Mitochondria from human term placenta. I. Isolation and assay conditions for oxidative phosphorylation.

Human term placental mitochondria were resolved by differential centrifugation into three fractions, heavy mitochondria, light mitochondria and a third, less dense fraction. Approximately equal amounts of mitochondrial protein were found in the three fractions. These mitochondrial preparations differed in physical properties. ATPase and "ADPase" content and oxidative capacities. Assay conditions were developed which permitted the polarographic measurement of respiration and coupled phosphorylation carried out by all three mitocondrial preparations despite the variable nucleotide-phosphate phosphatase activities present. With heavy mitochondria, rates of respiration were consistently higher than those previously reported for unfractionated placental mitochondria. Respiratory control ratios were comparable to those of mitochondria from other steroid hormone-producing endocrine tissues and ADP/O ratios approaching the theoretical maxima were obtained. Both lighter placental mitochondrial fractions displayed somewhat lower respiration rates and respiratory control but their primary defect was a selective uncoupling of the third site of energy conservation. Modification of isolation procedures were evaluated in terms of quantitative yield and functional activity of the three fractions.     

Mitochondria from human term placenta. II. Characterization of respiratory pathways and coupling mechanisms

Pathways of electron transport utilized for respiration in human term placental mitochondrial preparations were differentiated and characterized through the use of classical respiratory chain inhibitors and multiple sources of reducing equivalents. Mechanisms of associated energy conservation and utilization were examined in these preparations with uncouplers and inhibitors of phosphorylation.

Inhibition by rotenone, antimycin A and cyanide established the classical electron transport chain as the major pathway of respiration with glutamate and succinate as substrates. Approximately 20% of glutamate-supported respiration was insensitive to inhibitors and may proceed by the cytochrome P-450 linked pathway of electron transport. Approximately 50% of ascorbate-N,N,N′,N′-tetramethyl-p-phenylenediamine supported respiration was insensitive to 10⁻³ M cyanide and must utilize an undefined by-pass of cytochrome oxidase. A rotenone- and antimycin-insensitive, exterior pathway for NADH oxidation was demonstrated which could be artificially linked by exogenous cytochrome c to the cytochrome oxidase region of the classical electron transport system. Glycerol 3-phosphate also supported oxidative phosphorylation yielding ADP/O ratios of 2.

Respiration of placental mitochondria was stimulated by 2,4- dinitrophenol and gramicidin. With succinate, dinitrophenol-stimulated respiration exceeded that obtain-red in the presence of ADP. Oligomycin and atractyloside prevented the stimulation of respiration by ADP. Thus, respiration appeared coupled through normal mechanisms to ATP formation and ion transport. A preferential coupling of respiration to the energy-utilizing processes of steroid hormone biosynthesis may exist.     

Mitochondria from human term placenta. II. Characterization of respiratory pathways and coupling mechanisms.

Pathways of electron transport utilized for respiration in human term placental mitochondrial preparations were differentiated and characterized through the use of classical respiratory chain inhibitors and multiple sources of reducing equivalents. Mechanisms of associated energy conservation and utilization were examined in the preparations with uncouplers and inhibitors of phosphorylation. Inhibition by rotenone, antimycin A and cyanide established the classical electron transport chain as the major pathway of respiration with glutamate and succinate as substrates. Approximately 20% of glutamate-supported respiration was insensitive to inhibitors and may proceed by the cytochrome P-450 linked pathway of electron transport. Approximately 50% of ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine supported respiration was insensitive to 10-3 M cycanide and must utilize an undefined by-pass of cytochrome oxidase. A rotenone- and antimycin-insensitive, exterior pathway for NADH oxidation was demonstrated which could be artificially linked by exogenous cytochrome c to the cytochrome oxidase region of the classical electron transport system. Glycerol 3-phosphate also supported oxidative phosphorylation yielding ADP/O ratios of 2. Respiration of placental mitochondria was stimulated by 2,4-dinitrophenol and gramicidin. With succinate, dinitrophenol-stimulated respiration exceeded that obtained in the presence of ADP. Oligomycin and atractyloside prevented the stimulation of respiration by ADP. Thus, respiration appeared coupled through normal mechanisms to ATP formation and ion transport. A preferential coupling of respiration to the energy-utilizing processes of steroid hormone biosynthesis may exist.     

Isolation and characterization of a translation inhibitor from human term placenta

An inhibitor of protein synthesis has been isolated from free cytoplasmic ribonucleoprotein particles of human term placenta. The inhibitor is resistant to phenol, DNase, proteinase K, and heating at 100 degrees C, but is sensitive to alkaline hydrolysis. These data suggest that the inhibitor is RNA. Experiments provide evidence that this preparation contains no RNase contaminant and does not induce an RNase in this assay system. Three lines of evidence suggest that the inhibitor acts at the initiation of protein synthesis in the wheat germ translation system. First, a lag occurs before cessation of translation when the inhibitor is added to translating polyribosomes. This lag is identical to that seen upon the addition of aurintricarboxylic acid, a known inhibitor of initiation. Second, sucrose gradient analyses demonstrate that, when the inhibitor is present at the start of translation, 40 S complexes form, but neither 80 S complexes nor polyribosomes are seen. Third, gradient analyses show that, when the inhibitor is added to translating polyribosomes, 40 S complexes accumulate with a progressive loss of polyribosomes. Finally, the extent of inhibition depends upon the amount of wheat germ extract added to the reaction mixture and not the amount of mRNA present. This suggests an interaction between the inhibitor and a component of the wheat germ extract.     

