N-linked glycoprotein biosynthesis in the developing mouse embryo

We have developed microenzymic assays that have, for the first time, enabled analysis of several enzymes in the pathway for N-linked glycoprotein biosynthesis in pre- and peri-implantation mouse embryos. The in vitro activities of the glycosyl transferases responsible for the formation of N-acetylglucosaminylpyrophosphoryldolichol,N, N'-diacetyl-chitobiosylpyrophosphoryldolichol, mannosylphosphoryldolichol, and glucosylphosphoryldolichol were found to decrease after fertilization before increasing significantly at the blastocyst stage, a stage that was also found to be highly sensitive to the glycosylation inhibitor, tunicamycin. The observed elevation in the activities of these enzymes in blastocysts still occurred when ebbryos were cultured in alpha-amanitin, indicating that de novo mRNA synthesis is unnecessary for the observed increase in their activities. Thus, an elevated capacity for N-glycosylation exists at the blastocyst stage, a time when dramatic increases in cell-cell interactions are known to occur.     

Histamine induces CD86 expression and chemokine production by human immature dendritic cells

Mast cells and immature dendritic cells (DC) are in close contact in peripheral tissues. Upon activation, mast cells release histamine, a mediator involved in the immediate hypersensitivity reaction. We therefore tested whether histamine could affect human DC activation and maturation. Histamine induces CD86 expression on immature DC in a dose-dependent (significant at 10(-7) M) and transient manner (maximal after 24-h stimulation). Histamine also transiently up-regulates the expression of the costimulatory and accessory molecules, CD40, CD49d, CD54, CD80, and MHC class II. As a consequence, immature DC exposed for 24 h to histamine stimulate memory T cells more efficiently than untreated DC. In addition, histamine induces a potent production of IL-6, IL-8, monocyte chemoattractant protein 1, and macrophage-inflammatory protein 1alpha by immature DC and also up-regulates IL-1beta, RANTES, and macrophage-inflammatory protein 1beta but not TNF-alpha and IL-12 mRNA expression. Histamine activates immature DC through both the H1 and H2 receptors. However, histamine-treated DC do not have a phenotype of fully mature cells, as they do neither show significant changes in the expression of the chemokine receptors, CCR5, CCR7 and CXC chemokine receptor 4, nor expression of CD83 de novo. These data demonstrate that histamine activates immature DC and induces chemokine production, thereby suggesting that histamine, via stimulation of resident DC, may participate locally in T cell stimulation and in the late inflammatory reaction associated with allergic disorders.     

Heparan sulfate proteoglycan synthesis and metabolism by mouse uterine epithelial cells cultured in vitro

Characterization and metabolism of heparan sulfate glycosaminoglycans and proteoglycans (HSPGs) synthesized by primary cultures of mouse uterine epithelial cells are reported. HSPGs were detected in both the medium and in the cell-associated fraction, whereas glycosaminoglycans containing little or no protein (free glycosaminoglycans) were found primarily in the cell-associated fraction. The cell-associated HSPGs were relatively large (Kav = 0.1 on Superose 12), had a buoyant density in cesium chloride gradients of 1.45-1.55 g/ml, and contained heparan sulfate chains that fell into two size classes, exhibiting Kav values on Superose 12 of 0.2-0.5 and 0.7-0.8, respectively. The free glycosaminoglycan chains displayed a Kav on Superose 12 of 0.6-0.7. The secreted HSPGs were smaller (median Kav on Superose 12 of 0.28) than the cell-associated HSPGs. More than 90% of the cell-associated HSPGs contained hydrophobic portions, as evidenced by their ability to bind to octyl-Sepharose. In contrast, only 10-15% of the secreted HSPGs bound to octyl-Sepharose. HSPGs were detected at both apical and basal cell surfaces/extracellular matrices by indirect immunofluorescence in vitro and in utero and by accessibility to external proteases in vitro. It was estimated that 60-70% of the total cell-associated HSPGs were exposed at the cell surface. The HSPGs released from the cell surface by proteases were slightly smaller than the intact HSPGs and lacked the hydrophobic properties of the latter. These observations suggested that the cell surface HSPGs contain a small, hydrophobic domain that functions in the attachment of HSPGs to cells. The free glycosaminoglycans appeared to be primarily intracellular and were not secreted. The cell-associated HSPGs turned over rapidly (t1/2 = 1.5 h) and appeared to be the precursors to the free glycosaminoglycans. Metabolic turnover of the free glycosaminoglycan pool was a relatively slow process (t1/2 = 10-12 h).(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of a sialylated N-linked glycoprotein in insect cells

Under High Aspect Ratio Vessel (HARV) bioreactor culture conditions designed to simulate the microgravity of orbital space flight, insect tissue culture cells infected with a baculovirus expression vector produced a human glycoprotein with tri- and tetra-antennary complex N-linked oligosaccharides containing terminal sialic acid residues. The oligosaccharide structures were similar to those produced in human placental cells. Insect cells cultured in T-flasks only performed incomplete oligosaccharide processing. The mechanism of HARV-mediated changes in the eukaryotic N-linked glycosylation pathway was investigated and could be mimicked under T-flask growth conditions with the addition of N-acetylmannosamine to the culture medium. The significance of these investigations is discussed with respect to the production of recombinant therapeutic glycoproteins, insect physiology, and orbital space flight.     

