Effect of porcine placenta steroid extract on myogenic satellite cell proliferation, transdifferentiation, and lipid accumulation.

Intramuscular long-chain fatty acids (LCFAs) play an important role in energy production and initiation of mitochondrial oxidation of lipids. Herein, we report a natural porcine placenta steroid extract (PPSE) that stimulates transdifferentiation and lipid accumulation in bovine myogenic satellite cells (MSCs). The steroids hormones in PPSE were analyzed using enzyme-linked immunosorbant assay and presence of LCFA was established using gas chromatography. At 70% confluent growth, cells were treated with PPSE, LCFAs, transdifferentiation cocktail and commercially available steroid hormones. The working concentrations of all chemicals were manipulated similar to PPSE. The cells were observed for morphological changes and subjected to quantitative analysis of lipid deposition on Days 2, 4, and 6 of treatment. PPSE-treated MSCs exclusively transformed into lipid-accumulated adipose-like cells (ALCs). However, myotubes or adipocytes were formed in cells treated with other chemicals. Expression of different genes was studied to ascertain the molecular mechanism involved in ALC formation. CD36, fatty acid binding protein 4, and peroxisome proliferator-activated receptor-gamma were up-regulated. The expression of CD36 was established through immunocyto-chemical analysis. A viability assay was used to confirm the effect of PPSE on proliferation of MSCs. Hence, a natural steroid extract from porcine was found as a nontoxic mixture, which induces lipid accumulation and transdifferentiation of MSCs to ALCs. From the gene expression studies, it was established that the extract works almost in homogenous manner with other lipid inducers.

Effect of human placental extract on menopausal symptoms, fatigue, and risk factors for cardiovascular disease in middle-aged Korean women.

OBJECTIVE: In Korea, human placental extract (HPE) has recently been used to treat various diseases (chronic liver diseases, menopause syndrome, chronic fatigue, skin pigment diseases, etc.), but evidence-based studies are not yet sufficient. The aim of this study was to examine the effects of HPE on menopausal symptoms, fatigue, and risk factors for cardiovascular disease in middle-aged Korean women in a randomized controlled trial. DESIGN: Korean women, aged 40 to 64 years, with menopausal symptoms and fatigue were recruited as participants. The women were randomly assigned to a placebo group or an HPE group. The HPE group received subcutaneous injections of HPE in the abdomen for 8 weeks, whereas the placebo group received normal saline. Then, the Menopause Rating Scale, and Fatigue Severity Scale, and Visual Analog Scale were administered, and risk factors for cardiovascular disease were assessed. RESULTS: The Menopause Rating Scale total baseline score was not different between the two groups; however, the score of the HPE group decreased significantly at 8 weeks compared with that of the placebo group (P = 0.033). Fatigue Severity Scale and Visual Analog Scale scores of the placebo group did not change, whereas the scores of the HPE group decreased significantly during the study period (Fatigue Severity Scale, P = 0.002; Visual Analog Scale, P = 0.001). The baseline 17beta-estradiol level was not significantly different between the two groups, but the 17beta-estradiol level of the HPE group was significantly increased at 8 weeks compared with that of the placebo group (P = 0.031). No changes in risk factors for cardiovascular disease were observed in either group. CONCLUSIONS: Menopausal symptoms and fatigue in middle-aged Korean women improved after 8 weeks of HPE treatment, whereas risk factors for cardiovascular disease did not change during the study period.

Analysis of fluorescence excitation-emission matrix of multicomponent drugs. A case study with human placental extract used as wound healer.

