Phospholipase A activity in human and ovine uterine tissues

Phospholipase A was assayed in crude lysosomal fractions of human decidua, amnion, chorion and myometrium. Activity was present in all the tissues and was highest in decidua and amnion which contained enzymes with pH optima of 6.5 – 8.0 and 7.2 respectively. Comparison of the activities in tissues obtained before labor with others obtained during labor showed no differences. In sheep, the fetal membranes contained a greater activity of phospholipase A than placenta and myometrium. Stimulation of the fetal adrenals with corticotrophin caused a marked increase in activity in both amnion and chorioallantois. It is concluded that the human amnion and the ovine amnion and chorioallantois could participate in the biosynthesis of prostaglandins by releasing stored arachidonic acid.     

Process of the combination of alpha-lactalbumin with lysosomes in mammary epithelial cells

We previously reported that alpha-lactalbumin combines with the lysosomal membrane of mammary epithelial cells and that it acts to release lysosomal enzymes. However, the details of this combination within the cells remained undetermined. We now report that 125I-bovine-alpha-lactalbumin in the medium entered mouse mammary epithelial cells, and about 13 per cent of the alpha-lactalbumin that entered the cell bound to lysosomes. About 75 per cent of the alpha-lactalbumin that reached the lysosome was tightly bound to the lysosomal membrane. It appears that alpha-lactalbumin in the secretory vesicles does not migrate out, because murine and bovine whey did not induce the release of Golgi enzymes in vitro.     

Phospholipase A activity in human and ovine uterine tissues.

Phospholipase A was assayed in crude lysosomal fractions of human decidua, amnion, chorion and myometrium. Activity was present in all the tissues and was highest in decidua and amnion which contained enzymes with pH optima of 6.5-8.0 and 7.2 respectively. Comparison of the activities in tissues obtained before labor with others obtained during labor showed no differences. In sheep, the fetal membranes contained a greater activity of phospholipase A than placenta and myometrium. Stimulation of the fetal adrenals with corticotrophin caused a marked increase in activity in both amnion and chorioallantois. It is concluded that the human amnion and the ovine amnion and chorioallantois could participate in the biosynthesis of prostaglandins by releasing stored arachidonic acid.     

Cell type-specific regulation of fetal fibronectin expression in amnion: conservation of glucocorticoid responsiveness in human and nonhuman primates

The appearance of oncofetal fibronectin (FFN) in cervical and vaginal secretions is predictive of human labor. Levels of FFN in amnion increase with the onset of labor in rhesus monkeys. Since glucocorticoid (GC) levels in serum and amniotic fluid increase in association with parturition, we compared GC-mediated regulation of FFN expression in cultures of amnion epithelial cells and fibroblasts isolated from human and baboon amnions. Cells were maintained with and without dexamethasone (DEX), and levels of FFN in the conditioned media were determined by ELISA. We observed that DEX treatment suppressed FFN levels in both human and baboon amnion epithelial cells, whereas it increased FFN levels in amnion fibroblasts. DEX treatment reduced FFN levels in cytotrophoblasts from human placenta and increased FFN levels in placental fibroblasts. Northern blots revealed that DEX reduced levels of fibronectin (FN) mRNA in amnion epithelial cells and cytotrophoblasts, whereas it increased FN mRNA in amnion and placental fibroblasts. We conclude that GC differentially regulates FFN expression in epithelial and mesenchymal cells from amnion and placenta. In addition, this pattern of cell type-specific FFN regulation by GC is conserved in human and nonhuman primates and may be responsible for parturition-dependent changes in FFN expression in gestational tissues.     

Labor and inflammation increase the expression of oxytocin receptor in human amnion

The oxytocin/oxytocin receptor (OXT/OXTR) system plays an important role in the regulation of parturition. The amnion is a major source of prostaglandins and inflammatory cytokine synthesis, which increase both before and during labor. Amnion is a noncontractile tissue; therefore, the role played by OXT/OXTR in this tissue will be fundamentally different from the role played in myometrial contractions. In the present study, we demonstrate increased OXTR mRNA and protein concentrations in human amnion epithelial cells associated with the onset of labor. We show that incubation of primary human amnion epithelial cells with IL1B results in a rapid, transient up-regulation of OXTR mRNA expression, which peaks in prelabor samples after 6 h. Incubation of prelabor amnion epithelial cells with OXT results in a marked increase of prostaglandin E(2) synthesis, and we demonstrate that OXT activates the extracellular signal-regulated protein kinase signal transduction pathway to stimulate up-regulation of cyclo-oxygenase 2 in human amnion epithelial cells. The increased ability of human amnion to produce prostaglandins in response to OXT treatment suggests a complementary role for the OXT/OXTR system in the activation of human amnion and the onset of labor.     

