Sterol-phospholipid interaction in model membranes: role of C5-C6 double bond in cholesterol

Several double-bond isomers of cholesterol where the normal C5-C6 double bond (delta 5) has been moved to different positions in the ring skeleton, i.e., delta 1, delta 4, delta 7, delta 8(9), delta 8(14), and delta 14, have been synthesized and incorporated in phosphotidylcholine vesicles. In addition, dienes like delta 5,7, delta 7,14, and delta 8,14 have also been studied. Many of these cholesterol analogues are intermediates in the sterol biosynthesis in different organisms. The incorporation studied indicated that more than 90% of the sterol was present in the vesicles. The effect of these cholesterol analogues was studied by glucose permeability, electron spin resonance, and fluorescence polarization spectroscopy. These studies indicated that delta 14-cholesten-3 beta-ol was most effective in restricting glucose permeability or in increasing the order parameter but was still not as effective as cholesterol. This was followed by delta 8(14)- and delta 8(9)-cholesten-3 beta-ol. The delta 1, delta 4, and delta 7 analogues and the dienols were relatively less effective in condensing the membrane. These studies indicate that the double bond at C5-C6 in cholesterol is most effective for optimal sterol-phospholipid interaction and may have formed the basis of the migration of the double bond from rings C and D in sterols to C5-C6 during the evolution of cholesterol.     

Decidual cell-specific surface antigen(s) recognized by monoclonal antibodies: tissue and species distribution

Decidual cells are direct descendants of endometrial stromal cells and the ultimate progeny of bone marrow-derived precursors. In view of their bone marrow genealogy and demonstrated immunoregulatory role during pregnancy, this study attempted to identify a lineage-specific differentiation marker(s) on murine decidual cells with the hope of tracing their developmental pathway and exploring their familial relationship to other lymphomyeloid cells. Two protein A-binding, IgG2b isotype monoclonal antibodies (secreted by clones 16F12 and 2G4F8) were raised by immunizing virgin CBA mice with syngeneic decidual cells. The presence and the density of the antigenic marker(s) recognized by these antibodies were examined by radioautography on various cell types in single cell suspensions of the decidua, placenta, and lymphomyeloid organs after a sandwich labeling with hybridoma supernatants followed by 125I-protein A. Both antibodies appeared to recognize antigen(s) unique for the decidual cell lineage in mice, humans, and rats. The incidence of antigen-bearing decidual cells increased with gestational age in CBA, C3H, and CD1 mice between days 8 and 14, and in humans between 6 and 10.5 wk; in rats, however, some decline was noted between days 8 and 14. The binding was always higher with 16F12 than with 2G4F8 supernatants. No significant binding of either antibody to trophoblast cells of the placenta or leukocytes within the decidua was noted in any of the above mouse strains or species. Little or no labeling of any cell type was seen on lymphomyeloid cells of the virgin or pregnant CBA mice, but a consistent labeling of a rare blast-type cell in the blood was observed with both antibodies, raising the possibility that this cell may represent the circulating precursor of the decidual cell lineage. It remains to be investigated whether these antibodies are recognizing the same or different differentiation antigen(s) on the decidual cells, and whether a conservation of this antigen(s) during speciation signifies its functional importance.     

Three methods for producing fertile hemopoietic chimeras in mice

Three methods for producing semiallogeneic (F1----parental) hemopoietic chimeras with retained or regained fertility are detailed here. Prenatal (PN) chimeras were produced by injecting F1 ([BALB/c female x C3H/HeJ male] or [CBA/J female x C57BL/6 male]) fetal liver (days 13-18) or adult bone marrow cells (10(6)-10(7) cells/20 microliters/embryo) into the yolk-sac cavities of days 13-17 gestation BALB/c or CBA/J embryos, respectively, and allowing them to be born naturally. Neonatal (NN) chimeras were made by introducing F1 bone marrow cells (1-2 x 10(7) cells/0.25 ml) into newborn (less than 24 hr old) female mice through the anterior facial vein. Female mice were raised to maturity in both cases. Ovary-transplanted (OT) chimeras were made by first irradiating (9.5 Gy) and repopulating young female adult mice with 10(7) F1 bone marrow cells, followed by bilateral orthotopic transplantation of syngeneic ovarian tissue six weeks later. Females reconstituted with the above three methods were mated with normal syngeneic males and sacrificed at 11-16 days of pregnancy to evaluate hemopoietic chimerism. This was determined in all cases by a radioautographic evaluation of the extent of donor H-2 phenotype marker expression on splenic small lymphocytes, after an indirect labelling of single-cell suspensions with monospecific antibody and [125I]protein-A. Results indicate that hemopoietic chimerism was best in the PN group (0.3-78.1%, mean = 27.1); intermediate in the OT group (5.8-38.2%, mean = 18.1); and low in the NN group (0-14%, with one exception, which was 83.6%). Observed fertility was best for BALB/c host PN chimeras.(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishing a new genus,Chiharadinium gen. nov. (Peridiniales,Dinophyceae) for a tidal pool dinoflagellate formerly known as Scrippsiella hexapraecingula