The possibility of finger printing of multi component drugs, where a number of components are fluorescent, has been explored after generation of excitation-emission matrix (EEM) contour plots. With nicotinamide adenine dinucleotide, reduced form (NADH) and N-acetyl tryptophanamide (NATA) serving as reference compounds, the contour widths were found to be proportional to fluorophore concentration. EEM generated from different batches of processed human placental extract, used as wound healer, in presence and absence of extraneous fluorescence probes like 1-anilino 8-naphthalenesulphonic acid (1-ANS) or dansyl chloride were found to be highly consistent. Variation of contour widths of these EEM after excitation at 280 or 340 nm were essentially zero subject to the limit of detection of variation (n = 5). This high degree of consistency between batches has been supported from analysis with such independent techniques like UV-absorption spectra, Fourier Transformed IR spectra, TLC and reverse phase HPLC. The advantages of EEM analysis have been discussed.

Spectroscopic and chromatographic evidences of NADPH in human placental extract used as wound healer.

An aqueous extract of human placenta, which is used as wound healer, has been investigated in terms of fluorescence properties. When excited at 340 nm, it results in fluorescence emission having maxima around 436 nm, which is fairly specific for nicotinamide adenine dinucleotide, reduced form (NADH) and nicotinamide adenine dinucleotide phosphate, reduced form (NADPH). The excitation spectra, having emission at 440 nm, show patterns comparable to these nucleotides. Thin layer chromatography and reversed-phase (RP) HPLC confirm presence of only NADPH in the extract. The emission and excitation patterns of NADPH purified after HPLC resemble exactly with the reference compound. Its content has been estimated to be 0.018 +/- 0.003 mg/ml based on fluorescence emission with respect to a standard calibration curve (n=6). Biological functionality of NADPH in the extract has been confirmed by glutathione reductase assay (n=5).

TRH-like peptides in prostate gland and other tissues.

This minireview is aimed to recapitulate the occurrence of TRH-like peptides in the prostate gland and other tissues and to discuss their known functions in the organism. The hypothalamic thyrotropin-releasing hormone (TRH) was the first chemically defined hypophyseotropic hormone with the primary structure pGLU-HIS-PRO.NH2. However, the presence of extrahypothalamic TRH-immunoreactive peptides was reported in peripheral tissues including the gastrointestinal tract, placenta, neural tissues, male reproductive system and certain endocrine tissues. It was supposed that this TRH immunoreactivity can partially originate from TRH-homologous peptides and that these peptides have significant cross-reactions with the antibody specific against authentic TRH. This assumption was confirmed by the identification of prostatic TRH immunoreactivity as pyroGLU-GLU-PRO.NH2 using fast atom bombardment mass spectrometry and gas phase sequence analysis. TRH-like peptides are characterized by substitution of the basic amino acid histidine (related to authentic TRH) for neutral or acidic amino acids, such as glutamic acid, phenylalanine, glutamine or tyrosine. The physiological role of TRH-like peptides in peripheral tissues is not precisely known, but they possess a C-terminal amide group which is characteristic for many biologically active peptides. The occurrence of these peptides in the male reproductive system can influence male fertility. They are also closely related to circulating thyroid and steroid hormones. There might be an important connection of TRH-like peptides to the prostatic local autocrine/paracrine network mediated by extrahypothalamic TRH immunoreactivity corresponding to TRH-like peptides and extrapituitary thyrotropin (TSH) immunoreactivity also found in the prostatic tissue. A similar system of intraepithelial lymphocyte hormonal regulation due to the local paracrine network of TRH/TSH has been described in the gastrointestinal tract. The local network of TRH-like peptides/TSH may be involved in possible regulation of prostatic growth.

Characterization and quantitation of the active polynucleotide fraction (PDRN) from human placenta, a tissue repair stimulating agent.

The polydeoxyribonucleotide (PDRN) fraction is an extract which forms the active component in a new formulation of the drug Placentex (a tissue repair stimulating agent), obtained from human placenta through an original proprietory extraction method. From a comparison of the UV, NMR and IR spectra of this fraction (before and after nuclease treatment) with that of a similar standard (Sigma D1501), it was shown that the active substances in the PDRN fraction mainly consist of a mixture of DNA fragments. By gel electrophoresis, the molecular weights of the DNA fragments were shown to range from 50 to 2000 base pairs. Finally, an HPLC method is described, based on an anion-exchange material capable of determining the amount of PDRN in different batches of the extract, which varied from 80 to 90%.