Immunohistochemical localization of glucose transporters and insulin receptors in human fetal membranes at term

The localization has been investigated of the isoforms GLUT1, GLUT3 and GLUT4 of glucose transporter proteins as well as of insulin receptors. Fetal membranes (n=10) were examined by immunohistochemical methods at the light and electron microscopic levels using mono- and polyclonal antibodies. In all amnion epithelial cells, GLUT1 and GLUT3 antibodies were bound to the apical membrane. Very rarely the GLUT1 antibody also immunostained the basolateral membrane and reacted weakly with the endomembrane system and membranes of the lateral cell protrusions. Fibroblasts reacted with the antibodies against GLUT1, GLUT4 and insulin receptor, whereas they were labelled only in one case with GLUT3 antibody. Cytotrophoblast cells were only stained with antibodies against GLUT1 and GLUT3. Antibodies against GLUT4 only reacted with fibroblasts in the membranes. On amnion epithelial cells, weak immunoreactivity with insulin receptor antibodies was detected only at the electron microscopic level. The data indicate: (1) GLUT1 is located on all cells of the amnion, whereas GLUT3 is present in detectable amounts only on amnion epithelial cells and cytotrophoblast; (2) GLUT1 and GLUT3 on amnion epithelial cells are predominantly located on the apical surface; (3) GLUT4 and insulin receptors are not regularly expressed. We suggest that amnion epithelial cells cover their basal glucose requirements from the amniotic fluid and not from the maternal circulation.     

Modulation of lysosomal enzyme levels in cultured cells: effects of alterations in cell density, balanced growth, and endocytosis

Factors which influence lysosomal enzyme accumulation in cultured cells have been studied. In cell types of both fibroblast (3T6) and epithelial (HeLa) origin, acid phosphatase and β-N-acetylglucosaminidase activities increase with increasing cell density. However, in other cell lines such as BHK or chick embryo fibroblasts, little or no accumulation of lysosomal enzymes occurred with increased cell density. Increased lysosomal enzyme activity need not necessarily be accompanied by alterations in rate of cell growth, rate of pinocytosis, or amount of internalized degradable macromolecules. The stimulus for lysosomal enzyme accumulation appears to require cell contact, since sparsely plated cells do not exhibit lysosomal enzyme accumulation. In 3T6 cells, lysosomal enzymes also accumulate during “step-down” conditions, such as amino acid or serum depletion, or during unbalanced growth resulting from inhibition of cytokinesis or DNA synthesis. Increases in the specific activity of lysosomal enzymes which occur during step-down conditions or unbalanced growth require cell contact, since they are not seen in sparse cells, but are observed in medium- and high-density cells incubated in serum-free medium. Studies employing actinomycin D suggest that lysosomal enzyme levels are regulated primarily via control of enzyme synthesis, rather than enzyme degradation.     

Intermediate filament cytoskeleton of amnion epithelium and cultured amnion epithelial cells: expression of epidermal cytokeratins in cells of a simple epithelium

