Purification and Characterization of a Dark Red Skin Related Dimeric Polyphenol Oxidase from Huaniu Apples.

The distinct dark-red skin of Huaniu apples renders them attractive to customers. However, the mechanism that leads to the development of the color of the fruit is unclear. In this study, we found that compared with red Fuji (a bright-red apple cultivar), Huaniu apples had higher contents of (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-gallocatechin gallate (GCG), and procyanidins (PCs) B2 and C1 in the peel, which implies that the polymerization of the flavanols and PCs may be correlated with the dark-red skin of the fruit. Using EC as a substrate, we purified an enzyme from Huaniu peel. We performed protein sequencing and discovered that the enzyme was a polyphenol oxidase (PPO). The molecular weight of the enzyme was approximately 140 kDa, which we estimated by native-PAGE and SDS-PAGE, while it was 61 kDa by urea-SDS-PAGE, from which we discovered that the PPO was a dimer. We observed the lowest Km value for catechol (0.60 mM), and the best substrate was 4-methylcatechol, with a Vmax of 526.32 U mg-1 protein. EC is a suitable natural substrate, with a Km value of 1.17 mM, and 55.27% of the Vmax/Km of 4-methylcatechol. When we used EC as a substrate, the optimum temperature and pH of the PPO were 25 °C and 5.0, respectively. In summary, we purified a dimeric PPO from Huaniu apples that showed high activity to EC, which might catalyze the polymerization of flavanols and PCs and lead to the dark-red color development of the fruit.

A Noncovalent π-Stacked Porous Organic Molecular Framework for Selective Separation of Aromatics and Cyclic Aliphatics.

Selective separation using porous adsorbents is an energy-efficient alternative to traditional separation techniques. Stacked porous organic molecular frameworks (POMFs) capable of noncovalent π⋅⋅⋅π interactions are emerging as a new kind of adsorbents that facilitate green separation. Here we report a robust porous molecular crystal (TAPM-1), which is stabilized by multiple intermolecular π⋅⋅⋅π interactions. With its long-range π-stacking, TAPM-1 has excellent hydrophobicity, thermostability, recyclability, and high selectivity for aromatics over the corresponding cyclic aliphatics. This enables TAPM-1 to serve as the stationary phase in the high-resolution gas chromatographic separation of benzene and cyclohexane or toluene and methylcyclohexane.

GLDA and EDTA assisted phytoremediation potential of Sedum hybridum 'Immergrunchen' for Cd and Pb contaminated soil.

Exogenous application of chelants is a common way to enhance the phytoextraction of heavy metals. A pot experiment was conducted to investigate the influences of cadmium (Cd), lead (Pb), Cd and Pb, L-glutamic acid N, N-diacetic acid (GLDA) and ethylene diamine tetraacetate (EDTA) on the growth, Cd and Pb accumulation of Sedum hybridum 'Immergrunchen'. The results showed that Sedum hybridum 'Immergrunchen' had a high tolerance to Pb treatment, followed by Cd-Pb treatment. The plant was sensitive to Cd stress. EDTA treatment was more harmful to plant growth than that of GLDA treatment. The optimal Cd concentration of shoot and root reached 27.6 mg·kg-1 and 32.6 mg·kg-1, 757 mg·kg-1 and 1,025 mg·kg-1for Pb accumulation at 100-1,500 mg·kg-1. The maximum Cd and Pb phytoextraction from 3 mmol·kg-1 GLDA treatment were 1.40 and 1.73 times as much as that of the control, 1.21 and 1.02 times under 6 mmol·kg-1 EDTA treatment. Therefore, the enhanced phytoremediation of GLDA to Cd and Pb co-contaminated soil was better than that of EDTA. GLDA-assisted phytoextraction of Cd and Pb by Sedum hybridum 'Immergrunchen' can be considered as a promising way to phytoremediate Cd and Pb co-contaminated soil.

