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Lewis P Rubin

Publications and source records attributed to Lewis P Rubin.

13 recordsLinked to original sources
PubMed2006

Role of aspartyl-(asparaginyl) beta-hydroxylase in placental implantation: Relevance to early pregnancy loss.

Aspartyl-(asparaginyl) beta-hydroxylase (AAH) is a type 2 transmembrane protein with catalytic activity that hydroxylates epidermal growth factor-like domains of proteins that have a functional role in cell motility and invasion. Extravillous cytotrophoblasts (CTB) are motile and invasive unpolarized epithelial cells that mediate early implantation through interaction with the endometrium. This study characterizes the potential role of AAH in CTB implantation using human placentas from (1) terminated pregnancies (n = 11), (2) normal term deliveries (n = 21), (3) spontaneous abortuses (n = 21), and (4) small-for-gestational-age (SGA) term deliveries (n = 21). The SGA cases all had established clinical histories of intrauterine growth restriction or preeclampsia. Formalin-fixed, paraffin-embedded sections of placenta were immunostained using the 15C7 monoclonal antibody generated to recombinant AAH. In addition, snap-frozen or RNAlater-preserved specimens (Ambion, Austin, TX) were used for RNA analysis of AAH expression by real-time quantitative reverse transcriptase-polymerase chain reaction and protein analysis by Western blotting. The immunohistochemical staining studies demonstrated AAH expression in amniocytes, villous CTB, syncytiotrophoblast, extravillous CTB, decidua, and endometrial glands at all gestational ages and in all 4 groups. Higher levels of AAH immunoreactivity were observed in extravillous CTB compared with villous CTB. Immunohistochemical staining and RNA analysis demonstrated abundant AAH expression in placental trophoblastic cells as well as in decidua and endometrial glands, with reduced expression in spontaneous abortion and SGA, suggesting that AAH may serve as a biomarker of impaired implantation. The high levels of AAH in decidua and endometrial glands suggest a role for this molecule in "receptivity" of endometrium.

Abortion, Spontaneous↗
PubMed2006

Cooperation between MEF2 and PPARgamma in human intestinal beta,beta-carotene 15,15'-monooxygenase gene expression.

BACKGROUND: Vitamin A and its derivatives, the retinoids, are essential for normal embryonic development and maintenance of cell differentiation. beta, beta-carotene 15,15'-monooxygenase 1 (BCMO1) catalyzes the central cleavage of beta-carotene to all-trans retinal and is the key enzyme in the intestinal metabolism of carotenes to vitamin A. However, human and various rodent species show markedly different efficiencies in intestinal BCMO1-mediated carotene to retinoid conversion. The aim of this study is to identify potentially human-specific regulatory control mechanisms of BCMO1 gene expression. RESULTS: We identified and functionally characterized the human BCMO1 promoter sequence and determined the transcriptional regulation of the BCMO1 gene in a BCMO1 expressing human intestinal cell line, TC-7. Several functional transcription factor-binding sites were identified in the human promoter that are absent in the mouse BCMO1 promoter. We demonstrate that the proximal promoter sequence, nt -190 to +35, confers basal transcriptional activity of the human BCMO1 gene. Site-directed mutagenesis of the myocyte enhancer factor 2 (MEF2) and peroxisome proliferator-activated receptor (PPAR) binding elements resulted in decreased basal promoter activity. Mutation of both promoter elements abrogated the expression of intestinal cell BCMO1. Electrophoretic mobility shift and supershift assays and transcription factor co-expression in TC-7 cells showed MEF2C and PPARgamma bind to their respective DNA elements and synergistically transactivate BCMO1 expression. CONCLUSION: We demonstrate that human intestinal cell BCMO1 expression is dependent on the functional cooperation between PPARgamma and MEF2 isoforms. The findings suggest that the interaction between MEF2 and PPAR factors may provide a molecular basis for interspecies differences in the transcriptional regulation of the BCMO1 gene.

5' Flanking Region↗
PubMed2005

Integrins beta1, alpha6, and alpha3 contribute to mechanical strain-induced differentiation of fetal lung type II epithelial cells via distinct mechanisms.

