Improving Fab' fragment retention in an autonucleolytic Escherichia coli strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA.

PubMed

Schofield, Desmond M; Sirka, Ernestas; Keshavarz-Moore, Eli; Ward, John M; Nesbeth, Darren N

2017-12-01

To reduce unwanted Fab' leakage from an autonucleolytic Escherichia coli strain, which co-expresses OmpA-signalled Staphylococcal nuclease and Fab' fragment in the periplasm, by substituting in Serratial nuclease and the DsbA periplasm translocation signal as alternatives. We attempted to genetically fuse a nuclease from Serratia marcescens to the OmpA signal peptide but plasmid construction failed, possibly due to toxicity of the resultant nuclease. Combining Serratial nuclease to the DsbA signal peptide was successful. The strain co-expressing this nuclease and periplasmic Fab' grew in complex media and exhibited nuclease activity detectable by DNAse agar plate but its growth in defined medium was retarded. Fab' coexpression with Staphylococcal nuclease fused to the DsbA signal peptide resulted in cells exhibiting nuclease activity and growth in defined medium. In cultivation to high cell density in a 5 l bioreactor, DsbA-fused Staphylococcal nuclease co-expression coincided with reduced Fab' leakage relative to the original autonucleolytic Fab' strain with OmpA-fused staphylococcal nuclease. We successfully rescued Fab' leakage back to acceptable levels and established a basis for future investigation of the linkage between periplasmic nuclease expression and leakage of co-expressed periplasmic Fab' fragment to the surrounding growth media.

Simvastatin and Dipentyl Phthalate Lower Ex vivo Testicular Testosterone Production and Exhibit Additive Effects on Testicular Testosterone and Gene Expression Via Distinct Mechanistic Pathways in the Fetal Rat

EPA Science Inventory

Sex differentiation of the male reproductive tract in mammals is driven, in part, by fetal androgen production. In utero, some phthalate esters (PEs) alter fetal Leydig cell differentiation, reducing the expression of several genes associated with steroid synthesis/transport, and...

Influences of Reduced Expression of Maternal Bone Morphogenetic Protein 2 on Embryonic Development

PubMed Central

Singh, Ajeet P.; Castranio, Trisha; Scott, Greg; Guo, Dayong; Harris, Marie A.; Ray, Manas; Harris, Stephan E.; Mishina, Yuji

2009-01-01

Bone morphogenetic protein 2 (BMP2) was originally found by its osteoinductive ability, and recent genetic analyses have revealed that it plays critical roles during early embryogenesis, cardiogenesis, decidualization as well as skeletogenesis. During a course of evaluation of the conditional allele for Bmp2, we found that the presence of a neo cassette, a selection marker needed for gene targeting events in embryonic stem cells, in the 3’ untranslated region of exon 3 of Bmp2, reduced the expression levels of Bmp2 both in embryonic and maternal tissues. Some of the embryos that were genotyped as transheterozygous for the floxed allele with the neo cassette over the conventional null allele (fn/−) showed a lethal phenotype including defects in cephalic neural tube closure and ventral abdominal wall closure. Embryos exhibiting these abnormalities were increased when genotypes of the pregnant females were different; when expression levels of Bmp2 in maternal tissues were lower, a larger proportion of fn/− embryos exhibit these abnormalities. These results suggest that the expression levels of Bmp2 together in both in embryonic and maternal tissues influence the normal neural tube closure and body wall closure with different thresholds. PMID:18769073

Menadione (Vitamin K3) induces apoptosis of human oral cancer cells and reduces their metastatic potential by modulating the expression of epithelial to mesenchymal transition markers and inhibiting migration.

PubMed

Suresh, Shruthy; Raghu, Dinesh; Karunagaran, Devarajan

2013-01-01

Oral cancer is one of the most commonly occurring cancers worldwide, decreasing the patient's survival rate due to tumor recurrence and metastasis. Menadione (Vitamin K3) is known to exhibit cytotoxicity in various cancer cells but the present study focused on its effects on viability, apoptosis, epithelial to mesenchymal transition (EMT), anchorage independent growth and migration of oral cancer cells. The results show that menadione is more cytotoxic to SAS (oral squamous carcinoma) cells but not to non-tumorigenic HEK293 and HaCaT cells. Menadione treatment increased the expression of pro-apoptotic proteins, Bax and p53, with a concurrent decrease in anti-apoptotic proteins, Bcl-2 and p65. Menadione induced the expression of E-cadherin but reduced the expression of EMT markers, vimentin and fibronectin. Menadione also inhibited anchorage independent growth and migration in SAS cells. These findings reveal and confirm that menadione is a potential candidate in oral cancer therapy as it exhibits cytotoxic, antineoplastic and antimigratory effects besides effectively blocking EMT in oral cancer cells.

Alterations in the endocannabinoid system in the rat valproic acid model of autism.

PubMed

Kerr, D M; Downey, L; Conboy, M; Finn, D P; Roche, M

2013-07-15

The endocannabinoid system plays a crucial role in regulating emotionality and social behaviour, however it is unknown whether this system plays a role in symptoms associated with autism spectrum disorders. The current study evaluated if alterations in the endocannabinoid system accompany behavioural changes in the valproic acid (VPA) rat model of autism. Adolescent rats prenatally exposed to VPA exhibited impaired social investigatory behaviour, hypoalgesia and reduced lococmotor activity on exposure to a novel aversive arena. Levels of the endocananbinoids, anandamide (AEA) and 2-arachidonylglycerol (2-AG) in the hippocampus, frontal cortex or cerebellum were not altered in VPA- versus saline-exposed animals. However, the expression of mRNA for diacylglycerol lipase α, the enzyme primarily responsible for the synthesis of 2-AG, was reduced in the cerebellum of VPA-exposed rats. Furthermore, while the expression of mRNA for the 2-AG-catabolising enzyme monoacylglycerol lipase was reduced, the activity of this enzyme was increased, in the hippocampus of VPA-exposed animals. CB1 or CB2 receptor expression was not altered in any of the regions examined, however VPA-exposed rats exhibited reduced PPARα and GPR55 expression in the frontal cortex and PPARγ and GPR55 expression in the hippocampus, additional receptor targets of the endocannabinoids. Furthermore, tissue levels of the fatty acid amide hydrolase substrates, AEA, oleoylethanolamide and palmitoylethanolamide, were higher in the hippocampus of VPA-exposed rats immediately following social exposure. These data indicate that prenatal VPA exposure is associated with alterations in the brain's endocannabinoid system and support the hypothesis that endocannabinoid dysfunction may underlie behavioural abnormalities observed in autism spectrum disorders. Copyright © 2013 Elsevier B.V. All rights reserved.

Sodium lauryl sulphate alters the mRNA expression of lipid-metabolizing enzymes and PPAR signalling in normal human skin in vivo.

PubMed

Törmä, Hans; Berne, Berit

2009-12-01

Detergents irritate skin and affect skin barrier homeostasis. In this study, healthy skin was exposed to 1% sodium lauryl sulphate (SLS) in water for 24 h. Biopsies were taken 6 h to 8 days post exposure. Lipid patterns were stained in situ and real-time polymerase chain reaction (PCR) was used to examine mRNA expression of enzymes synthesizing barrier lipids, peroxisome proliferator-activated receptors (PPAR) and lipoxygenases. The lipid pattern was disorganized from 6 h to 3 days after SLS exposure. Concomitant changes in mRNA expression included: (i) reduction, followed by induction, of ceramide-generating beta-glucocerebrosidase, (ii) increase on day 1 of two other enzymes for ceramide biosynthesis and (iii) persistent reduction of acetyl-CoA carboxylase-B, a key enzyme in fatty acid synthesis. Surprisingly, the rate-limiting enzyme in cholesterol synthesis, HMG-CoA reductase, was unaltered. Among putative regulators of barrier lipids synthesis, PPARalpha and PPARgamma exhibited reduced mRNA expression, while PPARbeta/delta and LXRbeta were unaltered. Epidermal lipoxygenase-3, which may generate PPARalpha agonists, exhibited reduced expression. In conclusion, SLS induces reorganization of lipids in the stratum corneum, which play a role in detergents' destruction of the barrier. The changes in mRNA expression of enzymes involved in synthesizing barrier lipids are probably important for the restoration of the barrier.

Molecular evolution and expression of oxygen transport genes in livebearing fishes (Poeciliidae) from hydrogen sulfide rich springs.

PubMed

Barts, Nicholas; Greenway, Ryan; Passow, Courtney N; Arias-Rodriguez, Lenin; Kelley, Joanna L; Tobler, Michael

2018-04-01

Hydrogen sulfide (H 2 S) is a natural toxicant in some aquatic environments that has diverse molecular targets. It binds to oxygen transport proteins, rendering them non-functional by reducing oxygen-binding affinity. Hence, organisms permanently inhabiting H 2 S-rich environments are predicted to exhibit adaptive modifications to compensate for the reduced capacity to transport oxygen. We investigated 10 lineages of fish of the family Poeciliidae that have colonized freshwater springs rich in H 2 S-along with related lineages from non-sulfidic environments-to test hypotheses about the expression and evolution of oxygen transport genes in a phylogenetic context. We predicted shifts in the expression of and signatures of positive selection on oxygen transport genes upon colonization of H 2 S-rich habitats. Our analyses indicated significant shifts in gene expression for multiple hemoglobin genes in lineages that have colonized H 2 S-rich environments, and three hemoglobin genes exhibited relaxed selection in sulfidic compared to non-sulfidic lineages. However, neither changes in gene expression nor signatures of selection were consistent among all lineages in H 2 S-rich environments. Oxygen transport genes may consequently be predictable targets of selection during adaptation to sulfidic environments, but changes in gene expression and molecular evolution of oxygen transport genes in H 2 S-rich environments are not necessarily repeatable across replicated lineages.

Post-Traumatic Stress Avoidance is Attenuated by Corticosterone and Associated with Brain Levels of Steroid Receptor Co-Activator-1 in Rats

PubMed Central

Whitaker, Annie M.; Farooq, Muhammad A.; Edwards, Scott; Gilpin, Nicholas W.

2016-01-01

Individuals with post-traumatic stress disorder (PTSD) avoid trauma-related stimuli and exhibit blunted hypothalamic-pituitary-adrenal (HPA) axis activation at the time of stress. Our laboratory has established a rodent model of stress that mimics the avoidance symptom cluster of PTSD. Rats are classified as ‘Avoiders’ or ‘Non-Avoiders’ post-stress based on avoidance of a predator-odor paired context. Previously, we demonstrated that Avoiders exhibit an attenuated HPA stress response to predator odor. We hypothesized that corticosterone administration prior to stress would reduce magnitude and incidence of avoidance of a stress-paired context. Furthermore, we predicted that Avoiders would exhibit altered expression of GR signaling machinery elements, such as steroid receptor co-activator (SRC)-1. Male Wistar rats (n = 16) were pre-treated with corticosterone (25 mg/kg) or saline and exposed to predator odor stress paired with a context, and tested for avoidance 24 h later, A second group of corticosterone-naïve rats (n = 24) were stressed (or not stressed), indexed for avoidance 24 h later, and killed 48 h post-odor exposure for analysis of phosphorylated GR, FKBP51, and SRC-1 levels in the paraventricular nucleus (PVN), central amygdala (CeA) and ventral hippocampus (VH), all brain sites that express high quantities of GRs and regulate HPA function. Rats pre-treated with corticosterone exhibited lower magnitude and incidence of avoidance. Predator odor exposure also reduced SRC-1 expression in the PVN and CeA of Avoiders, and increased SRC-1 expression in the VH of Avoiders. SRC-1 expression in PVN, CeA, and VH was predicted by prior avoidance behavior. These results suggest that blunted HPA stress response may contribute to stress-induced neuroadaptations in central SRC-1 levels and behavioral dysfunction in Avoider rats. PMID:26482332

N-CAM Exhibits a Regulatory Function in Pathological Angiogenesis in Oxygen Induced Retinopathy

PubMed Central

Håkansson, Joakim; Ståhlberg, Anders; Wolfhagen Sand, Fredrik; Gerhardt, Holger; Semb, Henrik

2011-01-01

Background Diabetic retinopathy and retinopathy of prematurity are diseases caused by pathological angiogenesis in the retina as a consequence of local hypoxia. The underlying mechanism for epiretinal neovascularization (tuft formation), which contributes to blindness, has yet to be identified. Neural cell adhesion molecule (N-CAM) is expressed by Müller cells and astrocytes, which are in close contact with the retinal vasculature, during normal developmental angiogenesis. Methodology/Principal Findings Notably, during oxygen induced retinopathy (OIR) N-CAM accumulated on astrocytes surrounding the epiretinal tufts. Here, we show that N-CAM ablation results in reduced vascular tuft formation due to reduced endothelial cell proliferation despite an elevation in VEGFA mRNA expression, whereas retinal developmental angiogenesis was unaffected. Conclusion/Significance We conclude that N-CAM exhibits a regulatory function in pathological angiogenesis in OIR. This is a novel finding that can be of clinical relevance in diseases associated with proliferative vasculopathy. PMID:22043302

Downregulation in GATA4 and Downstream Structural and Contractile Genes in the db/db Mouse Heart

PubMed Central

Broderick, Tom L.; Jankowski, Marek; Wang, Donghao; Danalache, Bogdan A.; Parrott, Cassandra R.; Gutkowska, Jolanta

2012-01-01

Reduced expression of GATA4, a transcriptional factor for structural and cardioprotective genes, has been proposed as a factor contributing to the development of cardiomyopathy. We investigated whether the reduction of cardiac GATA4 expression reported in diabetes alters the expression of downstream genes, namely, atrial natriuretic peptide (ANP), B-type natriuretic, peptide (BNP), and α- and β-myosin heavy chain (MHC). db/db mice, a model of type 2 diabetes, with lean littermates serving as controls, were studied. db/db mice exhibited obesity, hyperglycemia, and reduced protein expression of cardiac GLUT4 and IRAP (insulin-regulated aminopeptidase), the structural protein cosecreted with GLUT4. Hearts from db/db mice had reduced protein expression of GATA4 (~35%) with accompanying reductions in mRNA expression of ANP (~40%), BNP (~85%), and α-MHC mRNA (~50%) whereas expression of β-MHC mRNA was increased by ~60%. Low GATA4 was not explained by an increased ligase or atrogin1 expression. CHIP protein content was modestly downregulated (27%) in db/db mice whereas mRNA and protein expression of the CHIP cochaperone HSP70 was significantly decreased in db/db hearts. Our results indicate that low GATA4 in db/db mouse heart is accompanied by reduced expression of GATA4-regulated cardioprotective and structural genes, which may explain the development of cardiomyopathy in diabetes. PMID:22474596

Rescue of protein expression defects may not be enough to abolish the pro-arrhythmic phenotype of long QT type 2 mutations.

PubMed

Perry, Matthew D; Ng, Chai Ann; Phan, Kevin; David, Erikka; Steer, Kieran; Hunter, Mark J; Mann, Stefan A; Imtiaz, Mohammad; Hill, Adam P; Ke, Ying; Vandenberg, Jamie I

2016-07-15

Most missense long QT syndrome type 2 (LQTS2) mutations result in Kv11.1 channels that show reduced levels of membrane expression. Pharmacological chaperones that rescue mutant channel expression could have therapeutic potential to reduce the risk of LQTS2-associated arrhythmias and sudden cardiac death, but only if the mutant Kv11.1 channels function normally (i.e. like WT channels) after membrane expression is restored. Fewer than half of mutant channels exhibit relatively normal function after rescue by low temperature. The remaining rescued missense mutant Kv11.1 channels have perturbed gating and/or ion selectivity characteristics. Co-expression of WT subunits with gating defective missense mutations ameliorates but does not eliminate the functional abnormalities observed for most mutant channels. For patients with mutations that affect gating in addition to expression, it may be necessary to use a combination therapy to restore both normal function and normal expression of the channel protein. In the heart, Kv11.1 channels pass the rapid delayed rectifier current (IKr ) which plays critical roles in repolarization of the cardiac action potential and in the suppression of arrhythmias caused by premature stimuli. Over 500 inherited mutations in Kv11.1 are known to cause long QT syndrome type 2 (LQTS2), a cardiac electrical disorder associated with an increased risk of life threatening arrhythmias. Most missense mutations in Kv11.1 reduce the amount of channel protein expressed at the membrane and, as a consequence, there has been considerable interest in developing pharmacological agents to rescue the expression of these channels. However, pharmacological chaperones will only have clinical utility if the mutant Kv11.1 channels function normally after membrane expression is restored. The aim of this study was to characterize the gating phenotype for a subset of LQTS2 mutations to assess what proportion of mutations may be suitable for rescue. As an initial screen we used reduced temperature to rescue expression defects of mutant channels expressed in Xenopus laevis oocytes. Over half (∼56%) of Kv11.1 mutants exhibited functional gating defects that either dramatically reduced the amount of current contributing to cardiac action potential repolarization and/or reduced the amount of protective current elicited in response to premature depolarizations. Our data demonstrate that if pharmacological rescue of protein expression defects is going to have clinical utility in the treatment of LQTS2 then it will be important to assess the gating phenotype of LQTS2 mutations before attempting rescue. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Reduced Pms2 expression in non-neoplastic flat mucosa from patients with colon cancer correlates with reduced apoptosis competence.

PubMed

Bernstein, Harris; Prasad, Anil; Holubec, Hana; Bernstein, Carol; Payne, Claire M; Ramsey, Lois; Dvorakova, Katerina; Wilson, Megan; Warneke, James A; Garewal, Harinder

2006-06-01

Pms2 protein is a component of the DNA mismatch repair complex responsible both for post-replication correction of DNA nucleotide mispairs and for early steps in apoptosis. Germline mutations in DNA mismatch repair genes give rise to hereditary non-polyposis colon cancer, which accounts for about 4% of colon cancers. However, little is known about the expression of mismatch repair proteins in relation to sporadic colon cancer, which accounts for the great majority of colon cancers. Multiple samples were taken from the non-neoplastic flat mucosa of colon resections from patients with no colonic neoplasia, a tubulovillous adenoma, or an adenocarcinoma. Expression of Pms2 was assessed using semiquantitative immunohistochemistry. Apoptosis was assessed in polychrome-stained epoxy sections using morphologic criteria. Samples from patients without colonic neoplasia had moderate to strong staining for Pms2 in cell nuclei at the base of crypts, while samples from 2 of the 3 colons with a tubulovillous adenoma, and from 6 of the 10 colons with adenocarcinomas, showed reduced Pms2 expression. Samples from patients with an adenocarcinoma that had reduced Pms2 expression also exhibited reduced apoptosis capability in nearby tissue samples, evidenced when this paired tissue was stressed ex vivo with bile acid. Reduced Pms2 expression in the colonic mucosa may be an early step in progression to colon cancer. This reduction may cause decreased mismatch repair, increased genetic instability, and/or reduced apoptotic capability. Immunohistochemical determination of reduced Pms2 expression, upon further testing, may prove to be a promising early biomarker of risk of progression to malignancy.

Post-traumatic stress avoidance is attenuated by corticosterone and associated with brain levels of steroid receptor co-activator-1 in rats.

PubMed

Whitaker, Annie M; Farooq, Muhammad A; Edwards, Scott; Gilpin, Nicholas W

2016-01-01

Individuals with post-traumatic stress disorder (PTSD) avoid trauma-related stimuli and exhibit blunted hypothalamic-pituitary-adrenal (HPA) axis activation at the time of stress. Our rodent model of stress mimics the avoidance symptom cluster of PTSD. Rats are classified as "Avoiders" or "Non-Avoiders" post-stress based on the avoidance of a predator-odor paired context. Previously, we found Avoiders exhibit an attenuated HPA stress response to predator odor. We hypothesized that corticosterone administration before stress would reduce the magnitude and incidence of stress-paired context avoidance. Furthermore, we also predicted that Avoiders would exhibit altered expression of glucocorticoid receptor (GR) signaling machinery elements, including steroid receptor co-activator (SRC)-1. Male Wistar rats (n = 16) were pretreated with corticosterone (25 mg/kg) or saline and exposed to predator-odor stress paired with a context and tested for avoidance 24 h later. A second group of corticosterone-naïve rats (n = 24) were stressed (or not), indexed for avoidance 24 h later, and killed 48 h post-odor exposure to measure phosphorylated GR, FKBP51 and SRC-1 levels in the paraventricular nucleus (PVN), central amygdala (CeA) and ventral hippocampus (VH), all brain sites that highly express GRs and regulate HPA function. Corticosterone pretreatment reduced the magnitude and incidence of avoidance. In Avoiders, predator-odor exposure led to lower SRC-1 expression in the PVN and CeA, and higher SRC-1 expression in the VH. SRC-1 expression in PVN, CeA and VH was predicted by prior avoidance behavior. Hence, a blunted HPA stress response may contribute to stress-induced neuroadaptations in central SRC-1 levels and behavioral dysfunction in Avoider rats.

Non-adaptive plasticity potentiates rapid adaptive evolution of gene expression in nature.

PubMed

Ghalambor, Cameron K; Hoke, Kim L; Ruell, Emily W; Fischer, Eva K; Reznick, David N; Hughes, Kimberly A

2015-09-17

Phenotypic plasticity is the capacity for an individual genotype to produce different phenotypes in response to environmental variation. Most traits are plastic, but the degree to which plasticity is adaptive or non-adaptive depends on whether environmentally induced phenotypes are closer or further away from the local optimum. Existing theories make conflicting predictions about whether plasticity constrains or facilitates adaptive evolution. Debate persists because few empirical studies have tested the relationship between initial plasticity and subsequent adaptive evolution in natural populations. Here we show that the direction of plasticity in gene expression is generally opposite to the direction of adaptive evolution. We experimentally transplanted Trinidadian guppies (Poecilia reticulata) adapted to living with cichlid predators to cichlid-free streams, and tested for evolutionary divergence in brain gene expression patterns after three to four generations. We find 135 transcripts that evolved parallel changes in expression within the replicated introduction populations. These changes are in the same direction exhibited in a native cichlid-free population, suggesting rapid adaptive evolution. We find 89% of these transcripts exhibited non-adaptive plastic changes in expression when the source population was reared in the absence of predators, as they are in the opposite direction to the evolved changes. By contrast, the remaining transcripts exhibiting adaptive plasticity show reduced population divergence. Furthermore, the most plastic transcripts in the source population evolved reduced plasticity in the introduction populations, suggesting strong selection against non-adaptive plasticity. These results support models predicting that adaptive plasticity constrains evolution, whereas non-adaptive plasticity potentiates evolution by increasing the strength of directional selection. The role of non-adaptive plasticity in evolution has received relatively little attention; however, our results suggest that it may be an important mechanism that predicts evolutionary responses to new environments.

Elimination of Kalrn Expression in POMC Cells Reduces Anxiety-Like Behavior and Contextual Fear Learning

PubMed Central

Mandela, Prashant; Yan, Yan; LaRese, Taylor; Eipper, Betty A.; Mains, Richard E.

2014-01-01

Kalirin, a Rho GDP/GTP exchange factor for Rac1 and RhoG, is known to play an essential role in the formation and maintenance of excitatory synapses and in the secretion of neuropeptides. Mice unable to express any of the isoforms of Kalrn in cells that produce POMC at any time during development (POMC cells) exhibited reduced anxiety-like behavior and reduced acquisition of passive avoidance behavior, along with sex-specific alteration in the corticosterone response to restraint stress. Strikingly, lack of Kalrn expression in POMC cells closely mimicked the effects of global Kalrn knockout on anxiety-like behavior and passive avoidance conditioning without causing the other deficits noted in Kalrn knockout mice. Our data suggest that deficits in excitatory inputs onto POMC neurons are responsible for the behavioral phenotypes observed. PMID:25014196

Xylitol synthesis mutant of xylose-utilizing zymomonas for ethanol production

DOEpatents

Viitanen, Paul V.; Chou, Yat-Chen; McCutchen, Carol M.; Zhang, Min

2010-06-22

A strain of xylose-utilizing Zymomonas was engineered with a genetic modification to the glucose-fructose oxidoreductase gene resulting in reduced expression of GFOR enzyme activity. The engineered strain exhibits reduced production of xylitol, a detrimental by-product of xylose metabolism. It also consumes more xylose and produces more ethanol during mixed sugar fermentation under process-relevant conditions.

EphA2 signaling is impacted by carcinoembryonic antigen cell adhesion molecule 1-L expression in colorectal cancer liver metastasis in a cell context-dependent manner.

PubMed

Arabzadeh, Azadeh; McGregor, Kevin; Breton, Valérie; Van Der Kraak, Lauren; Akavia, Uri David; Greenwood, Celia M T; Beauchemin, Nicole

2017-11-28

We have shown that carcinoembryonic antigen cell adhesion molecule 1 long isoform (CEACAM1-L) expression in MC38 metastatic colorectal cancer (CRC) cells results in liver metastasis inhibition via CCL2 and STAT3 signaling. But other molecular mechanisms orchestrating CEACAM1-L-mediated metastasis inhibition remain to be defined. We screened a panel of mouse and human CRC cells and evaluated their metastatic outcome after CEACAM1 overexpression or downregulation. An unbiased transcript profiling and a phospho-receptor tyrosine kinase screen comparing MC38 CEACAM1-L-expressing and non-expressing (CT) CRC cells revealed reduced ephrin type-A receptor 2 (EPHA2) expression and activity. An EPHA2-specific inhibitor reduced EPHA2 downstream signaling in CT cells similar to that in CEACAM1-L cells with decreased proliferation and migration. Human CRC patients exhibiting high CEACAM1 in combination with low EPHA2 expression benefited from longer time to first recurrence/metastasis compared to those with high EPHA2 expression. With the added interaction of CEACAM6 , we denoted that CEACAM1 high- and EPHA2 low-expressing patient samples with lower CEACAM6 expression also exhibited a longer time to first recurrence/metastasis. In HT29 human CRC cells, down-regulation of CEACAM1 along with CEA and CEACAM6 up-regulation led to higher metastatic burden. Overall, CEACAM1-L expression in poorly differentiated CRC can inhibit liver metastasis through cell context-dependent EPHA2-mediated signaling. However, CEACAM1's role should be considered in the presence of other CEACAM family members.

Over-expression of LeNCED1 in tomato (Solanum lycopersicum L.) with the rbcS3C promoter allows recovery of lines that accumulate very high levels of abscisic acid and exhibit severe phenotypes.

PubMed

Tung, Swee Ang; Smeeton, Rachel; White, Charlotte A; Black, Colin R; Taylor, Ian B; Hilton, Howard W; Thompson, Andrew J

2008-07-01

Previous work where 9-cis-epoxycarotenoid dioxygenase (NCED) was over-expressed using the constitutive Gelvin Superpromoter resulted in mild increases in abscisic acid (ABA) accumulation, accompanied by stomatal closure and increased water-use efficiency (WUE), but with apparently little impact on long-term biomass production. However, one of the negative effects of the over-expression of NCED using constitutive promoters in tomato was increased seed dormancy. Here we report the use of the rbcS3C promoter, from a gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), to drive LeNCED1 transgene expression in tomato in a light-responsive and circadian manner. In comparison to the constitutive promoter, the rbcS3C promoter allowed the generation of transgenic plants with much higher levels of ABA accumulation in leaves and sap, but the effect on seed dormancy was diminished. These plants displayed the expected reductions in stomatal conductance and CO(2) assimilation, but they also exhibited a severe set of symptoms that included perturbed cotyledon release from the testa, increased photobleaching in young seedlings, substantially reduced chlorophyll and carotenoid content, interveinal leaf flooding, and greatly reduced growth. These symptoms illustrate adverse consequences of long-term, very high ABA accumulation. Only more moderate increases in ABA biosynthesis are likely to be useful in the context of agriculture. Implications are discussed for the design of transgenic 'high ABA' plants that exhibit increased WUE but have minimal negative phenotypic effects.

Hepatic FGF21 expression is induced at birth via PPARalpha in response to milk intake and contributes to thermogenic activation of neonatal brown fat.

PubMed

Hondares, Elayne; Rosell, Meritxell; Gonzalez, Frank J; Giralt, Marta; Iglesias, Roser; Villarroya, Francesc

2010-03-03

Plasma FGF21 levels and hepatic FGF21 gene expression increase dramatically after birth in mice. This induction is initiated by suckling, requires lipid intake, is impaired in PPARalpha null neonates, and is mimicked by treatment with the PPARalpha activator, Wy14,643. Neonates exhibit reduced FGF21 expression in response to fasting, in contrast to the upregulation occurring in adults. Changes in FGF21 expression due to suckling or nutritional manipulations were associated with circulating free fatty acid and ketone body levels. We mimicked the FGF21 postnatal rise by injecting FGF21 into fasting neonates, and found that this enhanced the expression of genes involved in thermogenesis within brown fat, and increased body temperature. Brown adipocytes treated with FGF21 exhibited increased expression of thermogenic genes, higher total and uncoupled respiration, and enhanced glucose oxidation. We propose that the induction of FGF21 production by the liver mediates direct activation of brown fat thermogenesis during the fetal-to-neonatal transition. 2010 Elsevier Inc. All rights reserved.

E5 can be expressed in anal cancer and leads to epidermal growth factor receptor-induced invasion in a human papillomavirus 16-transformed anal epithelial cell line.

PubMed

Wechsler, Erin Isaacson; Tugizov, Sharof; Herrera, Rossana; Da Costa, Maria; Palefsky, Joel M

2018-05-01

We detected the first human papillomavirus (HPV)-16-immortalized anal epithelial cell line, known as AKC2 cells to establish an in vitro model of HPV-16-induced anal carcinogenesis. Consistent with detection of E6, E7 and E5 expression in anal cancer biopsies, AKC2 cells expressed high levels of all three HPV oncogenes. Also, similar to findings in anal cancer biopsies, epidermal growth factor receptor (EGFR) was overexpressed in AKC2 cells. AKC2 cells exhibited a poorly differentiated and invasive phenotype in three-dimensional raft culture and inhibition of EGFR function abrogated AKC2 invasion. Reducing E5 expression using E5-targeted siRNAs in AKC2 cells led to knockdown of E5 expression, but also HPV-16 E2, E6 and E7 expression. AKC2 cells treated with E5-targeted siRNA had reduced levels of total and phosphorylated EGFR, and reduced invasion. Rescue of E6/E7 expression with simultaneous E5 knockdown confirmed that E5 plays a key role in EGFR overexpression and EGFR-induced invasion.

GC-rich coding sequences reduce transposon-like, small RNA-mediated transgene silencing.

PubMed

Sidorenko, Lyudmila V; Lee, Tzuu-Fen; Woosley, Aaron; Moskal, William A; Bevan, Scott A; Merlo, P Ann Owens; Walsh, Terence A; Wang, Xiujuan; Weaver, Staci; Glancy, Todd P; Wang, PoHao; Yang, Xiaozeng; Sriram, Shreedharan; Meyers, Blake C

2017-11-01

The molecular basis of transgene susceptibility to silencing is poorly characterized in plants; thus, we evaluated several transgene design parameters as means to reduce heritable transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposon-like silencing of canola-biased PUFA synthase transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in a remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three crystal (Cry) proteins from Bacillus thuringiensis (Bt) tested the impact of CDS recoding using different codon bias tables. Transgenes with higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing, providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.

Expression and Function of Chemokines CXCL9-11 in Micturition Pathways in Cyclophosphamide (CYP)-Induced Cystitis and Somatic Sensitivity in Mice

PubMed Central

Guo, Michael; Chang, Phat; Hauke, Eric; Girard, Beatrice M.; Tooke, Katharine; Ojala, Jacqueline; Malley, Susan M.; Hsiang, Harrison; Vizzard, Margaret A.

2018-01-01

Changes in urinary bladder function and somatic sensation may be mediated, in part, by inflammatory changes in the urinary bladder including the expression of chemokines. Male and female C57BL/6 mice were treated with cyclophosphamide (CYP; 75 mg/kg, 200 mg/kg, i.p.) to induce bladder inflammation (4 h, 48 h, chronic). We characterized the expression of CXC chemokines (CXCL9, CXCL10 and CXCL11) in the urinary bladder and determined the effects of blockade of their common receptor, CXCR3, at the level urinary bladder on bladder function and somatic (hindpaw and pelvic) sensation. qRT-PCR and Enzyme-Linked Immunoassays (ELISAs) were used to determine mRNA and protein expression of CXCL9, CXCL10 and CXCL11 in urothelium and detrusor. In urothelium of female mice treated with CYP, CXCL9 and CXCL10 mRNA significantly (p ≤ 0.01) increased with CYP treatment whereas CXC mRNA expression in the detrusor exhibited both increases and decreases in expression with CYP treatment. CXC mRNA expression urothelium and detrusor of male mice was more variable with both significant (p ≤ 0.01) increases and decreases in expression depending on the specific CXC chemokine and CYP treatment. CXCL9 and CXCL10 protein expression was significantly (p ≤ 0.01) increased in the urinary bladder with 4 h CYP treatment in female mice whereas CXC protein expression in the urinary bladder of male mice did not exhibit an overall change in expression. CXCR3 blockade with intravesical instillation of AMG487 (5 mg/kg) significantly (p ≤ 0.01) increased bladder capacity, reduced voiding frequency and reduced non-voiding contractions in female mice treated with CYP (4 h, 48 h). CXCR3 blockade also reduced (p ≤ 0.01) hindpaw and pelvic sensitivity in female mice treated with CYP (4 h, 48 h). CXC chemokines may be novel targets for treating urinary bladder dysfunction and somatic sensitization resulting from urinary bladder inflammation. PMID:29681802

A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit

PubMed Central

2017-01-01

Experiments in synthetic biology and microbiology can benefit from protein expression systems with low cell-to-cell variability (noise) and expression levels precisely tunable across a useful dynamic range. Despite advances in understanding the molecular biology of microbial gene regulation, many experiments employ protein-expression systems exhibiting high noise and nearly all-or-none responses to induction. I present an expression system that incorporates elements known to reduce gene expression noise: negative autoregulation and bicistronic transcription. I show by stochastic simulation that while negative autoregulation can produce a more gradual response to induction, bicistronic expression of a repressor and gene of interest can be necessary to reduce noise below the extrinsic limit. I synthesized a plasmid-based system incorporating these principles and studied its properties in Escherichia coli cells, using flow cytometry and fluorescence microscopy to characterize induction dose-response, induction/repression kinetics and gene expression noise. By varying ribosome binding site strengths, expression levels from 55–10,740 molecules/cell were achieved with noise below the extrinsic limit. Individual strains are inducible across a dynamic range greater than 20-fold. Experimental comparison of different regulatory networks confirmed that bicistronic autoregulation reduces noise, and revealed unexpectedly high noise for a conventional expression system with a constitutively expressed transcriptional repressor. I suggest a hybrid, low-noise expression system to increase the dynamic range. PMID:29084263

Hepatic OATP Transporter and Thyroid Hormone Receptor Interplay Determines Cholesterol and Glucose Homeostasis

PubMed Central

Meyer zu Schwabedissen, Henriette E.; Ware, Joseph A.; Finkelstein, David; Chaudhry, Amarjit S.; Lemay, Sara; Leon-Ponte, Matilde; Strom, Stephen C.; Zaher, Hani; Schwarz, Ute I.; Freeman, David J.; Schuetz, Erin G.; Tirona, Rommel G.; Kim, Richard B.

2011-01-01

The role of Organic Anion Transporting Polypeptides (OATPs), particularly the members of OATP1B-subfamily, in hepatocellular handling of endogenous and exogenous compounds is an important and emerging area of research. Using a mouse model lacking Slco1b2, the murine ortholog of the OATP1B-subfamily, we previously demonstrated that genetic ablation causes reduced hepatic clearance capacity for substrates. In this report we focused on the physiological function of the hepatic OATP1B transporters. First we studied the influence of the Oatp1b2 deletion on bile acid metabolism showing that lack of the transporter results in a significantly reduced expression of Cyp7a1 the key enzyme of bile acid synthesis, resulting in elevated cholesterol levels after high dietary fat challenge. Furthermore, Slco1b2−/− mice exhibited delayed clearance after oral glucose challenge resulting from reduced hepatic glucose uptake. In addition to increased hepatic glycogen content, Slco1b2−/−exhibited reduced glucose output after pyruvate challenge. This is in accordance with reduced hepatic expression of PEPCK in knockout mice. We show this phenotype is due to the loss of liver-specific Oatp1b2-mediated hepatocellular thyroid hormone entry, which then leads to reduced transcriptional activation of target genes of hepatic thyroid hormone receptor (TR) including the prior mentioned Cyp7a1 and Pepck, but also Dio1 and Glut2. Importantly, we assessed human relevance using a cohort of archived human livers where OATP1B1 expression was noted to be highly associated with TR target genes, especially for GLUT2. Furthermore, GLUT2 expression was significantly decreased in livers harboring a common genetic polymorphism in SLCO1B1. Conclusion Our findings reveal that OATP1B-mediated hepatic thyroid hormone entry is a key determinant of cholesterol and glucose homeostasis. PMID:21538436

Do marmosets care to share? Oxytocin treatment reduces prosocial behavior toward strangers.

PubMed

Mustoe, Aaryn C; Cavanaugh, Jon; Harnisch, April M; Thompson, Breanna E; French, Jeffrey A

2015-05-01

Cooperatively-breeding and socially-monogamous primates, like marmosets and humans, exhibit high levels of social tolerance and prosociality toward others. Oxytocin (OXT) generally facilitates prosocial behavior, but there is growing recognition that OXT modulation of prosocial behavior is shaped by the context of social interactions and by other motivational states such as arousal or anxiety. To determine whether prosociality varies based on social context, we evaluated whether marmoset donors (Callithrix penicillata) preferentially rewarded pairmates versus opposite-sex strangers in a prosocial food-sharing task. To examine potential links among OXT, stress systems, and prosociality, we evaluated whether pretrial cortisol levels in marmosets altered the impact of OXT on prosocial responses. Marmosets exhibited spontaneous prosociality toward others, but they did so preferentially toward strangers compared to their pairmates. When donor marmosets were treated with marmoset-specific Pro(8)-OXT, they exhibited reduced prosociality toward strangers compared to marmosets treated with saline or consensus-mammalian Leu(8)-OXT. When pretrial cortisol levels were lower, marmosets exhibited higher prosociality toward strangers. These findings demonstrate that while marmosets show spontaneous prosocial responses toward others, they do so preferentially toward opposite-sex strangers. Cooperative breeding may be associated with the expression of prosociality, but the existence of a pair-bond between marmoset partners appears to be neither necessary nor sufficient for the expression of spontaneous prosocial responses. Furthermore, high prosociality toward strangers is significantly reduced in marmosets treated with Pro(8)-OXT, suggesting that OXT does not universally enhance prosociality, but, rather OXT modulation of prosocial behavior varies depending on social context. Copyright © 2015 Elsevier Inc. All rights reserved.

Do marmosets care to share? Oxytocin treatment reduces prosocial behavior toward strangers

PubMed Central

Mustoe, Aaryn C.; Cavanaugh, Jon; Harnisch, April M.; Thompson, Breanna E.; French, Jeffrey A.

2015-01-01

Cooperatively-breeding and socially-monogamous primates, like marmosets and humans, exhibit high levels of social tolerance and prosociality toward others. Oxytocin (OXT) generally facilitates prosocial behavior, but there is growing recognition that OXT modulation of prosocial behavior is shaped by the context of social interactions and by other motivational states such as arousal or anxiety. To determine whether prosociality varies based on social context, we evaluated whether marmoset donors (Callithrix penicillata) preferentially rewarded pairmates versus opposite-sex strangers in a prosocial food-sharing task. To examine potential links among OXT, stress systems, and prosociality, we evaluated whether pretrial cortisol levels in marmosets altered the impact of OXT on prosocial responses. Marmosets exhibited spontaneous prosociality toward others, but they did so preferentially toward strangers compared to their pairmates. When donor marmosets were treated with marmoset-specific Pro8-OXT, they exhibited reduced prosociality toward strangers compared to marmosets treated with saline or consensus-mammalian Leu8-OXT. When pretrial cortisol levels were lower, marmosets exhibited higher prosociality toward strangers. These findings demonstrate that while marmosets show spontaneous prosocial responses toward others, they do so preferentially toward opposite-sex strangers. Cooperative breeding may be associated with the expression of prosociality, but the existence of a pair-bond between marmoset partners appears to be neither necessary nor sufficient for the expression of spontaneous prosocial responses. Further, high prosociality toward strangers is significantly reduced in marmosets treated with Pro8-OXT, suggesting that OXT does not universally enhance prosociality, but, rather OXT modulation of prosocial behavior varies depending on social context. PMID:25934057

Comparative effect of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction on antioxidant enzymes activity in cellular ageing of human diploid fibroblasts

PubMed Central

2013-01-01

Background Human diploid fibroblasts (HDFs) undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular ageing. Even though beneficial effects of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction (TRF) have been reported, ongoing studies in relation to ageing is of interest to determine possible protective effects that may reverse the effect of ageing. The aim of this study was to evaluate the effect of P. betle, C. vulgaris and TRF in preventing cellular ageing of HDFs by determining the activity of antioxidant enzymes viz.; catalase, superoxide dismutase (SOD) and glutathione peroxidase. Methods Different passages of HDFs were treated with P. betle, C. vulgaris and TRF for 24 h prior to enzymes activity determination. Senescence-associated beta-galactosidase (SA β-gal) expression was assayed to validate cellular ageing. Results In cellular ageing of HDFs, catalase and glutathione peroxidase activities were reduced, but SOD activity was heightened during pre-senescence. P. betle exhibited the strongest antioxidant activity by reducing SA β-gal expression, catalase activities in all age groups, and SOD activity. TRF exhibited a strong antioxidant activity by reducing SA β-gal expression, and SOD activity in senescent HDFs. C. vulgaris extract managed to reduce SOD activity in senescent HDFs. Conclusion P. betle, C. vulgaris, and TRF have the potential as anti-ageing entities which compensated the role of antioxidant enzymes in cellular ageing of HDFs. PMID:23948056

Comparative effect of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction on antioxidant enzymes activity in cellular ageing of human diploid fibroblasts.

PubMed

Makpol, Suzana; Yeoh, Thong Wei; Ruslam, Farah Adilah Che; Arifin, Khaizurin Tajul; Yusof, Yasmin Anum Mohd

2013-08-16

Human diploid fibroblasts (HDFs) undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular ageing. Even though beneficial effects of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction (TRF) have been reported, ongoing studies in relation to ageing is of interest to determine possible protective effects that may reverse the effect of ageing. The aim of this study was to evaluate the effect of P. betle, C. vulgaris and TRF in preventing cellular ageing of HDFs by determining the activity of antioxidant enzymes viz.; catalase, superoxide dismutase (SOD) and glutathione peroxidase. Different passages of HDFs were treated with P. betle, C. vulgaris and TRF for 24 h prior to enzymes activity determination. Senescence-associated beta-galactosidase (SA β-gal) expression was assayed to validate cellular ageing. In cellular ageing of HDFs, catalase and glutathione peroxidase activities were reduced, but SOD activity was heightened during pre-senescence. P. betle exhibited the strongest antioxidant activity by reducing SA β-gal expression, catalase activities in all age groups, and SOD activity. TRF exhibited a strong antioxidant activity by reducing SA β-gal expression, and SOD activity in senescent HDFs. C. vulgaris extract managed to reduce SOD activity in senescent HDFs. P. betle, C. vulgaris, and TRF have the potential as anti-ageing entities which compensated the role of antioxidant enzymes in cellular ageing of HDFs.

Short-term regular aerobic exercise reduces oxidative stress produced by acute in the adipose microvasculature.

PubMed

Robinson, Austin T; Fancher, Ibra S; Sudhahar, Varadarajan; Bian, Jing Tan; Cook, Marc D; Mahmoud, Abeer M; Ali, Mohamed M; Ushio-Fukai, Masuko; Brown, Michael D; Fukai, Tohru; Phillips, Shane A

2017-05-01

High blood pressure has been shown to elicit impaired dilation in the vasculature. The purpose of this investigation was to elucidate the mechanisms through which high pressure may elicit vascular dysfunction and determine the mechanisms through which regular aerobic exercise protects arteries against high pressure. Male C57BL/6J mice were subjected to 2 wk of voluntary running (~6 km/day) for comparison with sedentary controls. Hindlimb adipose resistance arteries were dissected from mice for measurements of flow-induced dilation (FID; with or without high intraluminal pressure exposure) or protein expression of NADPH oxidase II (NOX II) and superoxide dismutase (SOD). Microvascular endothelial cells were subjected to high physiological laminar shear stress (20 dyn/cm 2 ) or static condition and treated with ANG II + pharmacological inhibitors. Cells were analyzed for the detection of ROS or collected for Western blot determination of NOX II and SOD. Resistance arteries from exercised mice demonstrated preserved FID after high pressure exposure, whereas FID was impaired in control mouse arteries. Inhibition of ANG II or NOX II restored impaired FID in control mouse arteries. High pressure increased superoxide levels in control mouse arteries but not in exercise mouse arteries, which exhibited greater ability to convert superoxide to H 2 O 2 Arteries from exercised mice exhibited less NOX II protein expression, more SOD isoform expression, and less sensitivity to ANG II. Endothelial cells subjected to laminar shear stress exhibited less NOX II subunit expression. In conclusion, aerobic exercise prevents high pressure-induced vascular dysfunction through an improved redox environment in the adipose microvasculature. NEW & NOTEWORTHY We describe potential mechanisms contributing to aerobic exercise-conferred protection against high intravascular pressure. Subcutaneous adipose microvessels from exercise mice express less NADPH oxidase (NOX) II and more superoxide dismutase (SOD) and demonstrate less sensitivity to ANG II. In microvascular endothelial cells, shear stress reduced NOX II but did not influence SOD expression.

Programmed hyperphagia secondary to increased hypothalamic SIRT1.

PubMed

Desai, Mina; Li, Tie; Han, Guang; Ross, Michael G

2014-11-17

Small for gestational age (SGA) offspring exhibit reduced hypothalamic neural satiety pathways leading to programmed hyperphagia and adult obesity. Appetite regulatory site, the hypothalamic arcuate nucleus (ARC) contains appetite (NPY/AgRP) and satiety (POMC) neurons. Using in vitro culture of hypothalamic neuroprogenitor cells (NPC) which form the ARC, we demonstrated that SGA offspring exhibit reduced NPC proliferation and neuronal differentiation. bHLH protein Hes1 promotes NPC self-renewal and inhibits differentiation by repressing neuronal differentiation genes (Mash1, neurogenin3). We hypothesized that Hes1/Mash1 and ultimately ARC neuronal differentiation and expression of NPY/POMC neurons are influenced by SIRT1 which is a nutrient sensor and a histone deacetylase. Control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to 21 (SGA). In vivo studies showed that SGA newborns and adult offspring had increased protein expression of hypothalamic/ARC SIRT1 and AgRP with decreased POMC. Additionally, SGA newborns had decreased expression of hypothalamic neurogenic factors with reduced in vivo NPC proliferation. In vitro culture of hypothalamic NPCs showed similar changes with elevated SIRT1 binding to Hes1 in SGA newborn. Silencing SIRT1 increased NPC proliferation and Hes1 and Tuj1expression in both Control and SGA NPCs. Although SGA NPC proliferation remained below that of Controls, it was higher than Control NPCs in the absence of SIRT1 siRNA. The direct impact of SIRT1 on NPC proliferation and differentiation were further confirmed with pharmacologic SIRT1 inhibitor and activator. Thus, in SGA newborns elevated SIRT1 induces premature differentiation of NPCs, reducing the NPC pool and cell proliferation. Copyright © 2014 Elsevier B.V. All rights reserved.

