Fabrication and optical characterization of large scale membrane containing InP/AlGaInP quantum dots.

Single-photon sources with a high extraction efficiency are a prerequisite for applications in quantum communication and quantum computation schemes. One promising approach is the fabrication of a quantum dot containing membrane structure in combination with a solid immersion lens and a metal mirror. We have fabricated an 80 nm thin semiconductor membrane with incorporated InP quantum dots in an AlGaInP double hetero barrier via complete substrate removal. In addition, a gold layer was deposited on one side of the membrane acting as a mirror. The optical characterization shows in detail that the unique properties of the quantum dots are preserved in the membrane structure.

An Ets motif in the proximal decidual prolactin promoter is essential for basal gene expression.

Transcriptional regulation of the prolactin gene in the decidua differs from that in the pituitary. Several lines of evidence strongly suggest this difference is due to regulation of the prolactin gene in the decidua and other extra-pituitary tissues by tissue-specific transcription factors, which activate distinct promoters to induce prolactin gene expression in extra-pituitary sites compared with the pituitary. The human decidua is a major site of extra-pituitary expression of the prolactin gene. Here we present evidence that the transcription factor Ets-1 is critical for basal expression of the decidua-type (or decidual) prolactin promoter. Overexpression of Ets-1 significantly induces decidual prolactin promoter activity in BeWo and JAR cells that express little or no endogenous Ets-1. Conversely, a dominant/negative mutant of Ets represses basal promoter activity. Although the proximal 1.5 kb of the decidual prolactin promoter contains six Ets motifs, only the Ets motif at nt -77/-71 is essential for basal gene expression. Mutation of the Ets motif at nt -77/-71 results in an approximately 90% decrease in promoter activity, while mutation of the other Ets motifs results in only small changes. Electrophoretic mobility shift assays demonstrate that Ets proteins in decidualized endometrial stromal cells bind this Ets motif in the decidual prolactin promoter. Ets protein expression increases up to 20-fold upon induction of decidualization in endometrial stromal cells under conditions in which expression of the prolactin gene is also induced. These studies provide strong evidence for a critical role of the Ets transcription factor in basal expression of the decidual prolactin promoter.

Identification of a decidua-specific enhancer on the human prolactin gene with two critical activator protein 1 (AP-1) binding sites.

Deletion analysis of the human PRL promoter in endometrial stromal cells decidualized in vitro revealed a 536-bp enhancer located between nucleotide (nt) -2,040 to -1,505 in the 5'-flanking region. The 536-bp enhancer fragment ligated into a thymidine kinase (TK) promoter-luciferase reporter plasmid conferred enhancer activity in decidual-type cells but not nondecidual cells. DNase I footprint analysis of decidualized endometrial stromal cells revealed three protected regions, FP1-FP3. Transfection of overlapping 100-bp fragments of the 536-bp enhancer indicated that FP1 and FP3 each conferred enhancer activity. Gel shift assays indicated that both FP1 and FP3 bind activator protein 1 (AP-1), and JunD and Fra-2 are components of the AP-1 complex in decidual fibroblasts. Mutation of the AP-1 binding site in either FP1 or FP3 decreased enhancer activity by approximately 50%, while mutation of both sites almost completely abolished activity. Coexpression of the 536-bp enhancer and A-fos, a dominant negative to AP-1, decreased enhancer activity by approximately 70%. Conversely, coexpression of Fra-2 in combination with JunD or c-Jun and p300 increased enhancer activity 6- to 10-fold. Introduction of JunD and Fra-2 into nondecidual cells is sufficient to confer enhancer activity. JunD and Fra-2 protein expression was markedly increased in secretory phase endometrium and decidua of early pregnancy (high PRL content) compared with proliferative phase endometrium (no PRL). These investigations indicate that the 5'-flanking region of the human PRL gene contains a decidua-specific enhancer between nt -2,040/-1,505 and AP-1 binding sites within this enhancer region are critical for activity.

