Abstract
RNA-binding proteins (RPBs) are deeply involved in fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far been dedicated to direct ...and global identification of bacterial RBPs. We have adapted the RNA interactome capture (RIC) technique, originally developed for eukaryotic systems, to globally identify RBPs in bacteria. RIC takes advantage of the base pairing potential of poly(A) tails to pull-down RNA–protein complexes. Overexpressing poly(A) polymerase I in Escherichia coli drastically increased transcriptome-wide RNA polyadenylation, enabling pull-down of crosslinked RNA–protein complexes using immobilized oligo(dT) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of a number of uncharacterized RBPs, including YhgF, which is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF RNA targets in vivo using CLIP-seq, verified specific binding in vitro, and reveal a putative role for YhgF in regulation of gene expression. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lay the foundation for further studies of the highly conserved RBP YhgF.
1 Perinatal Center, Department of Physiology, Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden; 2 Division of Bio-System Pharmacology, Department of ...Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan; 3 Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta, Georgia; and 4 Department of Obstetrics and Gynecology, University of Cincinnati College of Medicine, Cincinnati, Ohio
Submitted 24 June 2008
; accepted in final form 3 November 2008
The activity of placental amino acid transporters is decreased in intrauterine growth restriction (IUGR), but the underlying regulatory mechanisms have not been established. Inhibition of the mammalian target of rapamycin (mTOR) signaling pathway has been shown to decrease the activity of the system L amino acid transporter in human placental villous fragments, and placental mTOR activity is decreased in IUGR. In the present study, we used cultured primary trophoblast cells to study mTOR regulation of placental amino acid transporters in more detail and to test the hypothesis that mTOR alters amino acid transport activity by changes in transporter expression. Inhibition of mTOR by rapamycin significantly reduced the activity of system A (–17%), system L (–28%), and taurine (–40%) amino acid transporters. mRNA expression of isoforms of the three amino acid transporter systems in response to mTOR inhibition was measured using quantitative real-time PCR. mRNA expression of L -type amino acid transporter 1 (LAT1; a system L isoform) and taurine transporter was reduced by 13% and 50%, respectively; however, mTOR inhibition did not alter the mRNA expression of system A isoforms (sodium-coupled neutral amino acid transporter-1, -2, and -4), LAT2, or 4F2hc. Rapamycin treatment did not significantly affect the protein expression of any of the transporter isoforms. We conclude that mTOR signaling regulates the activity of key placental amino acid transporters and that this effect is not due to a decrease in total protein expression. These data suggest that mTOR regulates placental amino acid transporters by posttranslational modifications or by affecting transporter translocation to the plasma membrane.
system A; system L; taurine transporter
Address for reprint requests and other correspondence: S. Roos, Perinatal Center, Dept. of Physiology, Institute of Neuroscience and Physiology, Univ. of Gothenburg, PO Box 432, Gothenburg SE-405 30, Sweden (e-mail: sara.roos{at}gu.se )
Abstract Nutrient-sensing signaling pathways regulate cell metabolism and growth in response to altered nutrient levels and growth factor signaling. Because trophoblast cell metabolism and associated ...signaling influence fetal nutrient availability, trophoblast nutrient sensors may have a unique role in regulating fetal growth. We review data in support of a role for mammalian target of rapamycin complex 1 (mTORC1) in placental nutrient-sensing. Placental insulin/IGF-I signaling and fetal levels of oxygen, glucose and amino acids (AAs) are altered in pregnancy complications such as intrauterine growth restriction, and all these factors are well-established upstream regulators of mTORC1. Furthermore, mTORC1 is a positive regulator of placental AA transporters, suggesting that trophoblast mTORC1 modulates AA transfer across the placenta. In addition, placental mTORC1 signaling is also known to be modulated in pregnancy complications associated with altered fetal growth and in animal models in which maternal nutrient availability has been altered experimentally. Recently, significant progress has been made in identifying the molecular mechanisms by which mTORC1 senses AAs, a process requiring shuttling of mTOR to late endosomal and lysosomal compartments (LELs). We recently identified members of the proton-assisted amino acid transporter (PAT/SLC36) family as critical components of the AA-sensing system or ‘nutrisome’ that regulates mTORC1 on LEL membranes, placing AA transporters and their subcellular regulation both upstream and downstream of mTORC1-driven processes. We propose a model in which placental mTORC1 signaling constitutes a critical link between maternal nutrient availability and fetal growth, thereby influencing the long-term health of the fetus.
