A concentraçäo de interferon humano de membranas amnióticas: II - métodos químicos / Concentrations of the human amniotic membrane interferon: II - Chemical methods

Foi investigada a concentraçäo do interferon humano de membranas amnióticas (IFN-MA) por precipitaçäo através de métodos químicos. O IFN-MA foi produzido em âmnios infectados com o vírus da doença de Newcastle em meio contendo diferentes concentraçöes de soro. A atividade anti-vírica das preparaçöes foi titulada em células Vero infectadas com o vírus da encefalomiocardite de camundongo ou o vírus Sindbis. A precipitaçäo de IFN-MA, consideradas baixas. Empregando-se sulfato de âmnio em preparaçöes com 1//de soro, pode-se recuperar quantidades acima de 60//. O método mais adequado de concentraçäo de IFN-MA contendo 1//de soro foi a precipitaçäo com ácido tricloroactico e ressuspenso do matrial com tampäo clorto pH2, com recuperaçöes em torno de 100//da atividade. As tentativas de concentrar IFN-MA preparado sem soro por estes métodos näo permitiu uma precipitaçäo adequada

Purification of human anmiotic membrane interferon by affinity chromatography

A purificaçäo do interferon humano de membranas amnióticas foi realizada para se obter preparaçöes purificadas para sua caracterizaçäo físico-química, estudo de suas atividades biológicas e ensaios terapêuticos. O interferon foi produzido em doença de Newcastle ou o vírus Sendai. A determinaçäo da atividade anti-vírica foi feita em células Vero infectadas com o vírus da encefalomiocardite de camundongo. As preparaçöes de interferon foram concentradas por precipitaçäo por ácido tricloroactivo e cromatografadas em "Blue-Sepharose- concanavalina A, Sepharose ligada ao dipeptídeo triptofnio (trf) - triptofânio ao dipeptídeo triptofânio-tirosina (tyr). Com a "Blue-Sepharose", o pH ótimo de ligaçäo do interferon foi 5,4. A aplicaçäo em batelada mostrou que foram ligadas 37.000 unidades de interferon por ml de gel, sendo portanto, mais eficiente que a aplicaçäo em coluna, na qual somente foram ligadas 17.750 unidades por ml de gel. As fraçöes do pico de atividade foram purificadas 274 vezes, com 12//de recuperaçäo do interferon em um experimento, com um total de 78//de recuperaçäo e um fator de purificaçäo atingido foi de apenas 6,7; os ligantes Sepharose trp-trp e trp-tyr mostraram uma ligaçäo baixa do interferon, menor que 10.000 unidades por ml de gel

Human relaxin in the amnion, chorion, decidua parietalis, basal plate, and placental trophoblast by immunocytochemistry and northern analysis.

Immunocytochemistry and Northern analysis were used to show that relaxin is a product of intrauterine tissues of pregnancy. In addition, tissues from a patient without ovaries had similar results on both immunocytochemistry and Northern analysis as tissues from intact patients. The parietal decidua was clearly the major source of relaxin within the uterus and the relaxin mRNA (1.2 kilobases) from this tissue was detected with a 48-mer oligonucleotide probe designed to hybridize with both H1 and H2 relaxin gene transcripts. The mRNA isolated from the placental trophoblast was slightly smaller (1.1 kilobases), and the placental basal plate which has both maternal and fetal cells contained relaxin mRNAs of both sizes. Two monoclonal antibodies (Mabs) raised to synthetic human relaxin (H2) gave different patterns of localization in the fetal membranes, decidua and placenta. One Mab (RLX8) stained the chorionic cytotrophoblast in the fetal membranes and all of the cells in the placental basal plate. The other Mab (RLX6) stained the chorionic cytotrophoblast in some instances and selectively stained the decidua-like cells of the placental syncytiotrophoblast, whereas Mab RLX8 failed to detect this relaxin. Tissues obtained after spontaneous labor and delivery contained significantly less relaxin mRNA than tissues obtained at elective cesarean section without labor, but their hormone contents, as judged by immunocytochemistry, were not different. We conclude that the relaxin gene (H2) is expressed in intrauterine tissues, but that expression and hormone synthesis are not ubiquitous. Whether the relaxin gene H1 is expressed has not been determined.

Diamines and aminoalcohols: neutral solvents for native collagen.