Isolation and in vitro translation of human placental lactogen messenger RNA from human term placenta.

A messenger activity for HPL was identified in normal human term placentas. The mRNA was translated in rabbit reticulocyte cell-free system. The HPL synthesized was quantified by a specific immunoprecipitation and further identified by electrophoresis on sodium dodecyl sulfate polyacrylamide gel. The HPL synthesized in the reticulocyte lysate exhibited a molecular weight between 20,000 and 22,000 daltons similar to the active hormone. The messenger RNA activity for HPL corresponded to a sedimentation coefficient of 11-12 S. Furthermore the messenger activity for HPL was preferentially associated with membrane bound polyribosomes than with free polyribosomes.     

Isolation and characterization of trophoblasts from enzymatic explants of human term placenta

In the present study, we describe a new method of isolation and culture of human villous and extravillous trophoblasts from term placenta. The cultivation of trypsinized placental villous tissue explants, followed by the isolation of cells from outgrowth islets allows for obtaining a cytotrophoblast subpopulation that is free from contamination by other cell types. Compared to other methods, our protocol is mild, simple and effective, does not request costly reagents and provides isolation of the mononuclear cytotrophoblast cell populations free from contamination by other types of placental cells. The isolated cells proliferated and formed a pleomorphic monolayer, where cells fused into a small number of binuclear or polynuclear syncytiotrophoblasts. Isolated cytotrophoblast cells expressed the specific epithelial intermediate filament cytokeratin 7 (CK7), the epithelium-specific cell–cell adhesion molecule E-cadherin and were CD9-, CD45- and vimentin-negative. Cyto- and syncytiotrophoblasts obtained by this method can be used as a model or tool for the fundamental research of differentiation and function of human placental cells, and can provide a new understanding of drug distribution in placenta. Their combination with other in vitro cell models can be useful for studying a variety of other aspects concerning placental functions, which will provide new knowledge for understanding immunology, endocrinology and development of placenta.     

AMP-deaminase from human term placenta

AMP-deaminase from human term placenta was chromatographed on a phosphocellulose column and physico-chemical and immunological properties of the purified enzyme were investigated. At physiological pH7.0, in the absence of regulatory ligands (control conditions) studied AMP-deaminase manifested sigmoid-shaped substrate saturation kinetics, with half-saturation parameter (S_0.5) value of about 7 mM. Addition of important allosteric effectors (ATP, ADP or orthophosphate) modified kinetic properties of studied AMP-deaminase, influencing mainly the value of S_0.5 parameter. Micromolar concentrations of stearylo-CoA inhibited potently the enzyme making it no longer sensitive towards 1 mM ATP-induced activation. SDS-PAGE electrophoresis of the purified enzyme revealed presence of 68 kDa protein fragment, reacting with anti-(human) liver AMP-deaminase antibodies. Experimental results presented indicate that liver type of AMP-deaminase is an enzyme form present in human term placenta.     

Isolation and cellular properties of mesenchymal cells derived from the decidua of human term placenta

The clinical promise of cell-based therapies is generally recognized, and has driven an intense search for good cell sources. In this study, we isolated plastic-adherent cells from human term decidua vera, called decidua-derived-mesenchymal cells (DMCs), and compared their properties with those of bone marrow-derived-mesenchymal stem cells (BM-MSCs). The DMCs strongly expressed the mesenchymal cell marker vimentin, but not cytokeratin 19 or HLA-G, and had a high proliferative potential. That is, they exhibited a typical fibroblast-like morphology for over 30 population doublings. Cells phenotypically identical to the DMCs were identified in the decidua vera, and genotyping confirmed that the DMCs were derived from the maternal components of the fetal adnexa. Flow cytometry analysis showed that the expression pattern of CD antigens on the DMCs was almost identical to that on BM-MSCs, but some DMCs expressed the CD45 antigen, and over 50% of them also expressed anti-fibroblast antigen. In vitro, the DMCs showed good differentiation into chondrocytes and moderate differentiation into adipocytes, but scant evidence of osteogenesis, compared with the BM-MSCs. Gene expression analysis showed that, compared with BM-MSCs, the DMCs expressed higher levels of TWIST2 and RUNX2 (which are associated with early mesenchymal development and/or proliferative capacity), several matrix metalloproteinases (MMP1, 3, 10, and 12), and cytokines (BMP2 and TGFB2), and lower levels of MSX2, interleukin 26, and HGF. Although DMCs did not show the full multipotency of BM-MSCs, their higher proliferative ability indicates that their cultivation would require less maintenance. Furthermore, the use of DMCs avoids the ethical concerns associated with the use of embryonic tissues, because they are derived from the maternal portion of the placenta, which is otherwise discarded. Thus, the unique properties of DMCs give them several advantages for clinical use, making them an interesting and attractive alternative to MSCs for regenerative medicine.     