Programmed Hyperphagia secondary to Increased Hypothalamic SIRT1

PubMed Central

Desai, Mina; Li, Tie; Han, Guang; Ross, Michael G.

2014-01-01

Small for gestational age (SGA) offspring exhibit reduced hypothalamic neural satiety pathways leading to programmed hyperphagia and adult obesity. Appetite regulatory site, the hypothalamic arcuate nucleus (ARC) contains appetite (NPY/AgRP) and satiety (POMC) neurons. Using in vitro culture of hypothalamic neuroprogenitor cells (NPC) which form the ARC, we demonstrated that SGA offspring exhibit reduced NPC proliferation and neuronal differentiation. bHLH protein Hes1 promotes NPC self-renewal and inhibits differentiation by repressing neuronal differentiation genes (Mash1, neurogenin3). We hypothesized that Hes1/Mash1 and ultimately ARC neuronal differentiation and expression of NPY/POMC neurons are influenced by SIRT1 which is a nutrient sensor and a histone deacetylase. Control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to 21 (SGA). In vivo studies showed that SGA newborns and adult offspring had increased protein expression of hypothalamic/ARC SIRT1 and AgRP with decreased POMC. Additionally, SGA newborns had decreased expression of hypothalamic neurogenic factors with reduced in vivo NPC proliferation. In vitro culture of hypothalamic NPCs showed similar changes with elevated SIRT1 binding to Hes1 in SGA newborn. Silencing SIRT1 increased NPC proliferation and Hes1 and Tuj1expression in both Control and SGA NPCs. Although SGA NPC proliferation remained below that of Controls, it was higher than Control NPCs in the absence of SIRT1 siRNA. The direct impact of SIRT1 on NPC proliferation and differentiation were further confirmed with pharmacologic SIRT1 inhibitor and activator. Thus, in SGA newborns elevated SIRT1 induces premature differentiation of NPCs, reducing the NPC pool and cell proliferation. PMID:25245521

Reduced expression of the NMDA receptor-interacting protein SynGAP causes behavioral abnormalities that model symptoms of Schizophrenia.

PubMed

Guo, Xiaochuan; Hamilton, Peter J; Reish, Nicholas J; Sweatt, J David; Miller, Courtney A; Rumbaugh, Gavin

2009-06-01

Abnormal function of NMDA receptors is believed to be a contributing factor to the pathophysiology of schizophrenia. NMDAR subunits and postsynaptic-interacting proteins of these channels are abnormally expressed in some patients with this illness. In mice, reduced NMDAR expression leads to behaviors analogous to symptoms of schizophrenia, but reports of animals with mutations in core postsynaptic density proteins having similar a phenotype have yet to be reported. Here we show that reduced expression of the neuronal RasGAP and NMDAR-associated protein, SynGAP, results in abnormal behaviors strikingly similar to that reported in mice with reduced NMDAR function. SynGAP mutant mice exhibited nonhabituating and persistent hyperactivity that was ameliorated by the antipsychotic clozapine. An NMDAR antagonist, MK-801, induced hyperactivity in normal mice but SynGAP mutants were less responsive, suggesting that NMDAR hypofunction contributes to this behavioral abnormality. SynGAP mutants exhibited enhanced startle reactivity and impaired sensory-motor gating. These mice also displayed a complete lack of social memory and a propensity toward social isolation. Finally, SynGAP mutants had deficits in cued fear conditioning and working memory, indicating abnormal function of circuits that control emotion and choice. Our results demonstrate that SynGAP mutant mice have gross neurological deficits similar to other mouse models of schizophrenia. Because SynGAP interacts with NMDARs, and the signaling activity of this protein is regulated by these channels, our data in dicate that SynGAP lies downstream of NMDARs and is a required intermediate for normal neural circuit function and behavior. Taken together, these data support the idea that schizophrenia may arise from abnormal signaling pathways that are mediated by NMDA receptors.

Heterologous expression of Spathaspora passalidarum xylose reductase and xylitol dehydrogenase genes improved xylose fermentation ability of Aureobasidium pullulans.

PubMed

Guo, Jian; Huang, Siyao; Chen, Yefu; Guo, Xuewu; Xiao, Dongguang

2018-04-30

Aureobasidium pullulans is a yeast-like fungus that can ferment xylose to generate high-value-added products, such as pullulan, heavy oil, and melanin. The combinatorial expression of two xylose reductase (XR) genes and two xylitol dehydrogenase (XDH) genes from Spathaspora passalidarum and the heterologous expression of the Piromyces sp. xylose isomerase (XI) gene were induced in A. pullulans to increase the consumption capability of A. pullulans on xylose. The overexpression of XYL1.2 (encoding XR) and XYL2.2 (encoding XDH) was the most beneficial for xylose utilization, resulting in a 17.76% increase in consumed xylose compared with the parent strain, whereas the introduction of the Piromyces sp. XI pathway failed to enhance xylose utilization efficiency. Mutants with superior xylose fermentation performance exhibited increased intracellular reducing equivalents. The fermentation performance of all recombinant strains was not affected when glucose or sucrose was utilized as the carbon source. The strain with overexpression of XYL1.2 and XYL2.2 exhibited excellent fermentation performance with mimicked hydrolysate, and pullulan production increased by 97.72% compared with that of the parent strain. The present work indicates that the P4 mutant (using the XR/XDH pathway) with overexpressed XYL1.2 and XYL2.2 exhibited the best xylose fermentation performance. The P4 strain showed the highest intracellular reducing equivalents and XR and XDH activity, with consequently improved pullulan productivity and reduced melanin production. This valuable development in aerobic fermentation by the P4 strain may provide guidance for the biotransformation of xylose to high-value products by A. pullulans through genetic approach.

Elimination of Kalrn expression in POMC cells reduces anxiety-like behavior and contextual fear learning.

PubMed

Mandela, Prashant; Yan, Yan; LaRese, Taylor; Eipper, Betty A; Mains, Richard E

2014-07-01

Kalirin, a Rho GDP/GTP exchange factor for Rac1 and RhoG, is known to play an essential role in the formation and maintenance of excitatory synapses and in the secretion of neuropeptides. Mice unable to express any of the isoforms of Kalrn in cells that produce POMC at any time during development (POMC cells) exhibited reduced anxiety-like behavior and reduced acquisition of passive avoidance behavior, along with sex-specific alteration in the corticosterone response to restraint stress. Strikingly, lack of Kalrn expression in POMC cells closely mimicked the effects of global Kalrn knockout on anxiety-like behavior and passive avoidance conditioning without causing the other deficits noted in Kalrn knockout mice. Our data suggest that deficits in excitatory inputs onto POMC neurons are responsible for the behavioral phenotypes observed. Copyright © 2014 Elsevier Inc. All rights reserved.

Steroid signaling system responds differently to temperature and hormone manipulation in the red-eared slider turtle (Trachemys scripta elegans), a reptile with temperature-dependent sex determination.

PubMed

Ramsey, M; Crews, D

2007-01-01

Many reptiles, including the red-eared slider turtle (Trachemys scripta elegans), exhibit temperature-dependent sex determination (TSD). Temperature determines gonadal sex during the middle of embryogenesis, or the temperature-sensitive period (TSP), when gonadal sex is labile to both temperature and hormones--particularly estrogen. The biological actions of steroid hormones are mediated by their receptors as defined here as the classic transcriptional regulation of target genes. To elucidate estrogen action during sex determination, we examined estrogen receptor alpha (Esr1, hereafter referred to as ERalpha), estrogen receptor beta (Esr2, hereafter referred to as ERbeta), and androgen receptor (Ar, hereafter referred to as AR) expression in slider turtle gonads before, during and after the TSP, as well as following sex reversal via temperature or steroid hormone manipulation. ERalpha and AR levels spike at the female-producing temperature while ovarian sex is determined, but none of the receptors exhibited sexually dimorphic localization within the gonad prior to morphological differentiation. All three receptors respond differentially to sex-reversing treatments. When shifted to female-producing temperatures, embryos maintain ERalpha and AR expression while ERbeta is reduced. When shifted to male-producing temperatures, medullary expression of all three receptors is reduced. Feminization via estradiol (E(2)) treatment at a male-producing temperature profoundly changed the expression patterns for all three receptors. ERalpha and ERbeta redirected to the cortex in E(2)-created ovaries, while AR medullary expression was transiently reduced. Although warmer incubation temperature and estrogen result in the same endpoint (ovarian development), our results indicate different steroid signaling patterns between temperature- and estrogen-induced feminization. 2007 S. Karger AG, Basel

Fetal Liver Bisphenol A Concentrations and Biotransformation Gene Expression Reveal Variable Exposure and Altered Capacity for Metabolism in Humans

PubMed Central

Nahar, Muna S.; Liao, Chunyang; Kannan, Kurunthachalam; Dolinoy, Dana C.

2013-01-01

Widespread exposure to the endocrine active compound, bisphenol A (BPA), is well documented in humans. A growing body of literature suggests adverse health outcomes associated with varying ranges of exposure to BPA. In the current study, we measured the internal dose of free BPA and conjugated BPA and evaluated gene expression of bio-transformation enzymes specific for BPA metabolism in 50 first- and second-trimester human fetal liver samples. Both free BPA and conjugated BPA concentrations varied widely, with free BPA exhibiting three times higher concentrations than conjugated BPA concentrations. As compared to gender-matched adult liver controls, UDP-glucuronyltransferase, sulfotransferase, and steroid sulfatase genes exhibited reduced expression whereas β-glucuronidase mRNA expression remained unchanged in the fetal tissues. This study provides evidence that there is considerable exposure to BPA during human pregnancy and that the capacity for BPA metabolism is altered in the human fetal liver. PMID:23208979

The CNS glycoprotein Shadoo has PrPC-like protective properties and displays reduced levels in prion infections

PubMed Central

Watts, Joel C; Drisaldi, Bettina; Ng, Vivian; Yang, Jing; Strome, Bob; Horne, Patrick; Sy, Man-Sun; Yoong, Larry; Young, Rebecca; Mastrangelo, Peter; Bergeron, Catherine; Fraser, Paul E; Carlson, George A; Mount, Howard T J; Schmitt-Ulms, Gerold; Westaway, David

2007-01-01

The cellular prion protein, PrPC, is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS-expressed Doppel or internally deleted PrP (‘ΔPrP'). This paradigm has facilitated mapping of activity determinants in PrPC and implicated a cryptic PrPC-like protein, ‘π'. Shadoo (Sho) is a hypothetical GPI-anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N-terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrPC, but is low in cerebellar granular neurons (CGNs) containing PrPC and high in PrPC-deficient dendritic processes. In Prnp0/0 CGNs, Sho transgenes were PrPC-like in their ability to counteract neurotoxic effects of either Doppel or ΔPrP. Additionally, prion-infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for π, and since it engenders a PrPC-like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology. PMID:17703189

Berberine potently attenuates intestinal polyps growth in ApcMin mice and familial adenomatous polyposis patients through inhibition of Wnt signalling

PubMed Central

Zhang, Junfang; Cao, Hailong; Zhang, Bing; Cao, Hanwei; Xu, Xiuqin; Ruan, Hang; Yi, Tingting; Tan, Li; Qu, Rui; Song, Gang; Wang, Bangmao; Hu, Tianhui

2013-01-01

As a traditional anti-inflammatory Chinese herbal medicine, Alkaloid berberine has been recently reported to exhibit anti-tumour effects against a wide spectrum of cancer. However, the mechanism was largely unknown. Gene chip array reveals that with berberine treatment, c-Myc, the target gene of Wnt pathway, was down-regulated 5.3-folds, indicating that berberine might inhibit Wnt signalling. TOPflash analysis revealed that Wnt activity was significantly reduced after berberine treatment, and the mechanism of which might be that berberine disrupted β-catenin transfer to nucleus through up-regulating the expression of adenomatous polyposis coli (APC) gene and stabilized APC-β-catenin complex. Berberine administration in ApcMin/+ mice exhibited fewer and smaller polyps in intestine, along with reduction in cyclin D1 and c-Myc expression. In clinical practice, oral administration of berberine also significantly reduced the familial adenomatous polyposis patients' polyp size along with the inhibition of cyclin D1 expression in polyp samples. These observations indicate that berberine inhibits colon tumour formation through inhibition of Wnt/β-catenin signalling and berberine might be a promising drug for the prevention of colon cancer. PMID:24015932

A Critical Role for CD200R Signaling in Limiting the Growth and Metastasis of CD200+ Melanoma.

PubMed

Liu, Jin-Qing; Talebian, Fatemeh; Wu, Lisha; Liu, Zhihao; Li, Ming-Song; Wu, Laichu; Zhu, Jianmin; Markowitz, Joseph; Carson, William E; Basu, Sujit; Bai, Xue-Feng

2016-08-15

CD200 is a cell surface glycoprotein that functions through engaging CD200R on cells of the myeloid lineage and inhibits their functions. Expression of CD200 was implicated in a variety of human cancer cells, including melanoma cells; however, its roles in tumor growth and immunity are not clearly understood. In this study, we used CD200R-deficient mice and the B16 tumor model to evaluate this issue. We found that CD200R-deficient mice exhibited accelerated growth of CD200(+), but not CD200(-), B16 tumors. Strikingly, CD200R-deficient mice receiving CD200(+) B16 cells i.v. exhibited massive tumor growth in multiple organs, including liver, lung, kidney, and peritoneal cavity, whereas the growth of the same tumors in wild-type mice was limited. CD200(+) tumors grown in CD200R-deficient mice contained higher numbers of CD11b(+)Ly6C(+) myeloid cells, exhibited increased expression of VEGF and HIF1α genes with increased angiogenesis, and showed significantly reduced infiltration of CD4(+) and CD8(+) T cells, presumably as the result of reduced expression of T cell chemokines, such as CXCL9 and CXCL16. The liver from CD200R-deficient mice, under metastatic growth of CD200(+) tumors, contained significantly increased numbers of CD11b(+)Gr1(-) myeloid cells and Foxp3(+) regulatory T cells and reduced numbers of NK cells. Liver T cells also had a reduced capacity to produce IFN-γ or TNF-α. Taken together, we revealed a critical role for CD200R signaling in limiting the growth and metastasis of CD200(+) tumors. Thus, targeting CD200R signaling may potentially interfere with the metastatic growth of CD200(+) tumors, like melanoma. Copyright © 2016 by The American Association of Immunologists, Inc.

Crucial Role of Mesangial Cell-derived Connective Tissue Growth Factor in a Mouse Model of Anti-Glomerular Basement Membrane Glomerulonephritis

PubMed Central

Toda, Naohiro; Mori, Kiyoshi; Kasahara, Masato; Ishii, Akira; Koga, Kenichi; Ohno, Shoko; Mori, Keita P.; Kato, Yukiko; Osaki, Keisuke; Kuwabara, Takashige; Kojima, Katsutoshi; Taura, Daisuke; Sone, Masakatsu; Matsusaka, Taiji; Nakao, Kazuwa; Mukoyama, Masashi; Yanagita, Motoko; Yokoi, Hideki

2017-01-01

Connective tissue growth factor (CTGF) coordinates the signaling of growth factors and promotes fibrosis. Neonatal death of systemic CTGF knockout (KO) mice has hampered analysis of CTGF in adult renal diseases. We established 3 types of CTGF conditional KO (cKO) mice to investigate a role and source of CTGF in anti-glomerular basement membrane (GBM) glomerulonephritis. Tamoxifen-inducible systemic CTGF (Rosa-CTGF) cKO mice exhibited reduced proteinuria with ameliorated crescent formation and mesangial expansion in anti-GBM nephritis after induction. Although CTGF is expressed by podocytes at basal levels, podocyte-specific CTGF (pod-CTGF) cKO mice showed no improvement in renal injury. In contrast, PDGFRα promoter-driven CTGF (Pdgfra-CTGF) cKO mice, which predominantly lack CTGF expression by mesangial cells, exhibited reduced proteinuria with ameliorated histological changes. Glomerular macrophage accumulation, expression of Adgre1 and Ccl2, and ratio of M1/M2 macrophages were all reduced both in Rosa-CTGF cKO and Pdgfra-CTGF cKO mice, but not in pod-CTGF cKO mice. TGF-β1-stimulated Ccl2 upregulation in mesangial cells and macrophage adhesion to activated mesangial cells were decreased by reduction of CTGF. These results reveal a novel mechanism of macrophage migration into glomeruli with nephritis mediated by CTGF derived from mesangial cells, implicating the therapeutic potential of CTGF inhibition in glomerulonephritis. PMID:28191821

Crucial Role of Mesangial Cell-derived Connective Tissue Growth Factor in a Mouse Model of Anti-Glomerular Basement Membrane Glomerulonephritis.

PubMed

Toda, Naohiro; Mori, Kiyoshi; Kasahara, Masato; Ishii, Akira; Koga, Kenichi; Ohno, Shoko; Mori, Keita P; Kato, Yukiko; Osaki, Keisuke; Kuwabara, Takashige; Kojima, Katsutoshi; Taura, Daisuke; Sone, Masakatsu; Matsusaka, Taiji; Nakao, Kazuwa; Mukoyama, Masashi; Yanagita, Motoko; Yokoi, Hideki

2017-02-13

Connective tissue growth factor (CTGF) coordinates the signaling of growth factors and promotes fibrosis. Neonatal death of systemic CTGF knockout (KO) mice has hampered analysis of CTGF in adult renal diseases. We established 3 types of CTGF conditional KO (cKO) mice to investigate a role and source of CTGF in anti-glomerular basement membrane (GBM) glomerulonephritis. Tamoxifen-inducible systemic CTGF (Rosa-CTGF) cKO mice exhibited reduced proteinuria with ameliorated crescent formation and mesangial expansion in anti-GBM nephritis after induction. Although CTGF is expressed by podocytes at basal levels, podocyte-specific CTGF (pod-CTGF) cKO mice showed no improvement in renal injury. In contrast, PDGFRα promoter-driven CTGF (Pdgfra-CTGF) cKO mice, which predominantly lack CTGF expression by mesangial cells, exhibited reduced proteinuria with ameliorated histological changes. Glomerular macrophage accumulation, expression of Adgre1 and Ccl2, and ratio of M1/M2 macrophages were all reduced both in Rosa-CTGF cKO and Pdgfra-CTGF cKO mice, but not in pod-CTGF cKO mice. TGF-β1-stimulated Ccl2 upregulation in mesangial cells and macrophage adhesion to activated mesangial cells were decreased by reduction of CTGF. These results reveal a novel mechanism of macrophage migration into glomeruli with nephritis mediated by CTGF derived from mesangial cells, implicating the therapeutic potential of CTGF inhibition in glomerulonephritis.

Short-Term Hyperprolactinemia Reduces the Expression of Purinergic P2X7 Receptors during Allergic Inflammatory Response of the Lungs.

PubMed

Ochoa-Amaya, Julieta E; Queiroz-Hazarbassanov, Nicolle; Namazu, Lilian B; Calefi, Atilio S; Tobaruela, Carla N; Margatho, Rafael; Palermo-Neto, João; Ligeiro de Oliveira, Ana P; Felicio, Luciano F

2018-06-06

We have previously shown that domperidone-induced short-term hyperprolactinemia reduces the lung's allergic inflammatory response in an ovalbumin antigenic challenge model. Since purinergic receptor P2X7R activity leads to proinflammatory cytokine release and is possibly related to the pathogenesis of allergic respiratory conditions, the present study was designed to investigate a possible involvement of purinergic and prolactin receptors in this phenomenon. To induce hyperprolactinemia, domperidone was injected intraperitoneally in rats at a dose of 5.1 mg × kg-1 per day for 5 days. P2X7 expression was evaluated by lung immunohistochemistry while prolactin receptor expression in bronchoalveolar lavage leukocytes was analyzed through flow cytometry. Previous reports demonstrated that rats subjected to short-term hyperprolactinemia exhibited a decrease in leukocyte counts in bronchoalveolar lavage, especially granulocytes. Here, it is revealed that hyperprolactinemia promotes an increased expression of prolactin receptors in granulocytes. Also, increased expression of purinergic P2X7R observed in allergic animals was significantly reduced by hyperprolactinemia. Both purinergic and prolactin receptor expression changes occur during the anti-asthmatic effect of hyperprolactinemia. © 2018 S. Karger AG, Basel.

Deletion of connective tissue growth factor ameliorates peritoneal fibrosis by inhibiting angiogenesis and inflammation.

PubMed

Toda, Naohiro; Mori, Kiyoshi; Kasahara, Masato; Koga, Kenichi; Ishii, Akira; Mori, Keita P; Osaki, Keisuke; Mukoyama, Masashi; Yanagita, Motoko; Yokoi, Hideki

2018-06-01

Connective tissue growth factor (CTGF/CCN2) regulates the signalling of other growth factors and promotes fibrosis. CTGF is increased in mice and humans with peritoneal fibrosis. Inhibition of CTGF has not been examined as a potential therapeutic target for peritoneal fibrosis because systemic CTGF knockout mice die at the perinatal stage. To study the role of CTGF in peritoneal fibrosis of adult mice, we generated CTGF conditional knockout (cKO) mice by crossing CTGF floxed mice with RosaCreERT2 mice. We administered tamoxifen to Rosa-CTGF cKO mice to delete the CTGF gene throughout the body. We induced peritoneal fibrosis by intraperitoneal injection of chlorhexidine gluconate (CG) in wild-type and Rosa-CTGF cKO mice. Induction of peritoneal fibrosis in wild-type mice increased CTGF expression and produced severe thickening of the peritoneum. In contrast, CG-treated Rosa-CTGF cKO mice exhibited reduced thickening of the peritoneum. Peritoneal equilibration test revealed that the excessive peritoneal small-solute transport in CG-treated wild-type mice was normalized by CTGF deletion. CG-treated Rosa-CTGF cKO mice exhibited a reduced number of αSMA-, Ki67-, CD31- and MAC-2-positive cells in the peritoneum. Analyses of peritoneal mRNA showed that CG-treated Rosa-CTGF cKO mice exhibited reduced expression of Cd68, Acta2 (αSMA), Pecam1 (CD31) and Vegfa. These results indicate that a deficiency of CTGF can reduce peritoneal thickening and help to maintain peritoneal function by reducing angiogenesis and inflammation in peritoneal fibrosis. These results suggest that CTGF plays an important role in the progression of peritoneal fibrosis.

Elevated Na+/H+ exchanger-1 expression enhances the metastatic collective migration of head and neck squamous cell carcinoma cells.

PubMed

Kaminota, Teppei; Yano, Hajime; Shiota, Kohei; Nomura, Noriko; Yaguchi, Haruna; Kirino, Yui; Ohara, Kentaro; Tetsumura, Issei; Sanada, Tomoyoshi; Ugumori, Toru; Tanaka, Junya; Hato, Naohito

2017-04-22

Cancer cells can migrate as collectives during invasion and/or metastasis; however, the precise molecular mechanisms of this form of migration are less clear compared with single cell migration following epithelial-mesenchymal transition. Elevated Na + /H + exchanger1 (NHE1) expression has been suggested to have malignant roles in a number of cancer cell lines and in vivo tumor models. Furthermore, a metastatic human head and neck squamous cell carcinoma (HNSCC) cell line (SASL1m) that was isolated based on its increased metastatic potential also exhibited higher NHE1 expression than its parental line SAS. Time-lapse video recordings indicated that both cell lines migrate as collectives, although with different features, e.g., SASL1m was much more active and changed the direction of migration more frequently than SAS cells, whereas locomotive activities were comparable. SASL1m cells also exhibited higher invasive activity than SAS in Matrigel invasion assays. shRNA-mediated NHE1 knockdown in SASL1m led to reduced locomotive and invasive activities, suggesting a critical role for NHE1 in the collective migration of SASL1m cells. SASL1m cells also exhibited a higher metastatic rate than SAS cells in a mouse lymph node metastasis model, while NHE1 knockdown suppressed in vivo SASL1m metastasis. Finally, elevated NHE1 expression was observed in human HNSCC tissue, and Cariporide, a specific NHE1 inhibitor, reduced the invasive activity of SASL1m cells, implying NHE1 could be a target for anti-invasion/metastasis therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification and function analysis of contrary genes in Dupuytren's contracture.

PubMed

Ji, Xianglu; Tian, Feng; Tian, Lijie

2015-07-01

The present study aimed to analyze the expression of genes involved in Dupuytren's contracture (DC), using bioinformatic methods. The profile of GSE21221 was downloaded from the gene expression ominibus, which included six samples, derived from fibroblasts and six healthy control samples, derived from carpal-tunnel fibroblasts. A Distributed Intrusion Detection System was used in order to identify differentially expressed genes. The term contrary genes is proposed. Contrary genes were the genes that exhibited opposite expression patterns in the positive and negative groups, and likely exhibited opposite functions. These were identified using Coexpress software. Gene ontology (GO) function analysis was conducted for the contrary genes. A network of GO terms was constructed using the reduce and visualize gene ontology database. Significantly expressed genes (801) and contrary genes (98) were screened. A significant association was observed between Chitinase-3-like protein 1 and ten genes in the positive gene set. Positive regulation of transcription and the activation of nuclear factor-κB (NF-κB)-inducing kinase activity exhibited the highest degree values in the network of GO terms. In the present study, the expression of genes involved in the development of DC was analyzed, and the concept of contrary genes proposed. The genes identified in the present study are involved in the positive regulation of transcription and activation of NF-κB-inducing kinase activity. The contrary genes and GO terms identified in the present study may potentially be used for DC diagnosis and treatment.

Ablation of Sim1 Neurons Causes Obesity through Hyperphagia and Reduced Energy Expenditure

PubMed Central

Xi, Dong; Gandhi, Nilay; Lai, Meizan; Kublaoui, Bassil M.

2012-01-01

Single-minded 1 (Sim1) is a transcription factor necessary for development of the paraventricular nucleus of the hypothalamus (PVH). This nucleus is a critical regulator of appetite, energy expenditure and body weight. Previously we showed that Sim1+/− mice and conditional postnatal Sim1−/− mice exhibit hyperphagia, obesity, increased linear growth and susceptibility to diet-induced obesity, but no decrease in energy expenditure. Bilateral ablation of the PVH causes obesity due to hyperphagia and reduced energy expenditure. It remains unknown whether Sim1 neurons regulate energy expenditure. In this study, Sim1cre mice were bred to homozygous inducible diphtheria toxin receptor (iDTR) mice to generate mice expressing the simian DTR in Sim1 cells. In these mice, Sim1 neuron ablation was performed by intracerebroventricular (ICV) injection of diphtheria toxin. Compared to controls, mice with Sim1 neuron ablation became obese (with increased fat mass) on a chow diet due to increased food intake and reduced energy expenditure. In post-injection mice, we observed a strong inverse correlation between the degree of obesity and hypothalamic Sim1 expression. The reduction in baseline energy expenditure observed in these mice was accompanied by a reduction in activity. This reduction in activity did not fully account for the reduced energy expenditure as these mice exhibited decreased resting energy expenditure, decreased body temperature, decreased brown adipose tissue temperature, and decreased UCP1 expression suggesting an impairment of thermogenesis. In injected mice, hypothalamic gene expression of Sim1, oxytocin (OXT) and thyrotropin releasing hormone (TRH) was reduced by about 50%. These results demonstrate that Sim1 neurons in adult mice regulate both food intake and energy expenditure. Based on the body of work in the field, feeding regulation by Sim1 neurons likely occurs in both the PVH and medial amygdala, in contrast to energy expenditure regulation by Sim1 neurons, which likely is localized to the PVH. PMID:22558467

Ablation of Sim1 neurons causes obesity through hyperphagia and reduced energy expenditure.

PubMed

Xi, Dong; Gandhi, Nilay; Lai, Meizan; Kublaoui, Bassil M

2012-01-01

Single-minded 1 (Sim1) is a transcription factor necessary for development of the paraventricular nucleus of the hypothalamus (PVH). This nucleus is a critical regulator of appetite, energy expenditure and body weight. Previously we showed that Sim1(+/-) mice and conditional postnatal Sim1(-/-) mice exhibit hyperphagia, obesity, increased linear growth and susceptibility to diet-induced obesity, but no decrease in energy expenditure. Bilateral ablation of the PVH causes obesity due to hyperphagia and reduced energy expenditure. It remains unknown whether Sim1 neurons regulate energy expenditure. In this study, Sim1cre mice were bred to homozygous inducible diphtheria toxin receptor (iDTR) mice to generate mice expressing the simian DTR in Sim1 cells. In these mice, Sim1 neuron ablation was performed by intracerebroventricular (ICV) injection of diphtheria toxin. Compared to controls, mice with Sim1 neuron ablation became obese (with increased fat mass) on a chow diet due to increased food intake and reduced energy expenditure. In post-injection mice, we observed a strong inverse correlation between the degree of obesity and hypothalamic Sim1 expression. The reduction in baseline energy expenditure observed in these mice was accompanied by a reduction in activity. This reduction in activity did not fully account for the reduced energy expenditure as these mice exhibited decreased resting energy expenditure, decreased body temperature, decreased brown adipose tissue temperature, and decreased UCP1 expression suggesting an impairment of thermogenesis. In injected mice, hypothalamic gene expression of Sim1, oxytocin (OXT) and thyrotropin releasing hormone (TRH) was reduced by about 50%. These results demonstrate that Sim1 neurons in adult mice regulate both food intake and energy expenditure. Based on the body of work in the field, feeding regulation by Sim1 neurons likely occurs in both the PVH and medial amygdala, in contrast to energy expenditure regulation by Sim1 neurons, which likely is localized to the PVH.

Genome-wide gene expression profiling reveals aberrant MAPK and Wnt signaling pathways associated with early parthenogenesis.

PubMed

Liu, Na; Enkemann, Steven A; Liang, Ping; Hersmus, Remko; Zanazzi, Claudia; Huang, Junjiu; Wu, Chao; Chen, Zhisheng; Looijenga, Leendert H J; Keefe, David L; Liu, Lin

2010-12-01

Mammalian parthenogenesis could not survive but aborted during mid-gestation, presumably because of lack of paternal gene expression. To understand the molecular mechanisms underlying the failure of parthenogenesis at early stages of development, we performed global gene expression profiling and functional analysis of parthenogenetic blastocysts in comparison with those of blastocysts from normally fertilized embryos. Parthenogenetic blastocysts exhibited changes in the expression of 749 genes, of which 214 had lower expression and 535 showed higher expressions than fertilized embryos using a minimal 1.8-fold change as a cutoff. Genes important for placenta development were decreased in their expression in parthenote blastocysts. Some maternally expressed genes were up-regulated and paternal-related genes were down-regulated. Moreover, aberrantly increased Wnt signaling and reduced mitogen-activated protein kinase (MAPK) signaling were associated with early parthenogenesis. The protein level of extracellular signal-regulated kinase 2 (ERK2) was low in parthenogenetic blastocysts compared with that of fertilized blastocysts 120 h after fertilization. 6-Bromoindirubin-3'-oxime, a specific glycogen synthase kinase-3 (GSK-3) inhibitor, significantly decreased embryo hatching. The expression of several imprinted genes was altered in parthenote blastocysts. Gene expression also linked reduced expression of Xist to activation of X chromosome. Our findings suggest that failed X inactivation, aberrant imprinting, decreased ERK/MAPK signaling and possibly elevated Wnt signaling, and reduced expression of genes for placental development collectively may contribute to abnormal placenta formation and failed fetal development in parthenogenetic embryos.

In situ gene expression profiling of the thermoacidophilic alga Cyanidioschyzon in relation to visible and ultraviolet irradiance.

PubMed

Skorupa, Dana J; Castenholz, Richard W; Mazurie, Aurélien; Carey, Charles; Rosenzweig, Frank; McDermott, Timothy R

2014-06-01

Ultraviolet and high-intensity visible radiation generate reactive intermediates that damage phototrophic microorganisms. In Yellowstone National Park, the thermoacidophilic alga Cyanidioschyzon exhibits an annual seasonal biomass fluctuation referred to as 'mat decline', where algal viability decreases as ultraviolet and visible irradiances increase during summer. We examined the role irradiance might play in mat decline using irradiance filters that uncouple ultraviolet and visible effects along with custom microarrays to study gene expression in situ. Of the 6507 genes, 88% showed no response to ultraviolet or visible, implying that at the biomolecular level, these algae inhabit a chemostat-like environment and is consistent with the near constant aqueous chemistry measured. The remaining genes exhibited expression changes linked to ultraviolet exposure, to increased visible radiation, or to the apparent combined effects of ultraviolet and visible. Expression of DNA repetitive elements was synchronized, being repressed by visible but also influenced by ultraviolet. At highest irradiance levels, these algae reduced transcription of genes encoding functions involved with DNA replication, photosynthesis and cell cycle progression but exhibited an uptick in activities related to repairing DNA damage. This corroborates known physiological responses to ultraviolet and visible radiation, and leads us to provisionally conclude that mat decline is linked to photoinhibition. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

PACAP/Receptor System in Urinary Bladder Dysfunction and Pelvic Pain Following Urinary Bladder Inflammation or Stress

PubMed Central

Girard, Beatrice M.; Tooke, Katharine; Vizzard, Margaret A.

2017-01-01

Complex organization of CNS and PNS pathways is necessary for the coordinated and reciprocal functions of the urinary bladder, urethra and urethral sphincters. Injury, inflammation, psychogenic stress or diseases that affect these nerve pathways and target organs can produce lower urinary tract (LUT) dysfunction. Numerous neuropeptide/receptor systems are expressed in the neural pathways of the LUT and non-neural components of the LUT (e.g., urothelium) also express peptides. One such neuropeptide receptor system, pituitary adenylate cyclase-activating polypeptide (PACAP; Adcyap1) and its cognate receptor, PAC1 (Adcyap1r1), have tissue-specific distributions in the LUT. Mice with a genetic deletion of PACAP exhibit bladder dysfunction and altered somatic sensation. PACAP and associated receptors are expressed in the LUT and exhibit neuroplastic changes with neural injury, inflammation, and diseases of the LUT as well as psychogenic stress. Blockade of the PACAP/PAC1 receptor system reduces voiding frequency in preclinical animal models and transgenic mouse models that mirror some clinical symptoms of bladder dysfunction. A change in the balance of the expression and resulting function of the PACAP/receptor system in CNS and PNS bladder reflex pathways may underlie LUT dysfunction including symptoms of urinary urgency, increased voiding frequency, and visceral pain. The PACAP/receptor system in micturition pathways may represent a potential target for therapeutic intervention to reduce LUT dysfunction. PMID:29255407

A rice lectin receptor-like kinase that is involved in innate immune responses also contributes to seed germination

PubMed Central

Cheng, Xiaoyan; Wu, Yan; Guo, Jianping; Du, Bo; Chen, Rongzhi; Zhu, Lili; He, Guangcun

2013-01-01

Seed germination and innate immunity both have significant effects on plant life spans because they control the plant's entry into the ecosystem and provide defenses against various external stresses, respectively. Much ecological evidence has shown that seeds with high vigor are generally more tolerant of various environmental stimuli in the field than those with low vigor. However, there is little genetic evidence linking germination and immunity in plants. Here, we show that the rice lectin receptor-like kinase OslecRK contributes to both seed germination and plant innate immunity. We demonstrate that knocking down the OslecRK gene depresses the expression of α–amylase genes, reducing seed viability and thereby decreasing the rate of seed germination. Moreover, it also inhibits the expression of defense genes, and so reduces the resistance of rice plants to fungal and bacterial pathogens as well as herbivorous insects. Yeast two-hybrid and co-immunoprecipitation experiments revealed that OslecRK interacts with an actin-depolymerizing factor (ADF) in vivo via its kinase domain. Moreover, the rice adf mutant exhibited a reduced seed germination rate due to the suppression of α–amylase gene expression. This mutant also exhibited depressed immune responses and reduced resistance to biotic stresses. Our results thus provide direct genetic evidence for a common physiological pathway connecting germination and immunity in plants. They also partially explain the common observation that high-vigor seeds often perform well in the field. The dual effects of OslecRK may be indicative of progressive adaptive evolution in rice. PMID:24033867

Viability, biofilm formation, and MazEF expression in drug-sensitive and drug-resistant Mycobacterium tuberculosis strains circulating in Xinjiang, China.

PubMed

Zhao, Ji-Li; Liu, Wei; Xie, Wan-Ying; Cao, Xu-Dong; Yuan, Li

2018-01-01

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is one of the most common chronic infectious amphixenotic diseases worldwide. Prevention and control of TB are greatly difficult, due to the increase in drug-resistant TB, particularly multidrug-resistant TB. We speculated that there were some differences between drug-sensitive and drug-resistant MTB strains and that mazEF 3,6,9 toxin-antitoxin systems (TASs) were involved in MTB viability. This study aimed to investigate differences in viability, biofilm formation, and MazEF expression between drug-sensitive and drug-resistant MTB strains circulating in Xinjiang, China, and whether mazEF 3,6,9 TASs contribute to MTB viability under stress conditions. Growth profiles and biofilm-formation abilities of drug-sensitive, drug-resistant MTB strains and the control strain H37Rv were monitored. Using molecular biology experiments, the mRNA expression of the mazF 3, 6, and 9 toxin genes, the mazE 3, 6, and 9 antitoxin genes, and expression of the MazF9 protein were detected in the different MTB strains, H37RvΔ mazEF 3,6,9 mutants from the H37Rv parent strain were generated, and mutant viability was tested. Ex vivo culture analyses demonstrated that drug-resistant MTB strains exhibit higher survival rates than drug-sensitive strains and the control strain H37Rv. However, there was no statistical difference in biofilm-formation ability in the drug-sensitive, drug-resistant, and H37Rv strains. mazE 3,6 mRNA-expression levels were relatively reduced in the drug-sensitive and drug-resistant strains compared to H37Rv. Conversely, mazE 3,9 expression was increased in drug-sensitive strains compared to drug-resistant strains. Furthermore, compared with the H37Rv strain, mazF 3,6 expression was increased in drug-resistant strains, mazF 9 expression was increased in drug-sensitive strains, and mazF 9 exhibited reduced expression in drug-resistant strains compared with drug-sensitive strains. Protein expression of mazF9 was increased in drug-sensitive and drug-resistant strains compared to H37Rv, while drug-resistant strains exhibited reduced mazF9 expression compared to drug-sensitive strains. Compared to H37Rv, H37RvΔ mazEF 3,6,9-deletion mutants grew more slowly under both stress conditions, and their ability to survive in host macrophages was also weaker. Furthermore, the host macrophage-apoptosis rate was higher after infection with any of the H37RvΔ mazE F3,6,9 mutants than with the H37Rv strain. The increased viability of MTB drug-resistant strains compared with drug-sensitive strains is likely to be related to differential MazEF mRNA and protein expression. mazEF 3,6,9 TASs contribute to MTB viability under stress conditions.

Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

PubMed Central

Young, Christian D.; Lewis, Andrew S.; Rudolph, Michael C.; Ruehle, Marisa D.; Jackman, Matthew R.; Yun, Ui J.; Ilkun, Olesya; Pereira, Renata; Abel, E. Dale; Anderson, Steven M.

2011-01-01

Tumor cells exhibit an altered metabolism characterized by elevated aerobic glycolysis and lactate secretion which is supported by an increase in glucose transport and consumption. We hypothesized that reducing or eliminating the expression of the most prominently expressed glucose transporter(s) would decrease the amount of glucose available to breast cancer cells thereby decreasing their metabolic capacity and proliferative potential. Of the 12 GLUT family glucose transporters expressed in mice, GLUT1 was the most abundantly expressed at the RNA level in the mouse mammary tumors from MMTV-c-ErbB2 mice and cell lines examined. Reducing GLUT1 expression in mouse mammary tumor cell lines using shRNA or Cre/Lox technology reduced glucose transport, glucose consumption, lactate secretion and lipid synthesis in vitro without altering the concentration of ATP, as well as reduced growth on plastic and in soft agar. The growth of tumor cells with reduced GLUT1 expression was impaired when transplanted into the mammary fat pad of athymic nude mice in vivo. Overexpression of GLUT1 in a cell line with low levels of endogenous GLUT1 increased glucose transport in vitro and enhanced growth in nude mice in vivo as compared to the control cells with very low levels of GLUT1. These studies demonstrate that GLUT1 is the major glucose transporter in mouse mammary carcinoma models overexpressing ErbB2 or PyVMT and that modulation of the level of GLUT1 has an effect upon the growth of mouse mammary tumor cell lines in vivo. PMID:21826239

Neuroprotective effect of mesenchymal stem cell through complement component 3 downregulation after transient focal cerebral ischemia in mice.

PubMed

Jung, Hye-Seon; Jeong, Si-Yeon; Yang, Jiwon; Kim, So-Dam; Zhang, Baojin; Yoo, Hyun Seung; Song, Sun U; Jeon, Myung-Shin; Song, Yun Seon

2016-10-28

Bone marrow-derived mesenchymal stem cells (MSCs) are used in stroke treatment despite the poor understanding of its mode of action. The immune suppressive and anti-inflammatory properties of MSCs possibly play important roles in regulating neuroinflammation after stroke. We investigated whether MSCs reduce the inflammatory complement component 3 (C3) levels, thus, providing neuroprotection during stroke. Mice were subjected to transient focal cerebral ischemia (tFCI), after which MSCs were intravenously injected. The infarct volume of the brain was reduced in MSC-injected tFCI mice, and C3 expression was significantly reduced in both the brain and the blood. Additionally, the profiles of other inflammatory mediators demonstrated neuroprotective changes in the MSCs-treated group. In order to analyze the effect of MSCs on neurons during cerebral ischemia, primary cortical neurons were co-cultured with MSCs under oxygen-glucose deprivation (OGD). Primary neurons co-cultured with MSCs exhibited reduced levels of C3 expression and increased protection against OGD, indicating that treatment with MSCs reduces excessive C3 expression and rescues ischemia-induced neuronal damage. Our finding suggests that reduction of C3 expression by MSCs can help to ameliorate ischemic brain damage, offering a new neuroprotective strategy in stroke therapy. Copyright © 2016. Published by Elsevier Ireland Ltd.

Differential chemokine, chemokine receptor and cytokine expression in Epstein-Barr virus-associated lymphoproliferative diseases.

PubMed

Ohshima, Koichi; Karube, Kennosuke; Hamasaki, Makoto; Tutiya, Takeshi; Yamaguchi, Takahiro; Suefuji, Hiroaki; Suzuki, Keiko; Suzumiya, Junji; Ohga, Shouichi; Kikuchi, Masahiro

2003-08-01

T cell immunity plays an important role in the clinicopathology of Epstein-Barr virus (EBV)-associated diseases. Acute EBV-induced infectious mononucleosis (IM) is a common self-limiting disease, however, other EBV-associated diseases, including chronic active EBV infection (CAEBV), NK cell lymphoma (NKL), and Hodgkin's lymphoma (HL), exhibit distinct clinical features. Chemokines are members of a family of small-secreted proteins. The relationships between chemokines and the chemokine receptor (R) are thought to be important for selectivity of local immunity. Some chemokines, chemokine R and cytokines closely associate with the T cell subtypes, Th1 and Th2 T cells and cytotoxic cells. To clarify the role of T cell immunity in EBV-associated diseases, we conducted gene expression profiling, using chemokine, chemokine R and cytokine DNA chips. Compared to EBV negative non-specific lymphadenitis, CAEBV and NKL exhibited diffuse down- and up-regulation, respectively, of these gene profiles. IM had a predominantly Th1-type profile, whereas HL had a mixed Th1/Th2-type profile. Reduction of the Th1-type cytokine interferon gamma (IFN-gamma) in CAEBV was confirmed by Reverse transcriptase-polymerase chain reaction, whereas IFN-gamma expression was markedly enhanced in NKL, and moderately enhanced in IM. Compared to IM, CAEBV showed slight elevation of "regulated upon activation, normal T expressed and secreted" (RANTES), but almost all other genes assayed were down-regulated. NKL exhibited elevated expression of numerous genes, particularly IFN-gamma-inducible-10 (IP-10) and monokine induced by IFN-gamma (MIG). HL showed variable elevated and reduced expression of various genes, with increased expression of IL-13 receptor and MIG. Our study demonstrated the enormous potential of gene expression profiling for clarifying the pathogenesis of EBV-associated diseases.

Frequent Attenuation of the WWOX Tumor Suppressor in Osteosarcoma is Associated with Increased Tumorigenicity and Aberrant RUNX2 Expression

PubMed Central

Kurek, Kyle; Del Mare, Sara; Salah, Zaidoun; Abdeen, Suhaib; Sadiq, Hussain; Lee, Sukhee; Gaudio, Eugenio; Zanesi, Nicola; Jones, Kevin B.; DeYoung, Barry; Amir, Gail; Gebhardt, Mark; Warman, Matthew; Stein, Gary S.; Stein, Janet L.; Lian, Jane B.; Aqeilan, Rami I.

2011-01-01

The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor that is deleted or attenuated in most human tumors. Wwox-deficient mice develop osteosarcoma (OS), an aggressive bone tumor with poor prognosis that often metastasizes to lung. On the basis of these observations, we examined the status of WWOX in human OS specimens and cell lines. In human OS clinical samples, WWOX expression was absent or reduced in 58% of tumors examined (P< 0.0001). Compared to the primary tumors, WWOX levels frequently increased in tumors resected following chemotherapy. In contrast, tumor metastases to lung often exhibited reduced WWOX levels, relative to the primary tumor. In human OS cell lines having reduced WWOX expression, ectopic expression of WWOX inhibited proliferation and attenuated invasion in vitro, and suppressed tumorgenicity in nude mice. Expression of WWOX was associated with reduced RUNX2 expression in OS cell lines, whereas Runx2 levels were elevated in femurs of Wwox-deficient mice. Furthermore, WWOX reconstitution in HOS cells was associated with downregulation of RUNX2 levels and RUNX2 target genes, consistent with the ability of WWOX to suppress RUNX2 transactivation activity. In clinical samples, RUNX2 was expressed in the majority of primary tumors and undetectable in most tumors resected following chemotherapy, whereas most metastases were RUNX2 positive. Our results deepen the evidence of a tumor suppressor role for WWOX in OS, furthering its prognostic and therapeutic significance in this disease. PMID:20530675

Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests.

PubMed

Liu, Shu-Min; Li, Jie; Zhu, Jin-Qi; Wang, Xiao-Wei; Wang, Cheng-Shu; Liu, Shu-Sheng; Chen, Xue-Xin; Li, Sheng

2016-04-01

The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops. © 2015 Institute of Zoology, Chinese Academy of Sciences.

AGGRECAN MODULATION OF GROWTH PLATE MORPHOGENESIS

PubMed Central

Domowicz, Miriam S.; Cortes, Mauricio; Henry, Judith G.; Schwartz, Nancy B.