Mechanical forces regulate lung maturation in the fetus by promoting type II epithelial differentiation. However, the cell surface receptors that transduce these mechanical cues into cellular responses remain largely unknown. When distal lung type II epithelial cells isolated from embryonic day 19 rat fetuses were cultured on flexible plates coated with laminin, fibronectin, vitronectin, collagen, or elastin and exposed to a level of mechanical strain (5%) similar to that observed in utero, transmembrane signaling responses were induced under all conditions, as measured by ERK activation. However, mechanical stress maximally increased expression of the type II cell differentiation marker surfactant protein C when cells were cultured on laminin substrates. Strain-induced alveolar epithelial differentiation was inhibited by interfering with cell binding to laminin using soluble laminin peptides (IKVIV or YIGSR) or blocking antibodies against integrin beta1, alpha3, or alpha6. Additional studies were carried out with substrates coated directly with different nonactivating anti-integrin antibodies. Blocking integrin beta1 and alpha6 binding sites inhibited both cell adhesion and differentiation, whereas inhibition of alpha3 prevented differentiation without altering cell attachment. These data demonstrate that various integrins contribute to mechanical control of type II lung epithelial cell differentiation on laminin substrates. However, they may act via distinct mechanisms, including some that are independent of their cell anchoring role.

Animals↗
PubMed2005

Second-trimester maternal serum markers in twin pregnancy with complete mole: report of 2 cases.

We present second-trimester serum marker levels in cases of twin pregnancy with complete hydatidiform mole and a coexistent fetus (CHMCF). Second-trimester inhibin A (InhA) levels have not been previously reported in such cases. Second-trimester maternal serum screening, alpha-fetoprotein (AFP), unconjugated estriol (uE3), human chorionic gonadotropin (hCG), and InhA measurements combined with maternal age to estimate a patient's risk of Down syndrome during pregnancy was performed as a routine prenatal test in 2 cases of CHMCF. Hospital records containing serum marker data, patient history, pathology reports, and pregnancy outcome were reviewed. In cases of CHMCF, maternal serum AFP and uE3 levels were similar to those of a normal singleton pregnancy, whereas hCG and InhA levels were markedly increased. Second-trimester maternal serum marker profiles in cases of CHMCF are a composite of normal singleton and molar tissue secretions. We have found, for the first time, that second-trimester InhA levels are markedly increased in these cases. Serum marker levels may be useful in diagnosis of CHMCF, prenatal counseling, and evaluation of risk for persistent trophoblastic disease.

Adolescent↗
PubMed2005

Hyperoxia-induced apoptosis and Fas/FasL expression in lung epithelial cells.

Alveolar epithelial apoptosis is an important feature of hyperoxia-induced lung injury in vivo and has been described in the early stages of bronchopulmonary dysplasia (chronic lung disease of preterm newborn). Molecular regulation of hyperoxia-induced alveolar epithelial cell death remains incompletely understood. In view of functional involvement of Fas/FasL system in physiological postcanalicular type II cell apoptosis, we speculated this system may also be a critical regulator of hyperoxia-induced apoptosis. The aim of this study was to investigate the effects of hyperoxia on apoptosis and apoptotic gene expression in alveolar epithelial cells. Apoptosis was studied by TUNEL, electron microscopy, DNA size analysis, and caspase assays. Fas/FasL expression was determined by Western blot analysis and RPA. We determined that in MLE-12 cells exposed to hyperoxia, caspase-mediated apoptosis was the first morphologically and biochemically recognizable mode of cell death, followed by necrosis of residual adherent cells. The apoptotic stage was associated with a threefold upregulation of Fas mRNA and protein expression and increased susceptibility to direct Fas receptor activation, concomitant with a threefold increase of FasL protein levels. Fas gene silencing by siRNAs significantly reduced hyperoxia-induced apoptosis. In murine fetal type II cells, hyperoxia similarly induced markedly increased Fas/FasL protein expression, confirming validity of results obtained in transformed MLE-12 cells. Our findings implicate the Fas/FasL system as an important regulator of hyperoxia-induced type II cell apoptosis. Elucidation of regulation of hyperoxia-induced lung apoptosis may lead to alternative therapeutic strategies for perinatal or adult pulmonary diseases characterized by dysregulated type II cell apoptosis.

Animals↗
PubMed2004

Stromal cell-derived factor-1 (SDF-1) signalling regulates human placental trophoblast cell survival.

