Induction by cortisol of aminopeptidases production from the human placenta in tissue culture.

Human placental aminopeptidase M and A and post-proline endopeptidase are known to act as degrading enzymes of bioactive peptides such as angiotensin II, oxytocin and endogenous opioids. We tested the effects of cortisol on the activities of human placental aminopeptidase A and M and post-proline endopeptidase using short-cultured placental tissues. From 34.5 nM to 3.45 microM of cortisol significantly increased the activities of 3 enzymes. Our present data suggest a possible important role of cortisol in the growth of human placenta via induction of placental aminopeptidases.     

Fluctuation of gene expression for poly(ADP-ribose) synthetase during hemin-induced erythroid differentiation of human leukemia K562 cells and its reversion process

We have studied the regulation of gene expression for poly(ADP-ribose) synthetase during erythroid differentiation and its reversion process. When human leukemia K562 cells were incubated in the presence of 80 microM hemin, benzidine-positive cells appeared at day 2 and 90% of the cells became positive at day 6. However, RNA blot analysis reveals that mRNA for gamma-globin was already abundant in untreated K562 cells and the level of the message was slightly increased by hemin-treatment. Spectroscopic analysis and polyacrylamide gel electrophoresis of the induced cell extracts indicate that hemoglobin molecules were not detected in untreated cells, and increased successively up to day 6. The hemin-induced cells were thoroughly washed, and then recultured in the absence of hemin. The benzidine-positive cells mostly disappeared 3 days after the elimination of the inducer. During the hemin-induced erythroid differentiation, the activity and mRNA for poly(ADP-ribose) synthetase decreased to 50% and 20% of the initial level at day 3 and a low level of the gene expression was maintained afterwards, whereas the activity and mRNA returned to the initial value 1 day after hemin elimination. The results indicate that the hemin-induced erythroid differentiation of K562 cells is a reversible process and depression of the synthetase may be involved in the progress of differentiation.     

Spontaneous and sperm-induced activation of oocytes in LT/Sv strain mice

The oocytes of LT/Sv strain mice are unique in that a high proportion of them (∼40% in this study) are ovulated before reaching metaphase of the second meiotic division (metaphase II). The remaining oocytes of LT/Sv mice are ovulated at metaphase II, as in other strains of mice. When recently ovulated oocytes were cultured in vitro for 11–12 h, those ovulated at metaphase II remained at this stage, whereas those ovulated at metaphase of the first meiotic division (metaphase I) commonly resumed meiosis during in vitro aging. These oocytes extrude the polar body and form a diploid pronucleus. This oocyte activation is not coupled with cortical granule exocytosis. The oocytes ovulated at metaphase II are fully capable of normal fertilization, whereas those ovulated at metaphase I are not. Approximately 50% of metaphase I oocytes penetrated by spermatozoa remain at this stage, and sperm nuclei frequently undergo premature chromosome condensation. Only 13% of spermpenetrated metaphase I oocytes formed a diploid female pronucleus and a haploid male pronucleus by 4 h after insemination. These results demonstrate that the two types of ovulated LT/Sv oocytes have different potentials to undergo either spontaneous or sperm-induced activation.     

Serum levels of prolactin during the ovarian cycle,pregnancy, and lactation in the common marmoset (Callithrix jacchus)

A homologous double-antibody radioimmunoassay developed for humans was used to measure serum prolactin, progesterone, and estradiol in common marmosets. In the ovarian cycle of common marmosets, serum progesterone began to increase after an estradiol surge, attained a peak level, and then declined before the ensuing pre-ovulatory rise in estradiol. During the luteal phase, the change in serum concentrations of estradiol was synchronized with that of progesterone. During the ovarian cycle there was no consistent change in serum prolactin concentrations. During the last 75 days of pregnancy the prolactin level was higher than during the ovarian cycle and the first 70 days of pregnancy. Moreover, during lactation, mothers with suckling twin infants had a higher prolactin level than during the final stage of pregnancy.     

Characterization of three mouse monoclonal antibodies that react with high-molecular-mass glycopeptides isolated from F9 mouse teratocarcinoma cells