Differential effects of p-nitrophenyl-D-xylosides on mouse blastocysts and uterine epithelial cells

A number of studies have implicated glycoconjugates in cell recognition events associated with implantation of mammalian blastocysts into the uterus. We have found that p-nitrophenyl-D-xylosides inhibit mouse embryo attachment and outgrowth on monolayers of uterine epithelial cells when cocultured in vitro. Inhibition of attachment and trophoblast formation by alpha- and beta-xylosides was observed in embryos cultured on tissue culture plastic in serum containing medium or on monolayers of epithelial cells. The biochemical basis for this inhibition has been investigated. Consistent with their accepted mode of action, beta- but not alpha-D-xylosides greatly stimulated glycosaminoglycan chain production by uterine epithelial cells and likewise reduced proteoglycan assembly. In contrast, both alpha- and beta-anomers selectively inhibited embryo attachment and outgrowth without stimulating glycosaminoglycan chain production by embryos. The inhibitory effect of the xylosides on embryos was reversible and did not require concentrations that reduced the rate of protein synthesis. Both alpha- and beta-D-xylosides inhibited the synthesis of proteoglycans including heparan sulfate as well as certain other glycoconjugates by embryos. Collectively, these data indicate that proper assembly of glycoconjugates, including proteoglycans, is required for implantation-related processes, although the inhibition of embryo outgrowth by xylosides may be by an as yet uncharacterized mechanism.     

Cripto-1 induces apoptosis in HC-11 mouse mammary epithelial cells

Cripto-1 (CR-1) is an epidermal growth factor (EGF)-related protein. CR-1 can inhibit beta-casein and whey acidic protein expression in mouse mammary epithelial cells. The present study demonstrates that CR-1 can induce apoptosis in HC-11 mouse mammary epithelial cells, as measured by bis-benzimide stained nuclei, TUNEL assay and cell death ELISA. Apoptosis could be observed after 2 days of exposure of confluent HC-11 cells to CR-1 in the absence of the survival factors EGF and insulin, with maximum apoptosis occurring at 3 days. A reduction in poly(ADP-ribose) polymerase (PARP) expression and an increase in beta-catenin cleavage was found after 18 h of exposure to CR-1 suggesting that apoptosis was preceded by the induction of a caspase activity since the caspase inhibitor ZFAD.FMK could block the CR-1-induced reduction in PARP expression and CR-1-induced apoptosis. CR-1 was found to increase the expression of caspase-3-like protease. Although, the levels of p27kip1 and p21Bax did not change after exposure to CR-1 for 18 h, the levels of Bcl-xL became undetectable. These studies suggest that CR-1 promotes apoptosis by mediating the induction of caspase-3-like protease and downregulating the expression of Bcl-xL.     

Neoplastic transformation of mouse mammary epithelial cells by deregulated myc expression.

A spontaneously immortalized, nontumorigenic mouse mammary epithelial cell line (MMEC) was transfected with an activated myc construct by electroporation. Constitutive expression of myc in MMEC resulted in anchorage independence in soft agar and tumorigenicity in nude mice. The myc-expressing MMEC showed higher saturation density, faster growth rate, and partial abrogation of serum-derived growth factor(s) requirement compared with parent MMEC. Epidermal growth factor or transforming growth factor alpha stimulated the anchorage-independent growth, but not the anchorage-dependent growth, of MMEC-myc cells. Type 1 transforming growth factor beta, on the other hand, inhibited both the anchorage-independent and anchorage-dependent growth of MMEC-myc cells. These results demonstrate that deregulated expression of myc results in neoplastic transformation iin mammary epithelial cells. Accompanying the transformation is altered sensitivity to polypeptide growth factors.     

Development of morphological and functional polarity in primary cultures of immature rat uterine epithelial cells

The present study describes a culture environment in which luminal epithelial cells isolated from immature rat uteri and cultured on a matrix-coated permeable surface, with separate apical and basal secretory compartments, proliferate to confluence. Subsequently the cells undergo a process of differentiation accompanied by progressive development of functional polarity. Ultrastructural and immunocytochemical evidence verifies the ability of these primary cultures to regain polar organization, separate membrane domains, and form functional tight junctions as demonstrated by the development of transepithelial resistance. The appearance of uvomorulin is restricted to the lateral cell surface. Coordinated indices of functional polarity that develop progressively in post-confluent cultures include the preferential uptake of [35S]methionine from the basal surface and a rise in uterine epithelial cell secretory activity characterized by a progressive preference for apical secretion. The time dependent development of polarity was characterized by differences in the protein profiles of the apical and basolateral secretory compartments. The maintenance of hormone responsiveness by the cultured cells was validated by the secretion of two proteins identified as secretory markers of estrogen response in the intact uterus. The technique of culturing the cells on a matrix-coated permeable surface with separate secretory compartments produces a uterine epithelial cell that morphologically and functionally resembles its in situ equivalent. The culture method and analytical approach used in this present study may be applied to primary cultures of a variety of natural epithelia, which have hitherto proven resistant to more conventional culture methodologies.     

