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850 placental extracts were screened to detect the placental acid phosphatase variants among a few population groups of Andhra Pradesh, South India. Two new electrophoretic patterns were encountered, one among the Brahmins and the other among the Muslims.
The occurrence of methionine enkephalin (379 pg/g tissue), beta-endorphin (448 pg/g tissue) and Substance P (2.4 pg/g tissue) in human placental villus were demonstrated by sensitive and specific radioimmunoassays. Conditions for the bioassay of placental extracts for enkephalin-like activities using the rat vas deferens were described. Substance P did not interfere in this bioassay. Comparison of the enkephalin-like activities determined by bioassay and the content of beta-endorphin and methionine enkephalin determined by radioimmunoassays indicated that placental villus extracts contain other unidentified potent opioid-like peptides or substances. It is suggested that methionine enkephalin and/or beta-endorphin and Substance P regulate release of acetylcholine or hormones from placental villus. Alternatively, these peptides may regulate sensory transmission (pain impulses) locally from the distended uterus during pregnancy or from the vaginal tract during childbirth.
AMP-sepharose 4B has been widely used as a general ligand affinity chromatography for purification of AMP deaminase, 5'-nucleotidase, adenosine kinase and other adenine nucleotide metabolizing enzymes. Since these enzymes generally differ in their kinetic properties related to the values of Km for AMP and analogous compounds, it was assumed that there may be a specific elution pattern of some of the enzymes which would enable sequential elution from the column during a single run. Using 0.5 M NaCl, 10 mM ATP and 5 mM adenosine as eluting agents, it was possible to separate on AMP-sepharose column AMP deaminase "high Km" and "low Km" 5'-nucleotidase and adenosine kinase. Adenylate kinase, adenosine deaminase and nonspecific phosphatase did not bind to the column. Using human placental extract, AMP deaminase, "high Km" and "low Km" 5'-nucleotidase and adenosine kinase were purified 2.8, 2.9, 105 and 1240 fold, respectively. AMP deaminase and "high Km" 5'-nucleotidase were further separated using phosphocellulose column chromatography and the final purification was 227 and 143 fold, respectively. The specific activities of purified enzyme preparations were 9.1, 1.0, 0.4 and 0.5 mumols/min/mg protein of AMP deaminase, "high Km" 5'-nucleotidase and adenosine kinase, respectively. This approach provides a rapid method for initial purification of these enzymes from crude soluble extracts.
A radioimmunoassay (RIA) for the measurement of activin A, which is identical to erythroid differentiation factor (EDF), has been developed. A specific antiserum against activin A/EDF was raised in rabbits using a mixture of recombinant EDF and polyvinyl pyrrolidone. Of the compounds tested this polyclonal antibody cross-reacted only with bovine inhibin (3.2%) and human TGF-beta (4.2%). The least detectable value in this assay was 0.06 ng/tube. The within- and between-assay coefficients of variation at three different concentrations were 3.6-9.8% and 3.4-7.7%, respectively. Using this RIA, immunoreactive activin A/EDF levels in various biological fluids and tissues were examined. The dose-response curves of porcine follicular fluid and ovarian extract were parallel to the standard curve, and porcine follicular fluid contained high activin A/EDF immunoreactivity (1050 ng/ml). On gel chromatography of porcine follicular fluid, the major immunoreactivity was eluted in the same position as authentic activin A/EDF. Human placental extract and amniotic fluid had relatively high immunoreactive activin A/EDF levels (174 ng/g wet wt. and 63.9 ng/ml, respectively), but the dose-response curve of amniotic fluid was not parallel to the standard curve. Among rat tissues, the ovary showed the highest activin A/EDF immunoreactivity (163 ng/g wet wt.) much lower than that of porcine ovary (1020 ng/g wet wt.). Low immunoreactive activin A/EDF levels were detected in most parts of rat brain (8.7-14.2 ng/g wet wt.), except for the pituitary gland (70.0 ng/g wet wt.). The initial plasma half clearance time (t1/2) of exogenous activin A/EDF was 14 min in the rat and the plasma FSH concentration did not change significantly during this period. These results suggest that this RIA system has sufficient sensitivity and specificity to measure activin A/EDF concentrations in biological materials, and that the reproductive tissues are the main sources of activin A/EDF.