Stromal cell-derived factor-1 (SDF-1 or CXCL12) is the physiologic ligand for the chemokine receptor CXCR4. CXCR4-mediated signalling regulates cell migration and apoptosis in certain haematopoietic and neuronal cells. Using gene profiling, we determined that CXCR4 is the only chemokine receptor for which mRNA expression is regulated during trophoblast differentiation in vitro. Based on the known effects of CXCR4 ligation, we hypothesized that CXCR4 activation may regulate placental trophoblast cell survival (i.e. protection from apoptosis), an important mechanism for the establishment and maintenance of the uteroplacental barrier. Human cytotrophoblasts (CTBs) were cultured in defined media and treated with graded doses of SDF-1 (10-100 ng/ml) or with an anti-CXCR4 neutralizing antibody. Exposure to anti-CXCR4 antibody reduced CTB cell numbers by 25-40%. Treatment with SDF-1 decreased the proportions of apoptotic terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick-end labelling(+) cells (apoptotic index [AI] of 2.79+/-0.61% [control] versus 1.88+/-0.56% [SDF-1]; P<0.05) and caspase-activated cells (AI of 7.95+/-2.49% [control] versus 3.81+/-1.49% [SDF-1]; P<0.05). We determined that SDF-1 also activated the triple MAP Kinase isoforms ERK1/2 and p38 in trophoblasts. Immunocytochemistry confirmed SDF-1-induced nuclear translocation of phosphorylated ERK1/2. Blocking of ERK1/2 signalling with the specific inhibitor PD98059 reversed SDF-1-mediated inhibition of apoptosis (AI of 1.65+/-0.34 [SDF-1] versus 3.50+/-0.5 [SDF-1 + PD98059]; P<0.05), suggesting that SDF-1 acts through this pathway as a trophoblast survival factor. These results indicate that SDF-1/CXCR4 signalling stimulates anti-apoptotic pathways in cultured trophoblasts. This chemotactic ligand/receptor system may promote trophoblast survival during pregnancy. Alterations in SDF-1 and/or CXCR4 expression or function may be associated with specific pregnancy disorders.

Antibodies↗
PubMed2004

Arrested pulmonary alveolar cytodifferentiation and defective surfactant synthesis in mice missing the gene for parathyroid hormone-related protein.

Parathyroid hormone-related protein (PTHrP) and PTH/PTHrP receptor expression are developmentally regulated in lung epithelium and adepithelial mesenchyme, respectively. To test the hypothesis that PTHrP is a developmental regulator of terminal airway development, we investigated in vivo and in vitro models of alveolar cytodifferentiation using mice in which the gene encoding PTHrP was ablated by homologous recombination. We have determined that fetal and newborn PTHrP(-/-) lungs showed delayed mesenchymal-epithelial interactions, arrested type II cell differentiation, and reduced surfactant lamellar body formation and pulmonary surfactant production. Embryonic PTHrP(-/-) lung buds cultured in the absence of skeletal constriction or systemic compensating factors also exhibited delayed alveolar epithelial (type II cell) and mesenchymal cytodifferentiation, as well as a > 40% inhibition of surfactant phospholipid production (n = 3-5). Addition of exogenous PTHrP to embryonic PTHrP(-/-) lung cultures normalized interstitial cell morphology and surfactant phospholipid production. The importance of PTHrP as an endogenous regulatory molecule in mammalian lung development is supported by the findings that ablation of PTHrP expression in isolated developing lung is sufficient to disrupt normal development of the alveolar ducts and the centriacinar regions.

Animals↗
PubMed2004

Fas/FasL-mediated apoptosis in perinatal murine lungs.

Postcanalicular lung development is characterized by a time-specific increase in alveolar epithelial type II cell apoptosis. We have previously demonstrated that, in fetal rabbits, developmental type II cell apoptosis coincides with transient upregulation of the cell death regulator Fas ligand (FasL). The aims of this study were 1) to determine the spatiotemporal patterns of pulmonary apoptosis and Fas/FasL gene expression in the murine model [embryonic day 17 (E17) through postnatal day 5 (P5)], and 2) to investigate the functional involvement of the Fas/FasL system by determining the effect of Fas activation and inhibition on perinatal pulmonary apoptosis. The apoptotic activity of alveolar epithelial type II cells, determined by combined TUNEL labeling and anti-surfactant protein B immunohistochemistry, showed a dramatic increase during the perinatal transition (type II cell apoptotic index <0.1% at E17, 1.5% at P1-P3, and 0.3% at P5). This timing of enhanced type II cell apoptosis coincided with a robust 14-fold increase in Fas mRNA and protein levels and a threefold increase in FasL protein levels; both Fas and FasL immunolocalized to type II and bronchial epithelial cells. In vitro and in vivo exposure of fetal and postnatal murine type II cells to anti-Fas antibody induced a fourfold increase in apoptotic activity that was prevented by administration of a broad-spectrum caspase inhibitor; the pulmonary apoptotic activity of Fas-deficient lpr mice remained unchanged. Conversely, administration of a caspase inhibitor to newborn mice (P1) resulted in marked diminution of pulmonary apoptotic activity. These combined findings strongly implicate the Fas/FasL system as a critical regulator of perinatal type II cell apoptosis. The developmental time dependence of apoptosis-related events in the murine model should facilitate investigations of the regulation of perinatal pulmonary apoptotic gene expression.