Three IgM mouse monoclonal antibodies, NL-9, Thy-22, and HL-5, which were produced primarily against human hematopoietic cells, were tested for their reactivity with various mouse cell lines and were found to react predominantly with mouse embryonal carcinoma cells. Thy-22 reacted with 2-cell-stage mouse embryos, whereas the other two antibodies were not reactive at this stage. All three antibodies, however, reacted with 8-cell-stage embryos. At the blastocyst stage, Thy-22 reacted with the entire surface of the trophectoderm cells, whereas the reactivity of NL-9 and HL-5 was weaker and was polarized on the mural trophectoderm. Immunohistological examination of 6th-day mouse embryos using anti-complement immunofluorescence demonstrated that the embryonic ectoderm was positive for all three antibodies: the reaction of NL-9 and Thy-22 was uniformly distributed over these cells, whereas HL-5 predominantly stained the luminal aspects of the cells lining the proamniotic cavity. Visceral-endoderm cells and trophoblastic cells were positive with all three monoclonal antibodies, whereas the parietal endoderm, extraembryonic ectoderm, and ectoplacental cone were negative. In 19th-day fetuses and adult tissues, certain epithelial cells were stained by these three antibodies. The biochemical nature of the antigens detected was also investigated. Farr's assay showed that both NL-9 and Thy-22 precipitated approximately 10% of the high-molecular-mass glycopeptides isolated from F9 cells, while HL-5 reacted with about 5% of these glycopeptides. The reactivity of the three antibodies against the glycopeptides was completely inhibited by the presence of X-hapten-conjugated silica.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new radioimmunoassay for human chorionic gonadotropin using monoclonal antibody

A new radioimmunoassay (RIA) for human Chorionic Gonadotropin (hCG) was developed using murine monoclonal antibody to the beta-subunit of hCG (beta-hCG). The IgG fraction of the monoclonal antibody which did not react with 125I-beta-hCG was purified from hybridoma ascites, and covalently coupled to Sepharose 4B. This solid-phase antibody was incubated with standard hCG or serum sampled for 48 hours. The reaction medium was then removed by centrifugation and 125I-beta-hCG and anti-beta-hCG rabbit polyclonal antibody were added to the precipitate. The alcohol precipitation method was used for separating "bound" and "free" forms in the second reaction. The sensitivity for hCG in this assay system was 0.5 mIU/ml serum and the cross-reactivity with human Luteinizing Hormone (hLH) was 0.4%. This assay system was shown to be clinically applicable. Serial serum samples from two patients with trophoblastic disease were assayed and minute amounts of hCG, which could not be determined by conventional assay methods, could be assayed by this new RIA.     

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).     

Alteration of sperm thiol-disulfide status and capacitation in the guinea pig

Epididymal spermatozoa of the guinea pig were incubated under conditions known to promote a rapid synchronous capacitation in a large proportion of the spermatozoa (Ca²⁺-free medium with lysophosphatidylcholine, LC) or in Ca ²⁺-free medium without LC. To study the effects of altered thiol-disulfide status and content, incubations were conducted with reagents that maintain and increase thiol groups (DTT, GSH), maintain and increase disulfide groups (diamide, GSSG), or which irreversibly block thiol groups by alkylation (NEM). The permeable DTT inhibited LC-induced capacitation and at high concentrations diminished the percentage of acrosome reactions in capacitated spermatozoa. The permeable diamide exhibited a stimulatory effect upon capacitation. The largely impermeable GSH and GSSG exhibited effects similar to their respective permeable counterparts but their effects were moderate and required extremely high concentrations. The DTT inhibition of LC-induced capacitation was reversible by washing and a further 1 hr incubation. In this final incubation after removal of DTT by washing, LC was absent too so its stimulatory effect must have been accomplished prior to washing and in the presence of DTT. NEM-alkylation of the existing thiol population did not affect LC-induced capacitation but alkylation of the increased thiol population after prior DTT treatment was inhibitory of capacitation. These results suggest that the maintenance and/or formation of disulfide groups on enzymes or structural proteins may be a component of the capacitation process. In contrast, the formation and maintenance by alkylation of increased thiol groups but not the maintenance of existing thiol groups, is inhibitory of capacitation. The relevance of these findings to a role for a thiol-sensitive proteinase in capacitation is discussed.     

The structure of the phytoplankton community in the subsurface chlorophyll maxima in the western North Pacific Ocean

The phytoplankton taxonomic composition of the subsurface chlorophyllmaximum layer (SCM) was compared with that of the surface layerin July, September and November 1979 in the western North PacificOcean. Besides the use of chemical fixatives for preservablephytoplankton, serial dilution culture method was employed fornon-preservable flagellates and monads, i.e., fragile forms.The SCM was characterized by a high species diversity of preservablephytoplankton and by the numerical dominance of fragile forms.Among the fragile forms, Micromonas and Ochromonas were dominant.This dominance is consistent with their viability at low lightintensity. Cluster analysis revealed that, for blue-green algae,coccolithophorids, silicoflagellates and diatoms, a marked differenceexisted in species assemblages between the SCM and the surfacebut there was no distinct difference in dinoflagellate assemblages.The difference between both layers is discussed, as relatedto the mechanisms of formation of the SCM. ¹Present address: Nodai Research Institute, Tokyo Universityof Agriculture, 1-1-1, Sakuragaoka, Setagaya-ku, Tokyo 156,Japan.