Plasma immunoreactive substance P (iSP) was measured by radioimmunoassay in 12 paired samples of maternal and cord blood, and in placental extracts. The mean plasma iSP concentration in the cord vein was 42.5 pg/ml, as compared to 77.3 pg/ml in the maternal peripheral vein plasma. There was no difference in the mean iSP concentration in the cord vein and artery. Although no iSP was detected in placental tissue, the placenta could not be excluded as the source of iSP in cord blood in view of the rapid degradation of synthetic SP by the placental in vitro.
Incubation with thioglycollate destroyed biologic activity of oxytocin, but left immunologic activity intact. Incubation with plasma of pregnant women at term or with placental extract destroyed biologic and immunologic activities. Dissociation of biologic from immunologic sites is suggested.
A gonadotrophic substance was extracted from placentae and subplacentae of guinea-pigs. Biological and immunological assays of these tissues obtained on Days 10-63 of pregnancy showed the highest mean concentration of the gonadotrophin on Day 18. Extracts chromatographed on Sephadex G200 had components active in the radioimmunoassay system for hCG, hCG-alpha and hCG-beta subunits. The elution profiles of these extracts were similar to those obtained with human term placentae and purified hCG and its subunits. The guinea-pig placental extracts also cross-reacted with an antiserum to the beta subunit of ovine LH, as did hCG. The biological activity of placental and subplacental extracts and of the hCG standard as neutralized by an anti-hCG serum. There is a strong similarity between hCG and the substance extracted from guinea-pig placentae which may therefore have a luteotrophic function similar to that of hCG.
Monospecific antiserum was produced with the use of a placental extract showing high activity of a proteinase which hydrolyses a synthetic substrate, N-benzoyl-D,L-arginine p-nitroanilide, used for measuring tryptic activity. An immunological study showed that antibodies were not generated against the component with this activity but against an antigen which was not related to several well-known pregnancy-associated proteins. Pregnancy sera contained an antigen which immunologically was completely identical to the antigen of placental origin. Single radial immunodiffusion showed an elevated level of reactive antigen in pregnant subjects: 70% of 333 cases were positive. Cross-reacting antigen was also detected in sera as well as ascitic fluid from cases of advanced ovarian cancer. Sephadex G-200 gel filtration indicated that the antigen has an apparent molecular weight of 94000, and immunoelectrophoresis showed the molecule to be of beta-mobility. These properties suggest that the substance may represent an additional pregnancy-associated protein entity. Partial purification and some immunological properties of this antigen are described.
Numerous heparin-binding growth factors active in different types of cells have recently been shown to belong to the family of fibroblast growth factors. Because these factors are active in some types of epithelial cells, we tested the activity of basic fibroblast growth factor (bFGF) from bovine brain in human keratinocyte cultures. bFGF stimulated thymidine incorporation and cellular proliferation in these cultures with half-maximal activity at approximately 60 pg/ml (4 X 10(-12) M). Stimulation of thymidine incorporation was associated with increased nuclear labeling after 22 h in the presence of bFGF under the same conditions used in the thymidine incorporation assay. bFGF was nearly as effective as epidermal growth factor (EGF) in stimulating keratinocyte growth and substantially less effective than crude placental extract, and was not additive with EGF in stimulating thymidine incorporation or proliferation of cells. The findings indicate that bFGF is a potent growth factor for keratinocytes.