Using immunofluorescence microscopy and two-dimensional gel electrophoresis, we compared the cytoskeletal proteins expressed by human amnion epithelium in situ, obtained from pregnancies of from 10-wk to birth, with the corresponding proteins from cultured amnion epithelial cells and cultures of cells from the amniotic fluid of 16 week pregnancies. Epithelia of week 16 fetuses already display tissue-specific patterns of cytokeratin polypeptides which are similar, although not identical, to those of the corresponding adult tissues. In the case of the simple amnion epithelium, a complex and characteristic complement of cytokeratin polypeptides of Mr 58,000 (No. 5), 56,000 (No. 6), 54,000 (No. 7), 52,500 (No. 8), 50,000 (No. 14), 46,000 (No. 17), 45,000 (No. 18), and 40,000 (No. 19) is present by week 10 of pregnancy and is essentially maintained until birth, with the addition of cytokeratin No. 4 (Mr 59,000) and the disappearance of No. 7 (Mr 54,000) at week 16 of pregnancy. In full-term placentae, the amnion epithelium displays two morphologically distinct regions, i.e., a simple and a stratified epithelium, both of which express the typical amnion cytokeratin polypeptides. However, in addition the stratified epithelium also synthesizes large amounts of special epidermal cytokeratins such as No. 1 (Mr 68,000), 10 (Mr 56,500), and 11 (Mr 56,000). In culture amnion epithelial cells obtained from either 16-wk pregnancies or full-term placentae will continue to synthesize the amnion-typical cytokeratin pattern, except for a loss of detection of component No. 4. This pattern is considerably different from the cytokeratins synthesized by cultures of cells from amniotic fluids (cytokeratins No. 7, 8, 18, and 19, sometimes with trace amounts of No. 17) and from several so-called "amnion epithelial cell lines." In addition, amnion epithelial cells in situ as well as amnion epithelial cell cultures appear to be heterogeneous in that they possess some cells that co-express cytokeratins and vimentin. These observations lead to several important conclusions: In contrast to the general concept of recent literature, positively charged cytokeratins of the group No. 4-6 can be synthesized in a simple, i.e., one-layered epithelium. The change from simple to stratified amnion epithelium does not require a cessation of synthesis of cytokeratins of the simple epithelium type, but in this case keratins characteristic of the terminally differentiated epidermis (No. 1, 10, and 11) are also synthesized.(ABSTRACT TRUNCATED AT 400 WORDS)     

绵羊羊膜上皮细胞治疗兔桡骨缺损模型的研究

为了确定绵羊羊膜上皮细胞在体内向骨组织的分化能力,实验在分离培养绵羊羊膜上皮细胞并对其进行干细胞特性的鉴定的基础上,制作新西兰大白兔桡骨13mm骨缺损模型,随机分组对其进行注射绵羊羊膜上皮细胞实验。高剂量组:移植细胞5×107个;低剂量组:移植细胞5×106个;对照组:生理盐水。细胞移植后2、4、8周拍摄X光片观察骨缺损部位的缺损修复情况;相应时段取骨缺损部位新生骨进行组织学观察:分析骨小梁生成数量和骨的改建时期。实验结果显示,高剂量实验组在移入细胞第8周,骨缺损完全修复,且同期高剂量组新骨生成的数量和质量明显高于低剂量组,低剂量组优于对照组。由此可见,绵羊羊膜上皮细胞不仅可以在不同种动物间进行移植,而且对骨缺损有良好的修复能力。     

Activity of lysosomal hydrolases in human placenta, amnion, decidua and myometrium in the 2nd and 3rd trimester

Six lysosomal hydrolases were studied in tissues of human placenta, amnion, decidua, and myometrium obtained in the 2nd and 3rd trimester. No significant increase in the activity of any lysosomal hydrolase was found in the 3rd-trimester values compared with those obtained in the 2nd trimester. Thus, the increased activity of lysosomal hydrolases in maternal serum at term, previously described in several studies, seems not to be paralleled by a corresponding increase in the activity of these enzymes in the tissues of the pregnant uterus. We speculate that the increase in maternal serum towards term may reflect a macrophage activation initiated by elevated estrogen concentrations in the 3rd trimester.     

Lysosomal breakdown of erythrocytes in the sheep placenta

Summary The breakdown of erythrocytes within the lysosomal apparatus of trophoblastic epithelial cells of the sheep placenta was studied at the ultrastructural level. Acid phosphatase activity could be demonstrated in the interspace between the erythrocyte membrane and the lysosomal membrane, but not inside ingested erythrocytes. The erythrocyte plasma membrane remained observable until the final stage of the breakdown process. Together with a peripheral layer of indigestible hemoglobin it might form a barrier for further penetration of lysosomal enzymes into the ingested erythrocyte. The hemoglobin of the erythrocyte is suggested to diffuse through the erythrocyte plasma membrane into the interspace between this membrane and the lysosomal membrane. Subsequently, the hemoglobin is digested in the interspace or in fragments pinched off from erythrocyte-containing lysosomes (=erythrolysosomes). The fragmentation of erythrolysosomes is considered to be the most efficient mechanism for the breakdown of red blood cells in the trophoblastic epithelium of the sheep placenta. The method of entry of hydrolytic enzymes into erythrocyte-containing phagosomes is discussed.     