Gene induction and categorical reprogramming during in vitro human endometrial fibroblast decidualization.

Gene induction and categorical reprogramming during in vitro human endometrial fibroblast decidualization. Physiol Genomics 7: 135-148, 2001. First published September 21, 2001; 10.1152/physiolgenomics.00061.2001.-Human decidual fibroblasts undergo a differentiative commitment to the acquisition of endocrine, metabolic, and structural cell functions in a process known as decidualization. Decidualization is critical for embryo implantation and placental function. We characterized gene expression pattern kinetics during decidual fibroblast differentiation by microarray analysis. Of 6,918 genes analyzed, 121 genes were induced by more than twofold, 110 were downregulated, and 50 showed biphasic behavior. Dynamically regulated genes were could be fit into nine K-means algorithm-based kinetic pattern groups, and by biologic classification, into five categories: cell and tissue function, cell and tissue structure, regulation of gene expression, expressed sequence tag (EST), and "function unknown." Reprogramming of genes within specific functional groups and gene families was a prominent feature that consisted of simultaneous induction and downregulation of a set of genes with related function. We previously observed a conceptually similar process during fetal trophoblast differentiation, in which the same phenomena applied to different genes. Of the 569 dynamically regulated genes regulated by either model, only 81 of these were in common. These results suggest that reprogramming of gene expression within focused functional categories represents a fundamental aspect of cellular differentiation.

Activator protein-2 regulates human placental lactogen gene expression.

DNase I footprint analysis of the human placental lactogen-A (hPL-A) promoter using nuclear extracts from purified human trophoblast cells and BeWo choriocarcinoma cells revealed five protected regions within the proximal 325 bp. Two of the protected regions, FP4 (-289--267) and FP5 (-167--154), are homologous to regions on the human growth hormone (hGH) promoter that bind transcription factors AP-2 and/or NFI. Competitive gel shift assays and supershift assays indicated that FP4 forms complexes with activator protein-2 (AP-2) and nuclear factor I (NFI), while FP5 forms a complex with AP-2 alone. In transient transfection studies in human trophoblast cells, hPL promoter constructs containing point mutations in the AP-2 binding sites of FP4 and/or FP5 were 60-80% less active than plasmids containing the wild-type promoter. A mutation in the NFI binding site of FP4, however, had little effect on promoter activity in these cells. Overexpression of AP-2 in HepG2 cells co-transfected with the wild-type hPL promoter resulted in a significant increase in promoter activity. Taken together, these findings suggest a critical role for AP-2 in the regulation of hPL gene expression.

N5 endometrial stromal cell line: a model system to study decidual prolactin gene expression.

Prolactin gene expression in extrapituitary tissues, such as decidua and lymphocytes, is regulated by a distinct promoter approximately 6 kb upstream of the pituitary prolactin gene transcription start site. Here we describe studies in a human endometrial stromal cell line, N5, that was immortalized by transfection with an SV40 mutant and which expresses the prolactin gene driven by the extrapituitary promoter. The N5 cells have phenotypic features of primary cultures of decidualized human endometrial stromal cells and secrete low levels of prolactin and insulin-like growth factor binding protein-1 (IGFBP-1), both of which are markers of decidualized endometrial stromal cells. As in primary cultures of endometrial stromal cells, treatment of N5 cells with progesterone and estradiol alone or in combination with prostaglandin E2 stimulated the synthesis and release of prolactin. Transient transfection of the N5 cells with an expression vector containing - 2927/ + 66 bp of the decidual prolactin promoter coupled to a luciferase reporter gene resulted in a 20 to 25-fold increase in luciferase activity, a magnitude similar to that which occurs in primary cultures of endometrial stromal cells decidualized in vitro by treatment with progesterone and estradiol. Luciferase expression levels were similar in untreated N5 cells and N5 cells treated with progesterone and estradiol. Taken together, these results indicate that the N5 human endometrial stromal cell line has phenotypic characteristics of normal decidualized stromal cells and is a useful model to study regulation of decidual prolactin gene expression.