The response profiles of fetal sheep adrenals to tropic stimulation have been examined ih vivo and in vitro. Isolated adrenal cells from sheep fetuses in early pregnancy (Day 50) reduced cortisol in response to ACTH, dibutyryl cyclic AMP and GTP. The response was minimal on Day 100, but reappeared near term. 17 alpha-Hydroxyprogesterone was converted to cortisol by adrenals of all ages, but pregnenolone and progesterone were converted to cortisol only in early and late, but not mid-pregnancy. These studies suggested that the mid-gestation loss of fetal adrenal responsiveness was associated with post-receptor/adenylate cyclase events and involved loss of 17 alpha-hydroxylase activity. Fetal adrenal function was activated by exogenous ACTH in vivo, and was reflected in an increase in the ratio of cortisol to corticosterone in fetal plasma and in augmented cortisol output in vitro from dispersed fetal adrenal cells. The results were consistent with an effect of ACTH administration on 17 alpha-hydroxylation. Fetal pituitary cells, prostaglandin E2, alpha-MSH and term placental extract are other potential (sources of) corticotropins, although further studies are required to delineate the nature and origin of the active substances, and/or their primary sites of action.
Oral Submucous Fibrosis (OSMF) is a disease of unknown etiology. A total of 96 patients with Oral Submucous Fibrosis have received four regimens of treatment--local dexamethasone, local hyaluronidase, local combination of dexamethasone and hyaluronidase, and local placental extract. The patients were followed up for a period varying from 3 months to 2 years. The group of patients receiving hyaluronidase alone showed quicker improvement in symptoms although its combination with dexamethasone gave somewhat better longer-term results. A new regimen for the treatment of submucous fibrosis is recommended.
In a survey of 860 unselected human placental extracts, three variants of mitochondrial glutamic oxaloacetic transaminase were found, all of which were common enough to be considered polymorphisms. Family studies showed that this enzyme is under the control of nuclear rather than mitochondrial DNA.
The Fc receptor activity in placental extracts prepared using EDTA and 2-mercaptoethanol was assayed using an indirect hemagglutination technique with sheep erythrocytes sensitized with rabbit IgG. The agglutinating activity of the extract was not affected by storage at -70 degrees C, by rapid freezing and thawing, by treatment with periodic acid, formaldehyde, neuraminidase, trypsin, pronase, or phospholipase C. Papain abolished the activity, indicating that the receptor is a protein. Reduction and alkylation had no effect on the agglutinating activity, indicating that -S-S-bonds are not important for binding. In the presence of 0.6 M NaCl the agglutinating activity was abolished, indicating that electrostatic interactions are of significance. The solubilized Fc receptor shows so many similarities to the previously studied in situ Fc receptor that they are probably identical.
The placenta is an endocrinologically active organ and expresses an important number of polypeptide hormone genes. Although oxytocin (OT)-like immunoreactivity has been detected in placental extracts by RIA, the precise nature and origin of placental OT has remained unclear. In the present study, we examined OT gene expression in rat placental tissue at various stages of gestation using northern blot analysis, polymerase chain reaction, in situ hybridization, HPLC, and immunocytochemistry. Northern blot analysis of RNA extracted from rat placenta revealed a single type of OT gene transcript (0.66 kilobases) which differed in size from hypothalamic OT transcripts (0.75 kilobases). Deadenylation of placental and hypothalamic messenger RNA (mRNA) showed that this size difference was due to differences in poly(A) tail lengths. Polymerase chain reaction amplification of placental and hypothalamic complementary DNAs using four different exon-specific primers provided no evidence for the existence of any additional structural differences between hypothalamic and placental OT-gene transcripts. Quantitative evaluation of northern blots showed that OT mRNA abundance per microgram of total RNA was stage specific and declined by a factor of 6 from day 14 to day 21 of gestation. In contrast to the marked variation of mRNA abundance, the OT peptide content, as measured by RIA, underwent no significant change during the time period studied and varied between 0.37-0.51 ng/g wet tissue wt. Characterization of placental OT immunoreactivity by HPLC and gel filtration identified two peaks of immunoreactivity: one peak (70% of immunoreactivity) corresponded to synthetic OT; whereas the other peak (Mr 11,000, 30% of immunoreactivity) represented a noncovalent association between OT and another molecule, consistent with the formation of a neurophysin/OT complex. By in situ hybridization and immunocytochemistry, we localized OT mRNA and OT immunoreactivity to cells of the trophoblastic epithelium covering the septa of the labyrinth as well as to cytotrophoblastic elements and giant cells of the maternally derived basal zone of the placenta. Placental OT may act locally, may interact with uterine OT receptors, or may play a role in fetal development.