Sedum hybridum 'Immergrunchen', characterized by strong resistance to waterlogging, draught, salt and zinc, high propagating rate, long green period and high ornamental value, was used as the experimental material. Here, we investigated the best phytoremediation effect of Sedum hybridum 'Immergrunchen' to Cd-Pb stress, GLDA and EDTA assisted phytoremediation potential of Sedum hybridum 'Immergrunchen' for Cd and Pb contaminated soil under the best phytoremediation concentration. The results indicated that Cd and Pb phytoextraction under 3 mmol·kg⁻¹GLDA treatment increased by 39.8% and 73.4% compared with the control, while increased by 21.0% and 1.80% under 6 mmol·kg⁻¹EDTA. More importantly, Pb concentration of shoot in Sedum hybridum 'Immergrunchen' reached the criteria of hyperaccumulators under 3 mmol·kg⁻¹ GLDA treatment.

Bisphenol A stimulates steroidogenic acute regulatory protein expression via an unknown mechanism in adrenal cortical cells.

Bisphenol A (BPA) is one of the most widespread endocrine disrupting chemicals in the environment. Exposure to BPA is known to be associated with disruption of steroidogenesis in reproductive tissues, but little is known about its effects on the adrenal gland. We previously showed that prenatal BPA exposure resulted in elevated plasma corticosterone levels concomitant with increased adrenal levels of steroidogenic acute regulatory protein (StAR), the rate-limiting step in steroidogenesis, in adult female mouse offspring. However, the molecular mechanisms underlying the BPA-induced StAR protein expression in the adrenal gland remain unknown. Therefore, the current study was designed to address this important question using the human cortical cell line, H295A cells, as an in vitro model system. We found that: (1) BPA increased StAR protein levels in a dose-dependent manner; (2) both estrogen receptor alpha (ERα)- and ERß-specific agonists mimicked while the ER antagonist ICI abrogated the stimulatory effects of BPA on StAR protein levels; and (3) BPA did not alter StAR messenger RNA, 37kDa preprotein or protein half-life. Taken together, these findings demonstrate that BPA increases StAR protein levels through an unknown mechanism independent of StAR gene transcription, translation, and protein half-life. Furthermore, such effects are likely mediated by ERα and/or ERß.

Synthesis and characterization of π-extended "earring" subporphyrins.

A π-extended "earring" subporphyrin 3 was synthesized from ß,ß'-diiodosubporphyrin and diboryltripyrrane via a Suzuki-Miyaura coupling and following oxidation. Its Pd complex 3Pd was also synthesized and both of the compounds were fully characterized by 1H NMR, MS and X-ray single crystal diffraction. The 1H NMR spectra and single crystal structures revealed that aromatic ring current did not extend to the "ear" in both of the two compounds. Their UV-vis/NIR spectra were recorded and the absorption of both compounds is extended to the NIR region and that the absorption of 3Pd is further red-shifted and more intense.

Bisphenol A stimulates adrenal cortical cell proliferation via ERß-mediated activation of the sonic hedgehog signalling pathway.

We previously demonstrated that prenatal exposure to bisphenol A (BPA) resulted in increased adrenal gland weight independent of changes in plasma ACTH levels in adult mouse offspring. This finding suggested that BPA exposure likely had a direct effect on adrenal development. Given that (1) sonic hedgehog (Shh) signaling is essential for adrenal development; (2) deletion of the Shh gene in mice results in adrenal hypoplasia; (3) BPA is known to signal through estrogen receptor ß (ERß); and (4) ERß is highly expressed in adrenal glands; we hypothesized that BPA stimulates adrenal cell proliferation via ERß-mediated activation of the Shh pathway. To test this hypothesis, the human adrenal cell line, H295A cells, was used as an in vitro model system. Our main findings were: (1) BPA increased cell number and protein levels of proliferating cell nuclear antigen (PCNA; a universal marker of cell proliferation), cyclin D1 and D2 (key proliferation factors), as well as Shh and its key transcriptional regulator Gli1; (2) cyclopamine, a Shh pathway inhibitor, blocked these stimulatory effects of BPA on cell proliferation; (3) BPA increased the nuclear translocation of ERß; and (4) the ERß-specific agonist DPN mimicked while the ERß-specific antagonist PHTPP abrogated the stimulatory effects of BPA on cell proliferation and Shh signaling. Taken together, these findings demonstrate that BPA stimulates adrenal cell proliferation likely through ERß-mediated activation of the Shh signaling pathway. Thus, the present study provides novel insights into the molecular mechanisms underlying our previously reported BPA-induced aberrant adrenal phenotype.