Influencia de las condiciones de cultivo de placenta humana sobre la producción de medios condicionados placentarios / Influence of culture conditions on the production of human placent conditioned medium

En este trabajo se propone una técnica alternativa para la obtención de medios condicionados derivados de placenta humana. Se cultivaron homogenatos placentarios en Mc Coy's 5A suplementado con suero fetla bovino al 5 por ciento y 25mM de HEPES a 37oC. Luego de seis días, los cultivos fueron sacrificados, verificándose la esterilidad y la viabilidad celular. El sobrenadante fue centrifugado a 22.000 g 30 min a 4oC y esterilizado por filtración (0,22 um), denominándolo Medio Condicionado de Placenta Humana (MCPH). Se probó la actividad de los extractos, así obtenidos, en cultivos de linfocitos periféricos de mujeres con tres hijos, con la adición de MCPH al 5 por ciento ,10 por ciento y 20 por ciento y se determinó el índice de Transformación Blástica Linfocitaria (ITBL). La máxima estimulación se observó en los cultivos linfocitarios que contenían MCPH al 10 por ciento , siendo estos valores más uniformes que losa producidos con MCPH al 10 por ciento obtenidos por otra técnica. en conclusión, con esta metodología es posible obtener medios condicionados placentarios con una actividad estimulante más uniforme, independientemente de la placenta de la cual provengan

Influencia de las condiciones de cultivo de placenta humana sobre la producción de medios condicionados placentarios / Influence of culture conditions on the production of human placent conditioned medium

En este trabajo se propone una técnica alternativa para la obtención de medios condicionados derivados de placenta humana. Se cultivaron homogenatos placentarios en Mc Coys 5A suplementado con suero fetla bovino al 5 por ciento y 25mM de HEPES a 37oC. Luego de seis días, los cultivos fueron sacrificados, verificándose la esterilidad y la viabilidad celular. El sobrenadante fue centrifugado a 22.000 g 30 min a 4oC y esterilizado por filtración (0,22 um), denominándolo Medio Condicionado de Placenta Humana (MCPH). Se probó la actividad de los extractos, así obtenidos, en cultivos de linfocitos periféricos de mujeres con tres hijos, con la adición de MCPH al 5 por ciento ,10 por ciento y 20 por ciento y se determinó el índice de Transformación Blástica Linfocitaria (ITBL). La máxima estimulación se observó en los cultivos linfocitarios que contenían MCPH al 10 por ciento , siendo estos valores más uniformes que losa producidos con MCPH al 10 por ciento obtenidos por otra técnica. en conclusión, con esta metodología es posible obtener medios condicionados placentarios con una actividad estimulante más uniforme, independientemente de la placenta de la cual provengan(AU)

La placenta humana: estudio bibliográfico de su composición química y sus extractos / Human placenta: bibliographic study of its chemical composition and extracts

Se presentan los resultados de la revisión bibliográfica realizada hasta el año 1987 sobre la composición química de la placenta humana, así como de los diferentes extractos reportados que tienen aplicación en la terapéutica o en la elaboración de cosméticos. Una gran parte de la literatura consultada contempla solamente publicaciones referativas como el Chemical Abstracts. Debido a lo amplio del tema a tratar, sólo se profundiza en algunos tópicos que se han considerado los más importantes, mientras que en otros se han mencionado los trabajos sin detallar los mismos. La placenta es un órgano que contiene gran variedad de sustancias que forman un conjunto biológico bastante complejo. En este trabajo se exponen aquellas de mayor importancia de las tantas reportadas en la literatura

La placenta humana. estudio bibliográfico de su composición química y sus extractos / Huan placenta: bibliographic study of its chemical composition and extracts

