Urinary sex steroid hormone and placental leucine aminopeptidase concentration differences between live births and stillbirth of Bornean orangutans (Pongo pygmaeus).

BACKGROUND: Under the environment of pregnancy, the placenta assumes an important steroidogenic role in the maintenance of pregnancy. METHODS: Urinary placental leucine aminopeptidase (PLAP), estrone-3-glucuronide (E1 G), and pregnanediol-3-glucuronide (PdG) concentrations were compared among five pregnancies (four live births and one stillbirth) in four orangutans. RESULTS: The gestation period of the stillbirth (223 days) was shorter than that of the live births (239-254 days). In females who gave a live birth, average PLAP and E1 G concentrations increased until the delivery. Conversely, in the female who gave a stillbirth, PLAP concentration failed to increase, and E1 G concentration was significantly low in late pregnancy period. Regarding PdG concentrations, there was no significant difference among all pregnancies. CONCLUSIONS: This is the first study reporting a change in urinary PLAP, E1 G, and PdG concentrations during orangutan stillbirth and live birth pregnancies. The findings will assist in developing pregnancy screening tests.

Binding to and Inhibition of Insulin-Regulated Aminopeptidase by Macrocyclic Disulfides Enhances Spine Density.

Angiotensin IV (Ang IV) and related peptide analogs, as well as nonpeptide inhibitors of insulin-regulated aminopeptidase (IRAP), have previously been shown to enhance memory and cognition in animal models. Furthermore, the endogenous IRAP substrates oxytocin and vasopressin are known to facilitate learning and memory. In this study, the two recently synthesized 13-membered macrocyclic competitive IRAP inhibitors HA08 and HA09, which were designed to mimic the N terminus of oxytocin and vasopressin, were assessed and compared based on their ability to bind to the IRAP active site, and alter dendritic spine density in rat hippocampal primary cultures. The binding modes of the IRAP inhibitors HA08, HA09, and of Ang IV in either the extended or γ-turn conformation at the C terminus to human IRAP were predicted by docking and molecular dynamics simulations. The binding free energies calculated with the linear interaction energy method, which are in excellent agreement with experimental data and simulations, have been used to explain the differences in activities of the IRAP inhibitors, both of which are structurally very similar, but differ only with regard to one stereogenic center. In addition, we show that HA08, which is 100-fold more potent than the epimer HA09, can enhance dendritic spine number and alter morphology, a process associated with memory facilitation. Therefore, HA08, one of the most potent IRAP inhibitors known today, may serve as a suitable starting point for medicinal chemistry programs aided by MD simulations aimed at discovering more drug-like cognitive enhancers acting via augmenting synaptic plasticity.

Expression of placental leucine aminopeptidase is associated with a poor outcome in endometrial endometrioid adenocarcinoma.

OBJECTIVE: Placental leucine aminopeptidase (P-LAP) is a cell surface aminopeptidase (oxytocinase). We cloned P-LAP cDNA and found a widespread tissue distribution of P-LAP. Since P-LAP can degrade several small peptide hormones such as oxytocin, this enzyme may affect many cellular functions of carcinoma cells as well as normal cells. This study investigated whether the expression of P-LAP correlates with clinicopathologic factors and prognosis in patients with endometrial endometrioid adenocarcinoma. METHODS: Histologic sections of formalin-fixed, paraffin-embedded specimens from 99 primary endometrial carcinomas were stained for P-LAP using polyclonal P-LAP antibody. Disease-free survival and other clinicopathologic characteristics were analyzed according to the intensity of P-LAP staining. RESULTS: Of 99 cases, 69 (69.7%) showed specific P-LAP immunostaining. We found a positive correlation between the expression of P-LAP and histological grade (p < 0.01), surgical stage of the disease (p = 0.02), myometrial invasion (p = 0.01), lymph node involvement (p < 0.01), and vascular infiltration (p < 0.01). In patients who had strongly positive P-LAP staining, the disease-free interval was significantly lower than in patients who had negative or weakly positive P-LAP staining (p < 0.01). Multivariate analysis demonstrated that strongly immunoreactive P-LAP (odds ratio, 12.8; 95% confidence interval, 2.84-58.8; p < 0.01) and surgical stage (odds ratio, 8.78; 95% confidence interval, 2.77-27.8; p < 0.01) are independent prognostic factors. CONCLUSIONS: This study suggests P-LAP as an independent prognosticator of clinical outcome in patients with endometrial carcinoma. Therefore, assessment of the P-LAP status provides clinically useful prognostic information in patients with endometrial carcinoma.