Bisphenol A suppresses glucocorticoid target gene (ENaCγ) expression via a novel ERß/NF-κB/GR signalling pathway in lung epithelial cells.

We previously demonstrated that prenatal exposure to Bisphenol A (BPA) disrupts fetal lung maturation likely through the glucocorticoid signalling pathway, but the precise molecular mechanisms remain obscure. Given that BPA diminished the expression of epithelial sodium channel-γ (ENaCγ), a well-known glucocorticoid receptor (GR) target gene, in fetal lungs, we used this GR target gene to delineate the molecular pathway through which BPA exerts its effects on lung cells. The A549 lung epithelial cell line was used as an in vitro model system. As a first step, we validated our in vitro cell model by demonstrating a robust concentration-dependent suppression of ENaCγ expression following BPA exposure. We also showed that both dexamethasone and siRNA-mediated knockdown of GR expression blocked/abrogated the inhibitory effects of BPA on ENaCγ expression, suggesting that BPA repressed ENaCγ expression via inhibition of GR activity. Given the well-known antagonistic interactions between the pro-inflammatory transcriptional factor NF-κB and GR, we then showed that BPA inhibited GR activity through the activation of NF-κB. Lastly, since BPA is known to function as a pro-inflammatory factor via the estrogen receptor ß (ERß), we provided evidence that BPA signals through ERß to activate the NF-κB signalling pathway. Taken together, these findings demonstrate that BPA acts on ERß to activate the NF-κB signalling pathway, which in turn leads to diminished GR activity and consequent repression of ENaCγ expression in lung epithelial cells. Thus, our present study reveals a novel BPA signalling pathway that involves ERß, NF-κB and GR.

Prenatal exposure to bisphenol A disrupts adrenal steroidogenesis in adult mouse offspring.

The present study sought to determine if prenatal exposure to bisphenol A (BPA) alters adrenal steroidogenesis in adult offspring. Pregnant mice were exposed to BPA (25mg BPA/kg food pellet) via diet from day 7 to the end of pregnancy. At eight weeks of age, offsprings were sacrificed, blood samples and adrenal glands were collected for hormone assays and western blot analysis, respectively. We found that: (1) BPA increased adrenal gland weight in both males and females; (2) although BPA elevated plasma corticosterone levels in both sexes, it stimulated the expression of StAR and cyp11A1, the two rate-limiting factors in the steroidogenic pathway, only in female adrenal glands; and interestingly (3) BPA did not alter plasma ACTH levels or adrenal expression of the key steroidogenic transcription factor SF-1 in either sex. Taken together, the present study provides novel insights into the long-term consequences of developmental BPA exposure on adrenal steroidogenesis.

Prenatal exposure to bisphenol A alters mouse fetal pancreatic morphology and islet composition.

BACKGROUND: Exposure to bisphenol A (BPA), an endocrine disrupting chemical, during gestation is associated with a variety of metabolic dysfunctions in adulthood, including hyperinsulinemia, glucose intolerance and insulin resistance. These modifications in glucose homeostasis largely stem from alterations in pancreatic function. However, the effects of BPA on the fetal pancreas have never been explored. The present study addressed this important question by examining the effects of prenatal BPA exposure on the mouse fetal pancreatic development. MATERIALS AND METHODS: Pregnant mice were fed a BPA diet (25 mg BPA/kg diet) from embryonic day 7.5 (E7.5) to E18.5. At E18.5, fetal pancreata were collected and analyzed for morphological changes in the endocrine pancreas such as islet size, number and ß and α cell distribution. RESULTS: We showed that BPA exposed fetal pancreata had a greater number of islet-cell clusters (ICCs; <300 µm(2); p<0.05) compared with controls. Furthermore, immunohistochemical analysis revealed that prenatal BPA exposure increased both glucagon expression in islets and the numbers of glucagon-expressing islet-cell clusters (p<0.05). CONCLUSION: Considering that ICCs represent the initial stages of islet development in the fetal pancreas, our findings suggest that BPA promotes islet differentiation or delays the conversion of ICCs into mature islets. Moreover, the increase in glucagon expression suggests a potential alteration in the α:ß-cell ratio in islets, which may have significant implications for the fetal pancreas both structurally and functionally. This study provides novel insight into the effects of BPA exposure on the fetal pancreata, indicating alterations in glucagon expression in islets and ICCs.