Alpha-lactalbumin as a lysosomal enzyme-releasing factor

In the early stage of mammary gland involution, biochemically detectable lysosomal damage occurs. The mechanism(s) underlying this damage is not well understood. We found that alpha-lactalbumin from mouse milk induced the release of enzymes from the lysosomes of mouse mammary epithelial cells in vitro, and this induction also occurred with bovine alpha-lactalbumin. This enzyme release was accelerated by the addition of whey proteins with a molecular weight of 50 000 to 60 000. We also found that the lysosomal membrane of mammary epithelial cells had a strong affinity for alpha-lactalbumin.     

Degradation of nuclear DNA by DNase II-like acid DNase in cortical fiber cells of mouse eye lens

The eye lens is composed of fiber cells that differentiate from epithelial cells on its anterior surface. In concert with this differentiation, a set of proteins essential for lens function is synthesized, and the cellular organelles are degraded. DNase II-like acid DNase, also called DNase IIbeta, is specifically expressed in the lens, and degrades the DNA in the lens fiber cells. Here we report that DNase II-like acid DNase is synthesized as a precursor with a signal sequence, and is localized to lysosomes. DNase II-like acid DNase mRNA was found in cortical fiber cells but not epithelial cells, indicating that its expression is induced during the differentiation of epithelial cells into fiber cells. Immunohistochemical and immunocytochemical analyses indicated that DNase II-like acid DNase was colocalized with Lamp-1 in the lysosomes of fiber cells in a relatively narrow region bordering the organelle-free zone, and was often found in degenerating nuclei. A comparison by microarray analysis of the gene expression profiles between epithelial and cortical fiber cells of young mouse lens indicated that some genes for lysosomal enzymes (cathepsins and lipases) were strongly expressed in the fiber cells. These results suggest that the lysosomal system plays a role in the degradation of cellular organelles during lens cell differentiation.     

Inhibition of epithelial cell death in the secondary palate in vitro by alteration of lysosome function

The secondary palate in vivo and in vitro exhibits selective cell death at its medialedge epithelium (MEE) at a precise developmental age. This epithelial degeneration is mediated, in part, by MEE lysosomes. Previous studies in vitro (27) showed that the glutamine analogue, diazo-oxo-norleucine (DON), prevented MEE cell death by inhibiting glucosamine synthesis and thereby the glycosylation of proteins without affecting either the synthesis or activity of palatal lysosomal enzymes. In the present study, histochemical examination of MEE from DON treated day-15 rat palates demonstrated that acid phosphatase activity was restricted to Golgi saccules and associated vesicles as well as to lysosomes. Control MEE had reaction product in these structures and distributed diffusely throughout the cytoplasm of degenerating cells. DON treatment therefore appears to alter the intracellular distribution of lysosomal enzymes. Since DON treatment appears to have prevented MEE cell death by inhibiting glycosylation of proteins, glycosylation of lysosomal membranes or lysosomal enzymes may be essential for its role in programmed cell death.     

A novel method of imaging lysosomes in living human mammary epithelial cells

Cancer cells invade by secreting degradative enzymes which, under normal conditions, are sequestered in lysosomal vesicles. The ability to noninvasively label lysosomes and track lysosomal trafficking would be extremely useful to understand the mechanisms by which degradative enzymes are secreted in the presence of pathophysiological environments, such as hypoxia and acidic extracellular pH, which are frequently encountered in solid tumors. In this study, a novel method of introducing a fluorescent label into lysosomes of human mammary epithelial cells (HMECs) was evaluated. Highly glycosylated lysosomal membrane proteins were labeled with a newly synthesized compound, 5-dimethylamino-naphthalene-1-sulfonic acid 5-amino-3,4,6-trihydroxy-tetrahydro-pyran-2-ylmethyl ester (6-O-dansyl-GlcNH2). The ability to optically image lysosomes using this new probe was validated by determining the colocalization of the fluorescence from the dansyl group with immunofluorescent staining of two well-established lysosomal marker proteins, LAMP-1 and LAMP-2. The location of the dansyl group in lysosomes was also verified by using an anti-dansyl antibody in Western blots of lysosomes isolated using isopycnic density gradient centrifugation. This novel method of labeling lysosomes biosynthetically was used to image lysosomes in living HMECs perfused in a microscopy-compatible cell perfusion system.     

