Osamu Narita

Menotropin stimulation after prolonged gonadotropin releasing hormone agonist pretreatment for in vitro fertilization in patients with endometriosis

Two protocols were scheduled for in vitro fertilization and embryo transfer (IVF-ET) in patients with various stages of endometriosis who were resistant to conventional therapies. In the ultralong protocol (21 patients), gonadotropin releasing hormone agonist (Gn-RHa) was administered for at least 60 days prior to ovarian stimulation along with menotropin until human chorionic gonadotropin was injected. In the long protocol (11 patients), Gn-RHa was started at the midluteal phase and exogenous gonadotropin was commenced between the third and the seventh day of the menstrual cycle after pituitary suppression. The estradiol response and the number of retrieved oocytes, fertilized oocytes, cleaved oocytes, and transferred embryos were similar in both groups but the clinical pregnancy rate per transfer was superior in the ultralong protocol (67 vs 27%). The miscarriage rate was 14% (2/14) in the ultralong protocol. Prolonged Gn-RHa suppression of ovarian function before superovulation may overcome some causes of infertility in patients with endometriosis.

Purification and characterization of human placental aminopeptidase A.

Human placental aminopeptidase A (AAP) was purified 3,900-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100, then subjected to trypsin digestion, zinc sulfate fractionation, chromatographies with DE-52, Sephacryl S-300, and hydroxylapatite, affinity chromatography with Bestatin-Sepharose 4B, and finally immunoaffinity chromatography with the antibody against microsomal leucine aminopeptidase (LAP). Aminopeptidase A was completely separated from leucine aminopeptidase by the immunoaffinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 280,000 by gel filtration. The purified enzyme was most active at pH 7.1 with L-aspartyl-beta-naphthylamide (L-Asp-NA) as substrate; the Km value for this substrate was 4.0 mmol/l in the presence of Ca2+. Human placental aminopeptidase A was markedly activated by alkaline earth metals (Ca2+, Sr2+, Ba2+), but strongly inhibited by metal chelating agents such as EDTA and o-phenanthroline. The highest activity was observed with L-glutamyl-beta-naphthylamide, while only minimal hydrolysis was found with some neutral and basic amino acid beta-naphthylamides.

Post-proline endopeptidase in human placenta

Post-proline endopeptidase (EC 3.4.21.26) was found in human placenta, purified 3390-fold from it and briefly characterized. The post-proline endopeptidase could be completely separated from dipeptidyl peptidase IV (EC 3.4.14.5) by hydrophobic phenyl-Sepharose chromatography. The pH optimum of the enzyme was 6.7. The Km values for 7-(Succinyl-Gly-Pro)-4- methylcoumarinamide was 1.0 mM. The molecular weight of this enzyme was estimated to be 140 000 by gel filtration and 67 000 by dodecyl sulfate gel electrophoresis, indicating its dimeric structure. Human placental post-proline endopeptidase was suggested to be a thiol proteinase by inhibition studies.

Serum aminopeptidase A (AAP) in normal pregnancy and pregnancy complicated by pre-eclampsia

SummarySerum aminopeptidase A (AAP) activity was measured in normal pregnancy and pre-eclampsia. The AAP activity in normal pregnancy increased progressively with advancing gestation, reaching the highest value at the end of pregnancy. The AAP activity in pregnancy complicated by pre-eclampsia was lower than in normal pregnancy.

Angiotensin I-converting enzyme in human placenta.

Angiotensin I-converting enzyme (ACE, peptidyldipeptide hydrolase, kininase II, EC 3.4.15.I) from human placenta was purified 6297-fold and characterized. ACE could be extensively purified by affinity chromatography with Captopril (D-3-mercapto-2-methylpropanoyl-L-proline), an orally active antihypertensive agent and a potent inhibitor of this enzyme. Its molecular weight and subunit size were estimated to be 300 000 by high-performance gel permeation chromatography and 85 000 by sodium dodecyl sulphate gel electrophoresis, respectively, indicating its polymeric structure.

Purification and characterization of human placental microsomal aminopeptidase: immunological difference between placental microsomal aminopeptidase and pregnancy serum cystyl-aminopeptidase.