Wisp1 mediates Bmp3-stimulated mesenchymal stem cell proliferation.

Adipose tissue expansion, resulting from adipocyte hyperplasia and/or hypertrophy, is a hallmark of obesity. Adipocytes are derived from mesenchymal stem cells (MSCs) through adipogenesis, a process involving three key steps: proliferation, commitment and differentiation. Although studies have elaborated on the mechanisms regulating adipocyte commitment and differentiation, the factors that control MSC proliferation remain largely unknown. Previously, we demonstrated that bone morphogenetic protein 3 (Bmp3), the expression of which was upregulated in our rat model of hyperplasic visceral adiposity, potently stimulated MSC proliferation. In the present study, we investigate the molecular target of Bmp3. We conducted DNA microarray analysis on MSCs treated with and without Bmp3 and identified WNT1-inducible signaling pathway protein 1 (Wisp1) as a differentially expressed gene, whose expression was upregulated 3.7-fold by Bmp3. Wisp1 is a proliferative agent in various non-adipose cell types and is implicated in adipogenesis. Therefore, we tested the hypothesis that Wisp1 mediates Bmp3 stimulation of MSC proliferation. We showed that Bmp3 increased the expression of Wisp1 as early as 3 h following Bmp3 treatment in MSCs. Importantly, the upregulated Wisp1 expression preceded Bmp3-induced MSC proliferation, as determined by [(3)H]-thymidine incorporation. Furthermore, treatment of MSCs with recombinant Wisp1 led to a concentration-dependent increase in [(3)H]-thymidine incorporation with a maximal increase of 300%. In addition, siRNA-mediated knockdown of Wisp1 expression attenuated Bmp3-induced MSC proliferation. Taken together, our present findings reveal Wisp1 as a novel target of Bmp3 and suggest that the Bmp3/Wisp1 signaling pathway play a key role in MSC proliferation, and consequently adipogenesis.

Prenatal Exposure to Bisphenol A Disrupts Mouse Fetal Liver Maturation in a Sex-Specific Manner.

Bisphenol A (BPA) is one of the most prevalent endocrine disrupting chemicals in the environment. Developmental exposure to BPA is known to be associated with liver dysfunction and diseases, such as hepatic steatosis, liver tumors, metabolic syndrome, and altered hepatic gene expression, and DNA methylation profiles. However, the effects of BPA on rodent liver development are unknown. The present study was undertaken to address this important question using the mouse as an experimental model. Pregnant mice were exposed to BPA via diet from embryonic day 7.5 (E7.5) to E18.5. At E18.5, fetal livers were collected, and analyzed for changes in the expression of key hepatocyte maturation markers. We found the following significant alterations in BPA-exposed female but not male fetal livers: (a) levels of the mature hepatocyte markers, albumin and glycogen synthase proteins, were decreased (-65% and -40%, respectively); (b) levels of the immature hepatocyte marker, α-fetoprotein, were increased (+43%); (c) the level of C/EBP-α protein, the master transcription factor essential for hepatocyte maturation, was down-regulated (-50%); and (d) the level of PCNA protein (marker of proliferation) was elevated (+40%), while that of caspase-3 protein and activity (markers of apoptosis) was reduced (-40% and -55%, respectively), suggestive of a perturbed balance between cell proliferation and apoptosis in BPA-exposed female fetuses. Taken together, these findings demonstrate that prenatal exposure to BPA disrupts the mouse fetal liver maturation in a sex-specific manner, and suggest a fetal origin for BPA-induced hepatic dysfunction and diseases.

Prenatal exposure to bisphenol A disrupts mouse fetal lung development.