Amino acid transport across the human placenta is active, mediated by specific transporters in syncytiotrophoblast plasma membranes. Using functional criteria such as substrate specificity and sodium ...dependence, approximately 15 transport systems have been identified in the human placenta. Recently, the area of molecular biology of amino acid transporters has evolved rapidly and at least 25 cDNA clones coding for mammalian amino acid transporters or transporter subunits have been identified. The primary objective of this review is to integrate the available functional data on placental amino acid transport systems with recent molecular information on mammalian amino acid transporters. Furthermore, models for the mechanisms for net materno-fetal transfer of amino acids are discussed. Finally, the evidence to suggest that alterations in placental amino acid transport systems may play a crucial role in the regulation of fetal growth are presented briefly.
Abstract
In studies of human IVF, as compared to frozen embryo transfer (ET), fresh ET is associated with smaller infants and higher risk of small for gestational age infants. Recent observations ...suggest that ET using vitrified embryos is associated with higher pregnancy and live birth rates compared to fresh ET, but increased rates of large for gestational age infants. The mechanisms underlying these associations are largely unknown, and available evidence suggests that the influence of IVF, vitrification and the superovulated (SO) uterine environment on placental function and fetal growth is complex. This warrants further investigation given the prevalent practice in human IVF of both fresh ET into a SO uterine environment, and vitrification with ET into a more physiologic uterine environment. Using a mouse model that closely resembles human IVF, we investigated if vitrification of IVF embryos better preserves placental function and results in better pregnancy outcomes as compared to fresh ET because of transfer into a more physiologic endometrium. We found that the SO environment, independent of vitrification status, reduced implantation rates, inhibited placental mechanistic target of rapamycin signaling and induced placental stress signaling, resulting in fetal growth restriction (1.080 ± 0.05 g estrous fresh (n = 17 litters), 1.176 ± 0.05 g estrous vitrified (n = 12), 0.771 ± 0.06 g SO fresh (n = 15), 0.895 ± 0.08 g SO vitrified (n = 10), P < 0.0001). In addition, our study suggests that vitrification impairs the developmental potential of IVF blastocysts that resulted in a significantly smaller litter size (2.6 ± 2.3 fresh estrous vs 2.5 ± 2.4 fresh SO vs 1.6 ± 1.7 estrous vitrified vs 1.7 ± 1.8 SO vitrified, P = 0.019), with no effect on fetal growth or placental function at term. Our findings suggest that vitrification may negatively impact early embryonic viability, while the SO maternal uterine environment impairs both placental development and fetal growth in IVF.
Abstract Maternal obesity and gestational diabetes (GDM) are conditions associated with fetal overgrowth and excessive fat accumulation in the fetus, implicating an increased placental nutrient ...transfer in these pregnancies. Obese and GDM mothers have altered metabolism and hormone levels, including elevation of maternal circulatory lipids and pro-inflammatory cytokines. We tested the hypothesis that interleukin (IL)-6 and tumor necrosis factor (TNF)-α stimulate placental fatty acid transport, as these pro-inflammatory cytokines have been shown to affect lipid metabolism in other tissues. In cultured primary human trophoblast cells IL-6, but not TNF-α, stimulated fatty acid accumulation, as measured by BODIPY fluorescence. The increased fatty acid accumulation could not be explained by an increased expression of key components in placental fatty acid transport, such as adipophilin, fatty acid transport protein (FATP)1, FATP4, or lipoprotein lipase. In a cohort of lean and overweight/obese pregnant women, increasing maternal third trimester IL-6 plasma concentrations correlated with decreasing placental lipoprotein lipase activity. However, as no effect on lipoprotein lipase activity was observed in cultured trophoblast cells after exposure to either IL-6 or TNF-α, the correlation between maternal circulatory IL-6 levels and placental lipoprotein lipase activity at term is unlikely to represent a cause-and-effect relationship. In conclusion, high levels of IL-6 stimulate trophoblast fatty acid accumulation, which could contribute to an excessive nutrient transfer in conditions associated with elevated maternal IL-6 such as obesity and gestational diabetes.
The activity and expression of placental nutrient transporters are primary determinants for the supply of nutrients to the fetus, and these nutrients in turn regulate fetal growth. We developed an ...experimental system to assess amino acid uptake in single primary villous fragments to study hormonal regulation of the amino acid transporter system A in term human placenta. Validation of the method, using electron microscopy and studies of hormone production, indicated that fragments maintained ultrastructural and functional integrity for at least 3 h. The activity of system A was measured as the Na+-dependent uptake of methylaminoisobutyric acid (MeAIB), and the effect of 1 h incubation in various hormones was investigated. Uptake of MeAIB into villous fragments in the presence of Na+ was linear up to at least 30 min. Insulin (300 ng/ml, n = 14) increased system A activity by 56% (P < 0.05). This effect was also present at insulin concentrations in the physiological range (+47% at 0.6 ng/ml, n = 10, P < 0.05). Leptin (500 ng/ml, n = 14) increased Na+-dependent MeAIB uptake by 37% (P < 0.05). System A activity increased in a concentration-dependent fashion in response to leptin (n = 10). However, neither epidermal GF (600 ng/ml), cortisol (340 ng/ml), nor GH (500 ng/ml) altered system A activity significantly (n = 14). We conclude that primary single isolated villous fragments can be used in studies of hormonal regulation of nutrient uptake into the syncytiotrophoblast. These data suggest that leptin regulates system A, a key amino acid transporter.