A substantial fraction of interstitial collagens (types I, III, V and VI) can be dissolved from finely divided, connective tissues by homogenization in 0.5-1 M ethylene diamine and certain other organic amines neutralized by hydrochloric or other acids to pH 7-8.5. The amount of collagen solubilized from the tendons and other tissues of young animals is similar to that extracted by dilute acetic acid. In ethylene diamine hydrochloride solutions, native type I collagen denatures at 38 degrees and it has a sedimentation coefficient similar to that measured in acidic citrate solutions. From these amine hydrochloride solutions native collagen fibrils can be regenerated simply by tenfold dilution with water or by dialysis. The form of these precipitates is determined by temperature, pH, enzyme pretreatment of the collagen, and the dialysate salt concentrations and composition. Weak gels can be formed with less than 0.1 mg collagen per ml. Regenerated matrices containing types V and VI collagens, and with different fibril sizes can be used to support cell growth or to form sheets or hydrated gels possibly suitable for prosthetic uses.

Uterine estrogen receptors are increased by RU486 in late pregnant rhesus macaques but not after spontaneous labor.

Progesterone withdrawal as a mechanism for parturition in primates is controversial. The progesterone antagonist RU486, given in late pregnancy to rhesus monkeys at a dose of 47 mmol/kg.day (20 mg/kg.day), causes an increase in uterine activity, but not the expected increase in amniotic fluid prostaglandins or cervical dilatation. We, therefore, studied the effect of RU486 on estrogen receptor (ER) localization and concentration in reproductive tract tissues in rhesus monkeys during late gestation and after spontaneous labor at term. Distribution of ER in pregnant uterine tissues was studied by immunocytochemical techniques and quantified by a biochemical assay, both of which employed a monoclonal antibody specific for ER. ER was not present in amnion and chorion by immunocytochemical investigation; however, a significant increase in receptor staining was seen in decidua and myometrium after RU486 treatment compared to that in both pregnant control tissues and parturient tissues. Sucrose gradient assay of nuclear (n) and cytosolic (c) ER revealed a low level of ER (expressed as fmol of estradiol bound/mg of DNA) in pregnant and parturient decidua (pregnant: nER = 7.3 +/- 2.4, cER = 17.1 +/- 6.4; parturient, nER = 7.7 +/- 3.1, cER = 16.4 +/- 8.8) and myometrium (pregnant: nER = 21.7 +/- 4.1, cER = 20.8 +/- 5.3; parturient: nER = 30.0 +/- 2.8, cER = 10.7 +/- 6.7). In contrast, tissues collected from RU486-treated animals contained high levels of ER in decidua (nER = 52.3 +/- 16.8, cER = 240.5 +/- 145.3) and myometrium (nER = 77.0 +/- 19.2; cER = 66.5 +/- 31.6). We conclude that 1) the increase in ER in decidua and myometrium after RU486 treatment is the result of a decrease in the inhibitory action of progesterone on ER and documents the progesterone receptor antagonism by RU486 during induced myometrial contractility in late pregnant rhesus monkeys; 2) the absence of ER from amnion and chorion indicates that the normally observed increase in prostaglandin production by rhesus fetal membranes during labor is not mediated by ER; and 3) the absence of a change in the concentration of ER in decidua and myometrium from pregnant control monkeys and those in spontaneous labor indicates that an increase in ER (and, by inference, a withdrawal of receptor-mediated progesterone inhibition) is not part of the normal events in preparation for parturition in primates.

Connective tissue microfibrils. Isolation and characterization of three large pepsin-resistant domains of fibrillin.

Human amnion was solubilized using pepsin and the digest supernatant screened for fragments of fibrillin with a previously characterized monoclonal antibody (Sakai, L. Y., Keene, D. R., and Engvall, E. (1986) J. Cell Biol. 103, 2499-2509). One fragment (PF1), with an apparent molecular weight of 94,000, was isolated and characterized. Two other fragments, PF2 and PF3, were isolated and shown to be fragments of fibrillin by preparing a monospecific antisera to PF2 and a monoclonal antibody to PF3. Immunoelectron microscopy and immunoblotting showed that both antibodies were specific for fibrillin. Electron microscope pictures of rotary-shadowed PF1 and PF2 showed them to be short rod-shaped molecules while PF3 has a crab-like appearance and seems to be an aggregate of several fibrillin chain fragments. Amino-terminal amino acid sequencing of PF1 and PF2 gave single unique sequences. Each of the three antibodies used was specific for one fragment and peptide mapping of PF1 and PF2 showed that there was no significant amino acid sequence overlap. Aggregates of PF3 are described which provided insight into the assembly and macromolecular structure of fibrillin in microfibrils.

Epidermal growth factor receptors in rat placenta, amnion and yolk sac: characteristics of specific binding are dependent on gestational age.