Aging↗
PubMed2003

Mechanical stretch induces fetal type II cell differentiation via an epidermal growth factor receptor-extracellular-regulated protein kinase signaling pathway.

Mechanical forces are important for fetal alveolar epithelial cell differentiation. However, the signal transduction pathways regulating this process remain largely unknown. Based on the importance of the extracellular-regulated protein kinase (ERK) pathway in cell differentiation, we hypothesized that this cascade mediates stretch-induced fetal type II cell differentiation. We demonstrate that ERK1/2 was maximally activated (> 3-fold) after 15 min of cyclic stretch. Blockage of the ERK pathway with U0126 (a selective MEK1/2 inhibitor) significantly decreased stretch-inducible surfactant protein-C (SP-C) mRNA expression. We examined upstream activators of ERK1/2 and found that stretch induced phosphorylation of Raf-1 and activation of Ras. Moreover, GW5074, a selective c-Raf-1 inhibitor, decreased stretch-inducible SP-C mRNA accumulation. Mechanical stretch also stimulated epidermal growth factor receptor (EGFR) phosphorylation. Finally, blockage of the EGFR, either with tyrphostin AG1478 or neutralizing antibody, decreased stretch-inducible SP-C mRNA expression. We conclude that stretch, at least in part, induces differentiation of fetal epithelial cells via EGFR activation of the ERK pathway. These results suggest that EGFR may be a mechanosensor during fetal lung development. These findings may have significant implications for the design of strategies to accelerate lung maturation.

Animals↗
PubMed2003

Testing for hematologic disorders and complications.

This review summarizes state-of-the-art and emerging techniques in the antenatal diagnosis of fetal anemia and hemoglobinopathies. Fetal anemia may result from hemolytic disease, hemorrhage, suppression of erythropoiesis, infection (eg, parvovirus B19), or trauma. The clinical laboratory plays an essential role in the evaluation of these disorders by way of the use of various hematologic, biochemical, serologic, cytometric, and molecular genetics methods. Hemoglobinopathies are the most common class of single gene disorders worldwide. The authors have used the example of homozygous alpha-thalassemia major (Hb Barts disease) as a paradigmatic case for antenatal hemoglobinopathy screening. Perhaps the most familiar indication for hematologic screening in pregnancy is HDFN, most commonly in pregnancies previously sensitized to the RhD antigen. All pregnant women, regardless of their past medical or obstetric history or previous antibody screens, should have ABO/Rh blood typing and a red cell antibody screen performed at the first prenatal visit. Long-established methods for assaying FMH (KB method), microcytosis (hemogram with red cell indices), and blood group incompatibility (direct antigen test, serologies) remain critical for rapid, sensitive diagnosis. Analysis of fetal free DNA in maternal plasma holds the promise for rapid, ultrasensitive, and noninvasive detection of many fetal hematologic disorders.

Adult↗
PubMed2002

Cyclic mechanical stretch inhibits cell proliferation and induces apoptosis in fetal rat lung fibroblasts.

Development of the pulmonary air sacs is crucial for extrauterine survival. Late fetal lung development is characterized by a thinning of the mesenchyme, which brings pneumocytes and endothelial cells into apposition. We hypothesized that mechanical stretch, simulating fetal breathing movements, plays an important role in this remodeling process. Using a Flexercell Strain Unit, we analyzed the effects of intermittent stretch on cell proliferation and apoptosis activation in fibroblasts isolated from fetal rat lungs during late development. On day 19, intermittent stretch increased cells in G(0)/G(1) by 22% (P = 0.001) and decreased in S phase by 50% (P = 0.003) compared with unstretched controls. Cell proliferation analyzed by 5-bromo-2'-deoxyuridine incorporation showed a similar magnitude of cell cycle arrest (P = 0.04). At this same gestational age, stretch induced apoptosis by two- to threefold over controls, assayed by DNA flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick-end labeling, and caspase-3 activation. These results indicate that mechanical stretch of fibroblasts isolated during the canalicular stage inhibits cell cycle progression and activates apoptosis. These findings are cotemporal with the mesenchymal thinning that normally occurs in situ.

Animals↗