2009-01-01

Chick and mouse embryos with heritable deficiencies of aggrecan exhibit severe dwarfism and premature death, demonstrating the essential involvement of aggrecan in development. The aggrecan-deficient nanomelic (nm) chick mutant E12 fully formed growth plate (GP) is devoid of matrix and exhibits markedly altered cytoarchitecture, proliferative capacity, and degree of cell death. While differentiation of chondroblasts to pre-hypertrophic chondrocytes (IHH expression) is normal up to E6, the extended periosteum expression pattern of PTCH (a downstream effector of IHH) indicates altered propagation of IHH signaling, as well as accelerated down-regulation of FGFR3 expression, decreased BrdU incorporation and higher levels of ERK phosphorylation, all indicating early effects on FGF signaling. By E7 reduced IHH expression and premature expression of COL10A1 foreshadow the acceleration of hypertrophy observed at E12. By E8, exacerbated co-expression of IHH and COL10A1 lead to delayed separation and establishment of the two GPs in each element. By E9, increased numbers of cells express P-SMAD1/5/8, indicating altered BMP signaling. These results indicate that the IHH, FGF and BMP signaling pathways are altered from the very beginning of GP formation in the absence of aggrecan, thereby inducing premature hypertrophic chondrocyte maturation, leading to the nanomelic long bone growth disorder. PMID:19268444

A Novel Differentiation Therapy Approach to Reduce the Metastatic Potential of Basal, Highly Metastatic, Triple-Negative Breast Cancers

DTIC Science & Technology

2012-05-01

subset enriched in epithelial-to- mesenchymal transition and stem cell characteristics. Cancer Res 69: 4116–4124. Hoenerhoff MJ, Chu I, Barkan D, Liu ZY...expression of epithelial markers and loss of mesenchymal markers in MB- 231 cells (Task1a) Our analyses of m icroarray data com paring 231-Empty cells ...is considered a hallmark of EMT (Yang and Weinberg 2008). MB-231 cells lack E-cadherin expression and exhibit a more mesenchymal phenotype

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pols, Thijs W.H.; Ottenhoff, Roelof; Vos, Mariska

NR4A nuclear receptors are induced in the liver upon fasting and regulate hepatic gluconeogenesis. Here, we studied the role of nuclear receptor Nur77 (NR4A1) in hepatic lipid metabolism. We generated mice expressing hepatic Nur77 using adenoviral vectors, and demonstrate that these mice exhibit a modulation of the plasma lipid profile and a reduction in hepatic triglyceride. Expression analysis of >25 key genes involved in lipid metabolism revealed that Nur77 inhibits SREBP1c expression. This results in decreased SREBP1c activity as is illustrated by reduced expression of its target genes stearoyl-coA desaturase-1, mitochondrial glycerol-3-phosphate acyltransferase, fatty acid synthase and the LDL receptor,more » and provides a mechanism for the physiological changes observed in response to Nur77. Expression of LXR target genes Abcg5 and Abcg8 is reduced by Nur77, and may suggest involvement of LXR in the inhibitory action of Nur77 on SREBP1c expression. Taken together, our study demonstrates that Nur77 modulates hepatic lipid metabolism through suppression of SREBP1c activity.« less

The seed dormancy defect of Arabidopsis mutants lacking the transcript elongation factor TFIIS is caused by reduced expression of the DOG1 gene.

PubMed

Mortensen, Simon A; Grasser, Klaus D

2014-01-03

TFIIS is a transcript elongation factor that facilitates transcription by RNA polymerase II, as it assists the enzyme to bypass blocks to mRNA synthesis. Previously, we have reported that Arabidopsis plants lacking TFIIS exhibit reduced seed dormancy. Among the genes differentially expressed in tfIIs seeds, the DOG1 gene was identified that is a known QTL for seed dormancy. Here we have analysed plants that overexpress TFIIS in wild type background, or that harbour an additional copy of DOG1 in tfIIs mutant background. These experiments demonstrate that the down-regulation of DOG1 expression causes the seed dormancy phenotype of tfIIs mutants. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Ectopic Expression of Xylella fastidiosa rpfF Conferring Production of Diffusible Signal Factor in Transgenic Tobacco and Citrus Alters Pathogen Behavior and Reduces Disease Severity.

PubMed

Caserta, R; Souza-Neto, R R; Takita, M A; Lindow, S E; De Souza, A A

2017-11-01

The pathogenicity of Xylella fastidiosa is associated with its ability to colonize the xylem of host plants. Expression of genes contributing to xylem colonization are suppressed, while those necessary for insect vector acquisition are increased with increasing concentrations of diffusible signal factor (DSF), whose production is dependent on RpfF. We previously demonstrated that transgenic citrus plants ectopically expressing rpfF from a citrus strain of X. fastidiosa subsp. pauca exhibited less susceptibility to Xanthomonas citri subsp. citri, another pathogen whose virulence is modulated by DSF accumulation. Here, we demonstrate that ectopic expression of rpfF in both transgenic tobacco and sweet orange also confers a reduction in disease severity incited by X. fastidiosa and reduces its colonization of those plants. Decreased disease severity in the transgenic plants was generally associated with increased expression of genes conferring adhesiveness to the pathogen and decreased expression of genes necessary for active motility, accounting for the reduced population sizes achieved in the plants, apparently by limiting pathogen dispersal through the plant. Plant-derived DSF signal molecules in a host plant can, therefore, be exploited to interfere with more than one pathogen whose virulence is controlled by DSF signaling.

Engrailed-2 (En2) deletion produces multiple neurodevelopmental defects in monoamine systems, forebrain structures and neurogenesis and behavior

PubMed Central

Genestine, Matthieu; Lin, Lulu; Durens, Madel; Yan, Yan; Jiang, Yiqin; Prem, Smrithi; Bailoor, Kunal; Kelly, Brian; Sonsalla, Patricia K.; Matteson, Paul G.; Silverman, Jill; Crawley, Jacqueline N.; Millonig, James H.; DiCicco-Bloom, Emanuel

2015-01-01

Many genes involved in brain development have been associated with human neurodevelopmental disorders, but underlying pathophysiological mechanisms remain undefined. Human genetic and mouse behavioral analyses suggest that ENGRAILED-2 (EN2) contributes to neurodevelopmental disorders, especially autism spectrum disorder. In mouse, En2 exhibits dynamic spatiotemporal expression in embryonic mid-hindbrain regions where monoamine neurons emerge. Considering their importance in neuropsychiatric disorders, we characterized monoamine systems in relation to forebrain neurogenesis in En2-knockout (En2-KO) mice. Transmitter levels of serotonin, dopamine and norepinephrine (NE) were dysregulated from Postnatal day 7 (P7) to P21 in En2-KO, though NE exhibited the greatest abnormalities. While NE levels were reduced ∼35% in forebrain, they were increased 40–75% in hindbrain and cerebellum, and these patterns paralleled changes in locus coeruleus (LC) fiber innervation, respectively. Although En2 promoter was active in Embryonic day 14.5–15.5 LC neurons, expression diminished thereafter and gene deletion did not alter brainstem NE neuron numbers. Significantly, in parallel with reduced NE levels, En2-KO forebrain regions exhibited reduced growth, particularly hippocampus, where P21 dentate gyrus granule neurons were decreased 16%, suggesting abnormal neurogenesis. Indeed, hippocampal neurogenic regions showed increased cell death (+77%) and unexpectedly, increased proliferation. Excess proliferation was restricted to early Sox2/Tbr2 progenitors whereas increased apoptosis occurred in differentiating (Dcx) neuroblasts, accompanied by reduced newborn neuron survival. Abnormal neurogenesis may reflect NE deficits because intra-hippocampal injections of β-adrenergic agonists reversed cell death. These studies suggest that disruption of hindbrain patterning genes can alter monoamine system development and thereby produce forebrain defects that are relevant to human neurodevelopmental disorders. PMID:26220976

Gelsolin functions as a metastasis suppressor in B16-BL6 mouse melanoma cells and requirement of the carboxyl-terminus for its effect.

PubMed

Fujita, H; Okada, F; Hamada , J; Hosokawa, M; Moriuchi, T; Koya, R C; Kuzumaki, N

2001-09-01

Gelsolin, an actin-binding protein, is implicated as a critical regulator in cell motility. In addition, we have reported that cellular levels of gelsolin are decreased in various tumor cells, and overexpression of gelsolin by gene transfer suppresses tumorigenicity. We sought to assess the effects of gelsolin overexpression on metastasis and to determine the importance of a carboxyl-terminus that confers Ca(2+) dependency on gelsolin for effects of its overexpression. Expression vectors with cDNA encoding either full-length wild-type or His321 mutant form, isolated from a flat revertant of Ras-transformed cells and a carboxyl-terminal truncate, C-del of gelsolin, were transfected into a highly metastatic murine melanoma cell line, B16-BL6. Expression of introduced cDNA in transfectants was confirmed using Western blotting, 2-dimensional gel electrophoresis and reverse transcription-polymerase chain reaction (RT-PCR). We characterized phenotypes of transfectants, such as growth rate, colony formation in soft agar, cell motility and metastasis formation in vivo. Transfectants expressing the wild-type, His321 mutant and C-del gelsolin exhibited reduced growth ability in soft agar. Although expression of integrin beta1 or alpha4 on the cell surface of transfectants was not changed, wild-type and His321 mutant gelsolin, except for C-del gelsolin, exhibited retardation of cell spreading, reduced chemotatic migration to fibronectin and suppressed lung colonization in spontaneous metastasis assay. Gelsolin may function as a metastasis suppressor as well as a tumor suppressor gene. The carboxyl-terminus of gelsolin is important for retardation of cell spreading, reduced chemotasis and metastasis suppression. Copyright 2001 Wiley-Liss, Inc.

Perk Gene Dosage Regulates Glucose Homeostasis by Modulating Pancreatic β-Cell Functions

PubMed Central

Wang, Rong; Munoz, Elyse E.; Zhu, Siying; McGrath, Barbara C.; Cavener, Douglas R.

2014-01-01

Background Insulin synthesis and cell proliferation are under tight regulation in pancreatic β-cells to maintain glucose homeostasis. Dysfunction in either aspect leads to development of diabetes. PERK (EIF2AK3) loss of function mutations in humans and mice exhibit permanent neonatal diabetes that is characterized by insufficient β-cell mass and reduced proinsulin trafficking and insulin secretion. Unexpectedly, we found that Perk heterozygous mice displayed lower blood glucose levels. Methodology Longitudinal studies were conducted to assess serum glucose and insulin, intracellular insulin synthesis and storage, insulin secretion, and β-cell proliferation in Perk heterozygous mice. In addition, modulation of Perk dosage specifically in β-cells showed that the glucose homeostasis phenotype of Perk heterozygous mice is determined by reduced expression of PERK in the β-cells. Principal Findings We found that Perk heterozygous mice first exhibited enhanced insulin synthesis and secretion during neonatal and juvenile development followed by enhanced β-cell proliferation and a substantial increase in β-cell mass at the adult stage. These differences are not likely to entail the well-known function of PERK to regulate the ER stress response in cultured cells as several markers for ER stress were not differentially expressed in Perk heterozygous mice. Conclusions In addition to the essential functions of PERK in β-cells as revealed by severely diabetic phenotype in humans and mice completely deficient for PERK, reducing Perk gene expression by half showed that intermediate levels of PERK have a profound impact on β-cell functions and glucose homeostasis. These results suggest that an optimal level of PERK expression is necessary to balance several parameters of β-cell function and growth in order to achieve normoglycemia. PMID:24915520

Dietary Supplementation with n-3 Polyunsaturated Fatty Acids Reduces Torpor Use in a Tropical Daily Heterotherm.

PubMed

Vuarin, Pauline; Henry, Pierre-Yves; Perret, Martine; Pifferi, Fabien

Polyunsaturated fatty acids (PUFAs) are involved in a variety of physiological mechanisms, including heterothermy preparation and expression. However, the effects of the two major classes of PUFAs, n-6 and n-3, can differ substantially. While n-6 PUFAs enhance torpor expression, n-3 PUFAs reduce the ability to decrease body temperature. This negative impact of n-3 PUFAs has been revealed in temperate hibernators only. Yet because tropical heterotherms generally experience higher ambient temperature and exhibit higher minimum body temperature during heterothermy, they may not be affected as much by PUFAs as their temperate counterparts. We tested whether n-3 PUFAs constrain torpor use in a tropical daily heterotherm (Microcebus murinus). We expected dietary n-3 PUFA supplementation to induce a reduction in torpor use and for this effect to appear rapidly given the time required for dietary fatty acids to be assimilated into phospholipids. n-3 PUFA supplementation reduced torpor use, and its effect appeared in the first days of the experiment. Within 2 wk, control animals progressively deepened their torpor bouts, whereas supplemented ones never entered torpor but rather expressed only constant, shallow reductions in body temperature. For the rest of the experiment, the effect of n-3 PUFA supplementation on torpor use remained constant through time. Even though supplemented animals also started to express torpor, they exhibited higher minimum body temperature by 2°-3°C and spent two fewer hours in a torpid state per day than control individuals, on average. Our study supports the view that a higher dietary content in n-3 PUFAs negatively affects torpor use in general, not only in cold-acclimated hibernators.

Enhanced xylose fermentation by engineered yeast expressing NADH oxidase through high cell density inoculums.

PubMed

Zhang, Guo-Chang; Turner, Timothy L; Jin, Yong-Su

2017-03-01

Accumulation of reduced byproducts such as glycerol and xylitol during xylose fermentation by engineered Saccharomyces cerevisiae hampers the economic production of biofuels and chemicals from cellulosic hydrolysates. In particular, engineered S. cerevisiae expressing NADPH-linked xylose reductase (XR) and NAD + -linked xylitol dehydrogenase (XDH) produces substantial amounts of the reduced byproducts under anaerobic conditions due to the cofactor difference of XR and XDH. While the additional expression of a water-forming NADH oxidase (NoxE) from Lactococcus lactis in engineered S. cerevisiae with the XR/XDH pathway led to reduced glycerol and xylitol production and increased ethanol yields from xylose, volumetric ethanol productivities by the engineered yeast decreased because of growth defects from the overexpression of noxE. In this study, we introduced noxE into an engineered yeast strain (SR8) exhibiting near-optimal xylose fermentation capacity. To overcome the growth defect caused by the overexpression of noxE, we used a high cell density inoculum for xylose fermentation by the SR8 expressing noxE. The resulting strain, SR8N, not only showed a higher ethanol yield and lower byproduct yields, but also exhibited a high ethanol productivity during xylose fermentation. As noxE overexpression elicits a negligible growth defect on glucose conditions, the beneficial effects of noxE overexpression were substantial when a mixture of glucose and xylose was used. Consumption of glucose led to rapid cell growth and therefore enhanced the subsequent xylose fermentation. As a result, the SR8N strain produced more ethanol and fewer byproducts from a mixture of glucose and xylose than the parental SR8 strain without noxE overexpression. Our results suggest that the growth defects from noxE overexpression can be overcome in the case of fermenting lignocellulose-derived sugars such as glucose and xylose.

Cysteine, histidine and glycine exhibit anti-inflammatory effects in human coronary arterial endothelial cells.

PubMed

Hasegawa, S; Ichiyama, T; Sonaka, I; Ohsaki, A; Okada, S; Wakiguchi, H; Kudo, K; Kittaka, S; Hara, M; Furukawa, S

2012-02-01

The activation of nuclear factor-kappa B (NF-κB) in vascular endothelial cells may be involved in vascular pathogeneses such as vasculitis or atherosclerosis. Recently, it has been reported that some amino acids exhibit anti-inflammatory effects. We investigated the inhibitory effects of a panel of amino acids on cytokine production or expression of adhesion molecules that are involved in inflammatory diseases in various cell types. The activation of NF-κB was determined in human coronary arterial endothelial cells (HCAECs) because NF-κB modulates the production of many cytokines and the expression of adhesion molecules. We examined the inhibitory effects of the amino acids cysteine, histidine and glycine on the induction of NF-κB activation, expression of CD62E (E-selectin) and the production of interleukin (IL)-6 in HCAECs stimulated with tumour necrosis factor (TNF)-α. Cysteine, histidine and glycine significantly reduced NF-κB activation and inhibitor κBα (IκBα) degradation in HCAECs stimulated with TNF-α. Additionally, all the amino acids inhibited the expression of E-selectin and the production of IL-6 in HCAECs, and the effects of cysteine were the most significant. Our results show that glycine, histidine and cysteine can inhibit NF-κB activation, IκBα degradation, CD62E expression and IL-6 production in HCAECs, suggesting that these amino acids may exhibit anti-inflammatory effects during endothelial inflammation. © 2012 The Authors. Clinical and Experimental Immunology © 2012 British Society for Immunology.

Reduction of wobble-position GC bases in Corynebacteria genes and enhancement of PCR and heterologous expression.

PubMed

Sanli, G; Blaber, S I; Blaber, M

2001-01-01

Corynebacteria codon usage exhibits an overall GC content of 67%, and a wobble-position GC content of 88%. Escherichia coli, on the other hand has an overall GC content of 51%, and a wobble-position GC content of 55%. The high GC content of Corynebacteria genes results in an unfavorable codon preference for heterologous expression, and can present difficulties for polymerase-based manipulations due to secondary-structure effects. Since these characteristics are due primarily to base composition at the wobble-position, synthetic genes can, in principle, be designed to eliminate these problems and retain the wild-type amino acid sequence. Such genes would obviate the need for special additives or bases during in vitro polymerase-based manipulation and mutant host strains containing uncommon tRNA's for heterologous expression. We have evaluated synthetic genes with reduced wobble-position G/C content using two variants of the enzyme 2,5-diketo-D-gluconic acid reductase (2,5-DKGR A and B) from Corynebacterium. The wild-type genes are refractory to polymerase-based manipulations and exhibit poor heterologous expression in enteric bacteria. The results indicate that a subset of codons for five amino acids (alanine, arginine, glutamate, glycine and valine) contribute the greatest contribution to reduction in G/C content at the wobble-position. Furthermore, changes in codons for two amino acids (leucine and proline) enhance bias for expression in enteric bacteria without affecting the overall G/C content. The synthetic genes are readily amplified using polymerase-based methodologies, and exhibit high levels of heterologous expression in E. coli.

Hypoxia-inducible factor regulates alphavbeta3 integrin cell surface expression.

PubMed

Cowden Dahl, Karen D; Robertson, Sarah E; Weaver, Valerie M; Simon, M Celeste

2005-04-01

Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of alpha and beta aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt(-/-) and Hifalpha(-/-) TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin alphavbeta3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O(2)). Culturing B16F0 melanoma cells at 1.5% O(2) resulted in increased alphavbeta3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O(2) tension influence placental invasion and tumor migration by increasing cell surface expression of alphavbeta3 integrin.

Hypoxia-inducible Factor Regulates αvβ3 Integrin Cell Surface Expression

PubMed Central

Cowden Dahl, Karen D.; Robertson, Sarah E.; Weaver, Valerie M.; Simon, M. Celeste

2005-01-01

Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of α and β aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt-/- and Hifα-/- TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin αvβ3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O2). Culturing B16F0 melanoma cells at 1.5% O2 resulted in increased αvβ3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O2 tension influence placental invasion and tumor migration by increasing cell surface expression of αvβ3 integrin. PMID:15689487

Transgenic mice expressing mutant Pinin exhibit muscular dystrophy, nebulin deficiency and elevated expression of slow-type muscle fiber genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Hsu-Pin; Hsu, Shu-Yuan; Wu, Wen-Ai

Highlights: •Pnn CCD domain functions as a dominant negative mutant regulating Pnn expression and function. •Pnn CCD mutant Tg mice have a muscle wasting phenotype during development and show dystrophic histological features. •Pnn mutant muscles are susceptible to slow fiber type gene transition and NEB reduction. •The Tg mouse generated by overexpression of the Pnn CCD domain displays many characteristics resembling NEB{sup +/−} mice. -- Abstract: Pinin (Pnn) is a nuclear speckle-associated SR-like protein. The N-terminal region of the Pnn protein sequence is highly conserved from mammals to insects, but the C-terminal RS domain-containing region is absent in lower species.more » The N-terminal coiled-coil domain (CCD) is, therefore, of interest not only from a functional point of view, but also from an evolutionarily standpoint. To explore the biological role of the Pnn CCD in a physiological context, we generated transgenic mice overexpressing Pnn mutant in skeletal muscle. We found that overexpression of the CCD reduces endogenous Pnn expression in cultured cell lines as well as in transgenic skeletal muscle fibers. Pnn mutant mice exhibited reduced body mass and impaired muscle function during development. Mutant skeletal muscles show dystrophic histological features with muscle fibers heavily loaded with centrally located myonuclei. Expression profiling and pathway analysis identified over-representation of genes in gene categories associated with muscle contraction, specifically those related to slow type fiber. In addition nebulin (NEB) expression level is repressed in Pnn mutant skeletal muscle. We conclude that Pnn downregulation in skeletal muscle causes a muscular dystrophic phenotype associated with NEB deficiency and the CCD domain is incapable of replacing full length Pnn in terms of functional capacity.« less

The expression of proinflammatory genes in epidermal keratinocytes is regulated by hydration status.

PubMed

Xu, Wei; Jia, Shengxian; Xie, Ping; Zhong, Aimei; Galiano, Robert D; Mustoe, Thomas A; Hong, Seok J

2014-04-01

Mucosal wounds heal more rapidly, exhibit less inflammation, and are associated with minimal scarring when compared with equivalent cutaneous wounds. We previously demonstrated that cutaneous epithelium exhibits an exaggerated response to injury compared with mucosal epithelium. We hypothesized that treatment of injured skin with a semiocclusive dressing preserves the hydration of the skin and results in a wound healing phenotype that more closely resembles that of mucosa. Here we explored whether changes in hydration status alter epidermal gene expression patterns in rabbit partial-thickness incisional wounds. Using microarray studies on injured epidermis, we showed that global gene expression patterns in highly occluded versus non-occluded wounds are distinct. Many genes including IL-1β, IL-8, TNF-α (tumor necrosis factor-α), and COX-2 (cyclooxygenase 2) are upregulated in non-occluded wounds compared with highly occluded wounds. In addition, decreased levels of hydration resulted in an increased expression of proinflammatory genes in human ex vivo skin culture (HESC) and stratified keratinocytes. Hierarchical analysis of genes using RNA interference showed that both TNF-α and IL-1β regulate the expression of IL-8 through independent pathways in response to reduced hydration. Furthermore, both gene knockdown and pharmacological inhibition studies showed that COX-2 mediates the TNF-α/IL-8 pathway by increasing the production of prostaglandin E2 (PGE2). IL-8 in turn controls the production of matrix metalloproteinase-9 in keratinocytes. Our data show that hydration status directly affects the expression of inflammatory signaling in the epidermis. The identification of genes involved in the epithelial hydration pathway provides an opportunity to develop strategies to reduce scarring and optimize wound healing.

Inositol 1,4,5-trisphosphate receptor regulates replication, differentiation, infectivity and virulence of the parasitic protist Trypanosoma cruzi.

PubMed

Hashimoto, Muneaki; Enomoto, Masahiro; Morales, Jorge; Kurebayashi, Nagomi; Sakurai, Takashi; Hashimoto, Tetsuo; Nara, Takeshi; Mikoshiba, Katsuhiko

2013-03-01

In animals, inositol 1,4,5-trisphosphate receptors (IP3 Rs) are ion channels that play a pivotal role in many biological processes by mediating Ca(2+) release from the endoplasmic reticulum. Here, we report the identification and characterization of a novel IP3 R in the parasitic protist, Trypanosoma cruzi, the pathogen responsible for Chagas disease. DT40 cells lacking endogenous IP3 R genes expressing T. cruzi IP3 R (TcIP3 R) exhibited IP3 -mediated Ca(2+) release from the ER, and demonstrated receptor binding to IP3 . TcIP3 R was expressed throughout the parasite life cycle but the expression level was much lower in bloodstream trypomastigotes than in intracellular amastigotes or epimastigotes. Disruption of two of the three TcIP3 R gene loci led to the death of the parasite, suggesting that IP3 R is essential for T. cruzi. Parasites expressing reduced or increased levels of TcIP3 R displayed defects in growth, transformation and infectivity, indicating that TcIP3 R is an important regulator of the parasite's life cycle. Furthermore, mice infected with T. cruzi expressing reduced levels of TcIP3 R exhibited a reduction of disease symptoms, indicating that TcIP3 R is an important virulence factor. Combined with the fact that the primary structure of TcIP3 R has low similarity to that of mammalian IP3 Rs, TcIP3 R is a promising drug target for Chagas disease. © 2013 Blackwell Publishing Ltd.

Discovery and validation of a glioblastoma co-expressed gene module

PubMed Central

Dunwoodie, Leland J.; Poehlman, William L.; Ficklin, Stephen P.; Feltus, Frank Alexander

2018-01-01

Tumors exhibit complex patterns of aberrant gene expression. Using a knowledge-independent, noise-reducing gene co-expression network construction software called KINC, we created multiple RNAseq-based gene co-expression networks relevant to brain and glioblastoma biology. In this report, we describe the discovery and validation of a glioblastoma-specific gene module that contains 22 co-expressed genes. The genes are upregulated in glioblastoma relative to normal brain and lower grade glioma samples; they are also hypo-methylated in glioblastoma relative to lower grade glioma tumors. Among the proneural, neural, mesenchymal, and classical glioblastoma subtypes, these genes are most-highly expressed in the mesenchymal subtype. Furthermore, high expression of these genes is associated with decreased survival across each glioblastoma subtype. These genes are of interest to glioblastoma biology and our gene interaction discovery and validation workflow can be used to discover and validate co-expressed gene modules derived from any co-expression network. PMID:29541392

Discovery and validation of a glioblastoma co-expressed gene module.

PubMed

Dunwoodie, Leland J; Poehlman, William L; Ficklin, Stephen P; Feltus, Frank Alexander

2018-02-16

Tumors exhibit complex patterns of aberrant gene expression. Using a knowledge-independent, noise-reducing gene co-expression network construction software called KINC, we created multiple RNAseq-based gene co-expression networks relevant to brain and glioblastoma biology. In this report, we describe the discovery and validation of a glioblastoma-specific gene module that contains 22 co-expressed genes. The genes are upregulated in glioblastoma relative to normal brain and lower grade glioma samples; they are also hypo-methylated in glioblastoma relative to lower grade glioma tumors. Among the proneural, neural, mesenchymal, and classical glioblastoma subtypes, these genes are most-highly expressed in the mesenchymal subtype. Furthermore, high expression of these genes is associated with decreased survival across each glioblastoma subtype. These genes are of interest to glioblastoma biology and our gene interaction discovery and validation workflow can be used to discover and validate co-expressed gene modules derived from any co-expression network.

ASSESSING POSSIBLE ECOLOGICAL RISKS OF GENETICALLY MODIFIED CROPS: GENE EXPRESSION ASSAYS AND GENETIC MONITORING OF NON-TARGET ORGANISMS

EPA Science Inventory

Widespread planting of genetically modified crops with the Bt transgene pesticide has led to concern over non-target effects of Bt compounds in agroecosystems. While some research suggests that non-target organisms exposed to Bt toxin exhibit reduced fecundity and increased morta...

Dietary blueberries sttenuate atherosclerosis in apolipoprotein E-deficient mice by upregulating antioxidant enzymes expression

USDA-ARS?s Scientific Manuscript database

Blueberries (BB) contain high levels of polyphenols and exhibit high antioxidant capacity. In this study, protective effects of BB against atherosclerosis and possible underlying mechanisms in reducing oxidative stress were examined in ApoE deficient (apoE-/-) mice. ApoE-/- mice were fed AIN-93G die...

The influence of SV40 immortalization of human fibroblasts on p53-dependent radiation responses

NASA Technical Reports Server (NTRS)

Kohli, M.; Jorgensen, T. J.

1999-01-01

The simian virus 40 large tumor antigen (SV40 Tag) has been ascribed many functions critical to viral propagation, including binding to the mammalian tumor suppressor p53. Recent studies have demonstrated that SV40-transformed murine cells have functional p53. The status of p53 in SV40-immortalized human cells, however, has not been characterized. We have found that in response to ionizing radiation, p53-dependent p21 transactivation activity is present, albeit reduced, in SV40-immortalized cells and that this activity can be further reduced with either dominant negative p53 expression or higher SV40 Tag expression. Furthermore, overexpression of p53 in SV40-immortalized ataxia-telangiectasia (A-T) cells restores p53-dependent p21 induction to typical A-T levels. All SV40-immortalized cell lines exhibited an absence of G1 arrest. Moreover, all SV40-immortalized cell lines exhibited increased apoptosis relative to primary cells in response to ionizing radiation, suggesting that SV40 immortalization results in a unique phenotype with regard to DNA damage responses. Copyright 1999 Academic Press.

Regulation of the ovarian inflammatory response at ovulation by nuclear progesterone receptor.

PubMed

Akison, Lisa K; Robertson, Sarah A; Gonzalez, Macarena B; Richards, JoAnne S; Smith, C Wayne; Russell, Darryl L; Robker, Rebecca L

2018-06-01

The nuclear progesterone receptor (PGR) transcription factor is essential for ovulation; however, the exact mechanisms by which PGR controls ovulation are not known. The aim of this study was to determine whether PGR regulates inflammatory mediators in the ovary. Ovaries from mice lacking PGR (PRKO) and heterozygous PR+/- littermates were subjected to microarray analysis of a large panel of inflammatory genes. Immune cell subsets were detected by gene expression; and neutrophils by immunohistochemistry and chemotaxis assay. PRKO ovaries exhibited dysregulated expression of vasodilator (Edn1), cytokine (Il-6, Tgfb1), adhesion receptor (Cd34), apoptotic factor (Bax) and transcription factors (Nfkb2, Socs1, Stat3). Ptgs2 was also reduced in PRKO ovaries, but mRNA and protein were not different in granulosa cells. There were reduced neutrophils in ovaries of PRKO mice at ovulation; however, chemotaxis assays showed PRKO neutrophils migrate normally and that PRKO ovarian extracts exhibit chemotactic properties in vitro. Specific inflammatory mediators are altered in the ovaries of PRKO mice indicating that progesterone regulates features of inflammation at ovulation. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Engineering a Saccharomyces cerevisiae wine yeast that exhibits reduced ethanol production during fermentation under controlled microoxygenation conditions.

PubMed

Heux, Stéphanie; Sablayrolles, Jean-Marie; Cachon, Rémy; Dequin, Sylvie

2006-09-01

We recently showed that expressing an H(2)O-NADH oxidase in Saccharomyces cerevisiae drastically reduces the intracellular NADH concentration and substantially alters the distribution of metabolic fluxes in the cell. Although the engineered strain produces a reduced amount of ethanol, a high level of acetaldehyde accumulates early in the process (1 g/liter), impairing growth and fermentation performance. To overcome these undesirable effects, we carried out a comprehensive analysis of the impact of oxygen on the metabolic network of the same NADH oxidase-expressing strain. While reducing the oxygen transfer rate led to a gradual recovery of the growth and fermentation performance, its impact on the ethanol yield was negligible. In contrast, supplying oxygen only during the stationary phase resulted in a 7% reduction in the ethanol yield, but without affecting growth and fermentation. This approach thus represents an effective strategy for producing wine with reduced levels of alcohol. Importantly, our data also point to a significant role for NAD(+) reoxidation in controlling the glycolytic flux, indicating that engineered yeast strains expressing an NADH oxidase can be used as a powerful tool for gaining insight into redox metabolism in yeast.

Protective Effect of Quercetin against Oxidative Stress-Induced Cytotoxicity in Rat Pheochromocytoma (PC-12) Cells.

PubMed

Bao, Dengke; Wang, Jingkai; Pang, Xiaobin; Liu, Hongliang

2017-07-06

Oxidative stress has been implicated in the pathogenesis of many kinds of neurodegenerative disorders, particularly Parkinson's disease. Quercetin is a bioflavonoid found ubiquitously in fruits and vegetables, and has antioxidative activity. However, the underlying mechanism of the antioxidative effect of quercetin in neurodegenerative diseases has not been well explored. Here, we investigated the antioxidative effect and underlying molecular mechanisms of quercetin on PC-12 cells. We found that PC-12 cells pretreated with quercetin exhibited an increased cell viability and reduced lactate dehydrogenase (LDH) release when exposed to hydrogen peroxide (H₂O₂). The significantly-alleviated intracellular reactive oxygen species (ROS), malondialdehyde (MDA), and lipoperoxidation of the cell membrane of PC-12 cells induced by H₂O₂ were observed in the quercetin pretreated group. Furthermore, quercetin pretreatment markedly reduced the apoptosis of PC-12 cells and hippocampal neurons. The inductions of antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in PC-12 cells exposed to H₂O₂ were significantly reduced by preatment with quercetin. In addition, quercetin pretreatment significantly increased Bcl-2 expression, and reduced Bax, cleaved caspase-3 and p53 expressions. In conclusion, this study demonstrated that quercetin exhibited a protective effect against oxidative stress-induced apoptosis in PC-12 cells. Our findings suggested that quercetin may be developed as a novel therapeutic agent for neurodegenerative diseases induced by oxidative stress.

Melanoma Differentiation-associated Gene 7/IL-24 Exerts Cytotoxic Effects by Altering the Alternative Splicing of Bcl-x Pre-mRNA via the SRC/PKCδ Signaling Axis*

PubMed Central

Shapiro, Brian A.; Vu, Ngoc T.; Shultz, Michael D.; Shultz, Jacqueline C.; Mietla, Jennifer A.; Gouda, Mazen M.; Yacoub, Adly; Dent, Paul; Fisher, Paul B.; Park, Margaret A.; Chalfant, Charles E.

2016-01-01

Melanoma differentiation-associated gene 7 (MDA-7/IL-24) exhibits cytotoxic effects on tumor cells while sparing untransformed cells, and Bcl-x(L) is reported to efficiently block the induction of cell death by MDA-7/IL-24. The expression of Bcl-x(L) is regulated at the level of RNA splicing via alternative 5′ splice site selection within exon 2 to produce either the pro-apoptotic Bcl-x(s) or the anti-apoptotic Bcl-x(L). Our laboratory previously reported that Bcl-x RNA splicing is dysregulated in a large percentage of human non-small cell lung cancer (NSCLC) tumors. Therefore, we investigated whether the alternative RNA splicing of Bcl-x pre-mRNA was modulated by MDA-7/IL-24, which would suggest that specific NSCLC tumors are valid targets for this cytokine therapy. Adenovirus-delivered MDA-7/IL-24 (Ad.mda-7) reduced the viability of NSCLC cells of varying oncogenotypes, which was preceded by a decrease in the ratio of Bcl-x(L)/Bcl-x(s) mRNA and Bcl-x(L) protein expression. Importantly, both the expression of Bcl-x(L) and the loss of cell viability were “rescued” in Ad.mda-7-treated cells incubated with Bcl-x(s) siRNA. In addition, NSCLC cells ectopically expressing Bcl-x(s) exhibited significantly reduced Bcl-x(L) expression, which was again restored by Bcl-x(s) siRNA, suggesting the existence of a novel mechanism by which Bcl-x(s) mRNA restrains the expression of Bcl-x(L). In additional mechanistic studies, inhibition of SRC and PKCδ completely ablated the ability of MDA-7/IL-24 to reduce the Bcl-x(L)/(s) mRNA ratio and cell viability. These findings show that Bcl-x(s) expression is an important mediator of MDA-7/IL-24-induced cytotoxicity requiring the SRC/PKCδ signaling axis in NSCLC cells. PMID:27519412

Micro-RNA-126 Reduces the Blood Thrombogenicity in Diabetes Mellitus via Targeting of Tissue Factor.

PubMed

Witkowski, Marco; Weithauser, Alice; Tabaraie, Termeh; Steffens, Daniel; Kränkel, Nicolle; Witkowski, Mario; Stratmann, Bernd; Tschoepe, Diethelm; Landmesser, Ulf; Rauch-Kroehnert, Ursula

2016-06-01

Diabetes mellitus involves vascular inflammatory processes and is a main contributor to cardiovascular mortality. Notably, heightened levels of circulating tissue factor (TF) account for the increased thrombogenicity and put those patients at risk for thromboembolic events. Here, we sought to investigate the role of micro-RNA (miR)-driven TF expression and thrombogenicity in diabetes mellitus. Plasma samples of patients with diabetes mellitus were analyzed for TF protein and activity as well as miR-126 expression before and after optimization of the antidiabetic treatment. We found low miR-126 levels to be associated with markedly increased TF protein and TF-mediated thrombogenicity. Reduced miR-126 expression was accompanied by increased vascular inflammation as evident from the levels of vascular adhesion molecule-1 and fibrinogen, as well as leukocyte counts. With optimization of the antidiabetic treatment miR-126 levels increased and thrombogenicity was reduced. Using a luciferase reporter system, we demonstrated miR-126 to directly bind to the F3-3'-untranslated region, thereby reducing TF expression both on mRNA and on protein levels in human microvascular endothelial cells as well as TF mRNA and activity in monocytes. Circulating miR-126 exhibits antithrombotic properties via regulating post-transcriptional TF expression, thereby impacting the hemostatic balance of the vasculature in diabetes mellitus. © 2016 The Authors.

MiR-24 alleviates cardiomyocyte apoptosis after myocardial infarction via targeting BIM.

PubMed

Pan, L-J; Wang, X; Ling, Y; Gong, H

2017-07-01

Ischemia hypoxia induces cardiomyocyte (CM) apoptosis in the process of acute myocardial infarction (AMI). It was showed that pro-apoptosis factor BIM participates in regulating tumor cell apoptosis under ischemia or hypoxia condition, while its role in CM apoptosis after AMI is still unclear. It was revealed that miR-24 expression was significantly reduced in myocardial tissue after AMI. Bioinformatics analysis exhibits that miR-24 is targeted to the 3'-UTR of BIM. This study aims to investigate the role of miR-24 in mediating BIM expression and CM apoptosis. Dual-luciferase assay was used to confirm the targeted regulation between miR-24 and BIM. Cells were cultured under ischemia hypoxia for 12 h after transfection for 48 h. Cell apoptosis was tested by using flow cytometry. The caspase activity was detected by using spectrophotometry. Wistar rats were divided into four groups, including Sham, AMI, AMI + agomir-control, and AMI + agomir-24 groups. Cardiac function was evaluated by using echocardiography. CM apoptosis was determined by using TUNEL. Infarction area was measured by using evans blue staining. MiR-24 targeted suppressed BIM expression. MiR-24 mimic and/or si-BIM transfection significantly declined the BIM expression, inhibited caspase-9 and caspase-3 activities, and reduced cell apoptosis in H9C2 cells. MiR-24 expression was decreased, while BIM levels were up-regulated in myocardium after AMI. Agomir-24 injection down-regulated the BIM expression in myocardium, reduced CM apoptosis, narrowed infarction area, and improved cardiac function in rats. MiR-24 was reduced, whereas BIM was enhanced in the CM after AMI. MiR-24 up-regulation plays a critical role in decreasing BIM expression, reducing CM apoptosis, and improving cardiac function after AMI.

MicroRNA-153 Physiologically Inhibits Expression of Amyloid-β Precursor Protein in Cultured Human Fetal Brain Cells and Is Dysregulated in a Subset of Alzheimer Disease Patients*

PubMed Central

Long, Justin M.; Ray, Balmiki; Lahiri, Debomoy K.

2012-01-01

Regulation of amyloid-β (Aβ) precursor protein (APP) expression is complex. MicroRNAs (miRNAs) are expected to participate in the molecular network that controls this process. The composition of this network is, however, still undefined. Elucidating the complement of miRNAs that regulate APP expression should reveal novel drug targets capable of modulating Aβ production in AD. Here, we investigated the contribution of miR-153 to this regulatory network. A miR-153 target site within the APP 3′-untranslated region (3′-UTR) was predicted by several bioinformatic algorithms. We found that miR-153 significantly reduced reporter expression when co-transfected with an APP 3′-UTR reporter construct. Mutation of the predicted miR-153 target site eliminated this reporter response. miR-153 delivery in both HeLa cells and primary human fetal brain cultures significantly reduced APP expression. Delivery of a miR-153 antisense inhibitor to human fetal brain cultures significantly elevated APP expression. miR-153 delivery also reduced expression of the APP paralog APLP2. High functional redundancy between APP and APLP2 suggests that miR-153 may target biological pathways in which they both function. Interestingly, in a subset of human AD brain specimens with moderate AD pathology, miR-153 levels were reduced. This same subset also exhibited elevated APP levels relative to control specimens. Therefore, endogenous miR-153 inhibits expression of APP in human neurons by specifically interacting with the APP 3′-UTR. This regulatory interaction may have relevance to AD etiology, where low miR-153 levels may drive increased APP expression in a subset of AD patients. PMID:22733824

Adiponectin is required for maintaining normal body temperature in a cold environment.

PubMed

Wei, Qiong; Lee, Jong Han; Wang, Hongying; Bongmba, Odelia Y N; Wu, Chia-Shan; Pradhan, Geetali; Sun, Zilin; Chew, Lindsey; Bajaj, Mandeep; Chan, Lawrence; Chapkin, Robert S; Chen, Miao-Hsueh; Sun, Yuxiang

2017-10-23

Thermogenic impairment promotes obesity and insulin resistance. Adiponectin is an important regulator of energy homeostasis. While many beneficial metabolic effects of adiponectin resemble that of activated thermogenesis, the role of adiponectin in thermogenesis is not clear. In this study, we investigated the role of adiponectin in thermogenesis using adiponectin-null mice (Adipoq -/- ). Body composition was measured using EchoMRI. Metabolic parameters were determined by indirect calorimetry. Insulin sensitivity was evaluated by glucose- and insulin- tolerance tests. Core body temperature was measured by a TH-8 temperature monitoring system. Gene expression was assessed by real-time PCR and protein levels were analyzed by Western blotting and immunohistochemistry. The mitochondrial density of brown adipose tissue was quantified by calculating the ratio of mtDNA:total nuclear DNA. Under normal housing temperature of 24 °C and ad libitum feeding condition, the body weight, body composition, and metabolic profile of Adipoq -/- mice were unchanged. Under fasting condition, Adipoq -/- mice exhibited reduced energy expenditure. Conversely, under cold exposure, Adipoq -/- mice exhibited reduced body temperature, and the expression of thermogenic regulatory genes was significantly reduced in brown adipose tissue (BAT) and subcutaneous white adipose tissue (WAT). Moreover, we observed that mitochondrial content was reduced in BAT and subcutaneous WAT, and the expression of mitochondrial fusion genes was decreased in BAT of Adipoq -/- mice, suggesting that adiponectin ablation diminishes mitochondrial biogenesis and altered mitochondrial dynamics. Our study further revealed that adiponectin deletion suppresses adrenergic activation, and down-regulates β3-adrenergic receptor, insulin signaling, and the AMPK-SIRT1 pathway in BAT. Our findings demonstrate that adiponectin is an essential regulator of thermogenesis, and adiponectin is required for maintaining body temperature under cold exposure.

Quantitative Expression Analysis of SpA, FnbA and Rsp Genes in Staphylococcus aureus: Actively Associated in the Formation of Biofilms.

PubMed

Yeswanth, Sthanikam; Chaudhury, Abhijit; Sarma, Potukuchi Venkata Gurunadha Krishna

2017-12-01

In Staphylococcus aureus, adherence and secretory proteins play chief role in the formation of biofilms. This mode of growth exhibits resistance to a variety of antibiotics and spreads its infections. In the present study, secretary and adherence proteins, Protein-A, Fibronectin-binding protein-A (FnbA) and Rsp (a transcription regulator encoding proteolytic property) expression levels were evaluated at different stages of growth in S. aureus ATCC12600 a drug-sensitive strain and multidrug-resistant strains of S. aureus. Initially, the SpA, FnbA and Rsp genes of S. aureus ATCC12600 were cloned, sequenced, expressed and characterized. The proteolytic property of recombinant Rsp was conspicuously shown when this pathogen was grown in aerobic conditions correlating with reduced biofilm units. In anaerobic mode of growth, S. aureus exhibited a higher expression of SpA and FnbA in early and mid adherence phases and finally stabilized at 48 h of incubation. This expression was more pronounced in methicillin-resistant strains (LMV1-8 and D1-4) of S. aureus. In all these stages, Rsp gene expression was at the lowest level and these results concur with the increased biofilm units. The results of the present study explain proteins chiefly contribute in the formation of biofilms.

Expression of a soluble truncated Vargula luciferase in Escherichia coli

PubMed Central

Hunt, Eric A.; Moutsiopoulou, Angeliki; Broyles, David; Head, Trajen; Dikici, Emre; Daunert, Sylvia; Deo, Sapna K.

2017-01-01

Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties. PMID:28108349

EPO-cyclosporine combination therapy reduced brain infarct area in rat after acute ischemic stroke: role of innate immune-inflammatory response, micro-RNAs and MAPK family signaling pathway.

PubMed

Yuen, Chun-Man; Yeh, Kuo-Ho; Wallace, Christopher Glenn; Chen, Kuan-Hung; Lin, Hung-Sheng; Sung, Pei-Hsun; Chai, Han-Tan; Chen, Yung-Lung; Sun, Cheuk-Kwan; Chen, Chih-Hung; Kao, Gour-Shenq; Ko, Sheung-Fat; Yip, Hon-Kan

2017-01-01

This study tested the hypothesis that erythropoietin (EPO) and cyclosporine (CsA) could effectively reduce brain infarct area (BIA) in rat after acute ischemic stroke (AIS) through regulating inflammation, oxidative stress, MAPK family signaling and microRNA (miR-223/miR-30a/miR-383). Adult male Sprague-Dawley rats (n = 48) were equally divided into group 1 (sham control), group 2 (AIS), group 3 [AIS+EPO (5,000 IU/kg at 0.5/24/48 h, subcutaneous)] and group 4 [AIS+CsA (20.0 mg/kg at 0.5/24/48 h, intra-peritoneal)]. By 72 h, histopathology showed that BIA was largest in group 2 and smallest in group 1, and significantly larger in group 4 than group 3 (all P<0.0001). The three microRNAs expressed were higher in group 2 than in the other three groups (all P<0.04); between these three latter groups there were no significant differences. The protein expressions of MAPK family [phosphorylated (p)-ERK1/2, p-p38/p-JNK], inflammatory (iNOS/MMP-9/TNF-α/NF-κB/IL-12/MIP-1α/CD14/CD68/Ly6g), apoptotic (caspase-3/PARP/mitochondrial-Bax), oxidative-stress (NOX-1/NOX-2/oxidized protein) and mitochondrial-damaged (cytosolic cytochrome-C) biomarkers exhibited an identical pattern to BIA findings (all P<0.0001). The cellular expressions of brain edema (AQP4+), inflammation (CD11+/glial-fibrillary-acid protein+), and cellular damage (TUNEL assay/positive Periodic acid-Schiff stain) biomarkers exhibited an identical pattern, whereas the cellular-integrity markers (neuN+/MAP2+/doublecorin+) exhibited an opposite pattern to BIA (all P value <0.001). EPO-CsA therapy markedly reduced BIA mainly by suppressing the innate immune response to inflammation, oxidative stress, microRNAs (miR-223/miR-30a/miR-383) and MAPK family signaling.

Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth

NASA Technical Reports Server (NTRS)

Rashotte, A. M.; DeLong, A.; Muday, G. K.; Brown, C. S. (Principal Investigator)

2001-01-01

Auxin transport is required for important growth and developmental processes in plants, including gravity response and lateral root growth. Several lines of evidence suggest that reversible protein phosphorylation regulates auxin transport. Arabidopsis rcn1 mutant seedlings exhibit reduced protein phosphatase 2A activity and defects in differential cell elongation. Here we report that reduced phosphatase activity alters auxin transport and dependent physiological processes in the seedling root. Root basipetal transport was increased in rcn1 or phosphatase inhibitor-treated seedlings but showed normal sensitivity to the auxin transport inhibitor naphthylphthalamic acid (NPA). Phosphatase inhibition reduced root gravity response and delayed the establishment of differential auxin-induced gene expression across a gravity-stimulated root tip. An NPA treatment that reduced basipetal transport in rcn1 and cantharidin-treated wild-type plants also restored a normal gravity response and asymmetric auxin-induced gene expression, indicating that increased basipetal auxin transport impedes gravitropism. Increased auxin transport in rcn1 or phosphatase inhibitor-treated seedlings did not require the AGR1/EIR1/PIN2/WAV6 or AUX1 gene products. In contrast to basipetal transport, root acropetal transport was normal in phosphatase-inhibited seedlings in the absence of NPA, although it showed reduced NPA sensitivity. Lateral root growth also exhibited reduced NPA sensitivity in rcn1 seedlings, consistent with acropetal transport controlling lateral root growth. These results support the role of protein phosphorylation in regulating auxin transport and suggest that the acropetal and basipetal auxin transport streams are differentially regulated.

Identification of polycomb and trithorax group responsive elements in the regulatory region of the Drosophila homeotic gene Sex combs reduced

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gindhart, J.G. Jr.; Kaufman, T.C.

1995-02-01

The Drosophilia homeotic gene Sex combs reduced (Scr) is necessary for the establishment and maintenance of the morphological identity of the labial and prothoracic segments. In the early embryo, its expression pattern is established through the activity of several gap and segmentation gene products, as well as other transcription factors. Once established, the Polycomb group (Pc-G) and trithorax group (trx-G) gene products maintain the spatial pattern of Scr expression for the remainder of development. We report the identification of DNA fragments in the Scr regulatory region that may be important for its regulation by Polycomb and trithorax group gene products.more » When DNA fragments containing these regulatory sequences are subcloned into P-element vectors containing a white minigene, transformants containing these constructs exhibit mosaic patterns of pigmentation in the adult eye, indicating that white minigene expression is repressed in a clonally heritable manner. The size of pigmented and nonpigmented clones in the adult eye suggests that the event determining whether a cell in the eye anlagen will express white occurs at least as early as the first larval instar. The amount of white minigene repression is reduced in some Polycomb group mutants, whereas repression is enhanced in flies mutant for a subset of trithorax group loci. The repressor activity of one fragment, normally located in Scr Intron 2, is increased when it is able to homologously pair, a property consistent with genetic data suggesting that Scr exhibits transvection. Another Scr regulatory fragment, normally located 40 kb upstream of the Scr promoter, silences ectopic expression of an Scr-lacZ fusion gene in the embryo and does so in a Polycomb-dependent manner. We propose that the regulatory sequences located within these DNA fragments may normally mediate the regulation of Scr by proteins encoded by members of Polycomb and trithorax group loci. 98 refs., 6 figs., 4 tabs.« less

Unexpected Long-Term Protection of Adult Offspring Born to High-Fat Fed Dams against Obesity Induced by a Sucrose-Rich Diet

PubMed Central

Couvreur, Odile; Ferezou, Jacqueline; Gripois, Daniel; Serougne, Colette; Crépin, Delphine; Aubourg, Alain; Gertler, Arieh; Vacher, Claire-Marie; Taouis, Mohammed

2011-01-01

Background Metabolic and endocrine environment during early life is crucial for metabolic imprinting. When dams were fed a high fat diet (HF diet), rat offspring developed hypothalamic leptin resistance with lean phenotype when weaned on a normal diet. Interestingly, when grown on the HF diet, they appeared to be protected against the effects of HF diet as compared to offspring of normally fed dams. The mechanisms involved in the protective effect of maternal HF diet are unclear. Methodology/Principal Findings We thus investigated the impact of maternal high fat diet on offspring subjected to normal or high palatable diet (P diet) on metabolic and endocrine parameters. We compared offspring born to dams fed P or HF diet. Offspring born to dams fed control or P diet, when fed P diet exhibited a higher body weight, altered hypothalamic leptin sensitivity and metabolic parameters suggesting that maternal P diet has no protective effect on offspring. Whereas, maternal HF diet reduces body weight gain and circulating triglycerides, and ameliorates corpulence index of offspring, even when subjected to P diet. Interestingly, this protective effect is differently expressed in male and female offspring. Male offspring exhibited higher energy expenditure as mirrored by increased hypothalamic UCP-2 and liver AdipoR1/R2 expression, and a profound change in the arcuate nucleus astrocytic organization. In female offspring, the most striking impact of maternal HF diet is the reduced hypothalamic expression of NPY and POMC. Conclusions/Significance HF diet given during gestation and lactation protects, at least partially, offspring from excessive weight gain through several mechanisms depending upon gender including changes in arcuate nucleus astrocytic organization and increased hypothalamic UCP-2 and liver AdipoR1/2 expression in males and reduced hypothalamic expression of NPY and POMC in females. Taken together our results reveal new mechanisms involved in the protective effect of maternal HF diet. PMID:21464991

2-Deoxy-D-glucose treatment induces ketogenesis, sustains mitochondrial function, and reduces pathology in female mouse model of Alzheimer's disease.

PubMed

Yao, Jia; Chen, Shuhua; Mao, Zisu; Cadenas, Enrique; Brinton, Roberta Diaz

2011-01-01

Previously, we demonstrated that mitochondrial bioenergetic deficits preceded Alzheimer's disease (AD) pathology in the female triple-transgenic AD (3xTgAD) mouse model. In parallel, 3xTgAD mice exhibited elevated expression of ketogenic markers, indicating a compensatory mechanism for energy production in brain. This compensatory response to generate an alternative fuel source was temporary and diminished with disease progression. To determine whether this compensatory alternative fuel system could be sustained, we investigated the impact of 2-deoxy-D-glucose (2-DG), a compound known to induce ketogenesis, on bioenergetic function and AD pathology burden in brain. 6-month-old female 3xTgAD mice were fed either a regular diet (AIN-93G) or a diet containing 0.04% 2-DG for 7 weeks. 2-DG diet significantly increased serum ketone body level and brain expression of enzymes required for ketone body metabolism. The 2-DG-induced maintenance of mitochondrial bioenergetics was paralleled by simultaneous reduction in oxidative stress. Further, 2-DG treated mice exhibited a significant reduction of both amyloid precursor protein (APP) and amyloid beta (Aβ) oligomers, which was paralleled by significantly increased α-secretase and decreased γ-secretase expression, indicating that 2-DG induced a shift towards a non-amyloidogenic pathway. In addition, 2-DG increased expression of genes involved in Aβ clearance pathways, degradation, sequestering, and transport. Concomitant with increased bioenergetic capacity and reduced β-amyloid burden, 2-DG significantly increased expression of neurotrophic growth factors, BDNF and NGF. Results of these analyses demonstrate that dietary 2-DG treatment increased ketogenesis and ketone metabolism, enhanced mitochondrial bioenergetic capacity, reduced β-amyloid generation and increased mechanisms of β-amyloid clearance. Further, these data link bioenergetic capacity with β-amyloid generation and demonstrate that β-amyloid burden was dynamic and reversible, as 2-DG reduced activation of the amyloidogenic pathway and increased mechanisms of β-amyloid clearance. Collectively, these data provide preclinical evidence for dietary 2-DG as a disease-modifying intervention to delay progression of bioenergetic deficits in brain and associated β-amyloid burden.

Diet-induced obesity mediated by the JNK/DIO2 signal transduction pathway

PubMed Central

Vernia, Santiago; Cavanagh-Kyros, Julie; Barrett, Tamera; Jung, Dae Young; Kim, Jason K.; Davis, Roger J.

2013-01-01

The cJun N-terminal kinase (JNK) signaling pathway is a key mediator of metabolic stress responses caused by consuming a high-fat diet, including the development of obesity. To test the role of JNK, we examined diet-induced obesity in mice with targeted ablation of Jnk genes in the anterior pituitary gland. These mice exhibited an increase in the pituitary expression of thyroid-stimulating hormone (TSH), an increase in the blood concentration of thyroid hormone (T4), increased energy expenditure, and markedly reduced obesity compared with control mice. The increased amount of pituitary TSH was caused by reduced expression of type 2 iodothyronine deiodinase (Dio2), a gene that is required for T4-mediated negative feedback regulation of TSH expression. These data establish a molecular mechanism that accounts for the regulation of energy expenditure and the development of obesity by the JNK signaling pathway. PMID:24186979

Defective calcium inactivation causes long QT in obese insulin-resistant rat.

PubMed

Lin, Yen-Chang; Huang, Jianying; Kan, Hong; Castranova, Vincent; Frisbee, Jefferson C; Yu, Han-Gang

2012-02-15

The majority of diabetic patients who are overweight or obese die of heart disease. We suspect that the obesity-induced insulin resistance may lead to abnormal cardiac electrophysiology. We tested this hypothesis by studying an obese insulin-resistant rat model, the obese Zucker rat (OZR). Compared with the age-matched control, lean Zucker rat (LZR), OZR of 16-17 wk old exhibited an increase in QTc interval, action potential duration, and cell capacitance. Furthermore, the L-type calcium current (I(CaL)) in OZR exhibited defective inactivation and lost the complete inactivation back to the closed state, leading to increased Ca(2+) influx. The current density of I(CaL) was reduced in OZR, whereas the threshold activation and the current-voltage relationship of I(CaL) were not significantly altered. L-type Ba(2+) current (I(BaL)) in OZR also exhibited defective inactivation, and steady-state inactivation was not significantly altered. However, the current-voltage relationship and activation threshold of I(BaL) in OZR exhibited a depolarized shift compared with LZR. The total and membrane protein expression levels of Cav1.2 [pore-forming subunit of L-type calcium channels (LTCC)], but not the insulin receptors, were decreased in OZR. The insulin receptor was found to be associated with the Cav1.2, which was weakened in OZR. The total protein expression of calmodulin was reduced, but that of Cavβ2 subunit was not altered in OZR. Together, these results suggested that the 16- to 17-wk-old OZR has 1) developed cardiac hypertrophy, 2) exhibited altered electrophysiology manifested by the prolonged QTc interval, 3) increased duration of action potential in isolated ventricular myocytes, 4) defective inactivation of I(CaL) and I(BaL), 5) weakened the association of LTCC with the insulin receptor, and 6) decreased protein expression of Cav1.2 and calmodulin. These results also provided mechanistic insights into a remodeled cardiac electrophysiology under the condition of insulin resistance, enhancing our understanding of long QT associated with obese type 2 diabetic patients.

Characterization of Transcription Factor Gene OsDRAP1 Conferring Drought Tolerance in Rice

PubMed Central

Huang, Liyu; Wang, Yinxiao; Wang, Wensheng; Zhao, Xiuqin; Qin, Qiao; Sun, Fan; Hu, Fengyi; Zhao, Yan; Li, Zichao; Fu, Binying; Li, Zhikang

2018-01-01

HIGHLIGHTS Overexpressing and RNA interfering OsDRAP1 transgenic rice plants exhibited significantly improved and reduced drought tolerance, but accompanied with negative effects on development and yield. The dehydration responsive element binding (DREBs) genes are important transcription factors which play a crucial role in plant abiotic stress tolerances. In this study, we functionally characterized a DREB2-like gene, OsDRAP1 conferring drought tolerance (DT) in rice. OsDRAP1, containing many cis-elements in its promoter region, was expressed in all organs (mainly expressed in vascular tissues) of rice, and induced by a variety of environmental stresses and plant hormones. Overexpressing OsDRAP1 transgenic plants exhibited significantly improved DT; while OsDRAP1 RNA interfering plants exhibited significantly reduced DT which also accompanied with significant negative effects on development and yield. Overexpression of OsDRAP1 has a positive impact on maintaining water balance, redox homeostasis and vascular development in transgenic rice plants under drought stress. OsDRAP1 interacted with many genes/proteins and could activate many downstream DT related genes, including important transcription factors such as OsCBSX3 to response drought stress, indicating the OsDRAP1-mediated pathways for DT involve complex genes networks. All these results provide a basis for further complete understanding of the OsDRAP1 mediated gene networks and their related phenotypic effects. PMID:29449862

Thyroid hormone regulation of Sirtuin 1 expression and implications to integrated responses in fasted mice.

PubMed

Cordeiro, Aline; de Souza, Luana Lopes; Oliveira, Lorraine Soares; Faustino, Larissa Costa; Santiago, Letícia Aragão; Bloise, Flavia Fonseca; Ortiga-Carvalho, Tania Maria; Almeida, Norma Aparecida Dos Santos; Pazos-Moura, Carmen Cabanelas

2013-02-01

Sirtuin 1 (SIRT1), a NAD(+)-dependent deacetylase, has been connected to beneficial effects elicited by calorie restriction. Physiological adaptation to starvation requires higher activity of SIRT1 and also the suppression of thyroid hormone (TH) action to achieve energy conservation. Here, we tested the hypothesis that those two events are correlated and that TH may be a regulator of SIRT1 expression. Forty-eight-hour fasting mice exhibited reduced serum TH and increased SIRT1 protein content in liver and brown adipose tissue (BAT), and physiological thyroxine replacement prevented or attenuated the increment of SIRT1 in liver and BAT of fasted mice. Hypothyroid mice exhibited increased liver SIRT1 protein, while hyperthyroid ones showed decreased SIRT1 in liver and BAT. In the liver, decreased protein is accompanied by reduced SIRT1 activity and no alteration in its mRNA. Hyperthyroid and hypothyroid mice exhibited increases and decreases in food intake and body weight gain respectively. Food-restricted hyperthyroid animals (pair-fed to euthyroid group) exhibited liver and BAT SIRT1 protein levels intermediary between euthyroid and hyperthyroid mice fed ad libitum. Mice with TH resistance at the liver presented increased hepatic SIRT1 protein and activity, with no alteration in Sirt1 mRNA. These results suggest that TH decreases SIRT1 protein, directly and indirectly, via food ingestion control and, in the liver, this reduction involves TRβ. The SIRT1 reduction induced by TH has important implication to integrated metabolic responses to fasting, as the increase in SIRT1 protein requires the fasting-associated suppression of TH serum levels.

Expression of p27Kip1 and E-cadherin in Head and Neck Squamous Cell Carcinoma of Indonesian Patients.

PubMed

E I, Auerkari; V, Joewono; D R, Handjari; A T, Sarwono; A W, Suhartono; K, Eto; M A, Ikeda

2014-01-01

Cancer cells exhibit characteristic damage of DNA and its expression. The expression of the tumor suppressors E-cadherin and p27(Kip1) has been tested on 57 head and neck squamous cell carcinomas (HNSCC) of Indonesian subjects. HNSCC tumor samples including both primary and (unrelated) nodal cases were obtained from the archives of Indonesian hospitals, in accordance with acknowledged ethical requirements. Only modest correlation was found between reduced expression of E-cadherin or p27(Kip1) with increased malignancy of primary and nodal growth. The observed strong correlation regardless of malignancy between the expressed levels of E-cadherin and p27(Kip1) suggests that also in combination these would not help to better predict the outcome of HNSCC.

Down-regulation of hypothalamic pro-opiomelanocortin (POMC) expression after weaning is associated with hyperphagia-induced obesity in JCR rats overexpressing neuropeptide Y.

PubMed

Diané, Abdoulaye; Pierce, W David; Russell, James C; Heth, C Donald; Vine, Donna F; Richard, Denis; Proctor, Spencer D

2014-03-14

We hypothesised that hypothalamic feeding-related neuropeptides are differentially expressed in obese-prone and lean-prone rats and trigger overeating-induced obesity. To test this hypothesis, in the present study, we measured energy balance and hypothalamic neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) mRNA expressions in male JCR:LA-cp rats. We compared, in independent cohorts, free-feeding obese-prone (Obese-FF) and lean-prone (Lean-FF) rats at pre-weaning (10 d old), weaning (21-25 d old) and early adulthood (8-12 weeks). A group of Obese-pair-feeding (PF) rats pair-fed to the Lean-FF rats was included in the adult cohort. The body weights of 10-d-old Obese-FF and Lean-FF pups were not significantly different. However, when the pups were shifted from dams' milk to solid food (weaning), the obese-prone rats exhibited more energy intake over the days than the lean-prone rats and higher body and fat pad weights and fasting plasma glucose, leptin, insulin and lipid levels. These differences were consistent with higher energy consumption and lower energy expenditure. In the young adult cohort, the differences between the Obese-FF and Lean-FF rats became more pronounced, yielding significant age effects on most of the parameters of the metabolic syndrome, which were reduced in the Obese-PF rats. The obese-prone rats displayed higher NPY expression than the lean-prone rats at pre-weaning and weaning, and the expression levels did not differ by age. In contrast, POMC expression exhibited significant age-by-genotype differences. At pre-weaning, there was no genotype difference in POMC expression, but in the weanling cohort, obese-prone pups exhibited lower POMC expression than the lean-prone rats. This genotype difference became more pronounced at adulthood. Overall, the development of hyperphagia-induced obesity in obese-prone JCR rats is related to POMC expression down-regulation in the presence of established NPY overexpression.

Multiple regulatory mechanisms of hepatocyte growth factor expression in malignant cells with a short poly(dA) sequence in the HGF gene promoter.

PubMed

Sakai, Kazuko; Takeda, Masayuki; Okamoto, Isamu; Nakagawa, Kazuhiko; Nishio, Kazuto

2015-01-01

Hepatocyte growth factor (HGF) expression is a poor prognostic factor in various types of cancer. Expression levels of HGF have been reported to be regulated by shorter poly(dA) sequences in the promoter region. In the present study, the poly(dA) mononucleotide tract in various types of human cancer cell lines was examined and compared with the HGF expression levels in those cells. Short deoxyadenosine repeat sequences were detected in five of the 55 cell lines used in the present study. The H69, IM95, CCK-81, Sui73 and H28 cells exhibited a truncated poly(dA) sequence in which the number of poly(dA) repeats was reduced by ≥5 bp. Two of the cell lines exhibited high HGF expression, determined by reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The CCK-81, Sui73 and H28 cells with shorter poly(dA) sequences exhibited low HGF expression. The cause of the suppression of HGF expression in the CCK-81, Sui73 and H28 cells was clarified by two approaches, suppression by methylation and single nucleotide polymorphisms in the HGF gene. Exposure to 5-Aza-dC, an inhibitor of DNA methyltransferase 1, induced an increased expression of HGF in the CCK-81 cells, but not in the other cells. Single-nucleotide polymorphism (SNP) rs72525097 in intron 1 was detected in the Sui73 and H28 cells. Taken together, it was found that the defect of poly(dA) in the HGF promoter was present in various types of cancer, including lung, stomach, colorectal, pancreas and mesothelioma. The present study proposes the negative regulation mechanisms by methylation and SNP in intron 1 of HGF for HGF expression in cancer cells with short poly(dA).

Simvastatin and Dipentyl Phthalate Display Different Mechanisms of Action but Exhibit Dose Additive Effects on Fetal Testicular Testosterone Production in Sprague Dawley Rats

EPA Science Inventory

Sex differentiation of the male reproductive tract in mammals is driven, in part, by fetal androgen production. In utero exposure to some phthalate esters (PEs) alters fetal Leydig cell differentiation, reducing the expression of several genes associated with steroid synthesis/tr...

Reduced expression of the Ca(2+) transporter protein PMCA2 slows Ca(2+) dynamics in mouse cerebellar Purkinje neurones and alters the precision of motor coordination.

PubMed

Empson, Ruth M; Turner, Paul R; Nagaraja, Raghavendra Y; Beesley, Philip W; Knöpfel, Thomas

2010-03-15

Cerebellar Purkinje neurones (PNs) express high levels of the plasma membrane calcium ATPase, PMCA2, a transporter protein critical for the clearance of calcium from excitable cells. Genetic deletion of one PMCA2 encoding gene in heterozygous PMCA2 knock-out (PMCA2(+/-) mice enabled us to determine how PMCA2 influences PN calcium regulation without the complication of the severe morphological changes associated with complete PMCA2 knock-out (PMCA2(-/-) in these cells. The PMCA2(+/-) cerebellum expressed half the normal levels of PMCA2 and this nearly doubled the time taken for PN dendritic calcium transients to recover (mean fast and slow recovery times increased from 70 ms to 110 ms and from 600 ms to 1100 ms). The slower calcium recovery had distinct consequences for PMCA2(+/-) PN physiology. The PNs exhibited weaker climbing fibre responses, prolonged outward Ca(2+)-dependent K(+) current (mean fast and slow recovery times increased from 136 ms to 192 ms and from 595 ms to 1423 ms) and a slower mean frequency of action potential firing (7.4 Hz compared with 15.8 Hz). Our findings were consistent with prolonged calcium accumulation in the cytosol of PMCA2(+/-) Purkinje neurones. Although PMCA2(+/-) mice exhibited outwardly normal behaviour and little change in their gait pattern, when challenged to run on a narrow beam they exhibited clear deficits in hindlimb coordination. Training improved the motor performance of both PMCA2(+/-) and wild-type mice, although PMCA2(+/-) mice were always impaired. We conclude that reduced calcium clearance perturbs calcium dynamics in PN dendrites and that this is sufficient to disrupt the accuracy of cerebellar processing and motor coordination.

KLF4-dependent epigenetic remodeling modulates podocyte phenotypes and attenuates proteinuria

PubMed Central

Hayashi, Kaori; Sasamura, Hiroyuki; Nakamura, Mari; Azegami, Tatsuhiko; Oguchi, Hideyo; Sakamaki, Yusuke; Itoh, Hiroshi

2014-01-01

The transcription factor Kruppel-like factor 4 (KLF4) has the ability, along with other factors, to reprogram somatic cells into induced pluripotent stem (iPS) cells. Here, we determined that KLF4 is expressed in kidney glomerular podocytes and is decreased in both animal models and humans exhibiting a proteinuric. Transient restoration of KLF4 expression in podocytes of diseased glomeruli in vivo, either by gene transfer or transgenic expression, resulted in a sustained increase in nephrin expression and a decrease in albuminuria. In mice harboring podocyte-specific deletion of Klf4, adriamycin-induced proteinuria was substantially exacerbated, although these animals displayed minimal phenotypical changes prior to adriamycin administration. KLF4 overexpression in cultured human podocytes increased expression of nephrin and other epithelial markers and reduced mesenchymal gene expression. DNA methylation profiling and bisulfite genomic sequencing revealed that KLF4 expression reduced methylation at the nephrin promoter and the promoters of other epithelial markers; however, methylation was increased at the promoters of genes encoding mesenchymal markers, suggesting selective epigenetic regulation of podocyte gene expression. Together, these results suggest that KLF4 epigenetically modulates podocyte phenotype and function and that the podocyte epigenome can be targeted for direct intervention and reduction of proteinuria. PMID:24812666

Psoriatic T cells reduce epidermal turnover time and affect cell proliferation contributed from differential gene expression.

PubMed

Li, Junqin; Li, Xinhua; Hou, Ruixia; Liu, Ruifeng; Zhao, Xincheng; Dong, Feng; Wang, Chunfang; Yin, Guohua; Zhang, Kaiming

2015-09-01

Psoriasis is mediated primarily by T cells, which reduce epidermal turnover time and affect keratinocyte proliferation. We aimed to identify differentially expressed genes (DEG) in T cells from normal, five pairs of monozygotic twins concordant or discordant for psoriasis, to determine whether these DEG may account for the influence to epidermal turnover time and keratinocyte proliferation. The impact of T cells on keratinocyte proliferation and epidermal turnover time were investigated separately by immunohistochemistry and cultured with (3) H-TdR. mRNA expression patterns were investigated by RNA sequencing and verified by real-time reverse transcription polymerase chain reaction. After co-culture with psoriatic T cells, the expression of Ki-67, c-Myc and p53 increased, while expression of Bcl-2 and epidermal turnover time decreased. There were 14 DEG which were found to participate in the regulation of cell proliferation or differentiation. Psoriatic T cells exhibited the ability to decrease epidermal turnover time and affect keratinocyte proliferation because of the differential expression of PPIL1, HSPH1, SENP3, NUP54, FABP5, PLEKHG3, SLC9A9 and CHCHD4. © 2015 Japanese Dermatological Association.

The COP9 signalosome controls jasmonic acid synthesis and plant responses to herbivory and pathogens.

PubMed

Hind, Sarah R; Pulliam, Sarah E; Veronese, Paola; Shantharaj, Deepak; Nazir, Azka; Jacobs, Nekaiya S; Stratmann, Johannes W

2011-02-01

The COP9 signalosome (CSN) is a multi-protein complex that regulates the activities of cullin-RING E3 ubiquitin ligases (CRLs). CRLs ubiquitinate proteins in order to target them for proteasomal degradation. The CSN is required for proper plant development. Here we show that the CSN also has a profound effect on plant defense responses. Silencing of genes for CSN subunits in tomato plants resulted in a mild morphological phenotype and reduced expression of wound-responsive genes in response to mechanical wounding, attack by Manduca sexta larvae, and Prosystemin over-expression. In contrast, expression of pathogenesis-related genes was increased in a stimulus-independent manner in these plants. The reduced wound response in CSN-silenced plants corresponded with reduced synthesis of jasmonic acid (JA), but levels of salicylic acid (SA) were unaltered. As a consequence, these plants exhibited reduced resistance against herbivorous M. sexta larvae and the necrotrophic fungal pathogen Botrytis cinerea. In contrast, susceptibility to tobacco mosaic virus (TMV) was not altered in CSN-silenced plants. These data demonstrate that the CSN orchestrates not only plant development but also JA-dependent plant defense responses. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Consequences of reductive evolution for gene expression in an obligate endosymbiont.

PubMed

Wilcox, Jennifer L; Dunbar, Helen E; Wolfinger, Russell D; Moran, Nancy A

2003-06-01

The smallest cellular genomes are found in obligate symbiotic and pathogenic bacteria living within eukaryotic hosts. In comparison with large genomes of free-living relatives, these reduced genomes are rearranged and have lost most regulatory elements. To test whether reduced bacterial genomes incur reduced regulatory capacities, we used full-genome microarrays to evaluate transcriptional response to environmental stress in Buchnera aphidicola, the obligate endosymbiont of aphids. The 580 genes of the B. aphidicola genome represent a subset of the 4500 genes known from the related organism, Escherichia coli. Although over 20 orthologues of E. coli heat stress (HS) genes are retained by B. aphidicola, only five were differentially expressed after near-lethal heat stress treatments, and only modest shifts were observed. Analyses of upstream regulatory regions revealed loss or degradation of most HS (sigma32) promoters. Genomic rearrangements downstream of an intact HS promoter yielded upregulation of a functionally unrelated and an inactivated gene. Reanalyses of comparable experimental array data for E. coli and Bacillus subtilis revealed that genome-wide differential expression was significantly lower in B. aphidicola. Our demonstration of a diminished stress response validates reports of temperature sensitivity in B. aphidicola and suggests that this reduced bacterial genome exhibits transcriptional inflexibility.

Gender hormones and the progression of experimental polycystic kidney disease.

PubMed

Stringer, Kenneth D; Komers, Radko; Osman, Shukri A; Oyama, Terry T; Lindsley, Jessie N; Anderson, Sharon

2005-10-01

Male gender is a risk factor for progression of autosomal-dominant polycystic kidney disease (ADPKD), clinically and in the Han:SPRD rat model. Orchiectomy limits progression, but mechanisms of the detrimental effect of androgen, and/or beneficial effects of estrogen, are not known. This protocol tested the hypothesis that male gender (intact androgen status) promotes progression, while female gender (intact estrogen status) is protective; and that these disease-modifying effects are due to changes in expression of known fibrotic mediators. Studies were performed in male and female noncystic control (+/+) and cystic (+/-) rats subjected to orchiectomy, ovariectomy, or sham operation. At 12 weeks of age, renal function was measured. Blood and kidneys were taken for measurement of plasma and renal renin, endothelin (ET-1), endothelial nitric oxide synthase (eNOS), and vascular endothelial growth factor (VEGF), using biochemical, protein expression, and immunohistochemical methods. Cystic male rats exhibited significantly reduced glomerular filtration (GFR) and effective renal plasma flow (ERPF) rates, with suppression of plasma and renal renin, up-regulation of renal ET-1 and eNOS, and down-regulation of renal VEGF expression. Orchiectomy attenuated the fall in GFR and ERPF, while numerically limiting changes in eNOS and VEGF. Female rats exhibited less cystic growth, with normal renin status, lesser elevation of renal ET-1, and proportionately lesser changes in VEGF and eNOS. Ovariectomy led to higher blood pressure and reduced GFR and ERPF, with a trend toward upregulation of ET-1, and significant down-regulation of VEGF and eNOS. Female gender is protective, but ovariectomy attenuates the protective effect of female gender, in association with changes in renal expression of ET-1, VEGF, and eNOS. The accelerated disease in male rats can be attenuated by orchiectomy and consequent changes in expression of disease mediators.

Biobreeding rat islets exhibit reduced antioxidative defense and N-acetyl cysteine treatment delays type 1 diabetes

PubMed Central

Bogdani, Marika; Henschel, Angela M.; Kansra, Sanjay; Fuller, Jessica M.; Geoffrey, Rhonda; Jia, Shuang; Kaldunski, Mary L.; Pavletich, Scott; Prosser, Simon; Chen, Yi-Guang; Lernmark, Åke; Hessner, Martin J.

2014-01-01

Islet-level oxidative stress has been proposed as a trigger for type 1 diabetes (T1D), and release of cytokines by infiltrating immune cells further elevates reactive oxygen species (ROS), exacerbating β cell duress. To identify genes/mechanisms involved with diabeto-genesis at the β cell level, gene expression profiling and targeted follow-up studies were used to investigate islet activity in the biobreeding (BB) rat. Forty-day-old spontaneously diabetic lymphopenic BB DRlyp/lyp rats (before T cell insulitis) as well as nondiabetic BB DR+/+ rats, nondiabetic but lymphopenic F344lyp/lyp rats, and healthy Fischer (F344) rats were examined. Gene expression profiles of BB rat islets were highly distinct from F344 islets and under-expressed numerous genes involved in ROS metabolism, including glutathione S-transferase (GST) family members (Gstm2, Gstm4, Gstm7, Gstt1, Gstp1, and Gstk1), superoxide dismutases (Sod2 and Sod3), peroxidases, and peroxiredoxins. This pattern of under-expression was not observed in brain, liver, or muscle. Compared with F344 rats, BB rat pancreata exhibited lower GST protein levels, while plasma GST activity was found significantly lower in BB rats. Systemic administration of the antioxidant N-acetyl cysteine to DRlyp/lyp rats altered abundances of peripheral eosinophils, reduced severity of insulitis, and significantly delayed but did not prevent diabetes onset. We find evidence of β cell dysfunction in BB rats independent of T1D progression, which includes lower expression of genes related to antioxidative defense mechanisms during the pre-onset period that may contribute to overall T1D susceptibility. PMID:23111281

Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S

PubMed Central

Stewart, Andrew K.; Shmukler, Boris E.; Vandorpe, David H.; Rivera, Alicia; Heneghan, John F.; Li, Xiaojin; Hsu, Ann; Karpatkin, Margaret; O'Neill, Allison F.; Bauer, Daniel E.; Heeney, Matthew M.; John, Kathryn; Kuypers, Frans A.; Gallagher, Patrick G.; Lux, Samuel E.; Brugnara, Carlo; Westhoff, Connie M.

2011-01-01

Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wild-type RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li+ and 86Rb+, with secondarily increased 86Rb+ influx sensitive to ouabain and to bumetanide. Increased RhAG-associated 14C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li+, 86Rb+, and 14C-MA were pharmacologically distinct, and Li+ uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH4+ and Gd3+. RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH3/NH4+, but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA+). RhAG F65S-expressing oocytes exhibited near-wild-type responses to NH4Cl, but MA/MA+ elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li+ substitution or bath addition of 5 mM NH4Cl or MA/MA+. These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH3/NH4+ and MA/MA+; 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA+ transport, and decreased NH3/NH4+-associated depolarization; and 3) RhAG transports NH3/NH4+ and MA/MA+ by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte intracellular pH, membrane potential, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms. PMID:21849667

Ribosomal L1 domain and lysine-rich region are essential for CSIG/ RSL1D1 to regulate proliferation and senescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Liwei; Zhao, Wenting; Zheng, Quanhui

2016-01-15

The expression change of cellular senescence-associated genes is underlying the genetic foundation of cellular senescence. Using a suppressive subtractive hybridization system, we identified CSIG (cellular senescence-inhibited gene protein; RSL1D1) as a novel senescence-associated gene. CSIG is implicated in various process including cell cycle regulation, apoptosis, and tumor metastasis. We previously showed that CSIG plays an important role in regulating cell proliferation and cellular senescence progression through inhibiting PTEN, however, which domain or region of CSIG contributes to this function? To clarify this question, we investigated the functional importance of ribosomal L1 domain and lysine (Lys) -rich region of CSIG. Themore » data showed that expression of CSIG potently reduced PTEN expression, increased cell proliferation rates, and reduced the senescent phenotype (lower SA-β-gal activity). By contrast, neither the expression of CSIG N- terminal (NT) fragment containing the ribosomal L1 domain nor C-terminal (CT) fragment containing Lys-rich region could significantly altered the levels of PTEN; instead of promoting cell proliferation and delaying cellular senescence, expression of CSIG-NT or CSIG-CT inhibited cell proliferation and accelerated cell senescence (increased SA-β-gal activity) compared to either CSIG over-expressing or control (empty vector transfected) cells. The further immunofluorescence analysis showed that CSIG-CT and CSIG-NT truncated proteins exhibited different subcellular distribution with that of wild-type CSIG. Conclusively, both ribosomal L1 domain and Lys-rich region of CSIG are critical for CSIG to act as a regulator of cell proliferation and cellular senescence. - Highlights: • The ribosomal L1 domain and lysine-rich region of CSIG were expressed. • They are critical for CSIG to regulate proliferation and senescence. • CSIG and its domains exhibit different subcellular distribution.« less

ATP6AP2 over-expression causes morphological alterations in the hippocampus and in hippocampus-related behaviour.

PubMed

Bracke, A; Schäfer, S; von Bohlen Und Halbach, V; Klempin, F; Bente, K; Bracke, K; Staar, D; van den Brandt, J; Harzsch, S; Bader, M; Wenzel, U O; Peters, J; von Bohlen Und Halbach, O

2018-02-23

The (pro)renin receptor [(P)RR], also known as ATP6AP2 [ATPase 6 accessory protein 2], is highly expressed in the brain. ATP6AP2 plays a role in early brain development, adult hippocampal neurogenesis and in cognitive functions. Lack of ATP6AP2 has deleterious effects, and mutations of ATP6AP2 in humans are associated with, e.g. X-linked intellectual disability. However, little is known about the effects of over-expression of ATP6AP2 in the adult brain. We hypothesized that mice over-expressing ATP6AP2 in the brain might exhibit altered neuroanatomical features and behavioural responses. To this end, we investigated heterozygous transgenic female mice and confirmed increased levels of ATP6AP2 in the brain. Our data show that over-expression of ATP6AP2 does not affect adult hippocampal neurogenesis, exercise-induced cell proliferation, or dendritic spine densities in the hippocampus. Only a reduced ventricular volume on the gross morphological level was found. However, ATP6AP2 over-expressing mice displayed altered exploratory behaviour with respect to the hole-board and novel object recognition tests. Moreover, primary adult hippocampal neural stem cells over-expressing ATP6AP2 exhibit a faster cell cycle progression and increased cell proliferation. Together, in contrast to the known deleterious effects of ATP6AP2 depletion, a moderate over-expression results in moderate behavioural changes and affects cell proliferation rate in vitro.

The circadian clock stops ticking during deep hibernation in the European hamster

PubMed Central

Revel, Florent G.; Herwig, Annika; Garidou, Marie-Laure; Dardente, Hugues; Menet, Jérôme S.; Masson-Pévet, Mireille; Simonneaux, Valérie; Saboureau, Michel; Pévet, Paul

2007-01-01

Hibernation is a fascinating, yet enigmatic, physiological phenomenon during which body temperature and metabolism are reduced to save energy. During the harsh season, this strategy allows substantial energy saving by reducing body temperature and metabolism. Accordingly, biological processes are considerably slowed down and reduced to a minimum. However, the persistence of a temperature-compensated, functional biological clock in hibernating mammals has long been debated. Here, we show that the master circadian clock no longer displays 24-h molecular oscillations in hibernating European hamsters. The clock genes Per1, Per2, and Bmal1 and the clock-controlled gene arginine vasopressin were constantly expressed in the suprachiasmatic nucleus during deep torpor, as assessed by radioactive in situ hybridization. Finally, the melatonin rhythm-generating enzyme, arylalkylamine N-acetyltransferase, whose rhythmic expression in the pineal gland is controlled by the master circadian clock, no longer exhibits day/night changes of expression but constantly elevated mRNA levels over 24 h. Overall, these data provide strong evidence that in the European hamster the molecular circadian clock is arrested during hibernation and stops delivering rhythmic output signals. PMID:17715068

Incensole acetate reduces depressive-like behavior and modulates hippocampal BDNF and CRF expression of submissive animals.

PubMed

Moussaieff, Arieh; Gross, Moshe; Nesher, Elimelech; Tikhonov, Tatiana; Yadid, Gal; Pinhasov, Albert

2012-12-01

Incensole acetate (IA), a constituent of Boswellia resin ('frankincense'), was previously demonstrated to exhibit an antidepressive-like effect in the Forced Swim Test (FST) in mice following single dose administration (50 mg/kg). Here, we show that acute administration of considerably lower dose (10 mg/kg) IA to selectively bred mice, showing prominent submissive behavior, exerted significant antidepressant-like effects in the FST. Furthermore, chronic administration of 1 or 5 mg/kg per day of IA for three consecutive weeks dose- and time-dependently reduced the submissiveness of the mice in the Dominant-Submissive Relationship test, developed to screen the chronic effect of antidepressants. This behavioral effect was concomitant to reduced serum corticosterone levels, dose-dependent down-regulation of corticotropin releasing factor and up-regulation of brain derived neurotrophic factor transcripts IV and VI expression in the hippocampus. These data suggest that IA modulates the hypothalamic-pituitary-adrenal (HPA) axis and influences hippocampal gene expression, leading to beneficial behavioral effects supporting its potential as a novel treatment of depressive-like disorders.

Bit-1 is an essential regulator of myogenic differentiation

PubMed Central

Griffiths, Genevieve S.; Doe, Jinger; Jijiwa, Mayumi; Van Ry, Pam; Cruz, Vivian; de la Vega, Michelle; Ramos, Joe W.; Burkin, Dean J.; Matter, Michelle L.

2015-01-01

Muscle differentiation requires a complex signaling cascade that leads to the production of multinucleated myofibers. Genes regulating the intrinsic mitochondrial apoptotic pathway also function in controlling cell differentiation. How such signaling pathways are regulated during differentiation is not fully understood. Bit-1 (also known as PTRH2) mutations in humans cause infantile-onset multisystem disease with muscle weakness. We demonstrate here that Bit-1 controls skeletal myogenesis through a caspase-mediated signaling pathway. Bit-1-null mice exhibit a myopathy with hypotrophic myofibers. Bit-1-null myoblasts prematurely express muscle-specific proteins. Similarly, knockdown of Bit-1 expression in C2C12 myoblasts promotes early differentiation, whereas overexpression delays differentiation. In wild-type mice, Bit-1 levels increase during differentiation. Bit-1-null myoblasts exhibited increased levels of caspase 9 and caspase 3 without increased apoptosis. Bit-1 re-expression partially rescued differentiation. In Bit-1-null muscle, Bcl-2 levels are reduced, suggesting that Bcl-2-mediated inhibition of caspase 9 and caspase 3 is decreased. Bcl-2 re-expression rescued Bit-1-mediated early differentiation in Bit-1-null myoblasts and C2C12 cells with knockdown of Bit-1 expression. These results support an unanticipated yet essential role for Bit-1 in controlling myogenesis through regulation of Bcl-2. PMID:25770104

Circulating B cells in type 1 diabetics exhibit fewer maturation-associated phenotypes.

PubMed

Hanley, Patrick; Sutter, Jennifer A; Goodman, Noah G; Du, Yangzhu; Sekiguchi, Debora R; Meng, Wenzhao; Rickels, Michael R; Naji, Ali; Luning Prak, Eline T

2017-10-01

Although autoantibodies have been used for decades as diagnostic and prognostic markers in type 1 diabetes (T1D), further analysis of developmental abnormalities in B cells could reveal tolerance checkpoint defects that could improve individualized therapy. To evaluate B cell developmental progression in T1D, immunophenotyping was used to classify circulating B cells into transitional, mature naïve, mature activated, and resting memory subsets. Then each subset was analyzed for the expression of additional maturation-associated markers. While the frequencies of B cell subsets did not differ significantly between patients and controls, some T1D subjects exhibited reduced proportions of B cells that expressed transmembrane activator and CAML interactor (TACI) and Fas receptor (FasR). Furthermore, some T1D subjects had B cell subsets with lower frequencies of class switching. These results suggest circulating B cells exhibit variable maturation phenotypes in T1D. These phenotypic variations may correlate with differences in B cell selection in individual T1D patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Low expression of D2R and Wntless correlates with high motivation for heroin.

PubMed

Tacelosky, Diana M; Alexander, Danielle N; Morse, Megan; Hajnal, Andras; Berg, Arthur; Levenson, Robert; Grigson, Patricia S

2015-12-01

Drug overdose now exceeds car accidents as the leading cause of accidental death in the United States. Of those drug overdoses, a large percentage of the deaths are due to heroin and/or pharmaceutical overdose, specifically misuse of prescription opioid analgesics. It is imperative, then, that we understand the mechanisms that lead to opioid abuse and addiction. The rewarding actions of opioids are mediated largely by the mu-opioid receptor (MOR), and signaling by this receptor is modulated by various interacting proteins. The neurotransmitter dopamine also contributes to opioid reward, and opioid addiction has been linked to reduced expression of dopamine D2 receptors (D2R) in the brain. That said, it is not known if alterations in the expression of these proteins relate to drug exposure and/or to the "addiction-like" behavior exhibited for the drug. Here, we held total drug self-administration constant across acquisition and showed that reduced expression of the D2R and the MOR interacting protein, Wntless, in the medial prefrontal cortex was associated with greater addiction-like behavior for heroin in general and with a greater willingness to work for the drug in particular. In contrast, reduced expression of the D2R in the nucleus accumbens and hippocampus was correlated with greater seeking during signaled nonavailability of the drug. Taken together, these data link reduced expression of both the D2R and Wntless to the explicit motivation for the drug rather than to differences in total drug intake per se. (c) 2015 APA, all rights reserved).

Pleasure Experience and Emotion Expression in Patients with Schizophrenia

PubMed Central

CHU, Min-yi; LI, Xu; LV, Qin-yu; Yl, Zheng-hui; CHEUNG, Eric F. C.; CHAN, Raymond C. K.

2017-01-01

Background Impairments in emotional experience and expression have been observed in patients with schizophrenia. However, most previous studies have been limited to either emotional experience (especially anhedonia) or expression. Few studies have examined both the experience and expression of emotion in schizophrenia patients at the same time. Aims The present study aimed to examine pleasure experience and emotion expression in patients with schizophrenia. In particular, we specifically examined the relationship between emotion impairments (both pleasure experience and expression) and negative symptoms. Methods One hundred and fifty patients completed the Temporal Experience of Pleasure Scale and Emotional Expressivity Scale. Results Schizophrenia patients exhibited deficits in experiencing pleasure, but showed intact reported emotion expression. Patients with prominent negative symptoms showed reduced anticipatory pleasure, especially in abstract anticipatory pleasure. Conclusion The present findings suggest that patients with schizophrenia have deficits in pleasure experience, while their abilities to express emotion appear intact. Such deficits are more severe in patients with prominent negative symptoms. PMID:29276350

Autophagy influences the low-dose hyper-radiosensitivity of human lung adenocarcinoma cells by regulating MLH1.

PubMed

Wang, Qiong; Xiao, Zhuya; Lin, Zhenyu; Zhou, Jie; Chen, Weihong; Jie, Wuyun; Cao, Xing; Yin, Zhongyuan; Cheng, Jing

2017-06-01

To investigate the impact of autophagy on the low-dose hyper-radiosensitivity (HRS) of human lung adenocarcinoma cells via MLH1 regulation. Immunofluorescent staining, Western blotting, and electron microscopy were utilized to detect autophagy in A549 and H460 cells. shRNA was used to silence MLH1 expression. The levels of MLH1, mTOR, p-mTOR, BNIP3, and Beclin-1 were measured by real-time polymerase chain reaction (PCR) and Western blotting. A549 cells, which have low levels of MLH1 expression, displayed HRS/induced radioresistance (IRR). Conversely, the radiosensitivity of H460 cells, which express high levels of MLH1, conformed to the linear-quadratic (LQ) model. After down-regulating MLH1 expression, A549 cells showed increased HRS and inhibition of autophagy, whereas H460 cells exhibited HRS/IRR. The levels of mTOR, p-mTOR, and BNIP3 were reduced in cells harboring MLH1 shRNA, and the changes in the mTOR/p-mTOR ratio mirrored those in MLH1 expression. Low MLH1-expressing A549 cells may exhibit HRS. Both the mTOR/p-mTOR and BNIP3/Beclin-1 signaling pathways were found to be related to HRS, but only mTOR/p-mTOR is involved in the regulation of HRS via MLH1 and autophagy.

Expression of a soluble truncated Vargula luciferase in Escherichia coli.

PubMed

Hunt, Eric A; Moutsiopoulou, Angeliki; Broyles, David; Head, Trajen; Dikici, Emre; Daunert, Sylvia; Deo, Sapna K

2017-04-01

Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties. Copyright © 2017 Elsevier Inc. All rights reserved.

Cows with follicular fluid androgen excess exhibit anovulation and have altered circulating sex hormone binding globulin, gonadotropin secretion and plasma and follicular fluid composition

USDA-ARS?s Scientific Manuscript database

Our laboratory identified a group of cows with excess intrafollicular concentrations of androstenedione (A4; >30 fold), reduced calving rates, and theca gene expression profiles similar to women with Polycystic Ovary Syndrome (PCOS). Based on these previous studies, we hypothesized that High A4 cows...

Transgenic Mice Expressing a Truncated Form of CREB-Binding Protein (CBP) Exhibit Deficits in Hippocampal Synaptic Plasticity and Memory Storage

ERIC Educational Resources Information Center

Wood, Marcelo A.; Kaplan, Michael P.; Park, Alice; Blanchard, Edward J.; Oliveira, Ana M. M.; Lombardi, Thomas L.; Abel, Ted

2005-01-01

Deletions, translocations, or point mutations in the CREB-binding protein (CBP) gene have been associated with Rubinstein-Taybi Syndrome; a human developmental disorder characterized by retarded growth and reduced mental function. To examine the role of CBP in memory, transgenic mice were generated in which the CaMKII[alpha] promoter drives…

Novel clustering of items from the Autism Diagnostic Interview-Revised to define phenotypes within autism spectrum disorders

PubMed Central

Hu, Valerie W.; Steinberg, Mara E.