Human placental microsomal aminopeptidase (microsomal PAP) was purified 3,880-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100 and also trypsin digestion, and subjected to zinc sulfate fractionation, chromatographies with DE-52, hydroxylapatite, Sephacryl S-300 and lentil lectin-Sepharose 4B, and finally affinity chromatography with bestatin-Sepharose 4B. Microsomal PAP was separated from aminopeptidase A (AAP) by affinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 220,000 by high-performance liquid chromatography with an aqueous gel column. The purified enzyme gave almost a single band with a molecular mass of 140,000 by sodium dodecyl sulfate (SDS) gel electrophoresis. The isoelectric point of the enzyme was 5.2. The purified enzyme was most active at pH 8.0 with L-leucine-p-nitroanilide as substrate; the Km value for this substrate was 1.1 mmol/l. The microsomal PAP was immunologically different from the pregnancy serum cystyl aminopeptidase (serum PAP).

Plasma angiotensin I and serum placental leucine aminopeptidase (P-LAP) in pre-eclampsia.

SummaryA study was undertaken on serial measurements of plasma angiotensin I (A-I) and serum placental leucine aminopeptidase (P-LAP) activities in normal and pre-eclamptic pregnancy. There was the significant difference in A-I levels between normal and mild pre-eclamptic pregnancy at weeks 30, 35, and 37, between normal and severe pre-eclamptic pregnancy at week 37. There were no differences in serum P-LAP between normal and mild pre-eclamptic pregnancy up to week 33, but thereafter the levels for the mild pre-eclampsia were significantly higher than for the normal pregnancy. The P-LAP activity for the severe pre-eclampsia reached its maximum level at week 31. Around this week, the levels, for severe pre-eclampsia were significantly higher than in the normal pregnancy. After week 35, the activities decreased precipitously to week 40; the activities for severe pre-eclampsia in late pregnancy at weeks 39 and 40 were significantly lower than in normal pregnancy. The above data support the idea that P-LAP test is useful for prediction or diagnosis of pre-eclampsia.

Identification of two subtypes of protein kinase C in human placenta

Abstract A purified protein kinase C (PKC) has been isolated from term human placental tissue, which is phospholipid and Ca 2+ -dependent. Two subtypes of the enzyme were identified by hydroxyapatite column chromatography and using monoclonal antibodies with immunohistochemical techniques; subtype III is present in higher concentration than subtype II. Their ratio of 2.5 is very similar in second and third trimester placentas, but is higher, 6.5, in the first trimester. The subtype I was never expressed.

The ontogeny of growth hormone in the human fetal pituitary.

To examine the ontogeny of growth hormone synthesis and secretion in human fetus, growth hormone messenger ribonucleic acid was measured in 11 pituitaries from fetuses of 16 to 27 weeks of gestation by hybridization of cytosol ribonucleic acid with complementary deoxyribonucleic acid labeled with phosphate 32. Pituitary growth hormone content and serum growth hormone, thyroxine, and cortisol concentrations were assessed by radioimmunoassay. Growth hormone messenger ribonucleic acid content in the fetal pituitary increased from the early midtrimester, reaching a level 15.3 times higher at 27 weeks of gestation than the value at 16 weeks. However, growth hormone content in the pituitary showed no evident change during 16 to 21 weeks of gestation and started to increase after 22 weeks. Serum concentration of growth hormone was variable but always >50 ng/ml, and the maximal level was observed at 20 weeks of gestation (141 ng/ml). Although serum thyroxine concentration in the fetuses showed no correlation with pituitary growth hormone content or serum growth hormone concentration, serum concentration of cortisol was correlated positively with growth hormone content in the fetal pituitary. These results suggest that the maturation of the growth hormone synthesis and secretion system in the fetal pituitary occurs after 22 weeks of gestation and that cortisol may play some role in the ontogenesis of growth hormone.

Purification and properties of microsomal carboxypeptidase N (kininase I) in human placenta.

Carboxypeptidase N (kininase I, EC 3.4.17.3) was found in human placenta and purified 600-fold. The enzyme was solubilized from membrane fractions with Triton X-100 and was purified by affinity chromatography with histargin, a potent inhibitor of this enzyme. The pH optimum of the enzyme was 7.8. The Km values for L-hippuryl-L-lysine and bradykinin were 1.25 and 0.43 mmol/l, respectively. The apparent molecular mass (Mr) of the enzyme determined by gel filtration was estimated to be 280,000, which is identical to that of the human serum enzyme. We propose that the placenta is a major source of carboxypeptidase N and thus may be involved in the physiological control of fetal circulation by regulating the kallikrein-kinin and renin-angiotensin systems.