Immunohistochemical localization of placental leucine aminopeptidase/oxytocinase in normal human placental, fetal and adult tissues.

While oxytocinase is known to exist in pregnancy serum and placenta, the present study describes the expression of the mRNA for this enzyme in a wide variety of other human tissues. Northern blot analysis was used to detect the mRNA, with a probe derived from a cDNA for oxytocinase/placental leucine aminopeptidase (P-LAP). Both the distribution and localization of immunoreactive oxytocinase/P-LAP protein have been determined immunohistochemically by use of an anti-P-LAP antibody in normal placental, fetal and adult tissues. In placental tissues, only syncytiotrophoblasts were stained positively. In both fetal and adult tissues, positive staining was obtained in vascular endothelial cells, gastrointestinal mucosal cells, epithelial cells of hepato-biliary, pancreato-biliary, bronchial-alveolar and renal tubular systems as well as islet cells of pancreas and neurons in the central nervous systems. Sweat-gland cells, seminal vesicles and prostate gland in the adult, as well as adipocytes and skeletal muscle cells in the fetus were also stained. The widespread distribution of P-LAP suggests its involvement in a variety of physiological events not restricted to the regulation of the amounts of bioactive peptides such as arginine vasopressin (AVP) and oxytocin (OT) in pregnancy. The presence of P-LAP in syncytiotrophoblasts supports the idea that P-LAP in pregnancy serum is derived from the placenta.

[Results of fetal monitoring in pregnancy with enzymatic and neuroendocrinologic risk factors]. / Wyniki monitorowania rozwoju plodu w ciazy z wykladnikami enzymatycznego i neuroendokrynologicznego zagrozenia.

301 pregnant women after a successful infertility treatment was divided in to 4 groups depending on the first oxytocinase level less or more than 4 mumol/l/min after 32nd week of pregnancy, and ACTH-depot therapy. In conclusion, pregnancies with oxytocinase level less than 4 mumol/l/min after 32nd week ('enzymatic dangerous area') are neuroendocrinologic risk pregnancies and they are lasting statistically longer than pregnancies over this limit. The oxytocinase level in an "enzymatic dangerous area" alone, is not an indication to ACTH-depot therapy. A computer-aided method of birth prognosis and newborn's state allow to precisely determining delivery term and newborn's state independently on oxytocinase and iso-oxytocinase levels.

Human fetal membrane contribution to the prevention of parturition: uterotonin degradation.

This investigation was conducted to evaluate the potential capacity of the human fetal membranes-decidua parietalis, and in particular the chorion laeve, to degrade uterotonins that are produced in amnion, are present in amniotic fluid, or both. The four uterotonins that have been evaluated most frequently as myometrial contractants potentially involved in the initiation of human parturition are prostaglandins, oxytocin, endothelin-1, and platelet-activating factor. We assessed the levels of mRNA and the specific activities (SAs) of enkephalinase (the plasma membrane endopeptidase that degrades endothelins) and prostaglandin dehydrogenase (PGDH) in human fetal membranes, i.e. amnion and chorion leave, and in decidua parietalis. The SA of oxytocinase (which inactivates oxytocin) in these tissues also was determined. The SA of enkephalinase in chorion laeve from all anatomical sites (singleton and diamnionic-dichorionic twin placentae) in all pregnancies studied (mean +/- SEM, 95 +/- 7.9 ng/min.mg protein; n = 28) is similar to that in human fetal kidney (89.5 +/- 2.8; n = 6). Kidney tissue is believed to be one of the richest sources of enkephalinase. The SAs of enkephalinase in amnion (18.3 +/- 2.3 nmol/min.mg protein; n = 29) and in decidua parietalis (31.8 +/- 6.7; n = 20) also were high, but significantly less than that in chorion leave. The level of enkephalinase mRNA in chorion laeve in singleton pregnancies is high, as is the SA of enkephalinase (111.9 +/- 10.6 nmol/min.mg protein; n = 17). In paired chorion laeve tissues from five diamnionic-dichorionic twin placentae, the SAs of enkephalinase in reflected chorion laeve (74 +/- 12.8; P < 0.06 compared with singletons) and fused chorion laeve (64.8 +/- 6.5; P < 0.001 compared with singletons) were similar. The SA of PGDH in reflected chorion leave (46.3 +/- 6.9 nmol/min.mg protein; n = 19) was significantly greater than that in decidua (16 +/- 5.5; n = 15). There was a significant correlation between the levels of PGDH mRNA and PGDH enzyme SA. In fused chorion laeve of diamnionic-dichorionic twin placentae, the SA of PGDH (14.9 +/- 7.3; n = 4) was much less than that in reflected chorion laeve of the same twin pregnancy (70.5 +/- 14.7; n = 4). PGDH mRNA was not detectable in amnion tissue (n = 5) by northern analysis, and the SA of PGDH (< 1.2 +/- 1.0; n = 6) in amnion was undetectable or near the lower limit of assay detection.(ABSTRACT TRUNCATED AT 400 WORDS)