Developmental exposure to bisphenol A (BPA) is associated with lung dysfunction and diseases. However, it is unknown if this association has a fetal origin. The present study addressed this important question by examining the effects of BPA on fetal lung development. BPA was administered to pregnant mice via diet from embryonic day (E) 7.5 to E18.5. Fetal lungs were analyzed at E18.5 for changes in structure and expression of key molecular markers of lung maturation. Our main findings were as follows: BPA severely retards fetal lung maturation, as evidenced by diminished alveolar airspace (15% of control) and thickened septa, hallmarks of lung immaturity; this immaturity is characterized by aberrant alveolar epithelial type I cell differentiation because expression of the type I cell marker, aquaporin 5, but not type II cell markers, is dramatically reduced (16% of control); and the effects of BPA are likely mediated through the glucocorticoid signaling pathway because the expression of epithelial sodium channel γ and glutathione peroxidase, 2 well-known glucocorticoid target genes, is down-regulated in BPA-exposed fetal lungs, and, importantly, maternal dexamethasone administration rescues the lung immaturity phenotype. Taken together, these findings demonstrate that BPA disrupts fetal lung maturation, thus suggesting a fetal origin for BPA-induced lung diseases.

Bisphenol A disrupts gene expression in human placental trophoblast cells.

This study examined the effect of bisphenol A (BPA) on human placental gene expression using primary trophoblast cells as an in vitro model system. Trophoblast cells were isolated from human placentas at term, cultured and then exposed to environmentally relevant concentrations of BPA (0.1-2 µg/ml) for up to 24h, after which levels of 11ß-HSD2 mRNA, protein and activity were determined by standard radiometric conversion assay, western blotting, and qRT-PCR, respectively. The mRNA levels of several other prominent placental hormones/factors were also assessed by qRT-PCR. BPA dramatically increased levels of 11ß-HSD2 activity, protein and mRNA in a time- and concentration-dependent manner (> 4-fold). BPA also augmented aromatase, glucose transporter-1, CRH, and hCG mRNA levels while reducing the level of leptin mRNA. These findings demonstrate that BPA severely disrupts human placental gene expression in vitro, which suggests that exposure to BPA may contribute to altered placental function and consequent pregnancy complications.

Hypericin damages the ectatic capillaries in a Roman cockscomb model and inhibits the growth of human endothelial cells more potently than hematoporphyrin does through induction of apoptosis.

Hypericin (HY) is a promising photosensitizer (PS) for use in photodynamic therapy (PDT). Port-wine stains (PWSs) are congenital superficial dermal capillary malformations. In this study, we evaluated the photocytotoxic effects of HY for PDT in human vascular endothelial cells and a chicken cockscomb model. HY significantly inhibited the growth of human umbilical vein endothelial cells (HUVECs), as determined by colorimetric assays and morphological observation, and flow cytometry assays indicated induction of apoptosis and collapse of the mitochondrial membrane potential. In addition, HY more effectively inhibited growth of and induced apoptosis in HUVECs compared with hematoporphyrin (HP). Further experiments performed in a Roman chicken cockscomb model also showed a clear photocytotoxic effect on the cockscomb dermal capillary upon intravenous injection of HY. This effect may be due to the role of HY in the induction of apoptosis. Transmission electron microscopical analysis showed mitochondrial morphological changes such as incomplete ridges and swelling, and immunohistochemical assays showed an increase in the release of cytochrome c. In conclusion, HY exhibited a greater photocytotoxic activity than did HP toward the growth of endothelial cells and may thus represent a potent PS for PWS PDT.

Silver nanoparticle/chitosan oligosaccharide/poly(vinyl alcohol) nanofibers as wound dressings: a preclinical study.

In this study, a mixture of poly(vinyl alcohol) (PVA) and chitosan oligosaccharides (COS) was electrospun with silver nanoparticles (AgNPs) to produce fibrous mats for use in wound healing. The AgNPs were reduced by COS prior to electrospinning or Ag(+) was reduced via ultraviolet irradiation in nanofibers. The morphologies of the PVA/COS/AgNO3 and PVA/COS-AgNP nanofibers were analyzed by scanning electron microscopy. Formation of the AgNPs was investigated by field emission transmission electron microscopy, ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, and X-ray diffraction. We also evaluated the biocompatibility of the nanofibers, particularly their cytotoxicity to human skin fibroblasts and potential to cause primary skin irritation. The in vitro antibacterial activity and in vivo wound healing capacity of the nanofibers were also investigated. The nanofibers had a smooth surface with an average diameter of 130-192 nm. The diameters of the AgNPs were in the range of 15-22 nm. The nanofibers significantly inhibited growth of Escherichia coli and Staphylococcus aureus bacteria. PVA/COS-AgNP nanofibers accelerated the rate of wound healing over that of the control (gauze). The results of our in vitro and in vivo animal experiments suggest that PVA/COS-AgNP nanofibers should be of greater interest than PVA/COS/AgNO3 nanofibers for clinical use as a bioactive wound dressing.