Purification of human pregnancy-specific beta1-glycoprotein (SP1) and antigenically related proteins of sub-human primates (chimpanzee, rhesus monkey, cynomolgus and baboon) was achieved by means of an immunoadsorbent technique. The immunoglobulins of a rabbit antiserum to human SP1 were isolated on DEAE-cellulose and coupled to CNBr-activated Sepharose. This immunoadsorbent was used to bind human SP1, respectively monkey proteins immunochemically related to SP1 from placental extract fractions. After extensive washing the proteins were eluted by an acidic glycine buffer. Contaminating serum proteins could be removed by chromatography on hydroxyapatite columns. With this method it was possible to obtain SP1 and the antigenically related proteins of monkeys in good yield and in highly purified form. The proteins thus isolated from human and sub-human primate placentae were compared in their physicochemical and immunochemical properties. The amino acid and carbohydrate compositions of human SP1 and rhesus SP1 have been determined. In a biological test certain inhibitory effect of human SP1 on the mixed leukozyte culture (MLC) could be demonstrated.
The purpose of this work was to obtain placental extracts with the capacity of producing lymphocyte blastogenesis. Placental homogenates were incubated at 37 degrees C in McCoy's 5A supplemented with 5% fetal bovine serum and 25 mM Hepes. After a 6-day incubation period, the cultures were harvested and the sterility and cellular viability were controlled. The supernatant was centrifuged at 22,000g at 4 degrees C for 30 minutes. Sterilization was achieved by filtration (0.22 microns). This medium was denominated human placenta conditioned medium (HPCM-1). The other half of the original placenta was processed by the Burgess method and the extracts thus obtained were denominated HPCM-2. The activity of the extracts thus obtained was tested in peripheral lymphocyte cultures from women after the third pregnancy; 5, 10 and 20% HPCM-1 or HPCM-2 was added to these cultures and the index of lymphocyte blast transformation (ILBT) was determined. The highest stimulation occurred at 10% HPCM. The ILBT obtained with HPCM-1 at 10% was more constant than that obtained with HPCM-2 at the same concentration. In conclusion, it can be assumed that this methodology is appropriate to obtain conditioned media from human placenta with good blastogenic activity on peripheral lymphocytes in vitro.
Macromolecular forms of human placental lactogen have received little attention because it has been thought that such forms either compose only a small fraction of total immunoactive placental lactogen or are merely laboratory artifacts. We examined serum and placental tissue from women with normal pregnancy (first and third trimesters), serum and tissue from women with eutopic tumors (mole and choriocarcinoma), and serum from men with ectopic placental lactogen production. Samples were chromatographed on dextran gel (Sephadex G-100), and placental lactogen was measured in the fractions by radioimmunoassay. In all specimens examined, immunoactive placental lactogen was found at the void volume of the column (molecular weight greater than 150,000 daltons). This macromolecular placental lactogen comprised less than 4% of total placental lactogen in the third trimester, in mole, and in 16 of 18 first-trimester samples but was significantly higher, up to 19%, in the malignant cases. In two first-trimester placental extracts (but not in their matched sera) macromolecular placental lactogen was the dominant (greater than 45% of the total placental lactogen) immunoactive species. Authentic monomeric placental lactogen was not converted to macromolecular placental lactogen by repeated freezing and thawing. Third-trimester placental macromolecular placental lactogen was unstable; only 13% remained at the void on rechromatography. First-trimester placental macromolecular placental lactogen, on the other hand, was stable to rechromatography. The behavior of immunochemical dilutions of macromolecular placental lactogen from first-trimester placenta was similar to that of monomeric placental lactogen in the same sample. Macromolecular placental lactogen is probably not artifact, and it can comprise a large fraction of the total immunoactive placental lactogen in certain conditions.
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