Placental and plasma cystine aminopeptidase in pregnant animals.

The placental and plasma cystine aminopeptidase (CAP) in pregnant animals was examined on stability after the treatment with L-methionine, ethylene diamine tetra-acetic acid (EDTA) and heat. Inhibitory effects of these treatments on enzyme activities were different among CAPs from the animal species, however, significant correlation in those effects between placental and plasma CAPs was observed. These results suggested that plasma CAP might reflect placental CAP and seemed to be available for estimating maternal gestational conditions.

Decreased levels of amniotic fluid oxytocinase activity in preeclampsia.

Oxytocinase (EC 3.4.11.3) activity was determined in 80 amniotic fluid samples obtained from 40 normotensive primigravidas (median age 27 years) and 40 primigravidas (median age 29 years) with pregnancies complicated by preeclampsia between 32 and 39 weeks of gestation. The enzyme activity was significantly lower in preeclampsia than in normal pregnancy with matched gestations (p < 0.01). Considering the possible involvement of vasopressin and angiotensin II in preeclampsia, it is suggested that the enzyme which degrades these pressor hormones may play a role in the pathogenesis of hypertensive pregnancy.

Degradation of oxytocin by the human placenta: effect of selective inhibitors.

The hydrolysis of oxytocin by human placental subcellular fractions was studied in the presence of selective inhibitors by measuring liberated amino acids by high performance liquid chromatography (HPLC). Oxytocin degradation by microsomal and lysosomal fractions was inhibited by bestatin, amastatin and puromycin. The IC50 values of these inhibitors on oxytocin degradation by both fractions were similar to those of these inhibitors on the human placental aminopeptidase M measured by L-Leu-p-nitroanilide as a substrate (LAP activity), which we reported previously. However, purified aminopeptidase M from human placental microsomal fractions could not liberate any amino acid from oxytocin. Since phosphoramidon (1 mumol/l), a putative metalloendopeptidase inhibitor, and N-benzylcarbonyl-valyl-prolinal (Z-Val-prolinal) (14 mumol/l), a selective inhibitor of post-proline endopeptidase, could not significantly influence the degradation of oxytocin by either subcellular fractions, neither enzyme seems to be actively involved in oxytocin degradation. These results strongly suggested the existence of oxytocinase(s) other than the above three enzymes in microsomal and/or lysosomal fractions of human placenta.

Proteases in the human full-term placenta.