2009-01-01

Heterogeneity in phenotypic presentation of ASD has been cited as one explanation for the difficulty in pinpointing specific genes involved in autism. Recent studies have attempted to reduce the “noise” in genetic and other biological data by reducing the phenotypic heterogeneity of the sample population. The current study employs multiple clustering algorithms on 123 item scores from the Autism Diagnostic Interview-Revised (ADI-R) diagnostic instrument of nearly 2000 autistic individuals to identify subgroups of autistic probands with clinically relevant behavioral phenotypes in order to isolate more homogeneous groups of subjects for gene expression analyses. Our combined cluster analyses suggest optimal division of the autistic probands into 4 phenotypic clusters based on similarity of symptom severity across the 123 selected item scores. One cluster is characterized by severe language deficits, while another exhibits milder symptoms across the domains. A third group possesses a higher frequency of savant skills while the fourth group exhibited intermediate severity across all domains. Grouping autistic individuals by multivariate cluster analysis of ADI-R scores reveals meaningful phenotypes of subgroups within the autistic spectrum which we show, in a related (accompanying) study, to be associated with distinct gene expression profiles. PMID:19455643

HDM2 promotes WIP1-mediated medulloblastoma growth

PubMed Central

Buss, Meghan C.; Read, Tracy-Ann; Schniederjan, Matthew J.; Gandhi, Khanjan; Castellino, Robert C.

2012-01-01

Medulloblastoma is the most common malignant childhood brain tumor. The protein phosphatase and oncogene WIP1 is over-expressed or amplified in a significant number of primary human medulloblastomas and cell lines. In the present study, we examine an important mechanism by which WIP1 promotes medulloblastoma growth using in vitro and in vivo models. Human cell lines and intracerebellar xenografted animal models were used to study the role of WIP1 and the major TP53 regulator, HDM2, in medulloblastoma growth. Stable expression of WIP1 enhances growth of TP53 wild-type medulloblastoma cells, compared with cells with stable expression of an empty-vector or mutant WIP1. In an animal model, WIP1 enhances proliferation and reduces the survival of immunodeficient mice bearing intracerebellar xenografted human medulloblastoma cells. Cells with increased WIP1 expression also exhibit increased expression of HDM2. HDM2 knockdown or treatment with the HDM2 inhibitor Nutlin-3a, the active enantomer of Nutlin-3, specifically inhibits the growth of medulloblastoma cells with increased WIP1 expression. Nutlin-3a does not affect growth of medulloblastoma cells with stable expression of an empty vector or of mutant WIP1. Knockdown of WIP1 or treatment with the WIP1 inhibitor CCT007093 results in increased phosphorylation of known WIP1 targets, reduced HDM2 expression, and reduced growth specifically in WIP1 wild-type and high-expressing medulloblastoma cells. Combined WIP1 and HDM2 inhibition is more effective than WIP1 inhibition alone in blocking growth of WIP1 high-expressing medulloblastoma cells. Our preclinical study supports a role for therapies that target WIP1 and HDM2 in the treatment of medulloblastoma. PMID:22379189

Neuropeptide Y as a possible homeostatic element for changes in cortical excitability induced by repetitive transcranial magnetic stimulation.

PubMed

Jazmati, Danny; Neubacher, Ute; Funke, Klaus

2018-02-24

Repetitive transcranial magnetic stimulation (rTMS) is able to modify cortical excitability. Rat rTMS studies revealed a modulation of inhibitory systems, in particular that of the parvalbumin-expressing (PV+) interneurons, when using intermittent theta-burst stimulation (iTBS). The potential disinhibitory action of iTBS raises the questions of how neocortical circuits stabilize excitatory-inhibitory balance within a physiological range. Neuropeptide Y (NPY) appears to be one candidate. Analysis of cortical expression of PV, NPY and vesicular glutamate transporter type 1 (vGluT1) by immunohistochemical means at the level of cell counts, mean neuropil expression and single cell pre-/postsynaptic expression, with and without intraventricular NPY-injection. Our results show that iTBS not only reduced the number of neurons with high-PV expression in a dose-dependent fashion, but also increased the cortical expression of NPY, discussed to reduce glutamatergic transmission, and this was further associated with a reduced vGluT1 expression, an indicator of glutamateric presynaptic activity. Interneurons showing a low-PV expression exhibit less presynaptic vGluT1 expression compared to those with a high-PV expression. Intraventricular application of NPY prior to iTBS prevented the iTBS-induced reduction in the number of high-PV neurons, the reduction in tissue vGluT1 level and that presynaptic to high-PV cells. We conclude that NPY, possibly via a global but also slow homeostatic control of glutamatergic transmission, modulates the strength and direction of the iTBS effects, likely preventing pathological imbalance of excitatory and inhibitory cortical activity but still allowing enough disinhibition beneficial for plastic changes as during learning. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility.

PubMed

Natsuga, Ken; Cipolat, Sara; Watt, Fiona M

2016-01-01

Mice lacking three epidermal barrier proteins-envoplakin, periplakin, and involucrin (EPI-/- mice)-have a defective cornified layer, reduced epidermal γδ T cells, and increased dermal CD4(+) T cells. They are also resistant to developing skin tumors. The tumor-protective mechanism involves signaling between Rae-1 expressing keratinocytes and the natural killer group 2D receptor on immune cells, which also plays a role in host defenses against infection. Given the emerging link between bacteria and cancer, we investigated whether EPI-/- mice have an altered skin microbiota. The bacterial phyla were similar in wild-type and EPI-/- skin. However, bacteria were threefold more abundant in EPI-/- skin and penetrated deeper into the epidermis. The major epithelial defense mechanism against bacteria is production of antimicrobial proteins (AMPs). EPI-/- skin exhibited enhanced expression of antimicrobial peptides. However, reducing the bacterial load by antibiotic treatment or breeding mice under specific pathogen-free conditions did not reduce AMP expression or alleviate the abnormalities in T-cell populations. We conclude that the atopic characteristics of EPI-/- skin are a consequence of the defective barrier rather than a response to the increased bacterial load. It is therefore unlikely that the increase in skin microbiota contributes directly to the observed cancer resistance. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Alleviation of Ultraviolet B-Induced Photodamage by Coffea arabica Extract in Human Skin Fibroblasts and Hairless Mouse Skin

PubMed Central

Wu, Po-Yuan; Huang, Chi-Chang; Chu, Yin; Huang, Ya-Han; Lin, Ping; Liu, Yu-Han; Wen, Kuo-Ching; Lin, Chien-Yih; Hsu, Mei-Chich; Chiang, Hsiu-Mei

2017-01-01

Coffea arabica extract (CAE) containing 48.3 ± 0.4 mg/g of chlorogenic acid and a trace amount of caffeic acid was found to alleviate photoaging activity in human skin fibroblasts. In this study, polyphenol-rich CAE was investigated for its antioxidant and antiinflammatory properties, as well as for its capability to alleviate ultraviolet B (UVB)-induced photodamage in BALB/c hairless mice. The results indicated that 500 μg/mL of CAE exhibited a reducing power of 94.7%, ferrous ion chelating activity of 46.4%, and hydroxyl radical scavenging activity of 20.3%. The CAE dose dependently reduced UVB-induced reactive oxygen species (ROS) generation in fibroblasts. Furthermore, CAE inhibited the UVB-induced expression of cyclooxygenase-2 and p-inhibitor κB, and the translocation of nuclear factor-kappa B (NF-κB) to the nucleus of fibroblasts. In addition, CAE alleviated UVB-induced photoaging and photodamage in BALB/c hairless mice by restoring the collagen content and reduced UVB-induced epidermal hyperplasia. CAE also inhibited UVB-induced NF-κB, interleukin-6, and matrix metalloproteinase-1 expression in the hairless mouse skin. The results indicated that CAE exhibits antiphotodamage activity by inhibiting UV-induced oxidative stress and inflammation. Therefore, CAE is a candidate for use in antioxidant, antiinflammatory, and antiphotodamage products. PMID:28387707

Digital gene expression for non-model organisms

PubMed Central

Hong, Lewis Z.; Li, Jun; Schmidt-Küntzel, Anne; Warren, Wesley C.; Barsh, Gregory S.

2011-01-01

Next-generation sequencing technologies offer new approaches for global measurements of gene expression but are mostly limited to organisms for which a high-quality assembled reference genome sequence is available. We present a method for gene expression profiling called EDGE, or EcoP15I-tagged Digital Gene Expression, based on ultra-high-throughput sequencing of 27-bp cDNA fragments that uniquely tag the corresponding gene, thereby allowing direct quantification of transcript abundance. We show that EDGE is capable of assaying for expression in >99% of genes in the genome and achieves saturation after 6–8 million reads. EDGE exhibits very little technical noise, reveals a large (106) dynamic range of gene expression, and is particularly suited for quantification of transcript abundance in non-model organisms where a high-quality annotated genome is not available. In a direct comparison with RNA-seq, both methods provide similar assessments of relative transcript abundance, but EDGE does better at detecting gene expression differences for poorly expressed genes and does not exhibit transcript length bias. Applying EDGE to laboratory mice, we show that a loss-of-function mutation in the melanocortin 1 receptor (Mc1r), recognized as a Mendelian determinant of yellow hair color in many different mammals, also causes reduced expression of genes involved in the interferon response. To illustrate the application of EDGE to a non-model organism, we examine skin biopsy samples from a cheetah (Acinonyx jubatus) and identify genes likely to control differences in the color of spotted versus non-spotted regions. PMID:21844123

Writing About Past Failures Attenuates Cortisol Responses and Sustained Attention Deficits Following Psychosocial Stress

PubMed Central

DiMenichi, Brynne C.; Lempert, Karolina M.; Bejjani, Christina; Tricomi, Elizabeth

2018-01-01

Acute stress can harm performance. Paradoxically, writing about stressful events—such as past failures—has been shown to improve cognitive functioning and performance, especially in tasks that require sustained attention. Yet, there is little physiological evidence for whether writing about past failures or other negative events improves performance by reducing stress. In this experiment, we studied the effects of an acute psychosocial stressor, the Trier Social Stress Test, on attentional performance and salivary cortisol release in humans. Additionally, we investigated whether an expressive writing task could reduce the detrimental effects of stress, both on performance and physiological response. We found that when individuals were asked to write about a past failure before experiencing a stressor, they exhibited attenuated stress responses. Moreover, those who wrote about a past failure before being exposed to stress also exhibited better behavioral performance. Our results suggest that writing about a previous failure may allow an individual to experience a new stressor as less stressful, reducing its physiological and behavioral effects. PMID:29628878

Strand antagonism in RNAi: an explanation of differences in potency between intracellularly expressed siRNA and shRNA

PubMed Central

Jin, Xin; Sun, Tingting; Zhao, Chuanke; Zheng, Yongxiang; Zhang, Yufan; Cai, Weijing; He, Qiuchen; Taira, Kaz; Zhang, Lihe; Zhou, Demin

2012-01-01

Strategies to regulate gene function frequently use small interfering RNAs (siRNAs) that can be made from their shRNA precursors via Dicer. However, when the duplex components of these siRNA effectors are expressed from their respective coding genes, the RNA interference (RNAi) activity is much reduced. Here, we explored the mechanisms of action of shRNA and siRNA and found the expressed siRNA, in contrast to short hairpin RNA (shRNA), exhibits strong strand antagonism, with the sense RNA negatively and unexpectedly regulating RNAi. Therefore, we altered the relative levels of strands of siRNA duplexes during their expression, increasing the level of the antisense component, reducing the level of the sense component, or both and, in this way we were able to enhance the potency of the siRNA. Such vector-delivered siRNA attacked its target effectively. These findings provide new insight into RNAi and, in particular, they demonstrate that strand antagonism is responsible for making siRNA far less potent than shRNA. PMID:22039150

Interleukins 1alpha and 1beta secreted by some melanoma cell lines strongly reduce expression of MITF-M and melanocyte differentiation antigens.

PubMed

Kholmanskikh, Olga; van Baren, Nicolas; Brasseur, Francis; Ottaviani, Sabrina; Vanacker, Julie; Arts, Nathalie; van der Bruggen, Pierre; Coulie, Pierre; De Plaen, Etienne

2010-10-01

We report that melanoma cell lines expressing the interleukin-1 receptor exhibit 4- to 10-fold lower levels of mRNA of microphthalmia-associated transcription factor (MITF-M) when treated with interleukin-1beta. This effect is NF-kappaB and JNK-dependent. MITF-M regulates the expression of melanocyte differentiation genes such as MLANA, tyrosinase and gp100, which encode antigens recognized on melanoma cells by autologous cytolytic T lymphocytes. Accordingly, treating some melanoma cells with IL-1beta reduced by 40-100% their ability to activate such antimelanoma cytolytic T lymphocytes. Finally, we observed large amounts of biologically active IL-1alpha or IL-1beta secreted by two melanoma cell lines that did not express MITF-M, suggesting an autocrine MITF-M downregulation. We estimate that approximately 13% of melanoma cell lines are MITF-M-negative and secrete IL-1 cytokines. These results indicate that the repression of melanocyte-differentiation genes by IL-1 produced by stromal cells or by tumor cells themselves may represent an additional mechanism of melanoma immune escape.

Myeloid-Cell-Derived VEGF Maintains Brain Glucose Uptake and Limits Cognitive Impairment in Obesity.

PubMed

Jais, Alexander; Solas, Maite; Backes, Heiko; Chaurasia, Bhagirath; Kleinridders, André; Theurich, Sebastian; Mauer, Jan; Steculorum, Sophie M; Hampel, Brigitte; Goldau, Julia; Alber, Jens; Förster, Carola Y; Eming, Sabine A; Schwaninger, Markus; Ferrara, Napoleone; Karsenty, Gerard; Brüning, Jens C

2016-05-05

High-fat diet (HFD) feeding induces rapid reprogramming of systemic metabolism. Here, we demonstrate that HFD feeding of mice downregulates glucose transporter (GLUT)-1 expression in blood-brain barrier (BBB) vascular endothelial cells (BECs) and reduces brain glucose uptake. Upon prolonged HFD feeding, GLUT1 expression is restored, which is paralleled by increased expression of vascular endothelial growth factor (VEGF) in macrophages at the BBB. In turn, inducible reduction of GLUT1 expression specifically in BECs reduces brain glucose uptake and increases VEGF serum concentrations in lean mice. Conversely, myeloid-cell-specific deletion of VEGF in VEGF(Δmyel) mice impairs BBB-GLUT1 expression, brain glucose uptake, and memory formation in obese, but not in lean mice. Moreover, obese VEGF(Δmyel) mice exhibit exaggerated progression of cognitive decline and neuroinflammation on an Alzheimer's disease background. These experiments reveal that transient, HFD-elicited reduction of brain glucose uptake initiates a compensatory increase of VEGF production and assign obesity-associated macrophage activation a homeostatic role to restore cerebral glucose metabolism, preserve cognitive function, and limit neurodegeneration in obesity. Copyright © 2016 Elsevier Inc. All rights reserved.

GLUT4 in the endocrine pancreas--indicating an impact in pancreatic islet cell physiology?

PubMed

Bähr, I; Bazwinsky-Wutschke, I; Wolgast, S; Hofmann, K; Streck, S; Mühlbauer, E; Wedekind, D; Peschke, E

2012-06-01

The glucose transporter GLUT4 is well known to facilitate the transport of blood glucose into insulin-sensitive muscle and adipose tissue. In this study, molecular, immunohistochemical, and Western blot investigations revealed evidence that GLUT4 is also located in the mouse, rat, and human endocrine pancreas. In addition, high glucose decreased and insulin elevated the GLUT4 expression in pancreatic α-cells. In contrast, high glucose increased GLUT4 expression, whereas insulin led to a reduced expression level of the glucose transporter in pancreatic β-cells. In vivo experiments showed that in pancreatic tissue of type 2 diabetic rats as well as type 2 diabetic patients, the GLUT4 expression is significantly increased compared to the nondiabetic control group. Furthermore, type 1 diabetic rats exhibited reduced GLUT4 transcript levels in pancreatic tissue, whereas insulin treatment of type 1 diabetic animals enhanced the GLUT4 expression back to control levels. These data provide evidence for the existence of GLUT4 in the endocrine pancreas and indicate a physiological relevance of this glucose transporter as well as characteristic changes in diabetic disease. © Georg Thieme Verlag KG Stuttgart · New York.

Ankyrin-G isoform imbalance and interneuronopathy link epilepsy and bipolar disorder.

PubMed

Lopez, A Y; Wang, X; Xu, M; Maheshwari, A; Curry, D; Lam, S; Adesina, A M; Noebels, J L; Sun, Q-Q; Cooper, E C

2017-10-01

ANK3, encoding the adaptor protein Ankyrin-G (AnkG), has been implicated in bipolar disorder by genome-wide association studies. ANK3 has multiple alternative first exons, and a bipolar disorder-associated ANK3 variant has been shown to reduce the expression of exon 1b. Here we identify mechanisms through which reduced ANK3 exon 1b isoform expression disrupts neuronal excitation-inhibition balance. We find that parvalbumin (PV) interneurons and principal cells differentially express ANK3 first exon subtypes. PV interneurons express only isoforms containing exon 1b, whereas excitatory principal cells express exon 1e alone or both 1e and 1b. In transgenic mice deficient for exon 1b, PV interneurons lack voltage-gated sodium channels at their axonal initial segments and have increased firing thresholds and diminished action potential dynamic range. These mice exhibit an Ank3 gene dosage-dependent phenotype including behavior changes modeling bipolar disorder, epilepsy and sudden death. Thus ANK3's important association with human bipolar susceptibility may arise from imbalance between AnkG function in interneurons and principal cells and resultant excessive circuit sensitivity and output. AnkG isoform imbalance is a novel molecular endophenotype and potential therapeutic target.

Ankyrin-G isoform imbalance and interneuronopathy link epilepsy and bipolar disorder

PubMed Central

Lopez, Angel Y.; Wang, Xinjun; Xu, Mingxuan; Maheshwari, Atul; Curry, Daniel; Lam, Sandi; Adesina, Adekunle M.; Noebels, Jeffrey L.; Sun, Qian-Quan; Cooper, Edward C.

2016-01-01

ANK3, encoding the adaptor protein Ankyrin-G, has been implicated in bipolar disorder by genome wide association studies. ANK3 has multiple alternative first exons, and a bipolar disorder-associated ANK3 variant has been shown to reduce expression of exon 1b. Here we identify mechanisms through which reduced ANK3 exon 1b isoform expression disrupts neuronal excitation-inhibition balance. We find that parvalbumin interneurons and principal cells differentially express ANK3 first exon subtypes. Parvalbumin interneurons express only isoforms containing exon 1b, whereas excitatory principal cells express exon 1e alone, or both 1e and 1b. In transgenic mice deficient for exon 1b, parvalbumin interneurons lack voltage-gated sodium channels at their axonal initial segments and have increased firing thresholds and diminished action potential dynamic range. These mice exhibit an Ank3 gene dosage-dependent phenotype including behavior changes modeling bipolar disorder, epilepsy, and sudden death. Thus, ANK3’s important association with human bipolar susceptibility may arise from imbalance between ankyrin-G function in interneurons and principal cells and resultant excessive circuit sensitivity and output. Ankyrin-G isoform imbalance is a novel molecular endophenotype and potential therapeutic target. PMID:27956739

Insulin-like growth factor 1 signaling is essential for mitochondrial biogenesis and mitophagy in cancer cells.

PubMed

Lyons, Amy; Coleman, Michael; Riis, Sarah; Favre, Cedric; O'Flanagan, Ciara H; Zhdanov, Alexander V; Papkovsky, Dmitri B; Hursting, Stephen D; O'Connor, Rosemary

2017-10-13

Mitochondrial activity and metabolic reprogramming influence the phenotype of cancer cells and resistance to targeted therapy. We previously established that an insulin-like growth factor 1 (IGF-1)-inducible mitochondrial UTP carrier (PNC1/SLC25A33) promotes cell growth. This prompted us to investigate whether IGF signaling is essential for mitochondrial maintenance in cancer cells and whether this contributes to therapy resistance. Here we show that IGF-1 stimulates mitochondrial biogenesis in a range of cell lines. In MCF-7 and ZR75.1 breast cancer cells, IGF-1 induces peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) and PGC-1α-related coactivator (PRC). Suppression of PGC-1β and PRC with siRNA reverses the effects of IGF-1 and disrupts mitochondrial morphology and membrane potential. IGF-1 also induced expression of the redox regulator nuclear factor-erythroid-derived 2-like 2 (NFE2L2 alias NRF-2). Of note, MCF-7 cells with acquired resistance to an IGF-1 receptor (IGF-1R) tyrosine kinase inhibitor exhibited reduced expression of PGC-1β, PRC, and mitochondrial biogenesis. Interestingly, these cells exhibited mitochondrial dysfunction, indicated by reactive oxygen species expression, reduced expression of the mitophagy mediators BNIP3 and BNIP3L, and impaired mitophagy. In agreement with this, IGF-1 robustly induced BNIP3 accumulation in mitochondria. Other active receptor tyrosine kinases could not compensate for reduced IGF-1R activity in mitochondrial protection, and MCF-7 cells with suppressed IGF-1R activity became highly dependent on glycolysis for survival. We conclude that IGF-1 signaling is essential for sustaining cancer cell viability by stimulating both mitochondrial biogenesis and turnover through BNIP3 induction. This core mitochondrial protective signal is likely to strongly influence responses to therapy and the phenotypic evolution of cancer. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Lithocholic bile acid inhibits lipogenesis and induces apoptosis in breast cancer cells.

PubMed

Luu, Trang H; Bard, Jean-Marie; Carbonnelle, Delphine; Chaillou, Chloé; Huvelin, Jean-Michel; Bobin-Dubigeon, Christine; Nazih, Hassan

2018-02-01

It has amply been documented that mammary tumor cells may exhibit an increased lipogenesis. Biliary acids are currently recognized as signaling molecules in the intestine, in addition to their classical roles in the digestion and absorption of lipids. The aim of our study was to evaluate the impact of lithocholic acid (LCA) on the lipogenesis of breast cancer cells. The putative cytotoxic effects of LCA on these cells were also examined. The effects of LCA on breast cancer-derived MCF-7 and MDA-MB-231 cells were studied using MTT viability assays, Annexin-FITC and Akt phosphorylation assays to evaluate anti-proliferative and pro-apoptotic properties, qRT-PCR and Western blotting assays to assess the expression of the bile acid receptor TGR5 and the estrogen receptor ERα, and genes and proteins involved in apoptosis (Bax, Bcl-2, p53) and lipogenesis (SREBP-1c, FASN, ACACA). Intracellular lipid droplets were visualized using Oil Red O staining. We found that LCA induces TGR5 expression and exhibits anti-proliferative and pro-apoptotic effects in MCF-7 and MDA-MB-231 cells. Also, an increase in pro-apoptotic p53 protein expression and a decrease in anti-apoptotic Bcl-2 protein expression were observed after LCA treatment of MCF-7 cells. In addition, we found that LCA reduced Akt phosphorylation in MCF-7 cells, but not in MDA-MB-231 cells. We also noted that LCA reduced the expression of SREBP-1c, FASN and ACACA in both breast cancer-derived cell lines and that cells treated with LCA contained low numbers of lipid droplets compared to untreated control cells. Finally, a decrease in ERα expression was observed in MCF-7 cells treated with LCA. Our data suggest a potential therapeutic role of lithocholic acid in breast cancer cells through a reversion of lipid metabolism deregulation.

Drought response transcriptomes are altered in poplar with reduced tonoplast sucrose transporter expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xue, Liang-Jiao; Frost, Christopher J.; Tsai, Chung-Jui

Transgenic Populus tremula x alba (717-1B4) plants with reduced expression of a tonoplast sucrose efflux transporter, PtaSUT4, exhibit reduced shoot growth compared to wild type (WT) under sustained mild drought. The present study was undertaken to determine whether SUT4-RNAi directly or indirectly altered poplar predisposition and/or response to changes in soil water availability. While sucrose and hexose levels were constitutively elevated in shoot organs, expression responses to drought were most altered in the root tips of SUT4-RNAi plants. Prior to any drought treatment, constitutively elevated transcript levels of abscisic acid biosynthetic genes and bark/vegetative storage proteins suggested altered metabolism inmore » root tips of RNAi plants. Stronger drought-stimulation of stress-inducible genes encoding late-embryogenesis-abundant proteins in transgenic roots was consistent with increased vulnerability to soil drying. Transcript evidence suggested an RNAi effect on intercellular water trafficking by aquaporins in stem xylem during soil drying and recovery. Co-expression network analysis predicted altered integration of abscisic acid sensing/signaling with ethylene and jasmonate sensing/signaling in RNAi compared to WT roots. The overall conclusion is that steepened shoot-root sugar gradient in RNAi plants increased sensitivity of root tips to decreasing soil water availability.« less

Effect of Diet on Preference and Intake of Sucrose in Obese Prone and Resistant Rats

PubMed Central

Duca, Frank A.; Swartz, Timothy D.; Covasa, Mihai

2014-01-01

Increased orosensory stimulation from palatable diets and decreased feedback from gut signals have been proposed as contributing factors to obesity development. Whether altered taste functions associated with obesity are common traits or acquired deficits to environmental factors, such as a high-energy (HE)-diet, however, is not clear. To address this, we examined preference and sensitivity of increasing concentrations of sucrose solutions in rats prone (OP) and resistant (OR) to obesity during chow and HE feeding and measured lingual gene expression of the sweet taste receptor T1R3. When chow-fed, OP rats exhibited reduced preference and acceptance of dilute sucrose solutions, sham-fed less sucrose compared to OR rats, and had reduced lingual T1R3 gene expression. HE-feeding abrogated differences in sucrose preference and intake and lingual T1R3 expression between phenotypes. Despite similar sucrose intakes however, OP rats consumed significantly more total calories during 48-h two-bottle testing compared to OR rats. The results demonstrate that OP rats have an innate deficit for sweet taste detection, as illustrated by a reduction in sensitivity to sweets and reduced T1R3 gene expression; however their hyperphagia and subsequent obesity during HE-feeding is most likely not due to altered consumption of sweets. PMID:25329959

The scaffolding and signaling functions of a localization factor impact polar development

PubMed Central

Curtis, Patrick D.; Quardokus, Ellen M.; Lawler, Melanie L.; Guo, Xiaoyun; Klein, David; Chen, Joseph C.; Arnold, Randy J.; Brun, Yves V.

2012-01-01

SUMMARY In the differentiating alphaproteobacterium Caulobacter crescentus, organelle synthesis at cell poles is critical to forming different progeny after cell division. Coordination of polar organelle synthesis, including pili and holdfast, and flagellum ejection, is mediated in part by the scaffolding protein PodJ. At the time of cell division, PodJ undergoes regulated processing to a short form that persists at the flagellar pole of swarmer cells. This study analyzes how PodJ’s role in structural and signaling protein localization impacts organelle synthesis. A PodJ mutant with an internal deletion exhibits reduced sensitivity to pili-tropic phage ΦCbK, resulting from reduced pilA gene expression, which can be linked to altered signaling protein localization. The phage sensitivity defect of a ΔpodJ mutant can be partially suppressed by ectopic pilA expression. Induction of PodJ processing, by manipulation of podJ itself or controlled perP expression, resulted in decreased pilus biogenesis and, when coupled with a podJ mutation that reduced pilA expression, led to complete loss of phage sensitivity. As a whole, the results show that PodJ’s scaffolding role for structural and signaling proteins both contribute to flagellar pole organelle development. PMID:22512778

Drought response transcriptomes are altered in poplar with reduced tonoplast sucrose transporter expression

DOE PAGES

Xue, Liang-Jiao; Frost, Christopher J.; Tsai, Chung-Jui; ...

2016-09-19

Transgenic Populus tremula x alba (717-1B4) plants with reduced expression of a tonoplast sucrose efflux transporter, PtaSUT4, exhibit reduced shoot growth compared to wild type (WT) under sustained mild drought. The present study was undertaken to determine whether SUT4-RNAi directly or indirectly altered poplar predisposition and/or response to changes in soil water availability. While sucrose and hexose levels were constitutively elevated in shoot organs, expression responses to drought were most altered in the root tips of SUT4-RNAi plants. Prior to any drought treatment, constitutively elevated transcript levels of abscisic acid biosynthetic genes and bark/vegetative storage proteins suggested altered metabolism inmore » root tips of RNAi plants. Stronger drought-stimulation of stress-inducible genes encoding late-embryogenesis-abundant proteins in transgenic roots was consistent with increased vulnerability to soil drying. Transcript evidence suggested an RNAi effect on intercellular water trafficking by aquaporins in stem xylem during soil drying and recovery. Co-expression network analysis predicted altered integration of abscisic acid sensing/signaling with ethylene and jasmonate sensing/signaling in RNAi compared to WT roots. The overall conclusion is that steepened shoot-root sugar gradient in RNAi plants increased sensitivity of root tips to decreasing soil water availability.« less

Altered lipid and salt taste responsivity in ghrelin and GOAT null mice.

PubMed

Cai, Huan; Cong, Wei-Na; Daimon, Caitlin M; Wang, Rui; Tschöp, Matthias H; Sévigny, Jean; Martin, Bronwen; Maudsley, Stuart

2013-01-01

Taste perception plays an important role in regulating food preference, eating behavior and energy homeostasis. Taste perception is modulated by a variety of factors, including gastric hormones such as ghrelin. Ghrelin can regulate growth hormone release, food intake, adiposity, and energy metabolism. Octanoylation of ghrelin by ghrelin O-acyltransferase (GOAT) is a specific post-translational modification which is essential for many biological activities of ghrelin. Ghrelin and GOAT are both widely expressed in many organs including the gustatory system. In the current study, overall metabolic profiles were assessed in wild-type (WT), ghrelin knockout (ghrelin(-/-)), and GOAT knockout (GOAT(-/-)) mice. Ghrelin(-/-) mice exhibited decreased food intake, increased plasma triglycerides and increased ketone bodies compared to WT mice while demonstrating WT-like body weight, fat composition and glucose control. In contrast GOAT(-/-) mice exhibited reduced body weight, adiposity, resting glucose and insulin levels compared to WT mice. Brief access taste behavioral tests were performed to determine taste responsivity in WT, ghrelin(-/-) and GOAT(-/-) mice. Ghrelin and GOAT null mice possessed reduced lipid taste responsivity. Furthermore, we found that salty taste responsivity was attenuated in ghrelin(-/-) mice, yet potentiated in GOAT(-/-) mice compared to WT mice. Expression of the potential lipid taste regulators Cd36 and Gpr120 were reduced in the taste buds of ghrelin and GOAT null mice, while the salt-sensitive ENaC subunit was increased in GOAT(-/-) mice compared with WT mice. The altered expression of Cd36, Gpr120 and ENaC may be responsible for the altered lipid and salt taste perception in ghrelin(-/-) and GOAT(-/-) mice. The data presented in the current study potentially implicates ghrelin signaling activity in the modulation of both lipid and salt taste modalities.

Relevance of CCL3/CCR5 axis in oral carcinogenesis.

PubMed

da Silva, Janine Mayra; Moreira Dos Santos, Tálita Pollyanna; Sobral, Lays Martin; Queiroz-Junior, Celso Martins; Rachid, Milene Alvarenga; Proudfoot, Amanda E I; Garlet, Gustavo Pompermaier; Batista, Aline Carvalho; Teixeira, Mauro Martins; Leopoldino, Andréia Machado; Russo, Remo Castro; Silva, Tarcília Aparecida

2017-08-01

The chemokine CCL3 is a chemotactic cytokine crucial for inflammatory cell recruitment in homeostatic and pathological conditions. CCL3 might stimulate cancer progression by promoting leukocyte accumulation, angiogenesis and tumour growth. The expression of CCL3 and its receptors CCR1 and CCR5 was demonstrated in oral squamous cell carcinoma (OSCC), but their role was not defined. Here, the functions of CCL3 were assessed using a model of chemically induced tongue carcinogenesis with 4-nitroquinoline-1-oxide (4NQO). Lineages of OSCC were used to analyse the effects of CCL3 in vitro . The 4NQO-induced lesions exhibited increased expression of CCL3, CCR1 and CCR5. CCL3 -/- and CCR5 -/- mice presented reduced incidence of tongue tumours compared to wild-type (WT) and CCR1 -/- mice. Consistently, attenuated cytomorphological atypia and reduced cell proliferation were observed in lesions of CCL3 -/- and CCR5 -/- mice. OSCC from CCL3 -/- mice exhibited lower infiltration of eosinophils and reduced expression of Egf, Fgf1, Tgf-β1, Vegfa, Vegfb, Itga-4, Vtn, Mmp-1a, Mmp-2 and Mmp-9 than WT mice. In vitro , CCL3 induced invasion and production of CCL5, IL-6, MMP -2, -8, -9. Blockage of CCL3 in vitro using α-CCL3 or Evasin-1 (a CCL3-binding protein) impaired tumour cell invasion. In conclusion, CCL3/CCR5 axis has pro-tumourigenic effects in oral carcinogenesis. The induction of inflammatory and angiogenic pathways and eosinophils recruitment appear to be the underlying mechanism explaining these effects. These data reveal potential protective effects of CCL3 blockade in oral cancer.

N-acetylcysteine ameliorates contrast‑induced kidney injury in rats with unilateral hydronephrosis.

PubMed

Xia, Qiang; Liu, Chunxiao; Zheng, Xia

2018-02-01

The aim of the present study was to investigate the protective effects of N‑acetylcysteine (NAC) on contrast‑induced acute kidney injury in rats with unilateral hyronephrosis. Eighty‑two male Sprague Dawley rats were randomized to undergo sham operation (n=14) or unilateral ureteral obstruction (UUO) (n=68). After 3 weeks, the UUO animals were randomized to three groups: NAC gastric perfusion, UUO+iohexol+NAC (n=24); normal saline perfusion, UUO+iohexol (n=24); and controls, UUO (n=20). After 3 days, UUO+iohexol+NAC and UUO+iohexol rats were injected with iohexol. One day after contrast, half of the rats were sacrificed to assess the pathological changes to the kidneys, serum creatinine, serum neutrophil gelatinase‑associated lipocalin (NGAL), renal cell apoptosis rate and expression of apoptosis regulators Bcl‑2/Bax. The remaining rats underwent obstruction relief and were analyzed 3 weeks later. Compared with the controls, serum NGAL levels were high in UUO+iohexol rats 1 day following injection and 3 weeks after obstruction relief, but UUO+iohexol+NAC rats exhibited lower serum NGAL levels compared with UUO+iohexol rats (all P<0.05). Following modeling, UUO+iohexol rats exhibited a significantly higher apoptosis rate of renal tubular cells, higher expression of Bax mRNA, and lower ratio of Bcl‑2/Bax (all P<0.05). Three weeks after obstruction relief, UUO+iohexol+NAC rats exhibited a lower apoptosis rate, lower Bax mRNA expression, higher expression of Bcl‑2 mRNA and higher ratio of Bcl‑2/Bax (all P<0.05) compared with day 1 following drug administration. The prophylactic use of NAC reduced the apoptotic rate of renal tubular cells following contrast exposition, which was accompanied by changes in the expression of Bcl‑2/Bax mRNA.

Motor neurons and glia exhibit specific individualized responses to TDP-43 expression in a Drosophila model of amyotrophic lateral sclerosis.

PubMed

Estes, Patricia S; Daniel, Scott G; McCallum, Abigail P; Boehringer, Ashley V; Sukhina, Alona S; Zwick, Rebecca A; Zarnescu, Daniela C

2013-05-01

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by complex neuronal and glial phenotypes. Recently, RNA-based mechanisms have been linked to ALS via RNA-binding proteins such as TDP-43, which has been studied in vivo using models ranging from yeast to rodents. We have developed a Drosophila model of ALS based on TDP-43 that recapitulates several aspects of pathology, including motor neuron loss, locomotor dysfunction and reduced survival. Here we report the phenotypic consequences of expressing wild-type and four different ALS-linked TDP-43 mutations in neurons and glia. We show that TDP-43-driven neurodegeneration phenotypes are dose- and age-dependent. In motor neurons, TDP-43 appears restricted to nuclei, which are significantly misshapen due to mutant but not wild-type protein expression. In glia and in the developing neuroepithelium, TDP-43 associates with cytoplasmic puncta. TDP-43-containing RNA granules are motile in cultured motor neurons, although wild-type and mutant variants exhibit different kinetic properties. At the neuromuscular junction, the expression of TDP-43 in motor neurons versus glia leads to seemingly opposite synaptic phenotypes that, surprisingly, translate into comparable locomotor defects. Finally, we explore sleep as a behavioral readout of TDP-43 expression and find evidence of sleep fragmentation consistent with hyperexcitability, a suggested mechanism in ALS. These findings support the notion that although motor neurons and glia are both involved in ALS pathology, at the cellular level they can exhibit different responses to TDP-43. In addition, our data suggest that individual TDP-43 alleles utilize distinct molecular mechanisms, which will be important for developing therapeutic strategies.

Parkin-mediated mitophagy is downregulated in browning of white adipose tissue.

PubMed

Taylor, David; Gottlieb, Roberta A

2017-04-01

Browning of white adipose tissue (WAT) promotes increased energy expenditure through the action of uncoupling protein 1 (UCP1) and is an attractive target to promote weight loss in obesity. Lowering of mitochondrial membrane potential by UCP1 is uniquely beneficial in this context; in other tissues, reduced membrane potential promotes mitochondrial clearance via mitophagy. It is unknown how parkin-mediated mitophagy is regulated in beige adipocytes. The relationship between parkin expression and WAT browning was investigated in 3T3-L1 adipocytes and parkin-deficient male C57BL/6 mice in response to pharmacological browning stimuli. Rosiglitazone treatment in 3T3-L1 adipocytes promoted mitochondrial biogenesis, UCP1 expression, and mitochondrial uncoupling. Parkin expression was decreased and reduced mitochondrial-associated parkin, and p62 indicated a reduction in mitophagy activity. Parkin overexpression prevented mitochondrial remodeling in response to rosiglitazone. In CL 316,243-treated wild-type mice, decreased parkin expression was observed in subcutaneous inguinal WAT, where UCP1 was strongly induced. CL 316,243 treatment weakly induced UCP1 expression in the gonadal depot, where parkin expression was unchanged. In contrast, parkin-deficient mice exhibited robust UCP1 expression in gonadal WAT following CL 316,243 treatment. WAT browning was associated with a decrease in parkin-mediated mitophagy, and parkin expression antagonized browning of WAT. © 2017 The Obesity Society.

Reduction of CD147 surface expression on primary T cells leads to enhanced cell proliferation.

PubMed

Biegler, Brian; Kasinrerk, Watchara

2012-12-01

CD147 is a ubiquitously expressed membrane glycoprotein that has numerous functional associations in health and disease. However, the molecular mechanisms by which CD147 participates in these processes are unclear. Establishing physiologically relevant silencing of CD147 in primary T cells could provide clues essential for elucidating some aspects of CD147 biology. To date, achieving the knockdown of CD147 in primary T cells has remained elusive. Utilizing RNA interference and the Nucleofector transfection system, we were able to reduce the expression of CD147 in primary T cells. Comparison of basic functions, such as proliferation and CD25 expression, were then made between control populations and populations with reduced expression. Up-regulation of CD147 was found upon T-cell activation, indicating a role in T-cell responses. To better understand the possible importance of this up-regulation, we knocked down the expression of CD147 using RNA interference. When compared to control populations the CD147 knockdown populations exhibited increased proliferation. This alteration of cell proliferation, however, was not linked to a change in CD25 expression. We achieved reduction of CD147 surface expression in primary T cells by siRNA-mediated gene silencing. Our results point to CD147 having a possible negative regulatory role in T cell-mediated immune responses.

Human Fetal Testis Xenografts Are Resistant to Phthalate-Induced Endocrine Disruption

PubMed Central

Heger, Nicholas E; Hall, Susan J; Sandrof, Moses A; McDonnell, Elizabeth V; Hensley, Janan B; McDowell, Erin N; Martin, Kayla A; Gaido, Kevin W; Johnson, Kamin J

2012-01-01

Background: In utero exposure to endocrine-disrupting chemicals may contribute to testicular dysgenesis syndrome (TDS), a proposed constellation of increasingly common male reproductive tract abnormalities (including hypospadias, cryptorchidism, hypospermatogenesis, and testicular cancer). Male rats exposed in utero to certain phthalate plasticizers exhibit multinucleated germ cell (MNG) induction and suppressed steroidogenic gene expression and testosterone production in the fetal testis, causing TDS-consistent effects of hypospadias and cryptorchidism. Mice exposed to phthalates in utero exhibit MNG induction only. This disparity in response demonstrates a species-specific sensitivity to phthalate-induced suppression of fetal Leydig cell steroidogenesis. Importantly, ex vivo phthalate exposure of the fetal testis does not recapitulate the species-specific endocrine disruption, demonstrating the need for a new bioassay to assess the human response to phthalates. Objectives: In this study, we aimed to develop and validate a rat and mouse testis xenograft bioassay of phthalate exposure and examine the human fetal testis response. Methods: Fetal rat, mouse, and human testes were xenografted into immunodeficient rodent hosts, and hosts were gavaged with a range of phthalate doses over multiple days. Xenografts were harvested and assessed for histopathology and steroidogenic end points. Results: Consistent with the in utero response, phthalate exposure induced MNG formation in rat and mouse xenografts, but only rats exhibited suppressed steroidogenesis. Across a range of doses, human fetal testis xenografts exhibited MNG induction but were resistant to suppression of steroidogenic gene expression. Conclusions: Phthalate exposure of grafted human fetal testis altered fetal germ cells but did not reduce expression of genes that regulate fetal testosterone biosynthesis. PMID:22511013

Autolytic activity and molecular characteristics of Staphylococcus haemolyticus strains with induced vancomycin resistance.

PubMed

Kim, Jung Wook; Chung, Gyung Tae; Yoo, Jung Sik; Lee, Yeong Seon; Yoo, Jae Il

2012-10-01

The aim of this study was to investigate the molecular characteristics of induced vancomycin resistance in Staphylococcus haemolyticus. Autolytic properties and phenotypic characteristics of passage-selected vancomycin-resistant S. haemolyticus strains were examined. In addition, expression of autolysis-related genes (atl, lrgAB, sarA and lytS) was investigated using the RNase protection assay (RPA). The RPA results indicated that only the expression of the atl gene was significantly upregulated (2.5- to 6-fold increase) in vancomycin-intermediate and vancomycin-resistant strains. The vancomycin-resistant strains exhibited lower expression of murein hydrolase proteins and reduced autolytic activity compared with the parent strain. In addition, a reduced growth rate, cell wall thickening and higher survival rate in the presence of lysostaphin were observed in vancomycin-intermediate and vancomycin-resistant induced strains compared with the parent strain. In conclusion, altered autolytic properties, in particular upregulation of the atl gene, may contribute to vancomycin resistance in S. haemolyticus.

Endothelial insulin receptor restoration rescues vascular function in male insulin receptor haploinsufficient mice.

PubMed

Sengupta, Anshuman; Patel, Peysh A; Yuldasheva, Nadira Y; Mughal, Romana S; Galloway, Stacey; Viswambharan, Hema; Walker, Andrew M N; Aziz, Amir; Smith, Jessica; Ali, Noman; Mercer, Ben N; Imrie, Helen; Sukumar, Piruthivi; Wheatcroft, Stephen B; Kearney, Mark T; Cubbon, Richard M

2018-05-15

Reduced systemic insulin signaling promotes endothelial dysfunction and diminished endogenous vascular repair. We asked whether restoration of endothelial insulin receptor expression could rescue this phenotype. Insulin receptor haploinsufficient mice (IRKO) were crossed with mice expressing a human insulin receptor transgene in the endothelium (hIRECO), to produce IRKO-hIRECO progeny. No metabolic differences were noted between IRKO and IRKO-hIRECO in glucose- and insulin-tolerance tests. In contrast with control IRKO littermates, IRKO-hIRECO exhibited normal blood pressure and aortic vasodilatation in response to acetylcholine, comparable to parameters noted in wild-type littermates. These phenotypic changes were associated with enhanced basal- and insulin-stimulated nitric oxide production. IRKO-hIRECO also demonstrated normalized endothelial repair after denuding arterial injury, which was associated with rescued endothelial cell migration in vitro, but not with changes in circulating progenitor populations or culture-derived myeloid angiogenic cells. These data show that restoration of endothelial insulin receptor expression alone is sufficient to prevent the vascular dysfunction caused by systemically reduced insulin signaling.

Disruption of nucleotide excision repair by the human T-cell leukemia virus type 1 Tax protein.

PubMed

Kao, S Y; Marriott, S J

1999-05-01

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a transcriptional transactivator and viral oncogene. Since cellular transformation has been frequently linked to alterations in genome stability, we investigated the effect of Tax on nucleotide excision repair (NER), a prominent cellular DNA repair pathway. Cells expressing Tax exhibited a reduced capacity for NER as measured by unscheduled DNA synthesis and host cell reactivation assays. The cellular proliferating cell nuclear antigen (PCNA) gene product regulates DNA replication and repair pathways, including NER. Since Tax activates transcription of the PCNA promoter, we investigated whether this activity contributes to the reduction of NER. Tax increased endogenous PCNA protein expression, and analysis of Tax mutant proteins demonstrated that the reduction in NER correlated with Tax transactivation of PCNA gene expression. Direct overexpression of PCNA also reduced NER. We propose that overexpression of PCNA, and disruption of NER induced by Tax, predisposes cells to accumulate DNA damage and contributes to HTLV-1 transformation.

75 FR 82128 - Culturally Significant Objects Imported for Exhibition Determinations: “The Orient Expressed...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-29

... DEPARTMENT OF STATE [Public Notice: 7277] Culturally Significant Objects Imported for Exhibition Determinations: ``The Orient Expressed: Japan's Influence on Western Art, 1854-1918'' SUMMARY: Notice is hereby... hereby determine that the objects to be included in the exhibition ``The Orient Expressed: Japan's...

Transcriptomic imprints of adaptation to fresh water: parallel evolution of osmoregulatory gene expression in the Alewife

USGS Publications Warehouse

Velotta, Jonathan P.; Wegrzyn, Jill L.; Ginzburg, Samuel; Kang, Lin; Czesny, Sergiusz J.; O'Neill, Rachel J.; McCormick, Stephen; Michalak, Pawel; Schultz, Eric T.