Prenatal exposures have known adverse effects on maternal and neonatal outcomes. Professional societies recommend routine screening for environmental, occupational, and dietary exposures to reduce ...exposures and their associated sequelae.
Our objective was to determine the frequency of environmental exposure screening by obstetricians and gynecologists (OBGYNs) at initial patient visits.
Practicing OBGYNs were approached at the University of Colorado and by social media. The survey instrument queried demographics, environmental literacy, and screening practices. Statistical analysis was performed using Chi-square and two-sample t-test.
We received 312 online survey responses (response rate of 12%). Responding OBGYNs were predominantly female (96%), board-certified (78%), generalists (65%) with a mean age of 37.1 years. Fewer than half of physicians screened for the following factors: occupational exposures, environmental chemicals, air pollution, pesticide use, personal care products, household cleaners, water source, use of plastics for food storage, and lead and mercury exposure. Eighty five percent of respondents reported that they did not feel comfortable obtaining an environmental history and 58% respondents reported that they performed no regular screening of environmental exposures. A higher frequency of screening was associated with > 4 years of practice (p = 0.001), and having read the environmental committee opinion (p = <0.001).
The majority of OBGYNs did not incorporate screening for known environmental exposures into routine practice. Reading the environmental committee opinions was strongly and significantly associated with a higher rate of screening. Improving physician comfort in counseling patients may enhance screening for exposures that affect reproductive health.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Parkinson’s disease (PD) is a highly prevalent neurodegenerative disorder affecting the motor system. However, the correct diagnosis of PD and atypical parkinsonism may be difficult with high ...clinical uncertainty. There is an urgent need to identify reliable biomarkers using high-throughput, molecular-specific methods to improve current diagnostics. Here, we present a matrix-assisted laser desorption/ionization mass spectrometry imaging method that requires minimal sample preparation and only 1 μL of crude cerebrospinal fluid (CSF). The method enables analysis of hundreds of samples in a single experiment while simultaneously detecting numerous metabolites with subppm mass accuracy. To test the method, we analyzed CSF samples from 12 de novo PD patients (that is, newly diagnosed and previously untreated) and 12 age-matched controls. Within the identified molecules, we found neurotransmitters and their metabolites such as γ-aminobutyric acid, 3-methoxytyramine, homovanillic acid, serotonin, histamine, amino acids, and metabolic intermediates. Limits of detection were estimated for multiple neurotransmitters with high linearity (R 2 > 0.99) and sensitivity (as low as 16 pg/μL). Application of multivariate classification led to a highly significant (P < 0.001) model of PD prediction with a 100% classification rate, which was further thoroughly validated with a permutation test and univariate analysis. Molecules related to the neuromelanin pathway were found to be significantly increased in the PD group, indicated by their elevated relative intensities compared to the control group. Our method enables rapid detection of PD-related biomarkers in low sample volumes and could serve as a valuable tool in the development of robust PD diagnostics.
Background and Aim
Anastomotic leakage (AL) in colorectal resection and primary anastomosis is a common and feared complication. Fully covered self‐expandable metal stents (FCSEMSs) have been used ...for the treatment of AL. It is still unknown whether FCSEMSs affect anastomosis healing negatively by causing ischemia. In an animal study, we investigated the metabolic effects over a FCSEMS covering a stapled colon anastomosis.
Methods
Seven pigs were investigated using microdialysis after laparotomy, colon resection, and anastomosis with stent placement. Measurements were done at the proximal and distal ends of the anastomosis and at a reference catheter placed at the small intestine. Measurements of glucose, pyruvate, lactate, glycerol, and the lactate/pyruvate ratio (L/P) were carried out.
Results
Lactate and L/P were significantly higher at the oral part of the anastomosis, while glucose showed a small declining tendency. At the distal part of the anastomosis, glucose decreased significantly after the resection but did not reach zero. Lactate increased significantly whereas L/P increased only slightly. Glycerol levels were stable.
Conclusion
Colon resection caused initially hypermetabolism in the intestinal ends next to the resection site. This hypermetabolism neither deteriorated nor turned into ischemia during the initial postoperative course, but the start of hypoxemia could not be excluded during the study and after the placement of an FCSEMS.
Fully covered self‐expandable metal stents (FCSEMS) have been used for treatment of anastomotic leakage. It is still unknown if FCSEMS affect anastomosis healing negatively by causing ischemia. In our study, the colon resection and stent insertion caused hypermetabolism but not ischemia in the intestinal ends next to the resection site.
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