Studies of [125I]-EGF binding to the rat placenta, amnion and yolk sac were carried out on days 14, 17 and 20 of gestation. In the placenta EGF binding was detectable on all 3 days; in the amnion EGF binding was undetectably low on day 14 but was present on days 17 and 20, while in the yolk sac EGF binding was undetectably low on all 3 days. Although Scatchard analysis of EGF binding to placental tissue raised the possibility of high and low affinity receptors, a statistical analysis of the ligand binding data was consistent with the presence of only one type of EGF receptor. The overall affinity of the receptors did not change with stage of gestation. However, the concentration of EGF receptors was lower in placental tissue on day 17 than on days 14 or 20 of gestation; the receptor concentrations were similar on days 14 and 20. It is suggested that EGF binding to the placenta, amnion, and yolk sac may reflect the levels of cell proliferation in those tissues in the latter part of gestation.

The growth of human fibroblasts and A431 epidermoid carcinoma cells on gamma-irradiated human amnion collagen substrata.

Human fibroblasts and A431 human epidermoid carcinoma cells were cultured on gamma-irradiated human amnion collagen as well as on plastic dishes and non-irradiated collagen coated dishes. The morphology, attachment, growth and short-term cytotoxicity of these culture conditions have been determined. Both irradiated and non-irradiated amnion collagen enhanced the attachment and proliferation of fibroblasts as compared to the plastic dishes. No differences in these properties were observed for A431 cells cultured on irradiated collagen when compared with culture on non-irradiated collagen substrates. Cytotoxicity assays showed that irradiated and non-irradiated collagens were not cytotoxic for either fibroblasts or A431 cells. The results demonstrated that amnion collagen irradiated at doses of 0.25-2.0 Mrads is optimal for cell growth.

A high performance liquid chromatography method for the analysis of 35S-cystine: application to the diagnosis of cystinosis.

An HPLC method for the measurement of radioactively labelled cystine is described. This method has been applied to studies of the uptake and retention of 35S-cystine by cultured cells. Radioactive cystine was measured, as a proportion of the non-protein labelled products in cultured cells incubated with medium containing 35S-cystine. Cells from healthy individuals contained less than 7% cystine whereas cells from cases of cystinosis contained at least 19% cystine. The method has been applied to the prenatal diagnosis of cystinosis. The use of flow radioactivity detection provides the advantages of rapid diagnosis and quantitation of metabolites.

Micro extraction of DNA from whole blood and amniocytes.

We describe methods for extracting genomic DNA from a small amount of whole blood or cultured amniocytes. Nuclear DNA was extracted from whole blood spotted on blotting paper. Relatively large molecules of DNA with the average amount of 7-9 micrograms was extracted from 1 ml of blood spotted and stored for at most two years, being roughly 1/3 of that extracted directly from fresh whole blood. The estimated minimum amount of whole blood that gives a suitable autoradiogram of Southern hybridization was 0.3 ml. Another series of amounts of whole blood or an amniocyte suspension were molded in low-melting agarose into an 100 microliter gel block. The DNA extracted from a block that was made from at least 0.25 ml of whole blood, or from 1.25 x 10(5) amniocytes (equivalent to 1/8 of the number of confluent cells in a 25 cm2 culture flask) resulted in one suitable Southern analysis. Both methods described here are applicable to the diagnosis of newborns and/or fetuses at risk of a genetic disease and to the diagnosis of a patient from whom a large amount of blood material is difficult to obtain. These methods also make a long-way transportation of the materials possible.

Enzyme activation of human prolactin: a potential basis for a bioassay.

Small cultures of human amniotic cells were preincubated for 24 h. Human prolactin was then added to the medium. After a further short period of incubation the tubes were chilled, the medium removed and the cells rinsed with saline. The tubes then received cold Tris-sucrose and were frozen, to disrupt the cells. After thawing, adenosine triphosphatase (ATPase) and p-nitrophenyl phosphatase (PNPase) were measured. Buffer was added containing either ATP or PNP and the tubes were incubated for 30 min. Inorganic phosphate released from ATP and p-nitrophenol was measured spectrophotometrically. Prolactin stimulated both enzyme activities. The ATPase log dose-response curve was linear between approximately 12.5 and 200 mIU/l. It was inhibited by ouabain. Isobutyl-1-methylxanthine inhibited the ATPase but not the alkaline phosphatase activity. One of these human amniotic cell enzymes may provide the basis for a sensitive bioassay for human prolactin.

Circadian uterine activity in the pregnant rhesus macaque: do prostaglandins play a role?