Inhibition of Rho-kinase induces alphaB-crystallin expression in lens epithelial cells

The small heat shock protein, alphaB-crystallin, has been shown to interact with actin and intermediate filament proteins. However, little is known regarding the cellular mechanisms regulating such interactions. In this study, we explored the role of the Rho/Rho-kinase pathway in alphaB-crystallin distribution and expression in porcine lens epithelial cells. alphaB-crystallin was distributed uniformly throughout the cytoplasm and did not exhibit any unique redistribution in response to actin depolymerization induced by Rho/Rho-kinase inhibitors (C3-exoenzyme or Y-27632) or by overexpression of the dominant negative mutant of Rho-kinase (DNRK) in porcine lens epithelial cells. Interestingly, alphaB-crystallin levels markedly increased in lens epithelial cells treated with the inhibitors of Rho/Rho-kinase proteins (lovastatin, Y-27632 or DNRK) while a protein kinase C inhibitor (GF109203x) was found to have no effect. Further, Y-27632 showed a dose (2-50 microM) response effect on alphaB-crystallin induction. Nocodazole, a microtubule-depolymerizing agent, elicited an increase in alphaB-crystallin levels but latrunculin, an actin depolymerizing agent, did not show any significant effect. Pretreatment with cycloheximide or genistein blocked the Rho-kinase inhibitor-induced increase in alphaB-crystallin protein levels. Rho-kinase inhibitor-induced increases in alphaB-crystallin levels were found to be associated with activation of P38 mitogen-activated protein kinase (MAPK). These results suggest that Rho/Rho-kinase negatively regulates alphaB-crystallin expression, and this response appears to be dependent on tyrosine-protein kinase and P38 MAPK function. Finally, alphaB-crystallin induction appears to be better correlated with the direct inhibition of Rho/Rho-kinase than with actin depolymerization per se.     

孕激素诱导cyclin G1在小鼠子宫内膜上皮细胞的表达及其对细胞增殖的影响

本文在体外培养条件下研究卵巢激素诱导小鼠子宫内膜上皮细胞cyclin G1的表达及细胞增殖和细胞周期进程的变化,以探讨孕激素依赖的细胞周期调控因子cyclin G1对子宫内膜上皮细胞增殖的负调控作用.原代培养小鼠子宫内膜上皮细胞,待其生长汇合后分为4组:对照组(C组)、雌激素组(E组)、孕激素组(P组)、雌、孕激素共同作用组(EP组).加入相应激素作用24 h后,用细胞免疫化学方法检测各组细胞cyclin G1的表达水平:四甲基偶氮唑蓝(MTT)比色法检测各组细胞活力,间接观察子宫内膜上皮细胞的增殖情况;用流式细胞仪检测分布在细胞周期各时相的子宫内膜上皮细胞所占百分数.细胞免疫化学结果显示,cyclin G1在C组和E组子宫内膜上皮细胞上无明显表达,而在P组和EP组子宫内膜上皮细胞中表达明显,且定位于细胞核内.MTT法结果显示,与C组相比,E组细胞活力明显增高,而P组和EP组的细胞活力均明显下降,表明雌激素能促进子宫内膜上皮细胞增殖,而孕激素则具有抑制子宫内膜上皮细胞增殖的作用.流式细胞术检测显示,与C组相比,E组中处于S期的子宫内膜上皮细胞百分数增多;P组与EP组中处于S期的子宫内膜上皮细胞百分数明显减少,而处于G1期的细胞百分数和G2/M期的细胞百分数则明显增加.上述结果提示,孕激素依赖的cyclin G1可能通过阻滞细胞周期进程来参与孕激素对子宫内膜上皮细胞增殖的负调控作用.     

Failure of galectin-9 expression by uterine endometrial epithelial cells as a cause for preeclampsia

Preeclampsia is a severe pregnancy complication that originates in the placenta and is characterized by shallow trophoblast invasion into the spiral arteries. Immunological imbalances associated with abnormal uterine spiral arteries remodeling during pregnancy have been identified to contribute to the onset and progression of preeclampsia. Interferon (IFN)-γ has a bilateral role in mediating uterine spiral artery remodeling and may lead to preeclampsia under abnormal circumstances. Until recently, the mechanism that regulates the balance between IFN-γ-mediated artery remodeling and IFN-γ-induced Th1 cell activation is ambiguous; but recent studies suggest an important part for galectin-9 in the immune regulation. Therefore, we hypothesize that the galectin-9 expression by uterine endometrial epithelial cells plays a key role in the regulation of the dual function of IFN-γ. Engaging galectin-9 with its receptor on activated Th1 cells causes an inhibitory signal, resulting in apoptosis of Th1 cells and negatively regulates Th1 type immunity. We further hypothesize that failure of galectin-9 expression by endometrial epithelial cells may dampen the endovascular remodeling process and thus result in preeclampsia. This hypothesis proposes a new mechanism in the immunological balance at the uteroplacental interface. Also this hypothesis will help to find out new cause for preeclamspsia and provide new strategy for disease treatment.