To determine its accurate taxonomic position, a tidal pool bloom-forming dinoflagellate, Scrippsiella hexapraecingula was re-investigated using light, scanning and transmission electron microscopy together with a phylogenetic analysis based on concatenated ribosomal DNA sequences. The culture strains used in this study were established from intertidal rock pool samples taken from Jogashima, Kanagawa prefecture and Heisaura, Chiba prefecture, Japan and were identified as S. hexapraecingula originally described by Horiguchi and Chihara from a tidal pool in Hachijo Island, Tokyo, Japan in 1983. The thecal plate arrangement was determined as Po, X, 4′, 3a, 6″, 6c, 5s, 5″′, 2″″. The internal structure was investigated for the first time. The organism has typical dinoflagellate cellular organelles such as a dinokaryotic nucleus, mitochondria with tubular cristae, trichocysts and pusule. The chloroplast was single and connected to the central pyrenoid (stalked type). The eyespot found in the sulcus is of the B type with two rows of superficial intraplastidal lipid globules directly overlain by an extraplastidal single layer of crystalline bricks enveloped by a common membrane. The apical pore is plugged by a double-layered stub-like structure. Stalk building material for attachment covered the apical pore. Phylogenetic analysis indicated that S. hexapraecingula was most closely related to a freshwater dinoflagellate, Peridiniopsis borgei, the type species of the genus Peridiniopsis. However, clear differences exist between these two organisms, including their thecal plate arrangement, habitat and habit. As a result, a new genus, Chiharadinium Dawut & T. Horiguchi gen. nov. has been proposed rather than attempting to accommodate S. hexapraecingula in the genus Peridiniopsis. The new combination, Chiharadinium hexapraecingulum (T. Horiguchi & Chihara) Dawut & T. Horiguchi comb. nov. has been proposed.     

Localization of transforming growth factor-beta at the human fetal-maternal interface: role in trophoblast growth and differentiation.

We examined the localization of transforming growth factor (TGF)-beta in first-trimester and term human decidua and chorionic villi and explored the role of this factor on the proliferation and differentiation of cultured trophoblast cells. Two antibodies, 1D11.16.8, a mouse monoclonal neutralizing antibody capable of recognizing both TGF-beta 1 and TGF-beta 2 and CL-B1/29, a rabbit polyclonal antibody capable of recognizing TGF-beta 2, were used to immunolocalize TGF-beta in fixed, paraffin-embedded, or fixed, frozen sections of placenta and decidua, providing similar results. Intense labeling was observed in the extracellular matrix (ECM) of the first-trimester decidua and cytoplasm of term decidual cells. Syncytiotrophoblast cell cytoplasm as well as the ECM in the core of the chorionic villi of both first-trimester and term placentas exhibited a moderate degree of labeling. Strong cytoplasmic labeling was observed in the cytotrophoblastic shell of the term placenta. To examine the role of TGF-beta on trophoblast proliferation and differentiation, early passage cultures of first-trimester and primary cultures of term trophoblast cells were established and characterized on the basis of numerous immunocytochemical and functional markers. These cells expressed cytokeratin, placental alkaline phosphatase, urokinase-type plasminogen activator, and pregnancy-specific beta glycoprotein, but not factor VIII or 63D3; they also produced hCG and collagenase type IV. Exposure of first-trimester trophoblast cultures to TGF-beta 1 significantly inhibited proliferation in a dose-dependent manner. An antiproliferative effect was also noted in the presence of TGF-beta 2. These effects were abrogated in the presence of the neutralizing anti-TGF-beta antibody (1D11.16.8) in a concentration-dependent manner. In a 3-day culture, exogenous TGF-beta 1 stimulated formation of multinucleated cells by the first trimester as well as term trophoblast cells. Addition of neutralizing anti-TGF-beta antibody to first-trimester trophoblast cells stimulated proliferation beyond control levels in a 24-h culture and reduced formation of multinucleated cells in a 3-day culture, indicating the presence of endogenous TGF-beta activity. These results indicate that TGF-beta produced at the human fetal-maternal interface plays a major regulatory role in the proliferation and differentiation of the trophoblast.     

Regulated temporal and spatial expression of the calcium-binding proteins calcyclin and OPN (osteopontin) in mouse tissues during pregnancy.