Eight rhesus macaques between 127 and 132 days of gestation had catheters implanted into maternal femoral vessels and the amniotic fluid cavity and were placed in a vest-and-tether system for chronic catheter maintenance. Uterine activity was continuously recorded, and paired maternal arterial blood and amniotic fluid samples were collected at 0900 h (AM) and 2100 h (PM) until delivery and analyzed for prostaglandin metabolites (PGFM and PGEM-II). A circadian pattern in uterine contractility was observed, with peak activity occurring between 1900 and 0100 h (p less than 0.001). No significant AM-PM differences were observed in maternal plasma PGFM (240 +/- 24 AM vs. 273 +/- 35 PM) or PGEM-II (537 +/- 41 AM vs. 484 +/- 34 PM) or amniotic fluid PGFM (360 +/- 72 AM vs. 287 +/- 70 PM) or PGEM-II (1626 +/- 383 AM vs. 1771 +/- 431 PM). All values represent mean +/- SEM, pg/ml. Additional samples were collected at 3-h intervals for 24 h at selected times during the study. This more intensive sampling protocol also failed to reveal any significant time trends in maternal plasma or amniotic fluid prostaglandins. Despite the lack of AM-PM differences, amniotic fluid PGFM and PGEM-II increased significantly as delivery approached (p less than 0.01). It appears that circadian uterine activity is not related to changes in maternal plasma or amniotic fluid prostaglandins. Although prostaglandins are responsible for the progression of labor, other factors may be involved in the generation of uterine activity rhythms prior to the initiation of labor.

Umbilical cord is the major source of prostaglandin E2 in the gestational sac during term labor.

The umbilical cord is an amniotic structure histologically resembling amnion lining the basal plate and reflected chorion. Prostaglandin E2 is secreted by amnion and is present in amniotic fluid. This study measured prostaglandin E2 production by amnion from all three locations to determine the relative contributions of prostaglandin E2 to amniotic fluid at term. Total surface areas and weights of umbilical cord, basal placental plate, and reflected chorionic amnion were measured in afterbirths from 20 normal patients delivered at term by elective repeat cesarean section before the onset of labor or vaginally after spontaneous onset of labor. Subsequently, 2 cm lengths of umbilical cord and 8 cm2 disks of basal placental plate and reflected chorionic amnion were incubated in perfusion chambers, and prostaglandin E2 production was measured by radioimmunoassay. Umbilical cord accounted for the least surface area (16% to 17%) but greatest tissue mass (75% to 76%). Both basal placental plate and reflected chorionic amnion increased prostaglandin E2 production 2.3-fold and 4.1-fold, respectively, after labor versus before labor, whereas umbilical cord prostaglandin E2 output was unchanged. However, umbilical cord accounted for 66% and 44% of the total prostaglandin E2 output before labor (697 +/- 169 ng/hr) versus (1201 +/- 380 ng/hr) after labor. Thus, of the three amniotic locations, umbilical cord represents the principal site of prostaglandin E2 production within the gestational sac.

Molecular mechanism in the formation of a human ring chromosome 21.

We have characterized the structural rearrangements of a chromosome 21 that led to the de novo formation of a human ring chromosome 21 [r(21)]. Molecular cloning and chromosomal localization of the DNA regions flanking the ring junction provide evidence for a long arm to long arm fusion in formation of the r(21). In addition, the centromere and proximal long arm region of a maternal chromosome 21 are duplicated in the r(21). Therefore, the mechanism in formation of the r(21) was complex involving two sequential chromosomal rearrangements. (i) Duplication of the centromere and long arm of one maternal chromosome 21 occurred forming a rearranged intermediate. (ii) Chromosomal breaks in both the proximal and telomeric long arm regions on opposite arms of this rearranged chromosome occurred with subsequent reunion producing the r(21).

Lactoferrin in human amniotic fluid.

The concentration of human lactoferrin (LF) was measured by radioimmunoassay or non-competitive avidin--biotin assay in amniotic fluid, cord blood and in the decidua, trophoblast, fetal membranes and umbilical cord. Amniotic fluid was obtained by amniocentesis, and cord blood and tissue samples were taken after delivery or elective Caesarean section. No detectable concentration of LF was found in amniotic fluid before week 20 of pregnancy. A significant increase in the LF concentration was observed around week 30 and it remained high until term. In cord blood, an undetectable or low concentration of LF was measured. In tissue specimens the amount of LF was highest in the decidua (9-95 micrograms/g), a moderate concentration was assayed in the amniotic (2-37 micrograms/) and chorion (2-26 micrograms/g) membrane and in the trophoblast (5-35 micrograms/g). In the umbilical cord, the concentration was less than 1 microgram/g. These results suggest a decidual origin of LF. The role of LF during pregnancy is discussed.

ED-A sequence containing fibronectin in human amniotic fluid and amnion epithelial cells.