2017-01-01

Comparative approaches in physiological genomics offer an opportunity to understand the functional importance of genes involved in niche exploitation. We used populations of Alewife (Alosa pseudoharengus) to explore the transcriptional mechanisms that underlie adaptation to fresh water. Ancestrally anadromous Alewives have recently formed multiple, independently derived, landlocked populations, which exhibit reduced tolerance of saltwater and enhanced tolerance of fresh water. Using RNA-seq, we compared transcriptional responses of an anadromous Alewife population to two landlocked populations after acclimation to fresh (0 ppt) and saltwater (35 ppt). Our results suggest that the gill transcriptome has evolved in primarily discordant ways between independent landlocked populations and their anadromous ancestor. By contrast, evolved shifts in the transcription of a small suite of well-characterized osmoregulatory genes exhibited a strong degree of parallelism. In particular, transcription of genes that regulate gill ion exchange has diverged in accordance with functional predictions: freshwater ion-uptake genes (most notably, the ‘freshwater paralog’ of Na+/K+-ATPase α-subunit) were more highly expressed in landlocked forms, whereas genes that regulate saltwater ion secretion (e.g. the ‘saltwater paralog’ of NKAα) exhibited a blunted response to saltwater. Parallel divergence of ion transport gene expression is associated with shifts in salinity tolerance limits among landlocked forms, suggesting that changes to the gill's transcriptional response to salinity facilitate freshwater adaptation.

Attachment, invasion, chemotaxis, and proteinase expression of B16-BL6 melanoma cells exhibiting a low metastatic phenotype after exposure to dietary restriction of tyrosine and phenylalanine.

PubMed

Uhlenkott, C E; Huijzer, J C; Cardeiro, D J; Elstad, C A; Meadows, G G

1996-03-01

We previously reported that low levels of tyrosine (Tyr) and phenylalanine (Phe) alter the metastatic phenotype of B16-BL6 (BL6) murine melanoma and select for tumor cell populations with decreased lung colonizing ability. To more specifically characterize the effects of Tyr and Phe restriction on the malignant phenotype of BL6, we investigated in vitro attachment, invasion, proteinase expression, and chemotaxis of high and low metastatic BL6 variants. High metastatic variant cells were isolated from subcutaneous tumors of mice fed a nutritionally complete diet (ND cells) and low metastatic variant cells were isolated from mice fed a diet restricted in Tyr and Phe (LTP cells). Results indicate that attachment to reconstituted basement membrane (Matrigel) was significantly reduced in LTP cells as compared to ND cells. Attachment to collagen IV, laminin, and fibronectin were similar between the two variants. Invasion through Matrigel and growth factor-reduced Matrigel were significantly decreased in LTP cells as compared to ND cells. Zymography revealed the presence of M(r) 92,000 and M(r) 72,000 progelatinases, tissue plasminogen activator, and urokinase plasminogen activator in the conditioned medium of both variants; however, there were no differences in activity of these secreted proteinases between the two variants. Growth of the variants on growth factor-reduced Matrigel similarly induced expression of the M(r) 92,000 progelatinase. The variants exhibited similar chemotactic responses toward laminin. However, the chemotactic response toward fibronectin by LTP cells was significantly increased. MFR5, a monoclonal antibody which selectively blocks function of the alpha 5 chain of the alpha 5 beta 1 integrin, VLA-5, decreased the chemotactic response toward fibronectin of ND cells by 37%; the chemotactic response by LTP cells was reduced by 49%. This effect was specific for fibronectin-mediated chemotaxis since the chemotaxis toward laminin and invasion through Matrigel were not altered by the presence of MFR5. The surface expression of VLA-5 was significantly increased in LTP cells as compared to ND cells by flow cytometric analysis. These observations suggest that limitation of Tyr and Phe either directly modifies BL6 or selects for subpopulations with altered in vitro invasion, chemotaxis, and integrin expression.

Immediate early gene expression reveals interactions between social and nicotine rewards on brain activity in adolescent male rats

PubMed Central

Goenaga, Julianna; Hatch, Kayla N.; Henricks, Angela; Scott, Samantha; Hood, Lauren E.; Neisewander, Janet L.

2016-01-01

Smoking initiation predominantly occurs during adolescence, often in the presence of peers. Therefore, understanding the neural mechanisms underlying the rewarding effects of nicotine and social stimuli is vital. Using the conditioned place preference (CPP) procedure, we measured immediate early gene (IEG) expression in animals following exposure either to a reward-conditioned environment or to the unconditioned stimuli (US). Adolescent, male rats were assigned to the following CPP US conditions: (1) Saline + Isolated, (2) Nicotine + Isolated, (3) Saline + Social, or (4) Nicotine + Social. For Experiment 1, brain tissue was collected 90 min following the CPP expression test and processed for Fos immunohistochemistry. We found that rats conditioned with nicotine with or without a social partner exhibited CPP; however, we found no group differences in Fos expression in any brain region analyzed, with the exception of the nucleus accumbens core that exhibited a social-induced attenuation in Fos expression. For Experiment 2, brain tissue was collected 90 min following US exposure during the last conditioning session. We found social reward-induced increases in IEG expression in striatal and amydalar subregions. In contrast, nicotine reduced IEG expression in prefrontal and striatal subregions. Reward interactions were also found in the dorsolateral striatum, basolateral amygdala, and ventral tegmental area where nicotine alone attenuated IEG expression and social reward reversed this effect. These results suggest that in general social rewards enhance, whereas nicotine attenuates, activation of mesocorticolimbic regions; however, the rewards given together interact to enhance activation in some regions. The findings contribute to knowledge of how a social environment influences nicotine effects. PMID:27435419

Feline immunodeficiency virus OrfA alters gene expression of splicing factors and proteasome-ubiquitination proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sundstrom, Magnus; Chatterji, Udayan; Schaffer, Lana

2008-02-20

Expression of the feline immunodeficiency virus (FIV) accessory protein OrfA (or Orf2) is critical for efficient viral replication in lymphocytes, both in vitro and in vivo. OrfA has been reported to exhibit functions in common with the human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) accessory proteins Vpr and Tat, although the function of OrfA has not been fully explained. Here, we use microarray analysis to characterize how OrfA modulates the gene expression profile of T-lymphocytes. The primary IL-2-dependent T-cell line 104-C1 was transduced to express OrfA. Functional expression of OrfA was demonstrated by trans complementation of the OrfA-defectivemore » clone, FIV-34TF10. OrfA-expressing cells had a slightly reduced cell proliferation rate but did not exhibit any significant alteration in cell cycle distribution. Reverse-transcribed RNA from cells expressing green fluorescent protein (GFP) or GFP + OrfA were hybridized to Affymetrix HU133 Plus 2.0 microarray chips representing more than 47,000 genome-wide transcripts. By using two statistical approaches, 461 (Rank Products) and 277 (ANOVA) genes were identified as modulated by OrfA expression. The functional relevance of the differentially expressed genes was explored by Ingenuity Pathway Analysis. The analyses revealed alterations in genes critical for RNA post-transcriptional modifications and protein ubiquitination as the two most significant functional outcomes of OrfA expression. In these two groups, several subunits of the spliceosome, cellular splicing factors and family members of the proteasome-ubiquitination system were identified. These findings provide novel information on the versatile function of OrfA during FIV infection and indicate a fine-tuning mechanism of the cellular environment by OrfA to facilitate efficient FIV replication.« less

Excessive fluoride reduces Foxo1 expression in dental epithelial cells of the rat incisor.

PubMed

Gao, Jianghong; Ruan, Jianping; Gao, Liping

2014-10-01

Enamel fluorosis is characterized by hypomineralization, and forkhead box O1 (Foxo1) is essential for mouse enamel biomineralization. This study investigated the effect of fluoride on Foxo1 expression and its implications for enamel fluorosis. Mandibular incisors were extracted from Sprague Dawley rats treated for 3 months with water containing 0, 50, or 100 p.p.m. F⁻. Immunohistochemistry was used to localize and quantify FOXO1 expression in dental epithelial layer cells of the incisors. The effect of fluoride on expression of Foxo1, kallikrein-4 (Klk4), and amelotin (Amtn) mRNAs was analyzed by real-time RT-PCR, and western blotting was used to measure total and nuclear FOXO1 protein levels in mature dental epithelial cells. The results revealed that nuclear FOXO1 was mainly localized in the transition and the mature ameloblasts and exhibited weaker expression in the rats exposed to fluoride. In addition to the reduced levels of Foxo1, Klk4, and AmtnmRNAs, the protein levels of total and nuclearFOXO1 were decreased in the mature dental epithelial cells exposed to fluoride. Thus, excessive fluoride may have an effect on the expression levels of Foxo1 in dental epithelial cells and thereby affect hypomineralization of the enamel during fluorosis. © 2014 Eur J Oral Sci.

Increased SHP-1 Protein Expression by High Glucose Levels Reduces Nephrin Phosphorylation in Podocytes*

PubMed Central

Denhez, Benoit; Lizotte, Farah; Guimond, Marie-Odile; Jones, Nina; Takano, Tomoko; Geraldes, Pedro

2015-01-01

Nephrin, a critical podocyte membrane component that is reduced in diabetic nephropathy, has been shown to activate phosphotyrosine signaling pathways in human podocytes. Nephrin signaling is important to reduce cell death induced by apoptotic stimuli. We have shown previously that high glucose level exposure and diabetes increased the expression of SHP-1, causing podocyte apoptosis. SHP-1 possesses two Src homology 2 domains that serve as docking elements to dephosphorylate tyrosine residues of target proteins. However, it remains unknown whether SHP-1 interacts with nephrin and whether its elevated expression affects the nephrin phosphorylation state in diabetes. Here we show that human podocytes exposed to high glucose levels exhibited elevated expression of SHP-1, which was associated with nephrin. Coexpression of nephrin-CD16 and SHP-1 reduced nephrin tyrosine phosphorylation in transfected human embryonic kidney 293 cells. A single tyrosine-to-phenylalanine mutation revealed that rat nephrin Tyr1127 and Tyr1152 are required to allow SHP-1 interaction with nephrin. Overexpression of dominant negative SHP-1 in human podocytes prevented high glucose-induced reduction of nephrin phosphorylation. In vivo, immunoblot analysis demonstrated that nephrin expression and phosphorylation were decreased in glomeruli of type 1 diabetic Akita mice (Ins2+/C96Y) compared with control littermate mice (Ins2+/+), and this was associated with elevated SHP-1 and cleaved caspase-3 expression. Furthermore, immunofluorescence analysis indicated increased colocalization of SHP-1 with nephrin in diabetic mice compared with control littermates. In conclusion, our results demonstrate that high glucose exposure increases SHP-1 interaction with nephrin, causing decreased nephrin phosphorylation, which may, in turn, contribute to diabetic nephropathy. PMID:25404734

Acid-Sensing Ion Channels Expression, Identity and Role in the Excitability of the Cochlear Afferent Neurons

PubMed Central

González-Garrido, Antonia; Vega, Rosario; Mercado, Francisco; López, Iván A.; Soto, Enrique

2015-01-01

Acid-sensing ion channels (ASICs) are activated by an increase in the extracellular proton concentration. There are four genes (ASIC1-4) that encode six subunits, and they are involved in diverse neuronal functions, such as mechanosensation, learning and memory, nociception, and modulation of retinal function. In this study, we characterize the ASIC currents of spiral ganglion neurons (SGNs). These ASIC currents are primarily carried by Na+, exhibit fast activation and desensitization, display a pH50 of 6.2 and are blocked by amiloride, indicating that these are ASIC currents. The ASIC currents were further characterized using several pharmacological tools. Gadolinium and acetylsalicylic acid reduced these currents, and FMRFamide, zinc (at high concentrations) and N,N,N’,N’–tetrakis-(2-piridilmetil)-ethylenediamine increased them, indicating that functional ASICs are composed of the subunits ASIC1, ASIC2, and ASIC3. Neomycin and streptomycin reduced the desensitization rate of the ASIC current in SGNs, indicating that ASICs may contribute to the ototoxic action of aminoglycosides. RT-PCR of the spiral ganglion revealed significant expression of all ASIC subunits. By immunohistochemistry the expression of the ASIC1a, ASIC2a, ASIC2b, and ASIC3 subunits was detected in SGNs. Although only a few SGNs exhibited action potential firing in response to an acidic stimulus, protons in the extracellular solution modulated SGN activity during sinusoidal stimulation. Our results show that protons modulate the excitability of SGNs via ASICs. PMID:26733809

Down regulation of miR-124 in both Werner syndrome DNA helicase mutant mice and mutant Caenorhabditis elegans wrn-1 reveals the importance of this microRNA in accelerated aging

PubMed Central

Dallaire, Alexandra; Garand, Chantal; Paquet, Eric R.; Mitchell, Sarah J.; de Cabo, Rafael; Simard, Martin J.

2012-01-01

Small non-coding microRNAs are believed to be involved in the mechanism of aging but nothing is known on the impact of microRNAs in the progeroid disorder Werner syndrome (WS). WS is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN ortholog exhibit many phenotypic features of WS, including a pro-oxidant status and a shorter mean life span. Caenorhabditis elegans (C. elegans) with a nonfunctional wrn-1 DNA helicase also exhibit a shorter life span. Thus, both models are relevant to study the expression of microRNAs involved in WS. In this study, we show that miR-124 expression is lost in the liver of Wrn helicase mutant mice. Interestingly, the expression of this conserved miR-124 in whole wrn-1 mutant worms is also significantly reduced. The loss of mir-124 in C. elegans increases reactive oxygen species formation and accumulation of the aging marker lipofuscin, reduces whole body ATP levels and results in a reduction in life span. Finally, supplementation of vitamin C normalizes the median life span of wrn-1 and mir-124 mutant worms. These results suggest that biological pathways involving WRN and miR-124 are conserved in the aging process across different species. PMID:23075628

Deficiency in Nrf2 transcription factor decreases adipose tissue mass and hepatic lipid accumulation in leptin-deficient mice.

PubMed

Xu, Jialin; Donepudi, Ajay C; More, Vijay R; Kulkarni, Supriya R; Li, Liya; Guo, Liangran; Yan, Bingfang; Chatterjee, Tapan; Weintraub, Neal; Slitt, Angela L

2015-02-01

To evaluate whether Nrf2 deficiency impacts insulin resistance and lipid accumulation in liver and white adipose tissue. Lep(ob/ob) mice (OB) with targeted Nrf2 deletion (OB-Nrf2KO) were generated. Pathogenesis of obesity and type 2 diabetes was measured in C57BL/6J, Nrf2KO, OB, and OB-Nrf2KO mice. Hepatic lipid content, lipid clearance, and very low-density lipoprotein (VLDL) secretion were determined between OB and OB-Nrf2KO mice. OB-Nrf2KO mice exhibited decreased white adipose tissue mass and decreased adipogenic and lipogenic gene expression compared with OB mice. Nrf2 deficiency prolonged hyperglycemia in response to glucose challenge, which was paralleled by reduced insulin-stimulated Akt phosphorylation. In OB mice, Nrf2 deficiency decreased hepatic lipid accumulation, decreased peroxisome proliferator-activated receptor γ expression and nicotinamide adenine dinucleotide phosphate (NADPH) content, and enhanced VLDL secretion. However, this observation was opposite in lean mice. Additionally, OB-Nrf2KO mice exhibited increased plasma triglyceride content, decreased HDL-cholesterol content, and enhanced apolipoprotein B expression, suggesting Nrf2 deficiency caused dyslipidemia in these mice. Nrf2 deficiency in Lep(ob/ob) mice reduced white adipose tissue mass and prevented hepatic lipid accumulation but induced insulin resistance and dyslipidemia. This study indicates a dual role of Nrf2 during metabolic dysregulation-increasing lipid accumulation in liver and white adipose tissue but preventing lipid accumulation in obese mice. © 2014 The Obesity Society.

Def-6, a novel regulator of small GTPases in podocytes, acts downstream of atypical protein kinase C (aPKC) λ/ι.

PubMed

Worthmann, Kirstin; Leitges, Michael; Teng, Beina; Sestu, Marcello; Tossidou, Irini; Samson, Thomas; Haller, Hermann; Huber, Tobias B; Schiffer, Mario

2013-12-01

The atypical protein kinase C (aPKC) isotypes PKCλ/ι and PKCζ are both expressed in podocytes; however, little is known about differences in their function. Previous studies in mice have demonstrated that podocyte-specific loss of PKCλ/ι leads to a severe glomerular phenotype, whereas mice deficient in PKCζ develop no renal phenotype. We analyzed various effects caused by PKCλ/ι and PKCζ deficiency in cultured murine podocytes. In contrast to PKCζ-deficient podocytes, PKCλ/ι-deficient podocytes exhibited a severe actin cytoskeletal phenotype, reduced cell size, decreased number of focal adhesions, and increased activation of small GTPases. Comparative microarray analysis revealed that the guanine nucleotide exchange factor Def-6 was specifically up-regulated in PKCλ/ι-deficient podocytes. In vivo Def-6 expression is significantly increased in podocytes of PKCλ/ι-deficient mice. Cultured PKCλ/ι-deficient podocytes exhibited an enhanced membrane association of Def-6, indicating enhanced activation. Overexpression of aPKCλ/ι in PKCλ/ι-deficient podocytes could reduce the membrane-associated expression of Def-6 and rescue the actin phenotype. In the present study, PKCλ/ι was identified as an important factor for actin cytoskeletal regulation in podocytes and Def-6 as a specific downstream target of PKCλ/ι that regulates the activity of small GTPases and subsequently the actin cytoskeleton of podocytes. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Deletion of Tsc2 in Nociceptors Reduces Target Innervation, Ion Channel Expression, and Sensitivity to Heat

PubMed Central

Carlin, Dan; Golden, Judith P.; Monk, Kelly R.

2018-01-01

Abstract The mechanistic target of rapamycin complex 1 (mTORC1) is known to regulate cellular growth pathways, and its genetic activation is sufficient to enhance regenerative axon growth following injury to the central or peripheral nervous systems. However, excess mTORC1 activation may promote innervation defects, and mTORC1 activity mediates injury-induced hypersensitivity, reducing enthusiasm for the pathway as a therapeutic target. While mTORC1 activity is required for full expression of some pain modalities, the effects of pathway activation on nociceptor phenotypes and sensory behaviors are currently unknown. To address this, we genetically activated mTORC1 in mouse peripheral sensory neurons by conditional deletion of its negative regulator Tuberous Sclerosis Complex 2 (Tsc2). Consistent with the well-known role of mTORC1 in regulating cell size, soma size and axon diameter of C-nociceptors were increased in Tsc2-deleted mice. Glabrous skin and spinal cord innervation by C-fiber neurons were also disrupted. Transcriptional profiling of nociceptors enriched by fluorescence-associated cell sorting (FACS) revealed downregulation of multiple classes of ion channels as well as reduced expression of markers for peptidergic nociceptors in Tsc2-deleted mice. In addition to these changes in innervation and gene expression, Tsc2-deleted mice exhibited reduced noxious heat sensitivity and decreased injury-induced cold hypersensitivity, but normal baseline sensitivity to cold and mechanical stimuli. Together, these data show that excess mTORC1 activity in sensory neurons produces changes in gene expression, neuron morphology and sensory behavior. PMID:29766046

Reduced mitochondrial SOD displays mortality characteristics reminiscent of natural aging

PubMed Central

Paul, Anirban; Belton, Amy; Nag, Sanjay; Martin, Ian; Grotewiel, Michael S.; Duttaroy, Atanu

2009-01-01

Manganese superoxide dismutase (MnSOD or SOD2) is a key mitochondrial enzymatic antioxidant. Arguably the most striking phenotype associated with complete loss of SOD2 in flies and mice is shortened life span. To further explore the role of SOD2 in protecting animals from aging and age-associated pathology, we generated a unique collection of Drosophila mutants that progressively reduce SOD2 expression and function. Mitochondrial aconitase activity was substantially reduced in the Sod2 mutants, suggesting that SOD2 normally ensures the functional capacity of mitochondria. Flies with severe reductions in SOD2 expression exhibited accelerated senescence of olfactory behavior as well as precocious neurodegeneration and DNA strand breakage in neurons. Furthermore, life span was progressively shortened and age-dependent mortality was increased in conjunction with reduced SOD2 expression, while initial mortality and developmental viability were unaffected. Interestingly, life span and age-dependent mortality varied exponentially with SOD2 activity, indicating that there might normally be a surplus of this enzyme for protecting animals from premature death. Our data support a model in which disruption of the protective effects of SOD2 on mitochondria manifests as profound changes in behavioral and demographic aging as well as exacerbated age-related pathology in the nervous system. PMID:18078670

Mice with reduced brain-derived neurotrophic factor expression show decreased choline acetyltransferase activity, but regular brain monoamine levels and unaltered emotional behavior.

PubMed

Chourbaji, Sabine; Hellweg, Rainer; Brandis, Dorothee; Zörner, Björn; Zacher, Christiane; Lang, Undine E; Henn, Fritz A; Hörtnagl, Heide; Gass, Peter

2004-02-05

The "neurotrophin hypothesis" of depression predicts that depressive disorders in humans coincide with a decreased activity and/or expression of brain-derived neurotrophic factor (BDNF) in the brain. Therefore, we investigated whether mice with a reduced BDNF expression due to heterozygous gene disruption demonstrate depression-like neurochemical changes or behavioral symptoms. BNDF protein levels of adult BDNF(+/-) mice were reduced to about 60% in several brain areas investigated, including the hippocampus, frontal cortex, striatum, and hypothalamus. The content of monoamines (serotonin, norepinephrine, and dopamine) as well as of serotonin and dopamine degradation products was unchanged in these brain regions. By contrast, choline acetyltransferase activity was significantly reduced by 19% in the hippocampus of BDNF(+/-) mice, indicating that the cholinergic system of the basal forebrain is critically dependent on sufficient endogenous BDNF levels in adulthood. Moreover, BDNF(+/-) mice exhibited normal corticosterone and adrenocorticotropic hormone (ACTH) serum levels under baseline conditions and following immobilization stress. In a panel of behavioral tests investigating locomotor activity, exploration, anxiety, fear-associated learning, and behavioral despair, BDNF(+/-) mice were indistinguishable from wild-type littermates. Thus, a chronic reduction of BDNF protein content in adult mice is not sufficient to induce neurochemical or behavioral alterations that are reminiscent of depressive symptoms in humans.

Discovery and Characterization of the 3-Hydroxyacyl-ACP Dehydratase Component of the Plant Mitochondrial Fatty Acid Synthase System1[OPEN

PubMed Central

Okazaki, Yozo; Lithio, Andrew; Jin, Huanan

2017-01-01

We report the characterization of the Arabidopsis (Arabidopsis thaliana) 3-hydroxyacyl-acyl carrier protein dehydratase (mtHD) component of the mitochondrial fatty acid synthase (mtFAS) system, encoded by AT5G60335. The mitochondrial localization and catalytic capability of mtHD were demonstrated with a green fluorescent protein transgenesis experiment and by in vivo complementation and in vitro enzymatic assays. RNA interference (RNAi) knockdown lines with reduced mtHD expression exhibit traits typically associated with mtFAS mutants, namely a miniaturized morphological appearance, reduced lipoylation of lipoylated proteins, and altered metabolomes consistent with the reduced catalytic activity of lipoylated enzymes. These alterations are reversed when mthd-rnai mutant plants are grown in a 1% CO2 atmosphere, indicating the link between mtFAS and photorespiratory deficiency due to the reduced lipoylation of glycine decarboxylase. In vivo biochemical feeding experiments illustrate that sucrose and glycolate are the metabolic modulators that mediate the alterations in morphology and lipid accumulation. In addition, both mthd-rnai and mtkas mutants exhibit reduced accumulation of 3-hydroxytetradecanoic acid (i.e. a hallmark of lipid A-like molecules) and abnormal chloroplastic starch granules; these changes are not reversible by the 1% CO2 atmosphere, demonstrating two novel mtFAS functions that are independent of photorespiration. Finally, RNA sequencing analysis revealed that mthd-rnai and mtkas mutants are nearly equivalent to each other in altering the transcriptome, and these analyses further identified genes whose expression is affected by a functional mtFAS system but independent of photorespiratory deficiency. These data demonstrate the nonredundant nature of the mtFAS system, which contributes unique lipid components needed to support plant cell structure and metabolism. PMID:28202596

Comparative Studies on Behavioral, Cognitive and Biomolecular Profiling of ICR, C57BL/6 and Its Sub-Strains Suitable for Scopolamine-Induced Amnesic Models

PubMed Central

Karthivashan, Govindarajan; Park, Shin-Young; Kim, Joon-Soo; Cho, Duk-Yeon

2017-01-01

Cognitive impairment and behavioral disparities are the distinctive baseline features to investigate in most animal models of neurodegenerative disease. However, neuronal complications are multifactorial and demand a suitable animal model to investigate their underlying basal mechanisms. By contrast, the numerous existing neurodegenerative studies have utilized various animal strains, leading to factual disparity. Choosing an optimal mouse strain for preliminary assessment of neuronal complications is therefore imperative. In this study, we systematically compared the behavioral, cognitive, cholinergic, and inflammatory impairments of outbred ICR and inbred C57BL/6 mice strains subject to scopolamine-induced amnesia. We then extended this study to the sub-strains C57BL/6N and C57BL/6J, where in addition to the above-mentioned parameters, their endogenous antioxidant levels and cAMP response-element binding protein (CREB)/brain-derived neurotrophic factor (BDNF) protein expression were also evaluated. Compared with the ICR strain, the scopolamine-inflicted C57BL/6 strains exhibited a substantial reduction of spontaneous alternation and an approximately two-fold increase in inflammatory protein expression, compared to the control group. Among the sub-strains, scopolamine-treated C57BL/6N strains exhibited declined step-through latency, elevated acetylcholinesterase (AChE) activity and inflammatory protein expression, associated with reduced endogenous antioxidant levels and p-CREB/BDNF expression, compared to the control and tacrine-treated groups. This indicates that the C57BL/6N strains exhibit significantly enhanced scopolamine-induced neuronal impairment compared to the other evaluated strains. PMID:28792471

Comparative Studies on Behavioral, Cognitive and Biomolecular Profiling of ICR, C57BL/6 and Its Sub-Strains Suitable for Scopolamine-Induced Amnesic Models.

PubMed

Karthivashan, Govindarajan; Park, Shin-Young; Kim, Joon-Soo; Cho, Duk-Yeon; Ganesan, Palanivel; Choi, Dong-Kug

2017-08-09

Cognitive impairment and behavioral disparities are the distinctive baseline features to investigate in most animal models of neurodegenerative disease. However, neuronal complications are multifactorial and demand a suitable animal model to investigate their underlying basal mechanisms. By contrast, the numerous existing neurodegenerative studies have utilized various animal strains, leading to factual disparity. Choosing an optimal mouse strain for preliminary assessment of neuronal complications is therefore imperative. In this study, we systematically compared the behavioral, cognitive, cholinergic, and inflammatory impairments of outbred ICR and inbred C57BL/6 mice strains subject to scopolamine-induced amnesia. We then extended this study to the sub-strains C57BL/6N and C57BL/6J, where in addition to the above-mentioned parameters, their endogenous antioxidant levels and cAMP response-element binding protein (CREB)/brain-derived neurotrophic factor (BDNF) protein expression were also evaluated. Compared with the ICR strain, the scopolamine-inflicted C57BL/6 strains exhibited a substantial reduction of spontaneous alternation and an approximately two-fold increase in inflammatory protein expression, compared to the control group. Among the sub-strains, scopolamine-treated C57BL/6N strains exhibited declined step-through latency, elevated acetylcholinesterase (AChE) activity and inflammatory protein expression, associated with reduced endogenous antioxidant levels and p-CREB/BDNF expression, compared to the control and tacrine-treated groups. This indicates that the C57BL/6N strains exhibit significantly enhanced scopolamine-induced neuronal impairment compared to the other evaluated strains.

Redundancy in Kiss1 Expression Safeguards Reproduction in the Mouse

PubMed Central

Popa, Simina M.; Moriyama, Ryutaro M.; Caligioni, Claudia S.; Yang, Jasmine J.; Cho, Caroline M.; Concepcion, Tessa L.; Oakley, Amy E.; Lee, In Hae; Sanz, Elisenda; Amieux, Paul S.; Caraty, Alain; Palmiter, Richard D.; Navarro, Victor M.; Chan, Yee-Ming; Seminara, Stephanie B.; Clifton, Donald K.

2013-01-01

Kisspeptin (Kiss1) signaling to GnRH neurons is widely acknowledged to be a prerequisite for puberty and reproduction. Animals lacking functional genes for either kisspeptin or its receptor exhibit low gonadotropin secretion and infertility. Paradoxically, a recent study reported that genetic ablation of nearly all Kiss1-expressing neurons (Kiss1 neurons) does not impair reproduction, arguing that neither Kiss1 neurons nor their products are essential for sexual maturation. We posited that only minute quantities of kisspeptin are sufficient to support reproduction. If this were the case, animals having dramatically reduced Kiss1 expression might retain fertility, testifying to the redundancy of Kiss1 neurons and their products. To test this hypothesis and to determine whether males and females differ in the required amount of kisspeptin needed for reproduction, we used a mouse (Kiss1-CreGFP) that has a severe reduction in Kiss1 expression. Mice that are heterozygous and homozygous for this allele (Kiss1Cre/+ and Kiss1Cre/Cre) have ∼50% and 95% reductions in Kiss1 transcript, respectively. We found that although male Kiss1Cre/Cre mice sire normal-sized litters, female Kiss1Cre/Cre mice exhibit significantly impaired fertility and ovulation. These observations suggest that males require only 5% of normal Kiss1 expression to be reproductively competent, whereas females require higher levels for reproductive success. PMID:23736293

The collagen receptor DDR2 regulates proliferation and its elimination leads to dwarfism

PubMed Central

Labrador, Juan Pablo; Azcoitia, Valeria; Tuckermann, Jan; Lin, Calvin; Olaso, Elvira; Mañes, Santos; Brückner, Katja; Goergen, Jean-Louis; Lemke, Greg; Yancopoulos, George; Angel, Peter; Martínez-A, Carlos; Klein, Rüdiger

2001-01-01

The discoidin domain receptor 2 (DDR2) is a member of a subfamily of receptor tyrosine kinases whose ligands are fibrillar collagens, and is widely expressed in postnatal tissues. We have generated DDR2-deficient mice to establish the in vivo functions of this receptor, which have remained obscure. These mice exhibit dwarfism and shortening of long bones. This phenotype appears to be caused by reduced chondrocyte proliferation, rather than aberrant differentiation or function. In a skin wound healing model, DDR2–/– mice exhibit a reduced proliferative response compared with wild-type littermates. In vitro, fibroblasts derived from DDR2–/– mutants proliferate more slowly than wild-type fibroblasts, a defect that is rescued by introduction of wild-type but not kinase-dead DDR2 receptor. Together our results suggest that DDR2 acts as an extracellular matrix sensor to modulate cell proliferation. PMID:11375938

Following the 'tracks': Tramtrack69 regulates epithelial tube expansion in the Drosophila ovary through Paxillin, Dynamin, and the homeobox protein Mirror.

PubMed

Peters, Nathaniel C; Thayer, Nathaniel H; Kerr, Scott A; Tompa, Martin; Berg, Celeste A

2013-06-15

Epithelial tubes are the infrastructure for organs and tissues, and tube morphogenesis requires precise orchestration of cell signaling, shape, migration, and adhesion. Follicle cells in the Drosophila ovary form a pair of epithelial tubes whose lumens act as molds for the eggshell respiratory filaments, or dorsal appendages (DAs). DA formation is a robust and accessible model for studying the patterning, formation, and expansion of epithelial tubes. Tramtrack69 (TTK69), a transcription factor that exhibits a variable embryonic DNA-binding preference, controls DA lumen volume and shape by promoting tube expansion; the tramtrack mutation twin peaks (ttk(twk)) reduces TTK69 levels late in oogenesis, inhibiting this expansion. Microarray analysis of wild-type and ttk(twk) ovaries, followed by in situ hybridization and RNAi of candidate genes, identified the Phospholipase B-like protein Lamina ancestor (LAMA), the scaffold protein Paxillin, the endocytotic regulator Shibire (Dynamin), and the homeodomain transcription factor Mirror, as TTK69 effectors of DA-tube expansion. These genes displayed enriched expression in DA-tube cells, except lama, which was expressed in all follicle cells. All four genes showed reduced expression in ttk(twk) mutants and exhibited RNAi phenotypes that were enhanced in a ttk(twk)/+ background, indicating ttk(twk) genetic interactions. Although previous studies show that Mirror patterns the follicular epithelium prior to DA tubulogenesis, we show that Mirror has an independent, novel role in tube expansion, involving positive regulation of Paxillin. Thus, characterization of ttk(twk)-differentially expressed genes expands the network of TTK69 effectors, identifies novel epithelial tube-expansion regulators, and significantly advances our understanding of this vital developmental process. Copyright © 2013 Elsevier Inc. All rights reserved.

SCN3A deficiency associated with increased seizure susceptibility

PubMed Central

Lamar, Tyra; Vanoye, Carlos G.; Calhoun, Jeffrey; Wong, Jennifer C.; Dutton, Stacey B.B.; Jorge, Benjamin S.; Velinov, Milen; Escayg, Andrew; Kearney, Jennifer A.

2017-01-01

Mutations in voltage-gated sodium channels expressed highly in the brain (SCN1A, SCN2A, SCN3A, and SCN8A) are responsible for an increasing number of epilepsy syndromes. In particular, mutations in the SCN3A gene, encoding the pore-forming Nav1.3 α subunit, have been identified in patients with focal epilepsy. Biophysical characterization of epilepsy-associated SCN3A variants suggests that both gain- and loss-of-function SCN3A mutations may lead to increased seizure susceptibility. In this report, we identified a novel SCN3A variant (L247P) by whole exome sequencing of a child with focal epilepsy, developmental delay, and autonomic nervous system dysfunction. Voltage clamp analysis showed no detectable sodium current in a heterologous expression system expressing the SCN3A-L247P variant. Furthermore, cell surface biotinylation demonstrated a reduction in the amount of SCN3A-L247P at the cell surface, suggesting the SCN3A-L247P variant is a trafficking-deficient mutant. To further explore the possible clinical consequences of reduced SCN3A activity, we investigated the effect of a hypomorphic Scn3a allele (Scn3aHyp) on seizure susceptibility and behavior using a gene trap mouse line. Heterozygous Scn3a mutant mice (Scn3a+/Hyp) did not exhibit spontaneous seizures nor were they susceptible to hyperthermia-induced seizures. However, they displayed increased susceptibility to electroconvulsive (6 Hz) and chemiconvulsive (flurothyl and kainic acid) induced seizures. Scn3a+/Hyp mice also exhibited deficits in locomotor activity and motor learning. Taken together, these results provide evidence that loss-of-function of SCN3A caused by reduced protein expression or deficient trafficking to the plasma membrane may contribute to increased seizure susceptibility. PMID:28235671

Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing.

PubMed

Roy, Ankita; Goodman, Joshua H; Begum, Gulnaz; Donnelly, Bridget F; Pittman, Gabrielle; Weinman, Edward J; Sun, Dandan; Subramanya, Arohan R

2015-02-15

Sodium-coupled SLC12 cation chloride cotransporters play important roles in cell volume and chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These cotransporters are phosphorylated and activated indirectly by With-No-Lysine (WNK) kinases through their downstream effector kinases, Ste20- and SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1). Multiple WNK kinases can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Deletion of specific WNKs from cells that natively express a functional WNK-SPAK/OSR1 network will help resolve these knowledge gaps. Here, we outline a simple method to selectively knock out full-length WNK1 expression from mammalian cells using RNA-guided clustered regularly interspaced short palindromic repeats/Cas9 endonucleases. Two clonal cell lines were generated by using a single-guide RNA (sgRNA) targeting exon 1 of the WNK1 gene, which produced indels that abolished WNK1 protein expression. Both cell lines exhibited reduced endogenous WNK4 protein abundance, indicating that WNK1 is required for WNK4 stability. Consistent with an on-target effect, the reduced WNK4 abundance was associated with increased expression of the KLHL3/cullin-3 E3 ubiquitin ligase complex and was rescued by exogenous WNK1 overexpression. Although the morphology of the knockout cells was indistinguishable from control, they exhibited low baseline SPAK/OSR1 activity and failed to trigger regulatory volume increase after hypertonic stress, confirming an essential role for WNK1 in cell volume regulation. Collectively, our data show how this new, powerful, and accessible gene-editing technology can be used to dissect and analyze WNK signaling networks.

Resistin and interleukin-6 exhibit racially-disparate expression in breast cancer patients, display molecular association and promote growth and aggressiveness of tumor cells through STAT3 activation.

PubMed

Deshmukh, Sachin K; Srivastava, Sanjeev K; Bhardwaj, Arun; Singh, Ajay P; Tyagi, Nikhil; Marimuthu, Saravanakumar; Dyess, Donna L; Dal Zotto, Valeria; Carter, James E; Singh, Seema

2015-05-10

African-American (AA) women with breast cancer (BC) are diagnosed with more aggressive disease, have higher risk of recurrence and poorer prognosis as compared to Caucasian American (CA) women. Therefore, it is imperative to define the factors associated with such disparities to reduce the unequal burden of cancer. Emerging data suggest that inherent differences exist in the tumor microenvironment of AA and CA BC patients, however, its molecular bases and functional impact have remained poorly understood. Here, we conducted cytokine profiling in serum samples from AA and CA BC patients and identified resistin and IL-6 to be the most differentially-expressed cytokines with relative greater expression in AA patients. Resistin and IL-6 exhibited positive correlation in serum levels and treatment of BC cells with resistin led to enhanced production of IL-6. Moreover, resistin also enhanced the expression and phosphorylation of STAT3, and treatment of BC cells with IL-6-neutralizing antibody prior to resistin stimulation abolished STAT3 phosphorylation. In addition, resistin promoted growth and aggressiveness of BC cells, and these effects were mediated through STAT3 activation. Together, these findings suggest a crucial role of resistin, IL-6 and STAT3 in BC racial disparity.

Histone deacetylase 3 is required for maintenance of bone mass during aging

PubMed Central

McGee-Lawrence, Meghan E.; Bradley, Elizabeth W.; Dudakovic, Amel; Carlson, Samuel W.; Ryan, Zachary C.; Kumar, Rajiv; Dadsetan, Mahrokh; Yaszemski, Michael J.; Chen, Qingshan; An, Kai-Nan; Westendorf, Jennifer J.

2012-01-01

Histone deacetylase 3 (Hdac3) is a nuclear enzyme that removes acetyl groups from lysine residues in histones and other proteins to epigenetically regulate gene expression. Hdac3 interacts with bone-related transcription factors and co-factors such as Runx2 and Zfp521, and thus is poised to play a key role in the skeletal system. To understand the role of Hdac3 in osteoblasts and osteocytes, Hdac3 conditional knockout (CKO) mice were created with the Osteocalcin (OCN) promoter driving Cre expression. Hdac3 CKOOCN mice were of normal size and weight, but progressively lost trabecular and cortical bone mass with age. The Hdac3 CKOOCN mice exhibited reduced cortical bone mineralization and material properties and suffered frequent fractures. Bone resorption was lower, not higher, in the Hdac3 CKOOCN mice, suggesting that primary defects in osteoblasts caused the reduced bone mass. Indeed, reductions in bone formation were observed. Osteoblasts and osteocytes from Hdac3 CKOOCN mice showed increased DNA damage and reduced functional activity in vivo and in vitro. Thus, Hdac3 expression in osteoblasts and osteocytes is essential for bone maintenance during aging. PMID:23085085

The length of an internal poly(A) tract of hibiscus latent Singapore virus is crucial for its replication.

PubMed

Niu, Shengniao; Cao, Shishu; Huang, Li-Jing; Tan, Kelvin Chee-Leong; Wong, Sek-Man

2015-01-01

Hibiscus latent Singapore virus (HLSV) mutants were constructed to study roles of its internal poly(A) tract (IPAT) in viral replication and coat protein (CP) expression. Shortening of the IPAT resulted in reduced HLSV RNA accumulation and its minimal length required for HLSV CP expression in plants was 24 nt. Disruption of a putative long range RNA-RNA interacting structure between 5' and 3' untranslated regions of HLSV-22A and -24A resulted in reduced viral RNA and undetectable CP accumulation in inoculated leaves. Replacement of the IPAT in HLSV with an upstream pseudoknot domain (UPD) of Tobacco mosaic virus (TMV) or insertion of the UPD to the immediate downstream of a 24 nt IPAT in HLSV resulted in drastically reduced viral RNA replication. Plants infected with a TMV mutant by replacement of the UPD with 43 nt IPAT exhibited milder mosaic symptoms without necrosis. We have proposed a model for HLSV replication. Copyright © 2014 Elsevier Inc. All rights reserved.

DNA immunization against experimental genital herpes simplex virus infection.

PubMed

Bourne, N; Stanberry, L R; Bernstein, D I; Lew, D

1996-04-01

A nucleic acid vaccine, expressing the gene encoding herpes simplex virus (HSV) type 2 glycoprotein D (gD2) under control of the cytomegalovirus immediate-early gene promoter, was used to immunize guinea pigs against genital HSV-2 infection. The vaccine elicited humoral immune responses comparable to those seen after HSV-2 infection. Immunized animals exhibited protection from primary genital HSV-2 disease with little or no development of vesicular skin lesions and significantly reduced HSV-2 replication in the genital tract. After recovery from primary infection, immunized guinea pigs experienced significantly fewer recurrences and had significantly less HSV-2 genomic DNA detected in the sacral dorsal root ganglia compared with control animals. Thus, immunization reduced the burden of latent infection resulting from intravaginal HSV-2 challenge, and a nucleic acid vaccine expressing the HSV-2 gD2 antigen protected guinea pigs against genital herpes, limiting primary infection and reducing the magnitude of latent infection and the frequency of recurrent disease.

Klf8 regulates left-right asymmetric patterning through modulation of Kupffer's vesicle morphogenesis and spaw expression.

PubMed

Lin, Che-Yi; Tsai, Ming-Yuan; Liu, Yu-Hsiu; Lu, Yu-Fen; Chen, Yi-Chung; Lai, Yun-Ren; Liao, Hsin-Chi; Lien, Huang-Wei; Yang, Chung-Hsiang; Huang, Chang-Jen; Hwang, Sheng-Ping L

2017-07-17

Although vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Disruption of left-right axis asymmetric patterning often occurs in human genetic disorders. In zebrafish embryos, Kupffer's vesicle, like the mouse node, breaks symmetry by inducing asymmetric expression of the Nodal-related gene, spaw, in the left lateral plate mesoderm (LPM). Spaw then stimulates transcription of itself and downstream genes, including lft1, lft2, and pitx2, specifically in the left side of the diencephalon, heart and LPM. This developmental step is essential to establish subsequent asymmetric organ positioning. In this study, we evaluated the role of krüppel-like factor 8 (klf8) in regulating left-right asymmetric patterning in zebrafish embryos. Zebrafish klf8 expression was disrupted by both morpholino antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. Whole-mount in situ hybridization was conducted to evaluate gene expression patterns of Nodal signalling components and the positions of heart and visceral organs. Dorsal forerunner cell number was evaluated in Tg(sox17:gfp) embryos and the length and number of cilia in Kupffer's vesicle were analyzed by immunocytochemistry using an acetylated tubulin antibody. Heart jogging, looping and visceral organ positioning were all defective in zebrafish klf8 morphants. At the 18-22 s stages, klf8 morphants showed reduced expression of genes encoding Nodal signalling components (spaw, lft1, lft2, and pitx2) in the left LPM, diencephalon, and heart. Co-injection of klf8 mRNA with klf8 morpholino partially rescued spaw expression. Furthermore, klf8 but not klf8△zf overexpressing embryos showed dysregulated bilateral expression of Nodal signalling components at late somite stages. At the 10s stage, klf8 morphants exhibited reductions in length and number of cilia in Kupffer's vesicle, while at 75% epiboly, fewer dorsal forerunner cells were observed. Interestingly, klf8 mutant embryos, generated by a CRISPR-Cas9 system, showed bilateral spaw expression in the LPM at late somite stages. This observation may be partly attributed to compensatory upregulation of klf12b, because klf12b knockdown reduced the percentage of klf8 mutants exhibiting bilateral spaw expression. Our results demonstrate that zebrafish Klf8 regulates left-right asymmetric patterning by modulating both Kupffer's vesicle morphogenesis and spaw expression in the left LPM.

AIP1 mediates VEGFR-3-dependent angiogenic and lymphangiogenic responses

PubMed Central

Zhou, Huanjiao Jenny; Chen, Xiaodong; Liu, Renjing; Zhang, Haifeng; Wang, Yingdi; Jin, Yu; Liang, Xiaoling; Lu, Lin; Xu, Zhe; Min, Wang

2014-01-01

Objective To investigate the novel function of AIP1 in VEGFR-3 signaling, and VEGFR-3-dependent angiogenesis and lymphangiogenesis. Approach/Results AIP1, a signaling scaffold protein, is highly expressed in the vascular endothelium. We have previously reported that AIP1 functions as an endogenous inhibitor in pathological angiogenesis by blocking VEGFR-2 activity. Surprisingly, here we observe that mice with a global deletion of AIP1 (AIP1-KO) exhibit reduced retinal angiogenesis with less sprouting and fewer branches. Vascular endothelial cell (but not neuronal)-specific deletion of AIP1 causes similar defects in retinal angiogenesis. The reduced retinal angiogenesis correlates with reduced expression in VEGFR-3 despite increased VEGFR-2 levels in AIP1-KO retinas. Consistent with the reduced expression of VEGFR-3, AIP1-KO mice show delayed developmental lymphangiogenesis in neonatal skin and mesentery, and mount weaker VEGF-C-induced cornea lymphangiogenesis. In vitro, human lymphatic EC with AIP1 siRNA knockdown, retinal EC and lymphatic EC isolated from AIP1-KO all show attenuated VEGF-C-induced VEGFR-3 signaling. Mechanistically, we demonstrate that AIP1 via vegfr-3-specific miR-1236 increases VEGFR-3 protein expression, and by directly binding to VEGFR-3 enhances VEGFR-3 endocytosis and stability. Conclusion Our in vivo and in vitro results provide the first insight into the mechanism by which AIP1 mediates VEGFR-3-dependent angiogenic and lymphangiogenic signaling. PMID:24407031

Influence of maternal hyperthyroidism in the rat on the expression of neuronal and astrocytic cytoskeletal proteins in fetal brain.

PubMed

Evans, I M; Pickard, M R; Sinha, A K; Leonard, A J; Sampson, D C; Ekins, R P

2002-12-01

Maternal hypothyroidism during pregnancy impairs brain function in human and rat offspring, but little is known regarding the influence of maternal hyperthyroidism on neurodevelopment. We have previously shown that the expression of neuronal and glial differentiation markers in fetal brain is compromised in hypothyroid rat dam pregnancies and have now therefore extended this investigation to hyperthyroid rat dams. Study groups comprised partially thyroidectomised dams, implanted with osmotic pumps infusing either vehicle (TX dams) or a supraphysiological dose of thyroxine (T4) (HYPER dams), and euthyroid dams infused with vehicle (N dams). Cytoskeletal protein abundance was determined in fetal brain at 21 days of gestation by immunoblot analysis. Relative to N dams, circulating total T4 levels were reduced to around one-third in TX dams but were doubled in HYPER dams. Fetal brain weight was increased in HYPER dams, whereas litter size and fetal body weight were reduced in TX dams. Glial fibrillary acidic protein expression was similar in HYPER and TX dams, being reduced in both cases relative to N dams. alpha-Internexin (INX) abundance was reduced in HYPER dams and increased in TX dams, whereas neurofilament 68 (NF68) exhibited increased abundance in HYPER dams. Furthermore, INX was inversely related to - and NF68 directly related to - maternal serum total T4 levels, independently of fetal brain weight. In conclusion, maternal hyperthyroidism compromises the expression of neuronal cytoskeletal proteins in late fetal brain, suggestive of a pattern of accelerated neuronal differentiation.

