of this proteolytic enzyme inhibitor, binding studies showed a single class of low affinity receptors for the alpha 2M-T complex capable of binding 4.8 +/- 1.3 (SEM) micrograms of complex per mg of membrane protein. There was no binding of the native protein.

A monoclonal antibody based solid-phase enzyme-binding assay to measure levels of placental alkaline phosphatase in serum of women during pregnancy.

Monoclonal antibodies raised against highly purified placental alkaline phosphatase (PALP) reacted specifically with PALP and were found not to cross-react with the liver, kidney and intestinal enzymes. One of these antibodies (AP-3) was selected to develop an enzyme-binding assay to measure levels of PALP in the sera of 11 women throughout pregnancy. PALP activity rose sharply after 28 weeks of gestation reaching a peak at delivery followed by an immediate drop within 4 days to values approaching non-pregnant levels. The potential of using this assay as an index of placental function to monitor progress of a pregnancy is discussed.

Purification and properties of human placental cathepsin D.

Human placental cathepsin D has been purified 6000-fold and its properties characterized. Its molecular weight has been ascertained to be 42 000 by gel filtration and 43 300 by analytical ultracentrifugation. SDS gel electrophoresis in the presence of beta-mercaptoethanol cleaves the enzyme into two polypeptides of molecular weights 28 200 and 14 400. The placental enzyme resembles cathepsin D isolated from other mammalian tissues in many of its properties, including pH optimum. The higher degree of purification has led to a shift in the isoelectric points of the three isoenzymes from those recorded by other authors. Antibodies raised against cathepsin D in rabbits inhibit it at pH 5.0, and the inhibition is almost 100 per cent with adequate concentrations of monospecific antibody.