Se presentan los resultados de la revisión bibliográfica realizada hasta el año 1987 sobre la composición química de la placenta humana, así como de los diferentes extractos reportados que tienen aplicación en la terapéutica o en la elaboración de cosméticos. Una gran parte de la literatura consultada contempla solamente publicaciones referativas como el Chemical Abstracts. Debido a lo amplio del tema a tratar, sólo se profundiza en algunos tópicos que se han considerado los más importantes, mientras que en otros se han mencionado los trabajos sin detallar los mismos. La placenta es un órgano que contiene gran variedad de sustancias que forman un conjunto biológico bastante complejo. En este trabajo se exponen aquellas de mayor importancia de las tantas reportadas en la literatura

Immunoactive products of human placenta (III): Characterization of an inhibitor affecting lymphocyte proliferation.

We have investigated the effects of supernatants from human placenta explant cultures on lymphokine dependent T cell proliferation using mainly the CTLL-2 reference murine cell line. A profound, dose dependent, inhibition of both IL-2 and IL-4-dependent cell proliferation was observed. In addition, supernatants from human placental cultures inhibited B cell proliferation, IL-5-driven proliferation of B13 cells, and IL-6-induced proliferation of 7TD.1. Conversely, the supernatants from human placental cultures enhance the growth of human embryonic fibroblasts. Characterization of the material by HPLC yielded a 68-70 kDa peak at pH 7.4, but the peak activity was at a smaller molecular weight (20-25 kDa) if pH 2.9 acetic acid, KC1 buffer was used. The same fractions were inhibitory for IL-2 and IL-4 driven proliferation of CTLL-2 cells. We conclude that the substances tested are acting as a general growth inhibitor for lymphocytes.

[Human placental oxytocin and its increase by prostaglandin E2 and F2 alpha].

To investigate the physiological roles of human placental oxytocin (OT)-like substance, partial purification of human placental extract and placental tissue culture were carried out. 1. Both oxytocic immunoreaction in our own specific radioimmunoassay and bioaction on rat uterine contraction were observed in the larger (MW greater than or equal to 5,000) and the smaller molecular part (MW not equal to 1,000) in partially purified placental extracts by gel-filtration using Sephadex G-25. 2. Prostaglandin E2 and F2 alpha enhanced the immunoreactive OT in the cultured human term placental tissue at doses of 1-100 micrograms/ml. These findings are considered to be significant in basic studies of human placental OT-like substance.

The application of affinity chromatography for the separation of "high Km" and "low Km" 5'-nucleotidase and other AMP metabolizing enzymes.

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.

Evidence for "low Km" and "high Km" soluble 5'-nucleotidases in human tissues and rat liver.

Two kinetically distinct purine 5'-phosphomono-esterase activities were isolated from soluble fractions of human placenta, cultured human T- and B-lymphoblasts and rat liver using AMP-sepharose chromatography. We have defined these activities as "high Km" and "low Km" 5'-nucleotidase. The relative content of "high Km" and "low Km" activities in the tissues studied ranged from 2 to 264. The optimum pH of "low Km" 5'-nucleotidases ranged from 7.4 to 9.0, Km for AMP from 7 to 15 uM and for IMP from 10 to 26 uM. ATP and ADP were inhibitors of "low Km" enzymes with the apparent Ki values of 55 to 20 uM and 8 to 20 uM for ATP and ADP, respectively. "High Km" 5'-nucleotidases had an optimum pH at 6.5, Km for IMP of 0.3 to 0.5 mM and Km for AMP of 1.0 to 9.4 mM. "High Km" enzymes were activated by ATP with A0.5 values, of 1.7 to 2.3 mM at 100 microM IMP. The data indicate that soluble "low Km" and "high Km" 5'-nucleotidases coexist in mammalian cells and fulfill different functions. These observations suggest a complex system for the regulation of AMP and IMP dephosphorylation.