The ERK1/2 signaling pathway regulates 11beta-hydroxysteroid dehydrogenase type 2 expression in human trophoblast cells through a transcriptional mechanism.

The placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2; encoded by the HSD11B2 gene) plays a key role in fetal development, but its regulation is incompletely understood. We previously demonstrated that p38 MAPK was a positive regulator of placental 11beta-HSD2. However, it remains unknown if the other two MAPKs, ERK1/2 and JNK, were also involved. In the present study, we identified ERK1/2 as an important regulator of placental 11beta-HSD2. We showed that inhibition of ERK1/2 with the pharmacological inhibitor U0126 led to a 3-fold increase in 11beta-HSD2 activity, protein, and mRNA in primary human placental trophoblast cells. In contrast, the JNK inhibitor SP600125 had no effect. Furthermore, U0126 increased the HSD11B2 promoter activity by 300%, indicating that ERK1/2 regulates placental 11beta-HSD2 expression through a transcriptional mechanism. Importantly, siRNA-mediated knockdown of ERK1/2 caused a similar increase in 11beta-HSD2 protein. In addition, given that we previously showed that cadmium reduced placental 11beta-HSD2 expression via a transcriptional mechanism, but the signal transduction pathways involved remain unclear, we also addressed this question and found that treatment of trophoblast cells with cadmium led to rapid activation of ERK1/2. Importantly, U0126 completely abrogated the inhibitory effects of cadmium on placental 11beta-HSD2. Taken together, the present study not only identifies the ERK1/2 signaling pathway as a potent negative regulator of placental 11beta-HSD2 but also demonstrates that this pathway mediates cadmium repression of placental 11beta-HSD2. Thus, our present study reveals 11beta-HSD2 as an important target through which ERK1/2 may regulate human placental function and consequently fetal development.

Metallothionein-I- and -II-deficient mice display increased susceptibility to cadmium-induced fetal growth restriction.

Maternal cadmium exposure induces fetal growth restriction (FGR), but the underlying mechanisms remain largely unknown. The placenta is the main organ known to protect the fetus from environmental toxins such as cadmium. In this study, we examine the role of the two key placental factors in cadmium-induced FGR. The first is placental enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), which is known to protect the fetus from exposure to high cortisol levels and subsequently FGR, and the second the cadmium binding/sequestering proteins metallotheionein (MT)-I and -II. Using the MT-I/II(-/-) mouse model, pregnant mice were administered cadmium, following which pups and placentas were collected and examined. MT-I/II(-/-) pups exposed to cadmium were significantly growth restricted, but neither placental weight nor 11ß-HSD2 was altered. Although cadmium administration did not result in any visible structural changes in the placenta, increased apoptosis was detected in MT-I/II(-/-) placentas following cadmium exposure, with a significant increase in levels of both p53 and caspase 3 proteins. Additionally, glucose transporter (GLUT1) was significantly reduced in MT-I/II(-/-) placentas of pups exposed to cadmium, whereas zinc transporter (ZnT-1) remained unaltered. Taken together, these results demonstrate that MT-I/II(-/-) mice are more vulnerable to cadmium-induced FGR. The present data also suggest that increased apoptosis and reduced GLUT1 expression in the placenta contribute to the molecular mechanisms underlying cadmium-induced FGR.

Neuropeptide Y potentiates beta-adrenergic stimulation of lipolysis in 3T3-L1 adipocytes.