Progesterone-dependent decidualization of the human endometrium is mediated by cAMP.

Progesterone is a key factor in regulating endometrial cell decidualization, but the signal transduction pathways involved in mediating the effects of progesterone are not known. A role of the cAMP pathway in decidualization has been suggested by in vitro studies demonstrating that cAMP agonists can stimulate decidualization, in the absence of sex steroids. In this article, we have used an in vitro culture model of progesterone-dependent decidualization of human endometrial stromal cells to examine whether progesterone-induced decidualization is associated with activation of the cAMP signal transduction pathway in which the prolactin gene expression is a marker of decidualization. Following a lag period of approx 3 d, progesterone induced prolactin secretion and elevated intracellular cAMP levels. By d 15, cAMP and prolactin levels were approx 10- and 60-fold greater, respectively, than those on d 3. Changes in cAMP levels showed a positive correlation with prolactin secretion. Prostaglandin E2 (PGE2), which enhances progesterone-dependent decidualization, also increased both prolactin secretion and cAMP levels approx two- to fourfold on d 15 compared with d 3, whereas PGE2 alone, which does not induce decidualization, did not stimulate prolactin secretion or intracellular cAMP accumulation. Conversely, all-trans retinoic acid, which attenuates progesterone-dependent decidualization, significantly (p < 0.05) decreased both prolactin secretion and cAMP levels. Furthermore, the protein kinase A (PKA) inhibitor, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, significantly (p < 0.05) suppressed progesterone-dependent prolactin expression. Since activation of the PGE2 receptor subtype EP2 stimulates adenylate cyclase, reverse transcription-polymerase chain reaction (RT-PCR) analysis of endometrial cells was undertaken. Expression of EP2 mRNA was induced in cells treated with progesterone and estradiol alone or with PGE2, compared with untreated controls. The data suggest that the cAMP signal transduction cascade is activated during progesterone-dependent decidualization.

Retinoic acid suppresses in-vitro decidualization of human endometrial stromal cells.

All-trans retinoic acid (RA) has potent effects on cell differentiation and gene expression. Previous studies have demonstrated that human endometrial stromal cells express mRNA for retinoic acid receptors (RARs) and cellular RA-binding protein-II (CRABP-II). We examined whether RA regulates stromal cell differentiation (decidualization), a critical process in preparation of the uterus for blastocyst implantation. Decidualization was induced by incubating cultured stromal cells with medroxyprogesterone acetate (MPA) and oestradiol. Decidualization was defined by the induction of prolactin, insulin-like growth factor binding protein-1 (IGFBP-1), appearance of a differentiated phenotype and changes in fibronectin expression. RA treatment significantly (P < 0.05) suppressed prolactin and IGFBP-1 production associated with stromal cells decidualization. The formation of differentiated cells was inhibited by RA, and consistent with maintenance of the undifferentiated phenotype, fibronectin mRNA content was approximately 3.5-times greater than in the absence of RA. Upon induction of decidualization, the expression of mRNA for the major RA receptor sub-types (RAR-alpha, -beta and -gamma) was maintained while the relative amounts of CRABP-II mRNA progressively decreased with differentiation. With RA treatment, RAR-alpha and RAR-gamma mRNA concentrations were approximately 70 and 25% respectively of those in cells decidualized in the absence of RA. The effects of RA appear to be partially mediated by inhibition of cAMP action. RA suppressed intracellular cAMP concentrations induced by MPA and oestradiol to approximately 35% of those in cells without RA. Addition of 50 microM dibutyryl cAMP to stromal cells treated with MPA and oestradiol only partially reversed the suppression of decidualization and prolactin release by RA. In summary, we have demonstrated that in-vitro decidualization of human endometrial stromal cells induced by MPA and oestradiol treatment is suppressed by RA.