Importância da localizaçäo placentária na análise das artérias uteroplacentárias pela Dopplerfluxometria / The importance of the placental localization in the analysis of uteroplacental arteries by Dopplerfluxometry

Faz-se breve discussäo sobre as modificaçöes na análise da forma da velocidade da onda, pela Dopplerfluxometria, das artérias uterinas na gestaçäo e sua importância no diagnóstico precoce da toxemia e do crescimento intra-uterino retardado. Na análise do fluxo das artérias uteroplacentárias, chama-se a atençäo para a influência da localizaçäo da placenta

Platelet inhibitory activity in placentas from normal and abnormal pregnancies.

Retroplacental blood flow requires inhibition of coagulation in the absence of an endothelial lining. We confirmed that trophoblast releases an inhibitor of platelet aggregation which functions via degradation of adenosine diphosphate. This inhibitor appears to be deficient in some pregnancies with abruptio placentae and intrauterine growth retardation. Unimpeded retroplacental blood flow may depend upon the local inhibition of platelet aggregation. Placental tissue contains an inhibitor of platelet aggregation which appears to be an adenosine diphosphatase distinct from heat-stable alkaline phosphatase. Placental tissue from patients with abruptio placentae contains abnormally low amounts of this enzyme.

[Human placental tissue: a source of natural hematopoietic regulators]. / Le tissu placentaire humain: une source de régulateurs hématopoiétiques naturels.

In this paper, it has been shown that human placental tissular extracts are a potent source of natural haemopoietic growth factors. The colony-stimulating activities (CSA) recovered by extraction from washed placental pulp were active both on human and murine haemopoietic progenitors, from monocytic and granulocytic lineages. Crude tissular extracts contained CSA titers at least ten fold the titers usually found in placenta culture media. Placenta is the only human tissue easily available for the study of natural tissue-bound haemopoietic regulators. Extraction on an industrial scale, as proposed for the first time in this paper, should also benefit the identification and purification of new minor molecular classes of growth and maturation factors or inhibitors involved in human haemopoiesis.

Characterization of glycoprotein hormone free alpha-subunit from human pituitary and placenta extracts.

Standard alpha-subunit dissociated from glycoprotein hormones differs from individual (free) alpha-subunit found in sera or in cell culture media; secreted free alpha-subunit is larger, more acidic and lacks the ability of recombining in vitro with standard hCG-beta. It is unclear whether the large free alpha-subunit is only a secretory product or whether it is also present in tissue. Herein were studied the molecular size, the isoelectric pH, and the recombining activity of free alpha-subunit obtained from pituitary and placenta extracts. Sephadex exclusion chromatography showed the presence of both a large and a small form, and a changing large/small free alpha-subunit ratio in the various extracts. Most of the large form obtained from placenta extracts electrofocused into two peaks of pI 4.8 and 5.1. The large form showed no recombining activity with standard hCG-beta while the small free alpha-subunit recombined as well as did standard hCG-alpha. The observation of three common characteristics (a larger size, a pI 4.8, and a lack of recombining activity) suggests a similarity between the large secreted form and a fraction of the free alpha-subunit in tissue.

Biochemical fractionation of conditioned media containing factors which support in vitro proliferation of high proliferation potential colony forming cells.

Synergistic activity (SA), which is the capacity to support the in vitro proliferation of High Proliferation Potential Colony Forming Cells (HPP-CFC's), has been fractionated from conditioned media by standard biochemical techniques. On fractionation of both Wehi conditioned medium (Wehi CM) or human placental conditioned medium (HPCM) by several techniques, multiple peaks of SA were observed. Protein from HPCM expressing SA, ranged from 9,000-28,000 in molecular weight as determined by gel filtration. Chromatofocusing crude HPCM separated two activities possessing isoelectric points of 5.7 and 5.3. In crude Wehi CM, inhibitors or toxicity occasionally masked SA which partially adsorbed to Con-A sepharose and could be eluted with competing sugar. Further fractionation of this material on DEAE-sepharose separated three peaks of activity. Pooling the active fractions from the first two peaks gave a preparation that was up to 179 fold more active than the crude material.