In situ hybridization and northern/slot blot analyses were used to quantify the expression of calcyclin (2A9, 5B10), osteopontin (opn, secreted phosphoprotein, 2ar) and calmodulin mRNAs in mouse tissues that support pregnancy. High-to-moderate levels of the mRNAs of all three genes were detected at discrete locations in the uterus, decidua and placenta as a function of gestation time. Calmodulin expression was constant in these tissues; calcyclin mRNA was high during early pregnancy and declined after day 8-9 of gestation; and opn mRNA was undetectable before day 7, with maximal levels on days 9-12 in each of these tissues. Calcyclin, but not opn, expression was also observed in the chorioamnion after day 12. Calcyclin was expressed throughout the decidua on day 8 but became restricted to the primary (antimesometrial) decidual zone and decidua lateralis on day 9, and the decidua capsularis after day 9. By contrast, opn mRNA was localized on day 9 to the mesometrial triangle, which contains a large population of granulated metrial gland cells, and to the decidua basalis. These two genes may serve as markers for the two types of decidual tissue. We suggest that one function of OPN, which may be an indicator of cells in the decidua that have a bone marrow genealogy, is to mediate the flux of calcium from the maternal circulation to the developing embryo.     

Functional effects of enhancing or silencing adenosine A2b receptors in cardiac fibroblasts

Cardiac fibroblasts (CF) express adenosine (ADO) receptors, and pharmacological evidence suggests the possible involvement of the A2 (A2a and A2b) receptor (A2aR and A2bR) subtypes in inhibiting cell functions involved in fibrosis. The main objective of this study was to define the contributions of A2a and/or A2b receptors in modulating ADO-induced decreases in CF functions. For this purpose, CF were either treated pharmacologically or had the A2aR or A2bR levels modified through the use of recombinant adenovirus or siRNA. The assessment of mRNA expression in adult rat CF yielded evidence for A1R, A2bR, A2a), and A3R. Endogenously or exogenously enhanced ADO significantly inhibits CF proliferation, collagen, and protein synthesis. A2R and A2aR agonists, although capable of inhibiting CF protein and collagen synthesis, were unable to define the contributions derived from A2aR or A2bR. Overexpression of A2bR in CF yielded significant decreases in basal levels of collagen and protein synthesis and correlated with increases in cAMP levels. However, at higher doses of ADO receptor agonists, significant increases in protein and collagen synthesis were observed. CF with underexpression of A2bR yielded increases in protein and collagen synthesis. In contrast, A2aR underexpression did not modify ADO-induced decreases in CF protein or collagen synthesis. In conclusion, results derived from the molecular manipulation of receptor levels indicate that A2bR are critically involved in ADO-mediated inhibition of CF functions.     

Noncatalytic domain of uPA stimulates human extravillous trophoblast migration by using phospholipase C,phosphatidylinositol 3-kinase and mitogen-activated protein kinase

The serine protease urokinase-type plasminogen activator (uPA) promotes matrix degradation by many cell types, including the invasive extravillous trophoblast (EVT) of the human placenta. The noncatalytic amino-terminal end of uPA binds to uPA receptors (uPARs) expressed by these cells. A highly polarized expression of uPAR-bound uPA at the migration front of EVT cells in situ suggests a functional role of uPA:uPAR interaction in EVT cell migration. The present study examined whether uPA stimulates EVT cell migration, independent of proteolytic function, and investigated some of the signaling pathways involved. Using in vitro-propagated EVT cells in Transwell migration assays, both uPA and its noncatalytic amino-terminal fragment (ATF) were shown to stimulate migration through multiporous polycarbonate (pore size 8 microm) membranes. A uPAR-blocking antibody inhibited basal and ATF-stimulated migration. Migration was found to be stimulated by hypoxic conditions, which upregulates uPAR expression; this stimulation was abrogated with the uPAR-blocking antibody, indicating the role of endogenous uPA in EVT cell migration. Spectrofluorometric measurement of cytosolic calcium in cells treated with uPA and ATF demonstrated a rapid rise in [Ca2+](i), which was prevented by pretreatment of cells with thapsigargin, indicating a release from intracellular stores. Both basal and ATF-mediated migratory responses were suppressed in the presence of selective pharmacological inhibitors LY294002, U73122, and U0126, implicating the respective roles of phosphatidinylinositol 3-kinase (PI 3-K), phospholipase C (PLC), and MEK1/2 in basal and ATF-stimulated migratory capacity. Taken together, these results demonstrate that uPA:uPAR interaction stimulates EVT cell migration, independent of uPA enzymatic activity, using the mitogen-activated protein kinase pathway and calcium signaling events including the participation of PI 3-K and PLC. These findings are relevant to clinical conditions of aberrant trophoblast migration, including spontaneous abortion, preeclampsia, and choriocarcinoma.