Up-regulation of the human prolactin receptor in the endometrium.

In humans, uterine endometrial stromal cells differentiate (decidualize) into decidual cells that express prolactin (PRL). Decidual PRL expression continues throughout pregnancy, thus decidual cells lining fetal membranes of term placenta synthesize and secrete PRL. To examine the hypothesis that PRL may play an autocrine role in the decidual cells, we examined the expression of the PRL receptor (PRL-R) during in vitro decidualization of stromal cells and in term decidua. In endometrial stromal cells decidualized by treatment with 1 µM medroxyprogesterone and 10 nM estradiol for 3, 6, and 9 d, respectively, a 12.7 kb PRL-R transcript increased 3-3.5-fold, 16.5-17-fold, and 23.5-24-fold, respectively, compared with untreated controls, in duplicate experiments. Progesterone-dependent PRL-R and PRL expression were stimulated by 1 µ/M prostaglandin E(2). Term decidua expressed the long form of the PRL-R and five major PRL-R transcripts (12.7, 9.7, 7.0, 3.6, and 2.8 kb). In contrast, human liver expressed two major transcripts (12.7 and 9.7 kb) while hepG2 cells expressed a single 7.0-kb-sized transcript. These studies demonstrate that PRL-R expression is stimulated upon progesterone-induced PRL gene expression in endometrial stromal cells supporting the hypothesis that PRL may have an autocrine effect in the endometrium and decidua.

Laminin decreases PRL and IGFBP-1 expression during in vitro decidualization of human endometrial stromal cells.

The expression of laminin, a major constituent of endometrial cell basement membranes, is increased during differentiation of human endometrial stromal cells (decidualization). To determine whether laminin plays a role in decidualization, we studied the effects of laminin substrate on the synthesis and release of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), two major secretory proteins of decidualized stromal cells. Endometrial stromal cells were plated on laminin as well as several other extracellular matrix (ECM) proteins (types 1 and IV collagen or fibronectin) and on plastic, and cultured in media containing medroxyprogesterone acetate (MPA) and estradiol. Cells cultured on plastic or ECM proteins displayed similar morphological changes indicative of decidualization. However, the release of PRL and IGFBP-1 from cells cultured on plastic and ECM proteins (types 1 and IV collagen and fibronection) was approximately 2.1-fold and 2.8-fold greater respectively, than from cells cultured on laminin. The decrease in PRL and IGFBP-1 expression in cells cultured on laminin was not due to differences in initial cell attachment efficiency or final DNA content. In addition, laminin had no effect on the content of laminin protein or fibronectin mRNA levels, indicating that the effects of laminin on PRL and IGFBP-1 were specific. PGE2 stimulated the release of PRL and IGFBP-1 from cells cultured on laminin to levels comparable to those from cells cultured on plastic or other ECM proteins. This indicates that the decrease in PRL and IGFBP-1 release by laminin was not due to a generalized unresponsiveness. In contrast to the effects of laminin during decidualization, PRL expression was not altered by laminin in terminally differentiated decidual cells isolated at term. Our results support a role for laminin in selectively regulating PRL and IGFBP-1 gene expression during in vitro decidualization of human endometrial stromal cells.

An overview of endocrine function and dysfunction.

In more ways than is probably appreciated, critical care nurses deal with clinical situations that involve abnormalities in endocrine function. Endocrine emergencies frequently precipitate patient admissions to critical care units. More common, however, is the deleterious impact of critical illness and prescribed therapies on endocrine function. The manifestations of endocrine pathology are typically subtle and often go undetected until exacerbations and serious medical consequences ensue. To ensure prompt diagnosis and proper therapy, a general understanding of the endocrine function and hormone regulation is essential. It is also important that critical care nurses broaden their repertoire of assessment skills and become more attuned to the consequences of hormonal imbalance.

Endocrinopathies of hyperfunction: Cushing's syndrome and aldosteronism.