1. Human amniotic fluid fibronectin (aFn) was studied by using a monoclonal antibody 52DHl (DH) that recognizes the extra domain (ED-A) sequence of cellular Fn (cFn). 2. In immunoblotting the DH antibody reacted with a sharp polypeptide band at the top of the bulk of the diffuse aFn. Another monoclonal antibody 52BF12 (BF) against the cell binding site of Fn, recognized the whole aFn. 3. The ED-A sequence containing cFn (EcFn) formed a constant proportion in aFns from all amniotic fluid preparations studied. 4. In amniotic membranes the DH antibody revealed bright subepithelial immunofluorescence. 5. Also isolated and cultured human amnion epithelial cells were strongly positive in immunofluorescence and secreted EcFn into the culture medium as revealed by immunoblotting. 6. The results indicate that aFn is a composition of at least two different Fn subtypes of which the EcFn most probably originates from amnion epithelial cells.

Reduction of hydroxyproline content in the vessels of the human umbilical cord in premature rupture.

The hydroxyproline content of the amnion and the umbilical vessels obtained from cases in which the fetal membranes were instrumentally or prematurely ruptured was assayed. Hydroxyproline concentration (in micrograms/mg) in the latter group was 23.76, 16.79 and 17.86 in the amnion, artery and vein, whereas in the former the corresponding values were 42.20, 26.61 and 32.48. These results show that, in addition to a decrease in the amnion, hydroxyproline is lowered also in the umbilical vessels, suggesting that the reduction in the collagen in the fetal membranes is probably a particular manifestation of a general metabolic deficiency.

A "two-objective, one-area" procedure in absorption microphotometry and its application using an inverted microscope.

A 'two-objective, one-area' method and related equations are suggested to measure absorbance of microscopic stained objects. In such work, the measuring field invariably includes an image of the object and some clear area surrounding the image. The total intensity in the two areas is measured photometrically, using two different objectives, and substituted in the equation for absorbance. The equation is independent of the term representing intensity from the clear area and hence the error in the measurement of absorbance is reduced. The limitations of the 'two-objective, one-area' method are discussed and its pragmatic operation described with an experimental setup involving an inverted microscope. The method permits measurement of intensity in a part of a stained cell while the rest of the cell remains in the field of view. The method is applied to measure absorbance in Giemsa stained ascites cells and Feulgen stained liver and Human Amnion cells.

Amniotic fluid and decidual prolactin during pregnancy in rhesus macaques.

In rhesus macaques, the concentration of immunoreactive prolactin in the amniotic fluid remains low during most of the first trimester of pregnancy and then increases abruptly at 60-80 days of gestation. During the second half of pregnancy, large amounts of prolactin accumulate in the amniotic fluid. Much of this amniotic fluid prolactin may originate from the superficial endometrium (decidua). This hypothesis is supported by the increasing amounts of decidual prolactin (dPRL) measured in endometrium obtained at early (50 days), mid-(80 days), and late (greater than or equal to 150 days) gestation. In culture, late pregnancy endometrium released more dPRL than did early pregnancy endometrium. When tissues were cultured in medium without progesterone, the amounts of dPRL measured in the medium declined steadily over 6 days, regardless of the gestational age of the endometrium. dPRL was consistently measured in medium harvested from cultures that received either progesterone or medroxyprogesterone; however, progesterone did not induce an increase in the amounts of dPRL released by cultures prepared from early pregnancy endometrium. This suggests that factors in addition to progesterone may stimulate the increase in dPRL that occurs at midgestation in rhesus macaques.

Epidermolysis bullosa acquisita antigen is the globular carboxyl terminus of type VII procollagen.

Epidermolysis bullosa acquisita (EBA) is a severe, chronic blistering disease of the skin. EBA patients have circulating and tissue-bound autoantibodies to a large (Mr = 290,000) macromolecule that is localized within the basement membrane zone between the epidermis and dermis of skin, the site of blister formation. The "EBA antigen" is known to be distinct from laminin, heparan sulfate proteoglycan, fibronectin, the bullous pemphigoid antigen, elastin, and collagen types I, II, III, IV, and V. Sera from patients with EBA, two monoclonal antibodies to the EBA antigen, and a monoclonal antibody to the carboxyl terminus of type VII procollagen identically label human amnion and skin by immunofluorescent and immunoelectron microscopy. Western immunoblots of the EBA antigen extracted from skin and of type VII procollagen labeled with the above sera and antibodies are identical. None of the sera or antibodies labels Western blots of pepsinized type VII collagen which is missing the globular amino and carboxyl terminal domains. These data show that the EBA antigen is the carboxyl terminus of type VII procollagen.