Stimulation of the D5 dopamine receptor acidifies the lysosomal pH of retinal pigmented epithelial cells and decreases accumulation of autofluorescent photoreceptor debris

Optimal neuronal activity requires that supporting cells provide both efficient nutrient delivery and waste disposal. The incomplete processing of engulfed waste by their lysosomes can lead to accumulation of residual material and compromise their support of neurons. As most degradative lysosomal enzymes function best at an acidic pH, lysosomal alkalinization can impede enzyme activity and increase lipofuscin accumulation. We hypothesize that treatment to reacidify compromised lysosomes can enhance degradation. Here, we demonstrate that degradation of ingested photoreceptor outer segments by retinal pigmented epithelial cells is increased by stimulation of D5 dopamine receptors. D1/D5 receptor agonists reacidified lysosomes in cells alkalinized by chloroquine or tamoxifen, with acidification dependent on protein kinase A. Knockdown with siRNA confirmed acidification was mediated by the D5 receptor. Exposure of cells to outer segments increased lipofuscin-like autofluorescence, but SKF 81297 reduced autofluorescence. Likewise, SKF 81297 increased the activity of lysosomal protease cathepsin D in situ. D5DR stimulation also acidified lysosomes of retinal pigmented epithelial cells from elderly ABCA4(-/-) mice, a model of recessive Stargardt's retinal degeneration. In conclusion, D5 receptor stimulation lowers compromised lysosomal pH, enhancing degradation. The reduced accumulation of lipofuscin-like autofluorescence implies the D5 receptor stimulation may enable cells to better support adjacent neurons.     

Immunocytochemical demonstration of the lysosomal enzyme alpha-glucosidase in the brush border of human intestinal epithelial cells

In investigations on the intracellular transport route(s) of lysosomal enzymes in polarized epithelial cells, we used immunocytochemical methods to localize lysosomal alpha-glucosidase in human small-intestinal epithelial cells. Two monoclonal antibodies which can discriminate between different biosynthetic forms of this enzyme were used. One monoclonal antibody, 43D1, which recognizes all forms of the enzyme, showed labeling of the Golgi apparatus, the lysosomes and, unexpectedly, of the brush border of the cells. Multivesicular bodies were free of label. In contrast, monoclonal antibody 43G8, which recognizes all forms except the 110,000 Da precursor of alpha-glucosidase, showed labeling of the lysosomes only. This leads us to conclude that the 110,000 Da precursor form of alpha-glucosidase is present in the Golgi apparatus and the brush border of human small-intestinal epithelial cells. Moreover, biochemical experiments show that this precursor copurifies with sucrase, a typical brush-border marker, when a partially purified microvilli fraction is prepared.     

Characterization of laminin isoforms in human amnion

Epithelial cells of the human amnion have been reported to possess similar functions to many types of cells, such as hepatocytes, neurons, and pancreatic beta-cells. We reported previously that one of the hepatocyte-like functions of human amniotic epithelial cells was reinforced by the presence of basement membrane components. Laminin is one of the main components of the basement membrane; it critically contributes to cell differentiation. Laminin has several heterotrimer isoforms composed of an alpha-, a beta-, and a gamma-chain, and each type of chain has several types of subunit chains: alpha1-5, beta1-3, and gamma1-3. In this study, we characterized the laminin subunit chains in human amnion. Laminin is produced and secreted from adjacent epithelial cells, and therefore, the gene expression of laminin subunit chains in human amniotic epithelial cells was investigated by RT-PCR. Their localization was examined by immunohistochemical staining of frozen sections. The findings suggested that the basement membrane of the human amnion contains a broad spectrum of laminin isoforms, laminin-2, -4, -5, -6, -7, -10, -11. These findings will provide clues not only for understanding the physiological roles of the amnion and hAECs, but also for applying this tissue as a source of donor cells for cell transplantation therapy.     

The morphometry of the Sudan-III-positive granules in the cytoplasm of the human amniotic epithelium

Seventy-nine human amnions from different gestational ages were studied by new morphometric methods. The Sudan-III-positive granules in the cytoplasm of the amniotic epithelial cells were first observed in the reflected amnion from the second trimester of normal gestation. During the third trimester of normal gestation, the number of cytoplasmic granules increased remarkably with gestational age not only in the reflected amnion but also in the placental amnion. The number of granules was generally related to the increasing rates of anucleated cells and binucleated cells in the amniotic epithelium during the third trimester of normal gestation. During the third trimester of gestation, the number of granules was greater in the distant reflected amnion with its less adequate blood supply than in the placental amnion. A vast accumulation of the granules in the entire amnion was observed in cases of fetal death, in which blood supply had ceased. It is suggested, therefore, that the progressive accumulation of Sudan-III-positive cytoplasmic granules in the human amniotic epithelium during the third trimester of normal gestation is the result of cell degeneration.