Increased function of the adrenal cortex is a normal response in times of physiologic and psychologic stress. Adrenal cortical secretions (e.g., glucocorticoids, aldosterone) orchestrate a multitude of internal processes aimed at maintaining homeostasis and psychologic integrity. Many patients admitted to a critical care unit will manifest some increase, even minor, in adrenal function. However, excessive secretions of these hormones can have a lethal effect of fluid and electrolyte balance, energy metabolism, and immune function. Cushing's syndrome denotes a disorder characterized by increased circulating levels of glucocorticoids (primarily cortisol). An easily recognizable disorder, it may arise from pathology of the adrenal cortex or the anterior pituitary glands, ectopic secretions from a nonendocrine tumor, or from excessive doses of exogenously administered glucocorticoids. Cushing's syndrome is rarely an admitting diagnosis to critical care but is a disorder that can seriously affect recovery from coexisting illnesses if not treated. Aldosteronism, although rare, will often be diagnosed after admission to a critical care unit for management of troublesome hypertension, hypokalemia, congestive heart failure, and various dysrhythmias. Suspicion of the diagnosis should always arise when these manifestations occur, particularly when hypokalemia is refractory to potassium supplementation. Without timely diagnosis and treatment, these patients will succumb to lethal dysrhythmias.

Hyperglycemic emergencies.

Hyperglycemic emergencies are the most common endocrinopathies that require intensive care. It is estimated that between 10% and 15% of patients admitted to intensive care units experience complications of acute hyperglycemia. The common denominator of hyperglycemic emergencies is diabetes mellitus, a group of diseases in which, either because of beta-cell destruction of the pancreas or insulin receptor-site defects, there is a relative or absolute deficiency of insulin that results in hyperglycemia. In response to various precipitating factors, staggering hyperglycemia may develop in the form of diabetic ketoacidosis (DKA) or hyperglycemic hyperosmolar nonketotic syndrome (HHNK). The existence of DKA has been known since ancient times, and critical care nurses are familiar with the diagnosis. The more lethal disorder of HHNK was "rediscovered" in the 1950s and is occurring with greater frequency as clinical awareness of the condition grows and the elderly (who are at greatest risk for the disorder) populate critical care units in increasing numbers. Prevention is instrumental in abating deadly hyperglycemic emergencies. A positive outcome can be realized but only with timely diagnosis and prompt hormonal and fluid replacement.

The chicken urokinase-type plasminogen activator gene.

The chicken urokinase-type plasminogen activator (uPA) cDNA and gene have been isolated and the complete nucleotide sequence of each established. cDNA sequence and Northern blot RNA analysis indicate that the chicken uPA mRNA is approximately 2500 nucleotides in size and contains a large 3'-noncoding region (998 nucleotides). The predicted amino acid sequence of the chicken uPA primary translation product (434 residues) suggests a domain architecture comparable to the mammalian uPA proteins with the form: (i) signal peptide, (ii) growth factor domain (GF), (iii) kringle domain (K), and (iv) serine protease domain (C). The overall sequence identity between the chicken and human proteins is 43.1%, with 56.3, 48.5, and 45.6% identity in the GF, K, and C domains, respectively. The chicken uPA gene is similar to the mammalian uPA genes in both size (8158 base pairs between transcription initiation and polyadenylation sites) and organization (11 exons). However, the sequence of the chicken uPA gene is similar to the mammalian uPA genes only within the protein-coding portions of exons. The transcription initiation site is flanked by a remarkably G/C-rich region (77% between nucleotides -1 and -300) which contains a TATA element and several potential transcription factor Spl-binding sites. The promoter region also contains several repeat elements, including two 11-nucleotide repeats that encompass six potential transcription factor AP-2-binding sites. This work provides a foundation for exploring the mechanism(s) by which protein-tyrosine kinase pp60v-src and protein kinase C modulate uPA gene transcription.