Wheat miRNA TaemiR408 Acts as an Essential Mediator in Plant Tolerance to Pi Deprivation and Salt Stress via Modulating Stress-Associated Physiological Processes.

PubMed

Bai, Qianqian; Wang, Xiaoying; Chen, Xi; Shi, Guiqing; Liu, Zhipeng; Guo, Chengjin; Xiao, Kai

2018-01-01

MicroRNAs (miRNA) families act as critical regulators for plant growth, development, and responses to abiotic stresses. In this study, we characterized TaemiR408, a miRNA family member of wheat ( Triticum aestivum ), for the role in mediating plant responses to Pi starvation and salt stress. TaemiR408 targets six genes that encode proteins involving biochemical metabolism, microtubule organization, and signaling transduction. 5'- and 3'-RACE analyses confirmed the mRNA cleavage of target genes mediated by this wheat miRNA. TaemiR408 showed induced expression patterns upon Pi starvation and salt stress and whose upregulated expression was gradually repressed by the normal recovery treatments. The target genes of TaemiR408 exhibited reverse expression patterns to this miRNA, whose transcripts were downregulated under Pi starvation and salt stress and the reduced expression was recovered by the followed normal condition. These results suggest the regulation of the target genes under TaemiR408 through a cleavage mechanism. Tobacco lines with TaemiR408 overexpression exhibited enhanced stress tolerance, showing improved phenotype, biomass, and photosynthesis behavior compared with wild type under both Pi starvation and salt treatments, which closely associate increased P accumulation upon Pi deprivation and elevated osmolytes under salt stress, respectively. Phosphate transporter (PT) gene NtPT2 displays upregulated transcripts in the Pi-deprived TaemiR408 overexpressors; knockdown of this PT gene reduces Pi acquisition under low-Pi stress, confirming its role in improving plant Pi taken up. Likewise, NtPYL2 and NtSAPK3 , genes encoding abscisic acid (ABA) receptor and SnRK2 protein, respectively, exhibited upregulated transcripts in salt-challenged TaemiR408 overexpressors; knockdown of them caused deteriorated growth and lowered osmolytes amounts of plants upon salt treatment. Thus, TaemiR408 is crucial for plant adaptations to Pi starvation and salt stress through regulating Pi acquisition under low-Pi stress and remodel ABA signaling pathway and osmoprotects biosynthesis under salt stress.

Dietary tomato and lycopene impact androgen signaling- and carcinogenesis-related gene expression during early TRAMP prostate carcinogenesis

PubMed Central

Wan, Lei; Tan, Hsueh-Li; Thomas-Ahner, Jennifer M.; Pearl, Dennis K.; Erdman, John W.; Moran, Nancy E.; Clinton, Steven K.

2014-01-01

Consumption of tomato products containing the carotenoid lycopene is associated with a reduced risk of prostate cancer. To identify gene expression patterns associated with early testosterone-driven prostate carcinogenesis, which are impacted by dietary tomato and lycopene, wild type (WT) and transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were fed control or tomato- or lycopene-containing diets from 4-10 wk-of-age. Eight-week-old mice underwent sham surgery, castration, or castration followed by testosterone-repletion (2.5 mg/kg/d initiated 1 wk after castration). Ten-wk-old intact TRAMP mice exhibit early multifocal prostatic intraepithelial neoplasia (PIN). Of the 200 prostate cancer-related genes measured by quantitative NanoString®, 189 are detectable, 164 significantly differ by genotype, 179 by testosterone status, and 30 by diet type (P<0.05). In TRAMP, expression of Birc5, Mki67, Aurkb, Ccnb2, Foxm1, and Ccne2 is greater compared to WT and is decreased by castration. In parallel, castration reduces Ki67-positive staining (P<0.0001) compared to intact and testosterone-repleted TRAMP mice. Expression of genes involved in androgen metabolism/signaling pathways are reduced by lycopene feeding (Srd5a1) and by tomato-feeding (Srd5a2, Pxn, and Srebf1). Additionally, tomato-feeding significantly reduced expression of genes associated with stem cell features, Aldh1a and Ly6a, while lycopene-feeding significantly reduced expression of neuroendocrine differentiation-related genes, Ngfr and Syp. Collectively, these studies demonstrate a profile of testosterone-regulated genes associated with early stages of prostate carcinogenesis that are potential mechanistic targets of dietary tomato components. Future studies on androgen signaling/metabolism, stem cell features, and neuroendocrine differentiation pathways may elucidate the mechanisms by which dietary tomato and lycopene impact prostate cancer risk. PMID:25315431

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Youn-hee; Kim, Donghern; Dai, Jin

Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG → TCG) at codon 47 and the codon 72 polymorphism (CGC → CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 uponmore » acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer. - Highlights: • Short-term exposure of BEAS-2B cells to arsenic or Cr(VI) activates p53 and p21. • Chronic exposure of BEAS-2B cells to arsenic or Cr(VI) causes cell transformation and tumorigenesis. • Arsenic-transformed cells exhibit reduced activities of p53 and p21. • Cr(VI)-transformed cells exhibit increased activities of p53 and p21.« less

The Klebsiella pneumoniae YfgL (BamB) lipoprotein contributes to outer membrane protein biogenesis, type-1 fimbriae expression, anti-phagocytosis, and in vivo virulence

PubMed Central

Hsieh, Pei-Fang; Hsu, Chun-Ru; Chen, Chun-Tang; Lin, Tzu-Lung; Wang, Jin-Town

2016-01-01

ABSTRACT Klebsiella pneumoniae is an opportunistic pathogen that causes several kinds of infections, including pneumonia, bacteremia, urinary tract infection and community-acquired pyogenic liver abscess (PLA). Adhesion is the critical first step in the infection process. Our previous work demonstrated that the transcellular translocation is exploited by K. pneumoniae strains to migrate from the gut flora into other tissues, resulting in systemic infections. However, the initial stages of K. pneumoniae infection remain unclear. In this study, we demonstrated that a K. pneumoniae strain deleted for yfgL (bamB) exhibited reduced adherence to and invasion of host cells; changed biogenesis of major β-barrel outer membrane proteins; decreased transcriptional expression of type-1 fimbriae; and increased susceptibility to vancomycin and erythromycin. The yfgL deletion mutant also had reduced ability to against neutrophil phagocytosis; exhibited decreased induction of host IL-6 production; and was profoundly attenuated for virulence in a K. pneumoniae model of bacteremia. Thus, the K. pneumoniae YfgL lipoprotein mediates in outer membrane proteins biogenesis and is crucial for anti-phagocytosis and survival in vivo. These data provide a new insight for K. pneumoniae attachment and such knowledge could facilitate preventive therapies or alternative therapies against K. pneumoniae. PMID:27029012

Maternal-pup interaction disturbances induce long-lasting changes in the newborn rat pulmonary vasculature.

PubMed

Shifrin, Yulia; Sadeghi, Sina; Pan, Jingyi; Jain, Amish; Fajardo, Andres F; McNamara, Patrick J; Belik, Jaques

2015-11-15

The factors accounting for the pathological maintenance of a high pulmonary vascular (PV) resistance postnatally remain elusive, but neonatal stressors may play a role in this process. Cross-fostering in the immediate neonatal period is associated with adult-onset vascular and behavioral changes, likely triggered by early-in-life stressors. In hypothesizing that fostering newborn rats induces long-lasting PV changes, we evaluated them at 14 days of age during adulthood and compared the findings with animals raised by their biological mothers. Fostering resulted in reduced maternal-pup contact time when compared with control newborns. At 2 wk of age, fostered rats exhibited reduced pulmonary arterial endothelium-dependent relaxation secondary to downregulation of tissue endothelial nitric oxide synthase expression and tetrahydrobiopterin deficiency-induced uncoupling. These changes were associated with neonatal onset-increased ANG II receptor type 1 expression, PV remodeling, and right ventricular hypertrophy that persisted into adulthood. The pulmonary arteries of adult-fostered rats exhibited a higher contraction dose response to ANG II and thromboxane A2, the latter of which was abrogated by the oxidant scavenger Tempol. In conclusion, fostering-induced neonatal stress induces long-standing PV changes modulated via the renin-angiotensin system. Copyright © 2015 the American Physiological Society.

Arachidonic Acid: An Evolutionarily Conserved Signaling Molecule Modulates Plant Stress Signaling Networks[C][W

PubMed Central

Savchenko, Tatyana; Walley, Justin W.; Chehab, E. Wassim; Xiao, Yanmei; Kaspi, Roy; Pye, Matthew F.; Mohamed, Maged E.; Lazarus, Colin M.; Bostock, Richard M.; Dehesh, Katayoon

2010-01-01

Fatty acid structure affects cellular activities through changes in membrane lipid composition and the generation of a diversity of bioactive derivatives. Eicosapolyenoic acids are released into plants upon infection by oomycete pathogens, suggesting they may elicit plant defenses. We exploited transgenic Arabidopsis thaliana plants (designated EP) producing eicosadienoic, eicosatrienoic, and arachidonic acid (AA), aimed at mimicking pathogen release of these compounds. We also examined their effect on biotic stress resistance by challenging EP plants with fungal, oomycete, and bacterial pathogens and an insect pest. EP plants exhibited enhanced resistance to all biotic challenges, except they were more susceptible to bacteria than the wild type. Levels of jasmonic acid (JA) were elevated and levels of salicylic acid (SA) were reduced in EP plants. Altered expression of JA and SA pathway genes in EP plants shows that eicosapolyenoic acids effectively modulate stress-responsive transcriptional networks. Exogenous application of various fatty acids to wild-type and JA-deficient mutants confirmed AA as the signaling molecule. Moreover, AA treatment elicited heightened expression of general stress-responsive genes. Importantly, tomato (Solanum lycopersicum) leaves treated with AA exhibited reduced susceptibility to Botrytis cinerea infection, confirming AA signaling in other plants. These studies support the role of AA, an ancient metazoan signaling molecule, in eliciting plant stress and defense signaling networks. PMID:20935246

SOX12: a novel potential target for acute myeloid leukaemia.

PubMed

Wan, Haixia; Cai, Jiayi; Chen, Fangyuan; Zhu, Jianyi; Zhong, Jihua; Zhong, Hua

2017-02-01

The role of SRY-related high-mobility-group box (SOX) 12 in leukaemia progression and haematopoiesis remains elusive. This study aimed to examine the expression and function of SOX12 in acute myeloid leukaemia (AML) using human myeloid leukaemia samples and the acute myeloid cell line THP1. Mononuclear cells were isolated from the bone marrow of AML patients and healthy donors. SOX12 expression in haematopoietic cells was evaluated by reverse transcription polymerase chain reaction (RT-PCR). SOX12 short hairpin RNAs (shRNAs) were transduced into THP1 cells, and gene knockdown was confirmed by quantitative RT-PCR and Western blot analysis. SOX12 was preferentially expressed in CD34 + cells in AML patients. The THP1 cells transduced with SOX12 shRNAs exhibited significantly reduced SOX12 expression and cell proliferation. SOX12 knockdown had no effect on apoptosis, but it induced cell cycle arrest at G1 phase and reduced the number of colonies. The transduced THP1 and primary AML cells were reconstituted in non-obese diabetic-severe combined immunodeficient (NOD/SCID) mice, and their numbers were significantly reduced 6-12 weeks after transplantation. The mRNA and protein levels of β-catenin were significantly diminished following SOX12 knockdown, accompanied by a decrease in TCF/Wnt activity. SOX12 may be involved in leukaemia progression by regulating the expression of β-catenin and then interfering with TCF/Wnt pathway, which may be a target for AML. © 2016 John Wiley & Sons Ltd.

The impact of α-Lipoic acid on cell viability and expression of nephrin and ZNF580 in normal human podocytes.

PubMed

Leppert, Ulrike; Gillespie, Allan; Orphal, Miriam; Böhme, Karen; Plum, Claudia; Nagorsen, Kaj; Berkholz, Janine; Kreutz, Reinhold; Eisenreich, Andreas

2017-09-05

Human podocytes (hPC) are essential for maintaining normal kidney function and dysfunction or loss of hPC play a pivotal role in the manifestation and progression of chronic kidney diseases including diabetic nephropathy. Previously, α-Lipoic acid (α-LA), a licensed drug for treatment of diabetic neuropathy, was shown to exhibit protective effects on diabetic nephropathy in vivo. However, the effect of α-LA on hPC under non-diabetic conditions is unknown. Therefore, we analyzed the impact of α-LA on cell viability and expression of nephrin and zinc finger protein 580 (ZNF580) in normal hPC in vitro. Protein analyses were done via Western blot techniques. Cell viability was determined using a functional assay. hPC viability was dynamically modulated via α-LA stimulation in a concentration-dependent manner. This was associated with reduced nephrin and ZNF580 expression and increased nephrin phosphorylation in normal hPC. Moreover, α-LA reduced nephrin and ZNF580 protein expression via 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) inhibition. These data demonstrate that low α-LA had no negative influence on hPC viability, whereas, high α-LA concentrations induced cytotoxic effects on normal hPC and reduced nephrin and ZNF580 expression via NF-κB inhibition. These data provide first novel information about potential cytotoxic effects of α-LA on hPC under non-diabetic conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

Heat Shock Protein B1-Deficient Mice Display Impaired Wound Healing

PubMed Central

McNamee, Kay; Przybycien, Paulina M.; Lu, Xin; Williams, Richard O.; Bou-Gharios, George; Saklatvala, Jeremy; Dean, Jonathan L. E.

2013-01-01

There is large literature describing in vitro experiments on heat shock protein (hsp)B1 but understanding of its function in vivo is limited to studies in mice overexpressing human hspB1 protein. Experiments in cells have shown that hspB1 has chaperone activity, a cytoprotective role, regulates inflammatory gene expression, and drives cell proliferation. To investigate the function of the protein in vivo we generated hspB1-deficient mice. HspB1-deficient fibroblasts display increased expression of the pro-inflammatory cytokine, interleukin-6, compared to wild-type cells, but reduced proliferation. HspB1-deficient fibroblasts exhibit reduced entry into S phase and increased expression of cyclin-dependent kinase inhibitors p27kip1 and p21waf1. The expression of hspB1 protein and mRNA is also controlled by the cell cycle. To investigate the physiological function of hspB1 in regulating inflammation and cell proliferation we used an excisional cutaneous wound healing model. There was a significant impairment in the rate of healing of wounds in hspB1-deficient mice, characterised by reduced re-epithelialisation and collagen deposition but also increased inflammation. HspB1 deficiency augments neutrophil infiltration in wounds, driven by increased chemokine (C-X-C motif) ligand 1 expression. This appears to be a general mechanism as similar results were obtained in the air-pouch and peritonitis models of acute inflammation. PMID:24143227

Deletion of the Kv2.1 delayed rectifier potassium channel leads to neuronal and behavioral hyperexcitability

PubMed Central

Speca, David J.; Ogata, Genki; Mandikian, Danielle; Bishop, Hannah I.; Wiler, Steve W.; Eum, Kenneth; Wenzel, H. Jürgen; Doisy, Emily T.; Matt, Lucas; Campi, Katharine L.; Golub, Mari S.; Nerbonne, Jeanne M.; Hell, Johannes W.; Trainor, Brian C.; Sack, Jon T.; Schwartzkroin, Philip A.; Trimmer, James S.

2014-01-01

The Kv2.1 delayed rectifier potassium channel exhibits high-level expression in both principal and inhibitory neurons throughout the central nervous system, including prominent expression in hippocampal neurons. Studies of in vitro preparations suggest that Kv2.1 is a key yet conditional regulator of intrinsic neuronal excitability, mediated by changes in Kv2.1 expression, localization and function via activity-dependent regulation of Kv2.1 phosphorylation. Here we identify neurological and behavioral deficits in mutant (Kv2.1−/−) mice lacking this channel. Kv2.1−/− mice have grossly normal characteristics. No impairment in vision or motor coordination was apparent, although Kv2.1−/− mice exhibit reduced body weight. The anatomic structure and expression of related Kv channels in the brains of Kv2.1−/− mice appears unchanged. Delayed rectifier potassium current is diminished in hippocampal neurons cultured from Kv2.1−/− animals. Field recordings from hippocampal slices of Kv2.1−/− mice reveal hyperexcitability in response to the convulsant bicuculline, and epileptiform activity in response to stimulation. In Kv2.1−/− mice, long-term potentiation at the Schaffer collateral – CA1 synapse is decreased. Kv2.1−/− mice are strikingly hyperactive, and exhibit defects in spatial learning, failing to improve performance in a Morris Water Maze task. Kv2.1−/− mice are hypersensitive to the effects of the convulsants flurothyl and pilocarpine, consistent with a role for Kv2.1 as a conditional suppressor of neuronal activity. Although not prone to spontaneous seizures, Kv2.1−/− mice exhibit accelerated seizure progression. Together, these findings suggest homeostatic suppression of elevated neuronal activity by Kv2.1 plays a central role in regulating neuronal network function. PMID:24494598

Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma.

PubMed

Jayachandran, Aparna; Anaka, Matthew; Prithviraj, Prashanth; Hudson, Christopher; McKeown, Sonja J; Lo, Pu-Han; Vella, Laura J; Goding, Colin R; Cebon, Jonathan; Behren, Andreas

2014-07-30

Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis. We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype. This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process. We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process. Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation. Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells. Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion. Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube. In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies. These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance.

Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma

PubMed Central

Jayachandran, Aparna; Anaka, Matthew; Prithviraj, Prashanth; Hudson, Christopher; McKeown, Sonja J; Lo, Pu-Han; Vella, Laura J; Goding, Colin R; Cebon, Jonathan; Behren, Andreas

2014-01-01

Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis. We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype. This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process. We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process. Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation. Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells. Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion. Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube. In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies. These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance. PMID:25051363

Association of cytosolic sialidase Neu2 with plasma membrane enhances Fas-mediated apoptosis by impairing PI3K-Akt/mTOR-mediated pathway in pancreatic cancer cells.

PubMed

Nath, Shalini; Mandal, Chhabinath; Chatterjee, Uttara; Mandal, Chitra

2018-02-12

Modulation of sialylation by sialyltransferases and sialidases plays essential role in carcinogenesis. There are few reports on sialyltransferase, however, the contribution of cytosolic sialidase (Neu2) remains unexplored in pancreatic ductal adenocarcinoma (PDAC). We observed lower expression of Neu2 in different PDAC cells, patient tissues, and a significant strong association with clinicopathological characteristics. Neu2 overexpression guided drug-resistant MIAPaCa2 and AsPC1 cells toward apoptosis as evidenced by decreased Bcl2/Bax ratio, activation of caspase-3/caspase-6/caspase-8, PARP reduction, reduced CDK2/CDK4/CDK6, and cyclin-B1/cyclin-E with unaffected caspase-9. Neu2-overexpressed cells exhibited higher expression of Fas/CD95-death receptor, FasL, FADD, and Bid cleavage confirming extrinsic pathway-mediated apoptosis. α2,6-linked sialylation of Fas helps cancer cells to survive, which is a substrate for Neu2. Therefore, their removal should enhance Fas-mediated apoptosis. Neu2-overexpressed cells indeed showed increased enzyme activity even on membrane. Interestingly, this membrane-bound Neu2 exhibited enhanced association with Fas causing its desialylation and activation as corroborated by decreased association of Fas with α2,6-sialic acid-binding lectin. Additionally, enhanced cytosolic Neu2 inhibited the expression of several growth factor-mediated signaling molecules involved in PI3K/Akt-mTOR pathway probably through desialylation which in turn also causes Fas activation. Furthermore, Neu2-overexpressed cells exhibited reduced cell migration, invasion with decreased VEGF, VEGFR, and MMP9 levels. To the best of our knowledge, this is the first report of cytosolic Neu2 on membrane, its association with Fas, enhanced desialylation, activation, and Fas-mediated apoptosis. Taken together, our study ascertains a novel concept by which the function of Fas/CD95 could be modulated indicating a critical role of upstream Neu2 as a promising target for inducing apoptosis in pancreatic cancer.

Changes in Monocyte Functions of Astronauts

NASA Technical Reports Server (NTRS)

Kaur, I.; Simons, E.; Castro, V.; Ott, C. Mark; Pierson, Duane L.

2004-01-01

Monocyte cell numbers and functions, including phagocytosis, oxidative burst capacity, and degranulation and expression of related surface molecules, were studied in blood specimens from 25 astronauts and 9 healthy control subjects. Blood samples were obtained 10 days before a space flight, 3 hours after landing and 3 days after landing. The number of monocytes in astronauts did not change significantly among the three sample collection periods. Following space flight, the monocytes ability to phagocytize Escherichia coli, to exhibit an oxidative burst, and to degranulate was reduced as compared to monocytes from control subjects. These alterations in monocyte functions after space flight correlated with alterations in the expression of CD32 and CD64.

FAK Regulates Intestinal Epithelial Cell Survival and Proliferation during Mucosal Wound Healing

PubMed Central

Tilghman, Robert W.; Casanova, James E.; Bouton, Amy H.

2011-01-01

Background Following damage to the intestinal epithelium, restoration of epithelial barrier integrity is triggered by a robust proliferative response. In other tissues, focal adhesion kinase (FAK) regulates many of the cellular processes that are critical for epithelial homeostasis and restitution, including cell migration, proliferation and survival. However, few studies to date have determined how FAK contributes to mucosal wound healing in vivo. Methodology and Principal Findings To examine the role of FAK in intestinal epithelial homeostasis and during injury, we generated intestinal epithelium (IE)-specific conditional FAK knockout mice. Colitis was induced with dextran-sulfate-sodium (DSS) and intestinal tissues were analyzed by immunohistochemistry and immunoblotting. While intestinal development occurred normally in mice lacking FAK, FAK-deficient animals were profoundly susceptible to colitis. The loss of epithelial FAK resulted in elevated p53 expression and an increased sensitivity to apoptosis, coincident with a failure to upregulate epithelial cell proliferation. FAK has been reported to function as a mechanosensor, inducing cyclin D1 expression and promoting cell cycle progression under conditions in which tissue/matrix stiffness is increased. Collagen deposition, a hallmark of inflammatory injury resulting in increased tissue rigidity, was observed in control and FAK knockout mice during colitis. Despite this fibrotic response, the colonic epithelium in FAK-deficient mice exhibited significantly reduced cyclin D1 expression, suggesting that proliferation is uncoupled from fibrosis in the absence of FAK. In support of this hypothesis, proliferation of Caco-2 cells increased proportionally with matrix stiffness in vitro only under conditions of normal FAK expression; FAK depleted cells exhibited reduced proliferation concomitant with attenuated cyclin D1 expression. Conclusions In the colon, FAK functions as a regulator of epithelial cell survival and proliferation under conditions of mucosal injury and a mechanosensor of tissue compliance, inducing repair-driven proliferation in the colonic epithelium through upregulation of cyclin D1. PMID:21887232

Defective TFH Cell Function and Increased TFR Cells Contribute to Defective Antibody Production in Aging.

PubMed

Sage, Peter T; Tan, Catherine L; Freeman, Gordon J; Haigis, Marcia; Sharpe, Arlene H

2015-07-14

Defective antibody production in aging is broadly attributed to immunosenescence. However, the precise immunological mechanisms remain unclear. Here, we demonstrate an increase in the ratio of inhibitory T follicular regulatory (TFR) cells to stimulatory T follicular helper (TFH) cells in aged mice. Aged TFH and TFR cells are phenotypically distinct from those in young mice, exhibiting increased programmed cell death protein-1 expression but decreased ICOS expression. Aged TFH cells exhibit defective antigen-specific responses, and programmed cell death protein-ligand 1 blockade can partially rescue TFH cell function. In contrast, young and aged TFR cells have similar suppressive capacity on a per-cell basis in vitro and in vivo. Together, these studies reveal mechanisms contributing to defective humoral immunity in aging: an increase in suppressive TFR cells combined with impaired function of aged TFH cells results in reduced T-cell-dependent antibody responses in aged mice. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Small nucleoli are a cellular hallmark of longevity

PubMed Central

Tiku, Varnesh; Jain, Chirag; Raz, Yotam; Nakamura, Shuhei; Heestand, Bree; Liu, Wei; Späth, Martin; Suchiman, H. Eka. D.; Müller, Roman-Ulrich; Slagboom, P. Eline; Partridge, Linda; Antebi, Adam

2017-01-01

Animal lifespan is regulated by conserved metabolic signalling pathways and specific transcription factors, but whether these pathways affect common downstream mechanisms remains largely elusive. Here we show that NCL-1/TRIM2/Brat tumour suppressor extends lifespan and limits nucleolar size in the major C. elegans longevity pathways, as part of a convergent mechanism focused on the nucleolus. Long-lived animals representing distinct longevity pathways exhibit small nucleoli, and decreased expression of rRNA, ribosomal proteins, and the nucleolar protein fibrillarin, dependent on NCL-1. Knockdown of fibrillarin also reduces nucleolar size and extends lifespan. Among wildtype C. elegans, individual nucleolar size varies, but is highly predictive for longevity. Long-lived dietary restricted fruit flies and insulin-like-peptide mutants exhibit small nucleoli and fibrillarin expression, as do long-lived dietary restricted and IRS1 knockout mice. Furthermore, human muscle biopsies from individuals who underwent modest dietary restriction coupled with exercise also display small nucleoli. We suggest that small nucleoli are a cellular hallmark of longevity and metabolic health conserved across taxa. PMID:28853436

Small nucleoli are a cellular hallmark of longevity.

PubMed

Tiku, Varnesh; Jain, Chirag; Raz, Yotam; Nakamura, Shuhei; Heestand, Bree; Liu, Wei; Späth, Martin; Suchiman, H Eka D; Müller, Roman-Ulrich; Slagboom, P Eline; Partridge, Linda; Antebi, Adam

2016-08-30

Animal lifespan is regulated by conserved metabolic signalling pathways and specific transcription factors, but whether these pathways affect common downstream mechanisms remains largely elusive. Here we show that NCL-1/TRIM2/Brat tumour suppressor extends lifespan and limits nucleolar size in the major C. elegans longevity pathways, as part of a convergent mechanism focused on the nucleolus. Long-lived animals representing distinct longevity pathways exhibit small nucleoli, and decreased expression of rRNA, ribosomal proteins, and the nucleolar protein fibrillarin, dependent on NCL-1. Knockdown of fibrillarin also reduces nucleolar size and extends lifespan. Among wildtype C. elegans, individual nucleolar size varies, but is highly predictive for longevity. Long-lived dietary restricted fruit flies and insulin-like-peptide mutants exhibit small nucleoli and fibrillarin expression, as do long-lived dietary restricted and IRS1 knockout mice. Furthermore, human muscle biopsies from individuals who underwent modest dietary restriction coupled with exercise also display small nucleoli. We suggest that small nucleoli are a cellular hallmark of longevity and metabolic health conserved across taxa.

Peroxiredoxin Expression of Human Osteosarcoma Cells Is Influenced by Cold Atmospheric Plasma Treatment.

PubMed

Gümbel, Denis; Gelbrich, Nadine; Napp, Matthias; Daeschlein, Georg; Kramer, Axel; Sckell, Axel; Burchardt, Martin; Ekkernkamp, Axel; Stope, Matthias B

2017-03-01

To evaluate the potential involvement of redox-specific signalling pathways in cold atmospheric plasma (CAP)-induced apoptosis on human osteosarcoma cells. Osteosarcoma cell lines were treated with CAP with or without antioxidative agents and seeded in cell culture plates. Cell proliferation was determined by counting viable cells. Carrier gas-treated cells served as control. Peroxiredoxin (PRX) 1-3 expression and secretion were assessed. CAP treatment exhibited strongly attenuated proliferation rates. This effect was significantly attenuated by the addition of N-acetylcysteine (NAC). CAP-treated cells exhibited an increase of PRX 1 and 2 10 sec after treatment. The ratio of oxidized to reduced PRX1 and PRX2 was significantly altered with increasing cellular concentration of the oxidized dimer. Antioxidant supplementation with NAC increases proliferation of CAP-treated osteosarcoma cells, implicating an involvement of redox signalling. Activation of PRX1 and -2 indicate CAP affects redox homeostasis. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Wnt-Lrp5 Signaling Regulates Fatty Acid Metabolism in the Osteoblast

PubMed Central

Frey, Julie L.; Li, Zhu; Ellis, Jessica M.; Zhang, Qian; Farber, Charles R.; Aja, Susan; Wolfgang, Michael J.; Clemens, Thomas L.

2015-01-01

The Wnt coreceptors Lrp5 and Lrp6 are essential for normal postnatal bone accrual and osteoblast function. In this study, we identify a previously unrecognized skeletal function unique to Lrp5 that enables osteoblasts to oxidize fatty acids. Mice lacking the Lrp5 coreceptor specifically in osteoblasts and osteocytes exhibit the expected reductions in postnatal bone mass but also exhibit an increase in body fat with corresponding reductions in energy expenditure. Conversely, mice expressing a high bone mass mutant Lrp5 allele are leaner with reduced plasma triglyceride and free fatty acid levels. In this context, Wnt-initiated signals downstream of Lrp5, but not the closely related Lrp6 coreceptor, regulate the activation of β-catenin and thereby induce the expression of key enzymes required for fatty acid β-oxidation. These results suggest that Wnt-Lrp5 signaling regulates basic cellular activities beyond those associated with fate specification and differentiation in bone and that the skeleton influences global energy homeostasis via mechanisms independent of osteocalcin and glucose metabolism. PMID:25802278

Cloning, expression, purification, and characterization of glutaredoxin from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178.

PubMed

Wang, Quanfu; Hou, Yanhua; Shi, Yonglei; Han, Xiao; Chen, Qian; Hu, Zhiguo; Liu, Yuanping; Li, YuJin

2014-01-01

Glutaredoxins (Grxs) are small ubiquitous redox enzymes that catalyze glutathione-dependent reactions to reduce protein disulfide. In this study, a full-length Grx gene (PsGrx) with 270 nucleotides was isolated from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178. It encoded deduced 89 amino acid residues with the molecular weight 9.8 kDa. Sequence analysis of the amino acid sequence revealed the catalytic motif CPYC. Recombinant PsGrx (rPsGrx) stably expressed in E. coli BL21 was purified to apparent homogeneity by Ni-affinity chromatography. rPsGrx exhibited optimal activity at 30°C and pH 8.0 and showed 25.5% of the activity at 0°C. It retained 65.0% of activity after incubation at 40°C for 20 min and still exhibited 37.0% activity in 1.0 M NaCl. These results indicated that rPsGrx was a typical cold active protein with low thermostability.

A Novel Small Heat Shock Protein Gene, vis1, Contributes to Pectin Depolymerization and Juice Viscosity in Tomato Fruit1

PubMed Central

Ramakrishna, Wusirika; Deng, Zhiping; Ding, Chang-Kui; Handa, Avtar K.; Ozminkowski, Richard H.

2003-01-01

We have characterized a novel small heat shock protein gene, viscosity 1 (vis1) from tomato (Lycopersicon esculentum) and provide evidence that it plays a role in pectin depolymerization and juice viscosity in ripening fruits. Expression of vis1 is negatively associated with juice viscosity in diverse tomato genotypes. vis1 exhibits DNA polymorphism among tomato genotypes, and the alleles vis1-hta (high-transcript accumulator; accession no. AY128101) and vis1-lta (low transcript accumulator; accession no. AY128102) are associated with thinner and thicker juice, respectively. Segregation of tomato lines heterogeneous for vis1 alleles indicates that vis1 influences pectin depolymerization and juice viscosity in ripening fruits. vis1 is regulated by fruit ripening and high temperature and exhibits a typical heat shock protein chaperone function when expressed in bacterial cells. We propose that VIS1 contributes to physiochemical properties of juice, including pectin depolymerization, by reducing thermal denaturation of depolymerizing enzymes during daytime elevated temperatures. PMID:12586896

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iwasaki, Yuko; Iwasaki, Hitoshi; Yatoh, Shigeru

Transgenic mice expressing nuclear sterol regulatory element-binding protein-1a under the control of the insulin promoter were generated to determine the role of SREBP-1a in pancreatic {beta}-cells. Only low expressors could be established, which exhibited mild hyperglycemia, impaired glucose tolerance, and reduced plasma insulin levels compared to C57BL/6 controls. The islets isolated from the transgenic mice were fewer and smaller, and had decreased insulin content and unaltered glucagon staining. Both glucose- and potassium-stimulated insulin secretions were decreased. The transgenic islets consistently expressed genes for fatty acids and cholesterol synthesis, resulting in accumulation of triglycerides but not cholesterol. PDX-1, {beta}{epsilon}{tau}{alpha}2, MafA, andmore » IRS-2 were suppressed, partially explaining the loss and dysfunction of {beta}-cell mass. The transgenic mice on a high fat/high sucrose diet still exhibited impaired insulin secretion and continuous {beta}-cell growth defect. Therefore, nuclear SREBP-1a, even at a low level, strongly disrupts {beta}-cell mass and function.« less

p53 downregulates the Fanconi anaemia DNA repair pathway

PubMed Central

Jaber, Sara; Toufektchan, Eléonore; Lejour, Vincent; Bardot, Boris; Toledo, Franck

2016-01-01

Germline mutations affecting telomere maintenance or DNA repair may, respectively, cause dyskeratosis congenita or Fanconi anaemia, two clinically related bone marrow failure syndromes. Mice expressing p53Δ31, a mutant p53 lacking the C terminus, model dyskeratosis congenita. Accordingly, the increased p53 activity in p53Δ31/Δ31 fibroblasts correlated with a decreased expression of 4 genes implicated in telomere syndromes. Here we show that these cells exhibit decreased mRNA levels for additional genes contributing to telomere metabolism, but also, surprisingly, for 12 genes mutated in Fanconi anaemia. Furthermore, p53Δ31/Δ31 fibroblasts exhibit a reduced capacity to repair DNA interstrand crosslinks, a typical feature of Fanconi anaemia cells. Importantly, the p53-dependent downregulation of Fanc genes is largely conserved in human cells. Defective DNA repair is known to activate p53, but our results indicate that, conversely, an increased p53 activity may attenuate the Fanconi anaemia DNA repair pathway, defining a positive regulatory feedback loop. PMID:27033104

p53 downregulates the Fanconi anaemia DNA repair pathway.

PubMed

Jaber, Sara; Toufektchan, Eléonore; Lejour, Vincent; Bardot, Boris; Toledo, Franck

2016-04-01

Germline mutations affecting telomere maintenance or DNA repair may, respectively, cause dyskeratosis congenita or Fanconi anaemia, two clinically related bone marrow failure syndromes. Mice expressing p53(Δ31), a mutant p53 lacking the C terminus, model dyskeratosis congenita. Accordingly, the increased p53 activity in p53(Δ31/Δ31) fibroblasts correlated with a decreased expression of 4 genes implicated in telomere syndromes. Here we show that these cells exhibit decreased mRNA levels for additional genes contributing to telomere metabolism, but also, surprisingly, for 12 genes mutated in Fanconi anaemia. Furthermore, p53(Δ31/Δ31) fibroblasts exhibit a reduced capacity to repair DNA interstrand crosslinks, a typical feature of Fanconi anaemia cells. Importantly, the p53-dependent downregulation of Fanc genes is largely conserved in human cells. Defective DNA repair is known to activate p53, but our results indicate that, conversely, an increased p53 activity may attenuate the Fanconi anaemia DNA repair pathway, defining a positive regulatory feedback loop.

Alamandine reduces leptin expression through the c-Src/p38 MAP kinase pathway in adipose tissue.

PubMed

Uchiyama, Tsuyoshi; Okajima, Fumikazu; Mogi, Chihiro; Tobo, Ayaka; Tomono, Shoichi; Sato, Koichi

2017-01-01

Obesity is associated with an increased risk of diabetes mellitus, hypertension, and renal dysfunction. Angiotensin 1-7 and alamandine are heptameric renin angiotensin system peptide hormones. Further, alamandine levels increase with renal dysfunction. In the cardiovascular system, angiotensin 1-7 and alamandine produce similar improvements and counterbalance angiotensin II in regulating vascular function. We aimed to determine whether the effect of alamandine on leptin expression and secretion in adipocytes was similar to that of angiotensin 1-7. We studied isolated peri-renal visceral adipose tissue and peri-renal isolated visceral adipocytes from male Wistar rats. Angiotensin II from 0.01 to 10nM had no effect on leptin expression. Angiotensin 1-7 (1 nM) increased leptin secretion and expression, whereas alamandine (1 nM) decreased leptin secretion and expression in adipose tissue and isolated adipocytes and reduced blood leptin levels in vivo. These effects were mediated by Gq, c-Src, p38 mitogen-activated protein, and IκB activation. Additionally, alamandine induced nitric oxide expression via inducible nitric oxidase synthase and plasminogen activator inhibitor 1 expression in adipose tissue and isolated adipocytes. Angiotensin 1-7 and alamandine produced opposing effects on leptin expression and secretion in adipose tissue. This result suggests that the action of Mas (angiotensin 1-7 receptor) and Mas-related G-protein coupled receptor D in adipocytes exhibited opposing actions similar to angiotensin II type 1 and type 2 receptors.

Amino acids exhibit anti-inflammatory effects in human monocytic leukemia cell line, THP-1 cells.

PubMed

Hasegawa, Shunji; Ichiyama, Takashi; Sonaka, Ichiro; Ohsaki, Ayami; Hirano, Reiji; Haneda, Yasuhiro; Fukano, Reiji; Hara, Masami; Furukawa, Susumu

2011-11-01

The elemental diet is one of the effective therapies for inflammatory bowel disease. However, the mechanism remains unclear, and there have never been reports about the inhibitory effects of amino acids in human monocytes/macrophages. We investigated the inhibitory effects of amino acids on cytokine production or expression of adhesion molecules that are involved in inflammatory diseases, in human monocytes/macrophages. We examined the inhibitory effects of cysteine, histidine or glycine on the induction of nuclear factor-κB (NF-κB) activation, expression of intracellular adhesion molecule-1 (ICAM-1, CD54) and production of interleukin-8 (IL-8) in THP-1 cells, a human monocytic leukemia cell line, and peripheral blood mononuclear cells (PBMCs) stimulated with tumor necrosis factor-α (TNF-α). Cysteine, histidine and glycine significantly reduced the activation of NF-κB in THP-1 cells stimulated with TNF-α. In addition, cysteine and histidine significantly inhibited the expression of ICAM-1 and production of IL-8 in THP-1 cells and PBMCs. Our results suggest that cysteine and histidine exhibit anti-inflammatory effects in THP-1 cells, and may be responsible for the efficacy of treatment in inflammatory bowel diseases.

A primary cilia-dependent etiology for midline facial disorders

PubMed Central

Brugmann, Samantha A.; Allen, Nancy C.; James, Aaron W.; Mekonnen, Zesemayat; Madan, Elena; Helms, Jill A.

2010-01-01

Human faces exhibit enormous variation. When pathological conditions are superimposed on normal variation, a nearly unbroken series of facial morphologies is produced. When viewed in full, this spectrum ranges from cyclopia and hypotelorism to hypertelorism and facial duplications. Decreased Hedgehog pathway activity causes holoprosencephaly and hypotelorism. Here, we show that excessive Hedgehog activity, caused by truncating the primary cilia on cranial neural crest cells, causes hypertelorism and frontonasal dysplasia (FND). Elimination of the intraflagellar transport protein Kif3a leads to excessive Hedgehog responsiveness in facial mesenchyme, which is accompanied by broader expression domains of Gli1, Ptc and Shh, and reduced expression domains of Gli3. Furthermore, broader domains of Gli1 expression correspond to areas of enhanced neural crest cell proliferation in the facial prominences of Kif3a conditional knockouts. Avian Talpid embryos that lack primary cilia exhibit similar molecular changes and similar facial phenotypes. Collectively, these data support our hypothesis that a severe narrowing of the facial midline and excessive expansion of the facial midline are both attributable to disruptions in Hedgehog pathway activity. These data also raise the possibility that genes encoding ciliary proteins are candidates for human conditions of hypertelorism and FNDs. PMID:20106874

Recognition of facial expressions and prosodic cues with graded emotional intensities in adults with Asperger syndrome.

PubMed

Doi, Hirokazu; Fujisawa, Takashi X; Kanai, Chieko; Ohta, Haruhisa; Yokoi, Hideki; Iwanami, Akira; Kato, Nobumasa; Shinohara, Kazuyuki

2013-09-01

This study investigated the ability of adults with Asperger syndrome to recognize emotional categories of facial expressions and emotional prosodies with graded emotional intensities. The individuals with Asperger syndrome showed poorer recognition performance for angry and sad expressions from both facial and vocal information. The group difference in facial expression recognition was prominent for stimuli with low or intermediate emotional intensities. In contrast to this, the individuals with Asperger syndrome exhibited lower recognition accuracy than typically-developed controls mainly for emotional prosody with high emotional intensity. In facial expression recognition, Asperger and control groups showed an inversion effect for all categories. The magnitude of this effect was less in the Asperger group for angry and sad expressions, presumably attributable to reduced recruitment of the configural mode of face processing. The individuals with Asperger syndrome outperformed the control participants in recognizing inverted sad expressions, indicating enhanced processing of local facial information representing sad emotion. These results suggest that the adults with Asperger syndrome rely on modality-specific strategies in emotion recognition from facial expression and prosodic information.

Impacts of different expressions of PA-X protein on 2009 pandemic H1N1 virus replication, pathogenicity and host immune responses

PubMed Central

Lee, Jinhwa; Yu, Hai; Li, Yonghai; Ma, Jingjiao; Lang, Yuekun; Duff, Michael; Henningson, Jamie; Liu, Qinfang; Li, Yuhao; Nagy, Abdou; Bawa, Bhupinder; Li, Zejun; Tong, Guangzhi; Richt, Juergen A.; Ma, Wenjun

2017-01-01

Although several studies have investigated the functions of influenza PA-X, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on virus replication, pathogenicity and host response remains unclear. Herein, we generated two mutated viruses expressing a full-length or deficient PA-X protein based on the A/California/04/2009 (H1N1) virus that expresses a truncated PA-X to understand three different expressions of PA-X protein on virus replication, pathogenicity and host immune responses. The results showed that expression of either full-length or truncated PA-X protein enhanced viral replication and pathogenicity as well as reduced host innate immune response in mice by host shutoff activity when compared to the virus expressing the deficient PA-X form. Furthermore, the full-length PA-X expression exhibited a greater effect on virus pathogenicity than the truncated PA-X form. Our results provide novel insights of PA-X on viral replication, pathogenicity and host immune responses. PMID:28142079

A novel phenoxazine derivative suppresses surface IgM expression in DT40 B cell line

PubMed Central

Gao, Sanyang; Takano, Tomoko; Sada, Kiyonao; He, Jinsong; Noda, Chiseko; Hori-Tamura, Naoko; Tomoda, Akio; Yamamura, Hirohei

2002-01-01

2-amino-4, 4α-dihydro-4α, 7-dimethyl-3H-phenoxazine-3-one (Phx) has been demonstrated to be an actinomycin D-like phenoxazine, and to display anti-tumour activity. In this study, we report on the effect of Phx on B cell antigen receptor (BCR) and receptor-mediated signalling in DT40 B cells. Treatment of B cells with Phx for 12 h inhibited BCR-stimulated tyrosine phosphorylation of cellular proteins. B cells exposed to Phx exhibited down-regulation of surface IgM which is part of BCR. In contracts with actinomycin D, Phx rapidly reduced the expression of IgM without decreasing the expression of other signalling molecules. Analysis with confocal microscopy demonstrated that Phx treatment reduced IgM expression both at the cell surface and inside the cell. Treatment of B cells with Phx resulted in the reduction of IgM secretion. Since MG-132, a proteasomal inhibitor, restored IgM contents to the control levels, Phx has the specific effect of accelerating IgM degradation. These results suggest that Phx down-regulates the expression of IgM and inhibits BCR-mediated signalling and IgM secretion. Phx may be useful as an immunosuppressive agent for therapeutic purposes. PMID:12411404

Immediate early gene expression reveals interactions between social and nicotine rewards on brain activity in adolescent male rats.

PubMed

Bastle, Ryan M; Peartree, Natalie A; Goenaga, Julianna; Hatch, Kayla N; Henricks, Angela; Scott, Samantha; Hood, Lauren E; Neisewander, Janet L

2016-10-15

Smoking initiation predominantly occurs during adolescence, often in the presence of peers. Therefore, understanding the neural mechanisms underlying the rewarding effects of nicotine and social stimuli is vital. Using the conditioned place preference (CPP) procedure, we measured immediate early gene (IEG) expression in animals following exposure either to a reward-conditioned environment or to the unconditioned stimuli (US). Adolescent, male rats were assigned to the following CPP US conditions: (1) Saline+Isolated, (2) Nicotine+Isolated, (3) Saline+Social, or (4) Nicotine+Social. For Experiment 1, brain tissue was collected 90min following the CPP expression test and processed for Fos immunohistochemistry. We found that rats conditioned with nicotine with or without a social partner exhibited CPP; however, we found no group differences in Fos expression in any brain region analyzed, with the exception of the nucleus accumbens core that exhibited a social-induced attenuation in Fos expression. For Experiment 2, brain tissue was collected 90min following US exposure during the last conditioning session. We found social reward-induced increases in IEG expression in striatal and amydalar subregions. In contrast, nicotine reduced IEG expression in prefrontal and striatal subregions. Reward interactions were also found in the dorsolateral striatum, basolateral amygdala, and ventral tegmental area where nicotine alone attenuated IEG expression and social reward reversed this effect. These results suggest that in general social rewards enhance, whereas nicotine attenuates, activation of mesocorticolimbic regions; however, the rewards given together interact to enhance activation in some regions. The findings contribute to knowledge of how a social environment influences nicotine effects. Copyright © 2016 Elsevier B.V. All rights reserved.

Aquaporin family genes exhibit developmentally-regulated and host-dependent transcription patterns in the sea louse Caligus rogercresseyi.

PubMed

Farlora, Rodolfo; Valenzuela-Muñoz, Valentina; Chávez-Mardones, Jacqueline; Gallardo-Escárate, Cristian

2016-07-01

Aquaporins are small integral membrane proteins that function as pore channels for the transport of water and other small solutes across the cell membrane. Considering the important roles of these proteins in several biological processes, including host-parasite interactions, there has been increased research on aquaporin proteins recently. The present study expands on the knowledge of aquaporin family genes in parasitic copepods, examining diversity and expression during the ontogeny of the sea louse Caligus rogercresseyi. Furthermore, aquaporin expression was evaluated during the early infestation of Atlantic (Salmo salar) and Coho salmon (Oncorhynchus kisutch). Deep transcriptome sequencing data revealed eight full length and two partial open reading frames belonging to the aquaporin protein family. Clustering analyses with identified Caligidae sequences revealed three major clades of aquaglyceroporins (Cr-Glp), classical aquaporin channels (Cr-Bib and Cr-PripL), and unorthodox aquaporins (Cr-Aqp12-like). In silico analysis revealed differential expression of aquaporin genes between developmental stages and between sexes. Male-biased expression of Cr-Glp1_v1 and female-biased expression of Cr-Bib were further confirmed in adults by RT-qPCR. Additionally, gene expressions were measured for seven aquaporins during the early infestation stage. The majority of aquaporin genes showed significant differential transcription expressions between sea lice parasitizing different hosts, with Atlantic salmon sea lice exhibiting overall reduced expression as compared to Coho salmon. The observed differences in the regulation of aquaporin genes may reveal osmoregulatory adaptations associated with nutrient ingestion and metabolite waste export, exposing complex host-parasite relationships in C. rogercresseyi. Copyright © 2016 Elsevier B.V. All rights reserved.