Ribonuclease inhibitor from human placenta: rapid purification and assay.

The ribonuclease inhibitor from human placenta may be isolated in 65% yield (2.5 mg per placenta) in 2 days. The performance of the affinity chromatography on Sepharose-RNase A has been expedited through adaption of the spectrophotometric assay of ribonuclease toward 2',3'-cyclic cytidine monophosphate to determination of the inhibitor activity. The result of these improvements in procedure is increased availability of the ribonuclease inhibitor for study of its chemical properties (Blackburn, P., and Jailkhani, B.L. (1979) J. Biol. Chem. 254, 12488-12493) and for its inclusion into in vitro systems in which inhibition of mammalian neutral ribonucleases is desired.     

Isolation and characterization of thrombomodulin from human placenta

Protein C, a plasma protein, is activated by thrombin to a protease (protein Ca) that functions as a physiological anticoagulant. We have isolated thrombomodulin, a cofactor required for the rapid activation of protein C, from human placenta. The purification to near homogeneity was achieved using a crude Triton-solubilized protein fraction from a placental particulate fraction as starting material. Chromatography on DEAE-Sepharose removed 95% of the protein and achieved a 3-fold purification. Thrombomodulin was then isolated by affinity chromatography on a column of thrombin-Sepharose wherein the thrombin had been previously inactivated with diisopropyl fluorophosphate. The final preparation was purified 7,900-fold over the membrane extract with a yield of 7%. We obtained 0.88 mg of thrombomodulin from 100 g of membrane extract derived from 5 kg of placenta. The protein was nearly homogeneous as judged by electrophoresis on 10% acrylamide sodium dodecyl sulfate gels in the presence of 2-mercaptoethanol with an apparent Mr = 105,000. Western blot analysis without 2-mercaptoethanol gave an apparent Mr = 75,000. The protein stimulated the rate of protein C activation by thrombin 800-fold to 10 mol of Ca formed/min/mol of thrombin. Thrombin and thrombomodulin appear to form a 1:1 stoichiometric complex as judged from experiments where we measured the effect of varying the concentration of thrombomodulin with respect to thrombin and the converse, on rates of protein C activation. An antibody directed against rabbit lung thrombomodulin inhibited the human placenta protein by 66%, and the amino acid composition of the proteins from the two species was similar indicating that the proteins are closely related. The apparent Michaelis constant of the thrombin-thrombomodulin complex for protein C is 9.8 microM. The protein C activation reaction requires calcium ions and is maximal at 1 mM Ca2+; higher concentrations inhibited the reaction. Coagulation factor Va and factor Va light chain both stimulate the activity of human thrombomodulin 2- to 3-fold.     

Isolation and characterization of the transferrin receptor from human placenta.

The transferrin receptor has been isolated from human placenta using immunochromatography and affinity chromatography. The receptor is a glycoprotein and has a Mr = 90,000 on sodium dodecyl sulfate-gel electrophoresis in the presence of 2-mercaptoethanol. The isolated receptor is immunologically related to the transferrin receptor on the reticulocyte cell surface.     

Isolation and characterization of active ribosomal subunits from human placenta.

We have developed a method for the large-scale isolation of active ribosomal subunits from human placenta. The technique involves incubating crude ribosomes for 15 min at 37 degrees C with 0.2 mM puromycin in 50 mM Tris-HCl buffer, pH 7.6, 500 mM KCl and 3 mM MgCl2 followed by centrifugation at 5 degrees C in a BXV zonal rotor using an equivolumetric sucrose gradient in the same buffer, upon which 80--90% of all ribosomes are dissociated into subunits. The purified subunits differ in their chemical composition, the 60-S particle containing no more than 36% protein whereas the 40-S subunit consists of 43% protein. In poly(U)-directed protein synthesis, tested in a completely homologous cell-free system, one recombined couple polymerizes at 37 degrees C 12 to 17 phenylalanine residues at an initial rate of 0.7 residues per minute. However, free 80-S ribosomes obtained by puromycin treatment of the crude ribosomes and reassociation of the subunits without prior isolation, have an even higher incorporating activity (20--25 mol phenylalanine/mol of ribosome). At least 55% of the subunits were estimated to actively participate in the polyphenylalanine synthesis.     

Properties of Higher Plant Mitochondria. I. Isolation and Some Characteristics of Tightly-coupled Mitochondria from Dark-grown Mung Bean Hypocotyls

The mitochondria isolated from dark-grown mung bean hypocotyls oxidize succinate, l-malate, and externally added reduced nicotine adenine dinucleotide (NADH) with good respiratory control. While the pattern of respiration resembles that of animal mitochondria, there are 4 basic differences between the respiratory properties of mung bean and animal mitochondria: A) the ability to oxidize NADH, B) the pattern of succinate and malate oxidation, C) the rate of oxygen uptake, and D) the adenosine-5′-diphosphate to oxygen ratios.