Aminopeptidase A, not yet defined aminopeptidases and endopeptidases, dipeptidyl peptidase I, II and IV, gamma-glutamyl transferase and oxytocinase were investigated in the normal human full-term placenta using qualitative (catalytic) cytochemistry, isoelectric focusing, immunocytochemistry and kinetic fluorometry. Aminopeptidase A could be visualized cytochemically in the smooth muscle cells of the chorionic plate, stem villi and basal plate blood vessels. Aminopeptidases were found in connective tissue fibres of the chorionic plate, villous stroma, basal plate and paraplacenta. Dipeptidyl peptidase IV was detected at the same sites as the aminopeptidases and, in addition, in amniotic epithelial cells, fibroblasts of the villous stroma, endothelium of chorionic plate and villous blood vessels as well as in the basophilic cytotrophoblast cells (x-cells) of the basal plate and paraplacenta, and it possibly also occurred in some domains of the plasma membrane of the syncytiotrophoblast and cytotrophoblast cells. The x-cells surrounded the fetus in the form of a dipeptidyl peptidase IV-positive shell at the border to the mother. The enzyme represented the first specific marker for x-cells. Dipeptidyl peptidase I and II were primarily found in Hofbauer cells (macrophages) of the villous stroma, but also in the syncytiotrophoblast, other villous stromal cells and cells of the chorionic and basal plate. gamma-Glutamyl transferase was present in some connective tissue elements of the chorionic plate. Oxytocinase and endopeptidases were not detected. Isoelectric focusing of proteases revealed different molecular forms of dipeptidyl peptidase IV in the paraplacenta and villous tree, while the aminopeptidases shared the same pattern in both regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Predictive value analysis of measurements of human chorionic gonadotropin, pregnancy specific beta 1-glycoprotein, placental lactogen, and cystine aminopeptidase for the diagnosis of ectopic pregnancy.

The diagnostic utility of serum and urinary human chorionic gonadotropin (hCG) measurements and serum measurements of pregnancy-specific beta 1-glycoprotein (PSBG), placental lactogen, and cystine aminopeptidase (CAP) was prospectively studied in 51 consecutive patients with suspected ectopic pregnancy who underwent laparoscopy or laparotomy. CAP was not detected in the sera of any patient with ectopic pregnancy. False-positive results in each assay were found in patients with intrauterine pregnancy or missed abortion. The overall efficiency of the tests were: urine hCG slide test, 47%; urine hCG tube test, 80%; serum hCG, 86%; serum PSBG, 82%; and human placental lactogen, 60%. Although measurement of serum hCG by radioimmunoassay is the most accurate biochemical test in predicting the correct diagnosis in patients with suspected ectopic pregnancy, the relatively high efficiency of the urinary hCG tube test, coupled with its ease of performance, low cost, and rapid turnaround time, makes it the most practical screening test.

Adenomyosis: study in a Jewish female population.

In a series of 920 hysterectomies, performed during a three-year period at the Gynecological Clinic of Hasharon Hospital, adenomyosis weas found in 89 cases, an incidence of 9.06%. Although this result is in accordance with that reported by other authors, we found a marked difference between the incidence of adenomyosis in the two Jewish female groups under study: the Ashkenazic women (natives of European countries or U.S.A.), and the Sephardic women (natives of Near East countries, Yemen or North Africa); 69.66% of the adenomyosis cases were in the former group as against only 30.34% in the latter. A preoperative diagnosis of adenomyosis was made in 19.10% of the cases. This comparatively high percentage appears to be due to the routine examination of the serum activity of cystine aminopeptidase (CAP) and leucine aminopeptidase (LAP), performed in all patients before operation. Increased values of these enzymes indicate the presence of uterine or ovarian tumoral processes. It is suggested that the diagnosis of adenomyosis must be considered in cases of uterine bleeding, dysmenorrhea and increased CAP and LAP values discovered in multiparous women of around 50 years of age, even when the bimanual examination does not reveal any other pathologic condition.

Human placental oxytocinase and its relationship to pregnancy plasma oxytocinase.

Plasma "oxytocinase of pregnancy" and three placental "oxytocinase" fractions from human placental extracts were compared. On the basis of acrylamide-agarose chromatography, polyacrylamide gel electrophoresis, substrate specificity, heat liability and relative insensitiveness to L-methionine, it is concluded that placental enzyme activities, present in the second acrylamide-agarose peak, are identical with the plasma "pregnancy oxytocinase" and are to be regarded as its source. The hypothesis of a transplacental passage of placental peak II oxytocinase in the blood compartment of the mother seems well supported. Heat treatment of the placental extracts uncovered a minor activity behaving like Oya's microsomal oxytocinase, which is totally unlike the plasma oxytocinase and plays no part in the increased oxytocinase activity of human pregnancy plasma. A hitherto undescribed enzyme activity shares with the pregnancy oxytocinase its specificity towards L-leucyl-beta-naphthylamide and di-S-S-L-cysteinyl-beta-naphthylamide, its heat lability and realtive insensitiveness to L-methionine. However, this activity is carried by a protein of much lower molecular weight as judged by acrylamide-agarose chromatography, which shows only a single activity band on polyacrylamide gel electrophoresis.