Recently, we have shown that neuropeptide Y (NPY) is produced and upregulated in visceral adipose tissue of an early-life programmed rat model of central obesity. Moreover, we have demonstrated that NPY promotes proliferation of adipocyte precursor cells and contributes to the pathogenesis of obesity. However, the role of NPY in regulating adipocyte metabolism is poorly understood. The present study was designed to examine the effects of NPY on adipocyte metabolic function using 3T3-L1 adipocytes as an in vitro cell model system. We found that although it did not affect basal lipolysis, NPY potentiated isoproterenol (a ß-adrenergic receptor agonist) stimulated lipolysis. Furthermore, this potentiation occurred upstream of adenylyl cyclase, since NPY did not enhance forskolin (an activator of adenylyl cyclase) stimulated lipolysis. In addition, NPY also augmented isoproterenol-stimulated phosphorylation of hormone sensitive lipase. In contrast, NPY did not alter the expression of several key lipolytic and lipogenic enzymes/proteins. Taken together, our results revealed a novel cross talk between the NPY and ß-adrenergic signaling pathways in regulating lipolysis. Thus, the present findings add a new dimension to the dynamic role NPY plays in regulating energy balance.

Caffeine reduces 11ß-hydroxysteroid dehydrogenase type 2 expression in human trophoblast cells through the adenosine A(2B) receptor.

Maternal caffeine consumption is associated with reduced fetal growth, but the underlying molecular mechanisms are unknown. Since there is evidence that decreased placental 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) is linked to fetal growth restriction, we hypothesized that caffeine may inhibit fetal growth partly through down regulating placental 11ß-HSD2. As a first step in examining this hypothesis, we studied the effects of caffeine on placental 11ß-HSD2 activity and expression using our established primary human trophoblast cells as an in vitro model system. Given that maternal serum concentrations of paraxanthine (the primary metabolite of caffeine) were greater in women who gave birth to small-for-gestational age infants than to appropriately grown infants, we also studied the effects of paraxanthine. Our main findings were: (1) both caffeine and paraxanthine decreased placental 11ß-HSD2 activity, protein and mRNA in a concentration-dependent manner; (2) this inhibitory effect was mediated by the adenosine A(2B) receptor, since siRNA-mediated knockdown of this receptor prevented caffeine- and paraxanthine-induced inhibition of placental 11ß-HSD2; and (3) forskolin (an activator of adenyl cyclase and a known stimulator of 11ß-HSD2) abrogated the inhibitory effects of both caffeine and paraxanthine, which provides evidence for a functional link between exposure to caffeine and paraxanthine, decreased intracellular levels of cAMP and reduced placental 11ß-HSD2. Taken together, these findings reveal that placental 11ß-HSD2 is a novel molecular target through which caffeine may adversely affect fetal growth. They also uncover a previously unappreciated role for the adenosine A(2B) receptor signaling in regulating placental 11ß-HSD2, and consequently fetal development.

Cortisol induces aromatase expression in human placental syncytiotrophoblasts through the cAMP/Sp1 pathway.

One of the dominant effects of glucocorticoids in triggering parturition in certain animal species is to drive the placental conversion of progesterone to estrogen. However, in the human placenta, estrogen is formed using dehydroepiandrosterone from the fetal adrenal glands rather than progesterone as precursor. Although aromatization of dehydroepiandrosterone is crucial in estrogen synthesis in human placenta, it is not known whether glucocorticoids affect aromatase expression. Human term placental syncytiotrophoblasts were used to examine the effect of cortisol on aromatase expression. The signaling pathway and transcription factors involved were identified in this study. Results showed that cortisol induced aromatase expression in a concentration-dependent manner, which was mediated indirectly by glucocorticoid receptor and required the participation of other proteins. The induction of aromatase by cortisol could be blocked by either specificity protein 1 (Sp1) antagonist mithramycin or knockdown of Sp1 expression. The induction of aromatase and Sp1 by cortisol could be prevented by inhibitors of the cAMP pathway, whereas activators of the cAMP pathway induced Sp1 and aromatase expression as well as Sp1 binding to aromatase promoter. Concomitantly, cortisol treatment and activation of the cAMP pathway led to increased acetylation and decreased methylation of histone 3 at the aromatase promoter. In conclusion, cortisol stimulates aromatase expression through the cAMP/Sp1 pathway in human placental syncytiotrophoblasts. These findings reveal a novel role of cortisol in increasing the local level of estrogen within the placenta that would help transform the myometrium to a contractile state, thereby contributing to a cascade of events leading to human parturition.