DLC1-dependent parathyroid hormone-like hormone inhibition suppresses breast cancer bone metastasis.

PubMed

Wang, Yufeng; Lei, Rong; Zhuang, Xueqian; Zhang, Ning; Pan, Hong; Li, Gang; Hu, Jing; Pan, Xiaoqi; Tao, Qian; Fu, Da; Xiao, Jianru; Chin, Y Eugene; Kang, Yibin; Yang, Qifeng; Hu, Guohong

2014-04-01

Bone metastasis is a frequent complication of breast cancer that is often accelerated by TGF-β signaling; however, little is known about how the TGF-β pathway is regulated during bone metastasis. Here we report that deleted in liver cancer 1 (DLC1) is an important regulator of TGF-β responses and osteolytic metastasis of breast cancer cells. In murine models, breast cancer cells lacking DLC1 expression exhibited enhanced capabilities of bone metastasis. Knockdown of DLC1 in cancer cells promoted bone metastasis, leading to manifested osteolysis and accelerated death in mice, while DLC1 overexpression suppressed bone metastasis. Activation of Rho-ROCK signaling in the absence of DLC1 mediated SMAD3 linker region phosphorylation and TGF-β-induced expression of parathyroid hormone-like hormone (PTHLH), leading to osteoclast maturation for osteolytic colonization. Furthermore, pharmacological inhibition of Rho-ROCK effectively reduced PTHLH production and breast cancer bone metastasis in vitro and in vivo. Evaluation of clinical breast tumor samples revealed that reduced DLC1 expression was linked to elevated PTHLH expression and organ-specific metastasis to bone. Overall, our findings define a stroma-dependent paradigm of Rho signaling in cancer and implicate Rho-TGF-β crosstalk in osteolytic bone metastasis.

Deletion of the Ttf1 gene in differentiated neurons disrupts female reproduction without impairing basal ganglia function.

PubMed

Mastronardi, Claudio; Smiley, Gregory G; Raber, Jacob; Kusakabe, Takashi; Kawaguchi, Akio; Matagne, Valerie; Dietzel, Anja; Heger, Sabine; Mungenast, Alison E; Cabrera, Ricardo; Kimura, Shioko; Ojeda, Sergio R

2006-12-20

Thyroid transcription factor 1 (TTF1) [also known as Nkx2.1 (related to the NK-2 class of homeobox genes) and T/ebp (thyroid-specific enhancer-binding protein)], a homeodomain gene required for basal forebrain morphogenesis, remains expressed in the hypothalamus after birth, suggesting a role in neuroendocrine function. Here, we show an involvement of TTF1 in the control of mammalian puberty and adult reproductive function. Gene expression profiling of the nonhuman primate hypothalamus revealed that TTF1 expression increases at puberty. Mice in which the Ttf1 gene was ablated from differentiated neurons grew normally and had normal basal ganglia/hypothalamic morphology but exhibited delayed puberty, reduced reproductive capacity, and a short reproductive span. These defects were associated with reduced hypothalamic expression of genes required for sexual development and deregulation of a gene involved in restraining puberty. No extrapyramidal impairments associated with basal ganglia dysfunction were apparent. Thus, although TTF1 appears to fulfill only a morphogenic function in the ventral telencephalon, once this function is satisfied in the hypothalamus, TTF1 remains active as part of the transcriptional machinery controlling female sexual development.

[The molecular mechanisms and morphological manifestations of leiomyoma reduction induced by selective progesterone receptor modulators].

PubMed

Demura, T A; Revazova, Z V; Kogan, E A; Adamyan, L V

to investigate the molecular mechanisms and morphological substrate of reduced uterine leiomyoma in patients receiving the selective progesterone receptor modulator (SPRM) ulipristal acetate for 3 months, by estimating the immunohistochemical expression of the markers steroid receptor coactivator 1 (SRC-1), nuclear receptor corepressor 1 (NCoR-1), ER, PgR, Ki-67, p16, TGF-β, and VEGF in tumor tissue. The investigation enrolled 75 women with uterine leiomyoma, menorrhagias, and anemia. Group 1 included 40 patients who were treated with ulipristal for 3 months, followed by laparoscopic myomectomy. Group 2 consisted of 35 patients who underwent surgery without previous preparation. The intra- and postoperative parameters and molecular and morphological changes in the myomatous nodules were comparatively analyzed in both groups. After 3 months of therapy initiation, menorrhagia completely ceased, myomatous nodules decreased in size (p<0.05), hemoglobin levels were elevated (p<0.01), and total intraoperative blood loss and operative time decreased in all the patients in Group 1. The morphological substrate of partial leiomyoma reduction was leiomyocyte apoptosis and dystrophy, tumor stroma sclerosis and hyalinosis with diminished Ki-67 expression and elevated p16 in the smooth muscle cells, trophic nodular tissue disorders exhibited by vascular wall sclerosis and lower VEGF and TGF-β expression, and leiomyocyte hormonal reception dysregulation that made itself evident through the reduced expression of SRC-1 with the unchanged expression of PR and ER and the maintained level of NCoR-1. The molecular mechanisms of tumor reduction involved the reduced Ki-67 expression and elevated p16, lower VEGF and TGF-β, diminished SRC-1 expression with the maintained level of PR, ER, and NCoR-1. Overall, this is suggestive of enhanced apoptosis and reduced leiomyoma proliferation and angiogenesis induced by SPRM and indicative of the expediency of using ulipristal acetate as a preoperative agent for organ-sparing surgery in reproductive-aged patients with uterine myoma, menorrhagias, and anemia.

Impact of toll-like-receptor-9 (TLR9) deficiency on visceral adipose tissue adipokine expression during chronic DSS-induced colitis in mice.

PubMed

Karrasch, T; Schmid, A; Kopp, A; Obermeier, F; Hofmann, C; Schäffler, A

2015-02-01

Studies postulate an involvement of adipokines in inflammatory gastrointestinal diseases. Leptin-deficient ob/ob mice as well as TLR9-deficient mice have a more moderate course of chronic DSS-induced colitis (DSS-CC) and adipocytes do express functional TLR9 molecules. Adipokine mRNA expression in visceral adipose tissue of mice before and after the induction of DSS-CC was investigated. Experiments were performed in both TLR9(wt/wt) and TLR9(-/-) mice. In vitro, the effect of TLR9 blocking peptide on leptin and visfatin protein secretion was studied in 3T3-L1 adipocytes. Induction of DSS-CC led to an upregulation of leptin mRNA expression in TLR9(wt/wt) mice, while TLR9(-/-) animals showed a significant reduction of leptin expression even below baseline. While visfatin expression remained unchanged in TLR9(wt/wt) animals, TLR9(-/-) mice exhibited a significant induction during DSS-CC. CTRP-3 expression was reduced after colitis induction only in TLR9(-/-) animals. Of note, IL-6 expression levels remained unchanged, while CXCL1/KC and cyclophilin A expression was reduced in DSS-CC. Inhibition of TLR9 signaling by using TLR9 blocking peptide led to reduced leptin protein secretion into cell culture supernatants in 3T3-L1 adipocytes, while visfatin protein secretion was enhanced. DSS-CC leads to differential adipokine expression profiles in the visceral fat pad in TLR9(wt/wt) vs. TLR9(-/-) mice. In vitro, inhibition of TLR9 signaling induces visfatin secretion while inhibiting leptin secretion in adipocytes. Thus, visceral adipokines are regulated by intact TLR9 signaling pathway and a specific interplay between the leptin- and the TLR9-pathways might be of pathophysiological importance in chronic intestinal inflammation. © Georg Thieme Verlag KG Stuttgart · New York.

Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mueller, Edith E., E-mail: ed.mueller@salk.at; Mayr, Johannes A., E-mail: h.mayr@salk.at; Zimmermann, Franz A., E-mail: f.zimmermann@salk.at

2012-01-20

Highlights: Black-Right-Pointing-Pointer We examined OXPHOS and citrate synthase enzyme activities in HEK293 cells devoid of mtDNA. Black-Right-Pointing-Pointer Enzymes partially encoded by mtDNA show reduced activities. Black-Right-Pointing-Pointer Also the entirely nuclear encoded complex II and citrate synthase exhibit reduced activities. Black-Right-Pointing-Pointer Loss of mtDNA induces a feedback mechanism that downregulates complex II and citrate synthase. -- Abstract: Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complexmore » II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 {rho}{sup 0} cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in {rho}{sup 0} cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.« less

Increased Resting Intracellular Calcium Modulates NF-κB-dependent Inducible Nitric-oxide Synthase Gene Expression in Dystrophic mdx Skeletal Myotubes*

PubMed Central

Altamirano, Francisco; López, Jose R.; Henríquez, Carlos; Molinski, Tadeusz; Allen, Paul D.; Jaimovich, Enrique

2012-01-01

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by dystrophin mutations, characterized by chronic inflammation and severe muscle wasting. Dystrophic muscles exhibit activated immune cell infiltrates, up-regulated inflammatory gene expression, and increased NF-κB activity, but the contribution of the skeletal muscle cell to this process has been unclear. The aim of this work was to study the pathways that contribute to the increased resting calcium ([Ca2+]rest) observed in mdx myotubes and its possible link with up-regulation of NF-κB and pro-inflammatory gene expression in dystrophic muscle cells. [Ca2+]rest was higher in mdx than in WT myotubes (308 ± 6 versus 113 ± 2 nm, p < 0.001). In mdx myotubes, both the inhibition of Ca2+ entry (low Ca2+ solution, Ca2+-free solution, and Gd3+) and blockade of either ryanodine receptors or inositol 1,4,5-trisphosphate receptors reduced [Ca2+]rest. Basal activity of NF-κB was significantly up-regulated in mdx versus WT myotubes. There was an increased transcriptional activity and p65 nuclear localization, which could be reversed when [Ca2+]rest was reduced. Levels of mRNA for TNFα, IL-1β, and IL-6 were similar in WT and mdx myotubes, whereas inducible nitric-oxide synthase (iNOS) expression was increased 5-fold. Reducing [Ca2+]rest using different strategies reduced iNOS gene expression presumably as a result of decreased activation of NF-κB. We propose that NF-κB, modulated by increased [Ca2+]rest, is constitutively active in mdx myotubes, and this mechanism can account for iNOS overexpression and the increase in reactive nitrogen species that promote damage in dystrophic skeletal muscle cells. PMID:22549782

Specific expression of FOXP2 in cerebellum improves ultrasonic vocalization in heterozygous but not in homozygous Foxp2 (R552H) knock-in pups.

PubMed

Fujita-Jimbo, Eriko; Momoi, Takashi

2014-04-30

The R553H mutation has been found in the FOXP2 gene of patients with speech-language disorder. Foxp2(R552H) knock-in (KI) mice exhibit poor dendritic development of Purkinje cells in the cerebellum and impaired ultrasonic vocalization (USV), which is related to human speech and language; compared with wild-type mice, heterozygous Foxp2(R552H)-KI pups exhibit the reduced number of whistle-type USVs and the increased short-type ones, while homozygous pups exhibit only click-type USVs but no whistle-type or short-type ones. To make clear the relationship between the role of Foxp2 in the cerebellum and whistle-type USVs activity, we prepared transgenic (Tg) mice specifically expressing human FOXP2-myc in cerebellum (Pcp2-FOXP2-myc-Tg mice) by using purkinje cell protein-2 (Pcp2) promoter. FOXP2-myc expression in the cerebellum increased the relative numbers of whistle-type USVs in the heterozygous Foxp2(R552H)-KI pups and recovered their USVs but did not in the homozygous ones. Foxp2 in the cerebellum may pertain to the brain network engaged in whistle-type USVs activities including modification, but not their production. There may be common molecular contribution of Purkinje cells to human FOXP2-mediated speech-language and mouse Foxp2-mediated USVs. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Phosphatidylserine and GTPase activation control Cdc42 nanoclustering to counter dissipative diffusion.

PubMed

Sartorel, Elodie; Ünlü, Caner; Jose, Mini; Massoni-Laporte, Aurélie; Meca, Julien; Sibarita, Jean-Baptiste; McCusker, Derek

2018-04-18

The anisotropic organization of plasma membrane constituents is indicative of mechanisms that drive the membrane away from equilibrium. However, defining these mechanisms is challenging due to the short spatio-temporal scales at which diffusion operates. Here, we use high-density single protein tracking combined with photoactivation localization microscopy (sptPALM) to monitor Cdc42 in budding yeast, a system in which Cdc42 exhibits anisotropic organization. Cdc42 exhibited reduced mobility at the cell pole, where it was organized in nanoclusters. The Cdc42 nanoclusters were larger at the cell pole than those observed elsewhere in the cell. These features were exacerbated in cells expressing Cdc42-GTP, and were dependent on the scaffold Bem1, which contributed to the range of mobility and nanocluster size exhibited by Cdc42. The lipid environment, in particular phosphatidylserine levels, also played a role in regulating Cdc42 nanoclustering. These studies reveal how the mobility of a Rho GTPase is controlled to counter the depletive effects of diffusion, thus stabilizing Cdc42 on the plasma membrane and sustaining cell polarity. Movie S1 Movie S1 sptPALM imaging of live yeast expressing Pil1-mEOS expressed at the genomic locus. Pil1-mEOS was simultaneously photo-converted with a 405 nm laser and imaged with a 561 nm laser using HiLo illumination. Images were acquired at 20 ms intervals, of which 300 frames are shown at 7 frames per second.

Soluble guanylate cyclase generation of cGMP regulates migration of MGE neurons.

PubMed

Mandal, Shyamali; Stanco, Amelia; Buys, Emmanuel S; Enikolopov, Grigori; Rubenstein, John L R

2013-10-23

Here we have provided evidence that nitric oxide-cyclic GMP (NO-cGMP) signaling regulates neurite length and migration of immature neurons derived from the medial ganglionic eminence (MGE). Dlx1/2(-/-) and Lhx6(-/-) mouse mutants, which exhibit MGE interneuron migration defects, have reduced expression of the gene encoding the α subunit of a soluble guanylate cyclase (Gucy1A3). Furthermore, Dlx1/2(-/-) mouse mutants have reduced expression of NO synthase 1 (NOS1). Gucy1A3(-/-) mice have a transient reduction in cortical interneuron number. Pharmacological inhibition of soluble guanylate cyclase and NOS activity rapidly induces neurite retraction of MGE cells in vitro and in slice culture and robustly inhibits cell migration from the MGE and caudal ganglionic eminence. We provide evidence that these cellular phenotypes are mediated by activation of the Rho signaling pathway and inhibition of myosin light chain phosphatase activity.

Indian hedgehog roles in post-natal TMJ development and organization.

PubMed

Ochiai, T; Shibukawa, Y; Nagayama, M; Mundy, C; Yasuda, T; Okabe, T; Shimono, K; Kanyama, M; Hasegawa, H; Maeda, Y; Lanske, B; Pacifici, M; Koyama, E

2010-04-01

Indian hedgehog (Ihh) is essential for embryonic mandibular condylar growth and disc primordium formation. To determine whether it regulates those processes during post-natal life, we ablated Ihh in cartilage of neonatal mice and assessed the consequences on temporomandibular joint (TMJ) growth and organization over age. Ihh deficiency caused condylar disorganization and growth retardation and reduced polymorphic cell layer proliferation. Expression of Sox9, Runx2, and Osterix was low, as was that of collagen II, collagen I, and aggrecan, thus altering the fibrocartilaginous nature of the condyle. Though a disc formed, it exhibited morphological defects, partial fusion with the glenoid bone surface, reduced synovial cavity space, and, unexpectedly, higher lubricin expression. Analysis of the data shows, for the first time, that continuous Ihh action is required for completion of post-natal TMJ growth and organization. Lubricin overexpression in mutants may represent a compensatory response to sustain TMJ movement and function.

Epidermal dysplasia and abnormal hair follicles in transgenic mice overexpressing homeobox gene MSX-2.

PubMed

Jiang, T X; Liu, Y H; Widelitz, R B; Kundu, R K; Maxson, R E; Chuong, C M

1999-08-01

The homeobox gene Msx-2 is expressed specifically in sites of skin appendage formation. To explore its part in skin morphogenesis, we produced transgenic mice expressing Msx-2 under the control of the cytomegalovirus promoter. The skin of these transgenic mice was flaky, exhibiting desquamation and shorter hairs. Histologic analysis showed thickened epidermis with hyperproliferation, which was restricted to the basal layer. Hyperkeratosis was also evident. A wide zone of suprabasal cells were misaligned and coexpressed keratins 14 and 10. There was reduced expression of integrin beta 1 and DCC in the basal layer. Hair follicles were misaligned with a shrunken matrix region. The dermis showed increased cellularity and empty vacuoles. We suggest that Msx-2 is involved in the growth control of skin and skin appendages.

Omega-3 Fatty Acid Deficient Male Rats Exhibit Abnormal Behavioral Activation in the Forced Swim Test Following Chronic Fluoxetine Treatment: Association with Altered 5-HT1A and Alpha2A Adrenergic Receptor Expression

PubMed Central

Able, Jessica A.; Liu, Yanhong; Jandacek, Ronald; Rider, Therese; Tso, Patrick; McNamara, Robert K.

2014-01-01

Omega-3 fatty acid deficiency during development leads to enduing alterations in central monoamine neurotransmission in rat brain. Here we investigated the effects of omega-3 fatty acid deficiency on behavioral and neurochemical responses to chronic fluoxetine (FLX) treatment. Male rats were fed diets with (CON, n=34) or without (DEF, n=30) the omega-3 fatty acid precursor alpha-linolenic acid (ALA) during peri-adolescent development (P21-P90). A subset of CON (n=14) and DEF (n=12) rats were administered FLX (10 mg/kg/d) through their drinking water for 30 d beginning on P60. The forced swimming test (FST) was initiated on P90, and regional brain mRNA markers of serotonin and noradrenaline neurotransmission were determined. Dietary ALA depletion led to significant reductions in frontal cortex docosahexaenoic acid (DHA, 22:6n-3) composition in DEF (−26%, p=0.0001) and DEF+FLX (−32%, p=0.0001) rats. Plasma FLX and norfluoxetine concentrations did not different between FLX-treated DEF and CON rats. During the 15-min FST pretest, DEF+FLX rats exhibited significantly greater climbing behavior compared with CON+FLX rats. During the 5-min test trial, FLX treatment reduced immobility and increased swimming in CON and DEF rats, and only DEF+FLX rats exhibited significant elevations in climbing behavior. DEF+FLX rats exhibited greater midbrain, and lower frontal cortex, 5-HT1A mRNA expression compared with all groups including CON+FLX rats. DEF+FLX rats also exhibited greater midbrain alpha2A adrenergic receptor mRNA expression which was positively correlated with climbing behavior in the FST. These preclinical data demonstrate that low omega-3 fatty acid status leads to abnormal behavioral and neurochemical responses to chronic FLX treatment in male rats. PMID:24360505

A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity

PubMed Central

Song, De-Gang; Ye, Qunrui; Poussin, Mathilde; Liu, Lin; Figini, Mariangela; Powell, Daniel J.

2015-01-01

Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors in an HLA-independent manner. To date, various CARs have been constructed using mouse single chain antibody variable fragments (scFvs) of high affinity that are immunogenic in humans and have the potential to mediate “on-target” toxicity. Here, we developed and evaluated a fully human CAR comprised of the human C4 folate receptor-alpha (αFR)-specific scFv coupled to intracellular T cell signaling domains. Human T cells transduced to express the C4 CAR specifically secreted proinflammatory cytokine and exerted cytolytic functions when cultured with αFR-expressing tumors in vitro. Adoptive transfer of C4 CAR T cells mediated the regression of large, established human ovarian cancer in a xenogeneic mouse model. Relative to a murine MOv19 scFv-based αFR CAR, C4 CAR T cells mediated comparable cytotoxic tumor activity in vitro and in vivo but had lower affinity for αFR protein and exhibited reduced recognition of normal cells expressing low levels of αFR. Thus, T cells expressing a fully human CAR of intermediate affinity can efficiently kill antigen-expressing tumors in vitro and in vivo and may overcome issues of transgene immunogenicity and “on-target off-tumor” toxicity that plague trials utilizing CARs containing mouse-derived, high affinity scFvs. PMID:26101914

IFT88 influences chondrocyte actin organization and biomechanics.

PubMed

Wang, Z; Wann, A K T; Thompson, C L; Hassen, A; Wang, W; Knight, M M

2016-03-01

Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88(orpk)). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. IFT88(orpk) cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88(orpk) cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88(orpk) cells. Following membrane blebbing, IFT88(orpk) cells exhibited slower reformation of the actin cortex. IFT88(orpk) cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

NK Cells Restrain Spontaneous Antitumor CD8+ T Cell Priming through PD-1/PD-L1 Interactions with Dendritic Cells.

PubMed

Iraolagoitia, Ximena L Raffo; Spallanzani, Raul G; Torres, Nicolás I; Araya, Romina E; Ziblat, Andrea; Domaica, Carolina I; Sierra, Jessica M; Nuñez, Sol Y; Secchiari, Florencia; Gajewski, Thomas F; Zwirner, Norberto W; Fuertes, Mercedes B

2016-08-01

Despite the classical function of NK cells in the elimination of tumor and of virus-infected cells, evidence for a regulatory role for NK cells has been emerging in different models of autoimmunity, transplantation, and viral infections. However, this role has not been fully explored in the context of a growing tumor. In this article, we show that NK cells can limit spontaneous cross-priming of tumor Ag-specific CD8(+) T cells, leading to reduced memory responses. After challenge with MC57 cells transduced to express the model Ag SIY (MC57.SIY), NK cell-depleted mice exhibited a significantly higher frequency of SIY-specific CD8(+) T cells, with enhanced IFN-γ production and cytotoxic capability. Depletion of NK cells resulted in a CD8(+) T cell population skewed toward an effector memory T phenotype that was associated with enhanced recall responses and delayed tumor growth after a secondary tumor challenge with B16.SIY cells. Dendritic cells (DCs) from NK cell-depleted tumor-bearing mice exhibited a more mature phenotype. Interestingly, tumor-infiltrating and tumor-draining lymph node NK cells displayed an upregulated expression of the inhibitory molecule programmed death ligand 1 that, through interaction with programmed death-1 expressed on DCs, limited DC activation, explaining their reduced ability to induce tumor-specific CD8(+) T cell priming. Our results suggest that NK cells can, in certain contexts, have an inhibitory effect on antitumor immunity, a finding with implications for immunotherapy in the clinic. Copyright © 2016 by The American Association of Immunologists, Inc.

Naringin ameliorates endothelial dysfunction in fructose-fed rats.

PubMed

Malakul, Wachirawadee; Pengnet, Sirinat; Kumchoom, Chanon; Tunsophon, Sakara

2018-03-01

High fructose consumption is associated with metabolic disorders including hyperglycemia and dyslipidemia, in addition to endothelial dysfunction. Naringin, a flavonoid present in citrus fruit, has been reported to exhibit lipid lowering, antioxidant, and cardiovascular protective properties. Therefore, the present study investigated the effect of naringin on fructose-induced endothelial dysfunction in rats and its underlying mechanisms. Male Sprague-Dawley rats were given 10% fructose in drinking water for 12 weeks, whereas control rats were fed drinking water alone. Naringin (100 mg/kg) was orally administered to fructose fed rats during the last 4 weeks of the study. Following 12 weeks, blood samples were collected for measurement of blood glucose, serum lipid profile and total nitrate/nitrite (NOx). Vascular function was assessed by isometric tension recording. Aortic expression of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS), and nitrotyrosine were evaluated by western blot analysis. Fructose feeding induced increased levels of blood glucose, total cholesterol, triglyceride, and low density lipoprotein. In rat aortae, fructose reduced acethycholine-induced vasorelaxation, without affecting sodium nitroprusside-induced vasorelaxation. Treatment of fructose-fed rats with naringin restored fructose-induced metabolic alterations and endothelial dysfunction. Fructose-fed rats also exhibited decreased serum NOx level, reduced eNOS and p-eNOS protein expression, and enhanced nitrotyrosine expression in aortae. These alterations were improved by naringin treatment. The results of the present study suggested that naringin treatment preserves endothelium-dependent relaxation in aortae from fructose fed rats. This effect is primarily mediated through an enhanced NO bioavailability via increased eNOS activity and decreased NO inactivated to peroxynitrite in aortae.

Orexins Mediate Sex Differences in the Stress Response and in Cognitive Flexibility.

PubMed

Grafe, Laura A; Cornfeld, Amanda; Luz, Sandra; Valentino, Rita; Bhatnagar, Seema

2017-04-15

Women are twice as likely as men to experience stress-related psychiatric disorders. The biological basis of these sex differences is poorly understood. Orexins are altered in anxious and depressed patients. Using a rat model of repeated stress, we examined whether orexins contribute to sex differences in outcomes relevant to stress-related psychiatric diseases. Behavioral, neural, and endocrine habituation to repeated restraint stress and subsequent cognitive flexibility was examined in adult male and female rats. In parallel, orexin expression and activation were determined in both sexes, and chromatin immunoprecipitation was used to determine transcription factors acting at the orexin promoter. Designer receptors exclusively activated by designer drugs were used to inhibit orexin activation throughout repeated restraint to determine if the stress-related impairments in female rats could be reduced. Female rats exhibited impaired habituation to repeated restraint with subsequent deficits in cognitive flexibility compared with male rats. Increased orexin expression and activation were observed in female rats compared with male rats. The higher expression of orexin messenger RNA in female rats was due to actions of glucocorticoid receptors on the orexin promoter, as determined by chromatin immunoprecipitation. Inhibition of orexins using designer receptors exclusively activated by designer drugs in female rats throughout repeated restraint abolished their heightened hypothalamic-pituitary-adrenal responsivity and reduced stress-induced cognitive impairments. Orexins mediate the impairments in adaptations to repeated stress and in subsequent cognitive flexibility exhibited by female rats and provide evidence for a broader role for orexins in mediating functions relevant to stress-related psychiatric diseases. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Prehypertensive treatment with losartan, however not amlodipine, leads to long-term effects on blood pressure and reduces the risk of stroke in spontaneously hypertensive stroke-prone rats.

PubMed

Zhang, Liangmin; He, Dehua; Lin, Jinxiu

2016-02-01

The current study investigated the efficacy of losartan and amlodipine in protecting spontaneously hypertensive stroke-prone (SHRSP) rats against the risk of stroke. SHRSP rats were administered losartan, amlodipine or the vehicle for 6 weeks. There were no significant differences in systolic blood pressure (SBP) in rats treated with losartan or amlodipine, however, following drug withdrawal, rats treated with losartan maintained reduced SBP for a longer time compared with rats treated with amlodipine. In addition, rats treated with losartan exhibited thinner vascular walls and improved systolic and diastolic function. Clinical stroke scores in the losartan group were significantly reduced compared with those in the amlodipine and vehicle groups. However, rats treated with losartan exhibited higher levels of angiotensin II and lower levels of aldosterone in the serum and brain cortex compared with the vehicle and amlodipine-treated rats. Furthermore, losartan significantly reduced the abnormal expression of angiotensin II receptors type 1 and 2 in SHRSP rats, whilst amlodipine did not. These results suggest that losartan may be more efficacious than amlodipine in ameliorating blood pressure deterioration and reducing stroke risk in SHRSP rats via regulation of the renin angiotensin system.

Anethole potentiates dodecanol's fungicidal activity by reducing PDR5 expression in budding yeast.

PubMed

Fujita, Ken-Ichi; Ishikura, Takayuki; Jono, Yui; Yamaguchi, Yoshihiro; Ogita, Akira; Kubo, Isao; Tanaka, Toshio

2017-02-01

trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum and a weaker antimicrobial potency than other available antibiotics. When combined with polygodial, nagilactone E, and n-dodecanol, anethole has been shown to exhibit synergistic antifungal activity against a budding yeast, Saccharomyces cerevisiae, and a human opportunistic pathogenic yeast, Candida albicans. However, the mechanism underlying this synergistic effect of anethole has not been characterized. We studied this mechanism using dodecanol-treated S. cerevisiae cells and focusing on genes related to multidrug efflux. Although dodecanol transiently reduced the number of colony forming units, this recovered to levels similar to those of untreated cells with continued incubation beyond 24h. Reverse transcription polymerase chain reaction analysis revealed overexpression of an ATP-binding cassette (ABC) transporter gene, PDR5, in addition to a slight increase in PDR11, PDR12, and PDR15 transcriptions in dodecanol-treated cells. In the presence of anethole, these effects were attenuated and the fungicidal activity of dodecanol was extended. Dodecanol showed longer lasting fungicidal activity against a Δpdr5. In addition, Δpdr3 and Δlge1, lack transcription factors of PDR5 and PDR3, were partly and completely susceptible to dodecanol, respectively. Furthermore, combination of anethole with fluconazole was also found to exhibit synergy on C. albicans. These results indicated that although anethole reduced the transcription of several transporters, PDR5 expression was particularly relevant to dodecanol efflux. Anethole is expected to be a promising candidate drug for the inhibition of efflux by reducing the transcription of several ABC transporters. Copyright © 2016 Elsevier B.V. All rights reserved.

ZitB (YbgR), a Member of the Cation Diffusion Facilitator Family, Is an Additional Zinc Transporter in Escherichia coli

PubMed Central

Grass, Gregor; Fan, Bin; Rosen, Barry P.; Franke, Sylvia; Nies, Dietrich H.; Rensing, Christopher

2001-01-01

The Escherichia coli zitB gene encodes a Zn(II) transporter belonging to the cation diffusion facilitator family. ZitB is specifically induced by zinc. ZitB expression on a plasmid rendered zntA-disrupted E. coli cells more resistant to zinc, and the cells exhibited reduced accumulation of 65Zn, suggesting ZitB-mediated efflux of zinc. PMID:11443104

Snail1 induced in breast cancer cells in 3D collagen I gel environment suppresses cortactin and impairs effective invadopodia formation.

PubMed

Lee, Mi-Sook; Kim, Sudong; Kim, Baek Gil; Won, Cheolhee; Nam, Seo Hee; Kang, Suki; Kim, Hye-Jin; Kang, Minkyung; Ryu, Jihye; Song, Haeng Eun; Lee, Doohyung; Ye, Sang-Kyu; Jeon, Noo Li; Kim, Tai Young; Cho, Nam Hoon; Lee, Jung Weon

2014-09-01

Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFβ1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents. Copyright © 2014 Elsevier B.V. All rights reserved.

Zoledronic acid overcomes chemoresistance by sensitizing cancer stem cells to apoptosis.

PubMed

Rouhrazi, H; Turgan, N; Oktem, G

2018-01-01

Unlike low tumorigenic bulk tumor cells (non-CSCs), cancer stem cells (CSCs) are a subset of tumor cells that can self-renew and differentiate into different cancer subtypes. CSCs are considered responsible for tumor recurrence, distant metastasis, angiogenesis, and drug or radiation resistance. CSCs also are resistant to apoptosis. Zoledronic acid (ZA) is a third generation bisphosphonate that reduces cell proliferation and exhibits anti-tumor effects by inducing cell death in some malignancies; however, the effects of ZA on CSCs are unclear. We investigated the anti-cancer effects of ZA on two epithelial cancer cell lines, prostate DU-145 and breast MCF7, focusing primarily on induction and activation of apoptosis. Cluster of differentiation (CD) 133 + /CD44 + prostate CSCs and CD 44 + /CD24 breast CSCs were isolated from the DU-145 human prostate cancer and MCF-7 human breast cancer cell lines, respectively, using FACSAria flow cytometry cell sorting. CSCs and non-CSCs were exposed to increasing concentrations of ZA for 24, 48 and 72 h to determine the IC 50 dose. Annexin-V assay for detecting cell death and cell cycle was performed using the Muse™ Cell Analyzer. Prostate CSCs and non-CSCs were assayed by quantitative reverse transcription PCR (qRT-PCR) array for detecting 84 key apoptosis related genes. Gene regulation at the protein level was investigated by immunofluorescence. ZA caused a dose- and time-dependent decrease in cell viability. Treatment with ZA resulted in a concomitant increase in apoptosis and cell cycle arrest at S-phase in CSCs. Significant over/under-expressions were detected in seven of the genes of ZA-treated DU-145 CSCs cells. Expressions of CASP9, CASP4, BAX and BAD genes increased, while the expressions of BIRC3, BIRC2 and BCL2 genes decreased. In the DU-145 non-CSCs, five genes exhibited changes in gene expression after ZA treatment, two exhibited increased expression (CASP7 and BAD) and three exhibited decreased expression (BIRC3, BIRC2 and BCL2). ZA caused cell death of drug resistant breast MCF-7 and prostate DU-145 cancer stem cells by activating apoptosis. ZA can facilitate the intrinsic pathway of apoptosis in human prostate CSCs by down-regulating anti-apoptotic genes and up-regulating pro-apoptotic genes. ZA may be an effective therapeutic agent for targeting chemoresistance in CSCs.

Divergent patterns of age-dependence in ornamental and reproductive traits in the collared flycatcher.

PubMed

Evans, Simon R; Gustafsson, Lars; Sheldon, Ben C

2011-06-01

Sexual ornaments are predicted to honestly signal individual condition. We might therefore expect ornament expression to show a senescent decline, in parallel with late-life deterioration of other characters. Conversely, life-history theory predicts the reduced residual reproductive value of older individuals will favor increased investment in sexually attractive traits. Using a 25-year dataset of more than 5000 records of breeding collared flycatchers (Ficedula albicollis) of known age, we quantify cross-sectional patterns of age-dependence in ornamental plumage traits and report long-term declines in expression that mask highly significant positive age-dependency. We partition this population-level age-dependency into its between- and within-individual components and show expression of ornamental white plumage patches exhibits within-individual increases with age in both sexes, consistent with life-history theory. For males, ornament expression also covaries with life span, such that, within a cohort, ornamentation indicates survival. Finally, we compared longitudinal age-dependency of reproductive traits and ornamental traits in both sexes, to assess whether these two trait types exhibit similar age-dependency. These analyses revealed contrasting patterns: reproductive traits showed within-individual declines in late-life females consistent with senescence; ornamental traits showed the opposite pattern in both males and females. Hence, our results for both sexes suggest that age-dependent ornament expression is consistent with life-history models of optimal signaling and, unlike reproductive traits, proof against senescence. © 2011 The Author(s). Evolution© 2011 The Society for the Study of Evolution.

Vibration stimulates vocal mucosa-like matrix expression by hydrogel-encapsulated fibroblasts.

PubMed

Kutty, Jaishankar K; Webb, Ken

2010-01-01

The composition and organization of the vocal fold extracellular matrix (ECM) provide the viscoelastic mechanical properties that are required to sustain high-frequency vibration during voice production. Although vocal injury and pathology are known to produce alterations in matrix physiology, the mechanisms responsible for the development and maintenance of vocal fold ECM are poorly understood. The objective of this study was to investigate the effect of physiologically relevant vibratory stimulation on ECM gene expression and synthesis by fibroblasts encapsulated within hyaluronic acid hydrogels that approximate the viscoelastic properties of vocal mucosa. Relative to static controls, samples exposed to vibration exhibited significant increases in mRNA expression levels of HA synthase 2, decorin, fibromodulin and MMP-1, while collagen and elastin expression were relatively unchanged. Expression levels exhibited a temporal response, with maximum increases observed after 3 and 5 days of vibratory stimulation and significant downregulation observed at 10 days. Quantitative assays of matrix accumulation confirmed significant increases in sulphated glycosaminoglycans and significant decreases in collagen after 5 and 10 days of vibratory culture, relative to static controls. Cellular remodelling and hydrogel viscosity were affected by vibratory stimulation and were influenced by varying the encapsulated cell density. These results indicate that vibration is a critical epigenetic factor regulating vocal fold ECM and suggest that rapid restoration of the phonatory microenvironment may provide a basis for reducing vocal scarring, restoring native matrix composition and improving vocal quality. 2009 John Wiley & Sons, Ltd.

Suppression of Allene Oxide Cyclase in Hairy Roots of Medicago truncatula Reduces Jasmonate Levels and the Degree of Mycorrhization with Glomus intraradices1[w

PubMed Central

Isayenkov, Stanislav; Mrosk, Cornelia; Stenzel, Irene; Strack, Dieter; Hause, Bettina

2005-01-01

During the symbiotic interaction between Medicago truncatula and the arbuscular mycorrhizal (AM) fungus Glomus intraradices, an endogenous increase in jasmonic acid (JA) occurs. Two full-length cDNAs coding for the JA-biosynthetic enzyme allene oxide cyclase (AOC) from M. truncatula, designated as MtAOC1 and MtAOC2, were cloned and characterized. The AOC protein was localized in plastids and found to occur constitutively in all vascular tissues of M. truncatula. In leaves and roots, MtAOCs are expressed upon JA application. Enhanced expression was also observed during mycorrhization with G. intraradices. A partial suppression of MtAOC expression was achieved in roots following transformation with Agrobacterium rhizogenes harboring the MtAOC1 cDNA in the antisense direction under control of the cauliflower mosaic virus 35S promoter. In comparison to samples transformed with 35S∷uidA, roots with suppressed MtAOC1 expression exhibited lower JA levels and a remarkable delay in the process of colonization with G. intraradices. Both the mycorrhization rate, quantified by fungal rRNA, and the arbuscule formation, analyzed by the expression level of the AM-specific gene MtPT4, were affected. Staining of fungal material in roots with suppressed MtAOC1 revealed a decreased number of arbuscules, but these did not exhibit an altered structure. Our results indicate a crucial role for JA in the establishment of AM symbiosis. PMID:16244141

Hormone-induced protection against mammary tumorigenesis is conserved in multiple rat strains and identifies a core gene expression signature induced by pregnancy.

PubMed

Blakely, Collin M; Stoddard, Alexander J; Belka, George K; Dugan, Katherine D; Notarfrancesco, Kathleen L; Moody, Susan E; D'Cruz, Celina M; Chodosh, Lewis A

2006-06-15

Women who have their first child early in life have a substantially lower lifetime risk of breast cancer. The mechanism for this is unknown. Similar to humans, rats exhibit parity-induced protection against mammary tumorigenesis. To explore the basis for this phenomenon, we identified persistent pregnancy-induced changes in mammary gene expression that are tightly associated with protection against tumorigenesis in multiple inbred rat strains. Four inbred rat strains that exhibit marked differences in their intrinsic susceptibilities to carcinogen-induced mammary tumorigenesis were each shown to display significant protection against methylnitrosourea-induced mammary tumorigenesis following treatment with pregnancy levels of estradiol and progesterone. Microarray expression profiling of parous and nulliparous mammary tissue from these four strains yielded a common 70-gene signature. Examination of the genes constituting this signature implicated alterations in transforming growth factor-beta signaling, the extracellular matrix, amphiregulin expression, and the growth hormone/insulin-like growth factor I axis in pregnancy-induced alterations in breast cancer risk. Notably, related molecular changes have been associated with decreased mammographic density, which itself is strongly associated with decreased breast cancer risk. Our findings show that hormone-induced protection against mammary tumorigenesis is widely conserved among divergent rat strains and define a gene expression signature that is tightly correlated with reduced mammary tumor susceptibility as a consequence of a normal developmental event. Given the conservation of this signature, these pathways may contribute to pregnancy-induced protection against breast cancer.

Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique

Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4{sup +} T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependentmore » phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. - Highlights: • Jurkat T cells expressing the HIV-1 envelope fuse with THP-1 monocytes. • Heterokaryons display a dominant myeloid phenotype and monocyte function. • Heterokaryons exhibit activation features in the absence of activation agents. • Activation is not due to cell-cell interaction but requires cell-cell fusion. • The activated monocyte-like phenotype is mediated by TLR2/TLR4 signaling.« less

The Putative PAX8/PPARγ Fusion Oncoprotein Exhibits Partial Tumor Suppressor Activity through Up-Regulation of Micro-RNA-122 and Dominant-Negative PPARγ Activity.

PubMed

Reddi, Honey V; Madde, Pranathi; Milosevic, Dragana; Hackbarth, Jennifer S; Algeciras-Schimnich, Alicia; McIver, Bryan; Grebe, Stefan K G; Eberhardt, Norman L

2011-01-01

In vitro studies have demonstrated that the PAX8/PPARγ fusion protein (PPFP), which occurs frequently in follicular thyroid carcinomas (FTC), exhibits oncogenic activity. However, paradoxically, a meta-analysis of extant tumor outcome studies indicates that 68% of FTC-expressing PPFP are minimally invasive compared to only 32% of those lacking PPFP (χ(2) = 6.86, P = 0.008), suggesting that PPFP favorably impacts FTC outcomes. In studies designed to distinguish benign thyroid neoplasms from thyroid carcinomas, the previously identified tumor suppressor miR-122, a major liver micro-RNA (miR) that is decreased in hepatocellular carcinoma, was increased 8.9-fold (P < 0.05) in all FTC versus normal, 9.2-fold in FTC versus FA (P < 0.05), and 16.8-fold (P < 0.001) in FTC + PPFP versus FTC - PPFP. Constitutive expression of PPFP in the FTC-derived cell line WRO (WRO-PPFP) caused a 5-fold increase of miR-122 expression (P < 0.05) and a striking 5.1-fold reduction (P < 0.0001) in tumor progression compared to WRO-vector cells in a mouse xenograft model. Constitutive expression of either miR-122 or a dominant-negative PPARγ mutant in WRO cells was less effective than PPFP at inhibiting xenograft tumor progression (1.8-fold [P < 0.001] and 1.7-fold [P < 0.03], respectively). PPFP-induced up-regulation of miR-122 expression was independent of its known dominant-negative PPARγ activity. Up-regulation of miR-122 negatively regulates ADAM-17, a known downstream target, in thyroid cells, suggesting an antiangiogenic mechanism in thyroid carcinoma. This latter inference is directly supported by reduced CD-31 expression in WRO xenografts expressing PPFP, miR-122, and DN-PPARγ. We conclude that, in addition to its apparent oncogenic potential in vitro, PPFP exhibits paradoxical tumor suppressor activity in vivo, mediated by multiple mechanisms including up-regulation of miR-122 and dominant-negative inhibition of PPARγ activity.

A Cascade of Sequentially Expressed Sucrose Transporters in the Seed Coat and Endosperm Provides Nutrition for the Arabidopsis Embryo[OPEN

PubMed Central

Chen, Li-Qing; Lin, I Winnie; Qu, Xiao-Qing; Sosso, Davide; McFarlane, Heather E.; Londoño, Alejandra; Samuels, A. Lacey; Frommer, Wolf B.

2015-01-01

Developing plant embryos depend on nutrition from maternal tissues via the seed coat and endosperm, but the mechanisms that supply nutrients to plant embryos have remained elusive. Sucrose, the major transport form of carbohydrate in plants, is delivered via the phloem to the maternal seed coat and then secreted from the seed coat to feed the embryo. Here, we show that seed filling in Arabidopsis thaliana requires the three sucrose transporters SWEET11, 12, and 15. SWEET11, 12, and 15 exhibit specific spatiotemporal expression patterns in developing seeds, but only a sweet11;12;15 triple mutant showed severe seed defects, which include retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a “wrinkled” seed phenotype. In sweet11;12;15 triple mutants, starch accumulated in the seed coat but not the embryo, implicating SWEET-mediated sucrose efflux in the transfer of sugars from seed coat to embryo. This cascade of sequentially expressed SWEETs provides the feeding pathway for the plant embryo, an important feature for yield potential. PMID:25794936

Effect of flagella expression on adhesion of Achromobacter piechaudii to chalk surfaces.

PubMed

Nejidat, A; Saadi, I; Ronen, Z

2008-12-01

To examine flagella role and cell motility in adhesion of Achromobacter piechaudii to chalk. Transmission electron microscopy revealed that stationary cells have thicker and longer flagella than logarithmic cells. SDS-PAGE analysis showed that flagellin was more abundant in stationary cells than logarithmic ones. Sonication or inhibition of flagellin synthesis caused a 30% reduction in adhesion to chalk. Preincubation of chalk with flagella extracts reduced adhesion, by 50%. Three motility mutants were isolated. Mutants 94 and 153 were nonmotile, expressed normal levels of flagellin, have regular flagella and exhibited reduced adhesion. Mutant 208 expressed low levels of flagellin, no flagella and a spherical cell shape but with normal adhesion capacity. Multiple cell surface factors affect the adhesion efficiency to chalk. Flagella per se through physical interaction and through cell motility contribute to the adhesion process. The adhesion behaviour of mutant 208 suggests that cell shape can compensate for flagellar removal and motility. Physiological status affects bacterial cell surface properties and hence adhesion efficiency to chalk. This interaction is essential to sustain biodegradation activities and thus, remediation of contaminated chalk aquifers.

Monoacylglycerol lipase promotes metastases in nasopharyngeal carcinoma.

PubMed

Hu, Wen-Rong; Lian, Yi-Fan; Peng, Li-Xia; Lei, Jin-Ju; Deng, Cheng-Cheng; Xu, Miao; Feng, Qi-Sheng; Chen, Li-Zhen; Bei, Jin-Xin; Zeng, Yi-Xin

2014-01-01

Monoacylglycerol lipase (MAGL) is a serine hydrolase that hydrolyzes monoacylglycerides into free fatty acids and glycerol. It has recently been found to be involved in cancer progression through the free fatty acid or endocannabinoid network after studies on its function in the endocannabinoid system. Here, we determined a role for MAGL in nasopharyngeal carcinoma (NPC), which is known for its high metastatic potential. Among the different NPC cells we tested, MAGL was highly expressed in high metastatic NPC cells, whereas low metastatic potential NPC cells exhibited lower expression of MAGL. Overexpression of MAGL in low metastatic NPC cells enhanced their motile behavior and metastatic capacity in vivo. Conversely, knockdown of MAGL reduced the motility of highly metastatic cells, reducing their metastatic capacity in vivo. Growth rate was not influenced by MAGL in either high or low metastatic cells. MAGL expression was associated with the epithelial-mesenchymal transition (EMT) proteins, such as E-cadherin, vimentin and Snail. It was also related to the sidepopulation (SP) of NPC cells. Our findings establish that MAGL promotes metastases in NPC through EMT, and it may serve as a target for the prevention of NPC metastases.

Hypothalamic transcriptional expression of the kisspeptin system and sex steroid receptors differs among polycystic ovary syndrome rat models with different endocrine phenotypes.

PubMed

Marcondes, Rodrigo Rodrigues; Carvalho, Kátia Cândido; Giannocco, Gisele; Duarte, Daniele Coelho; Garcia, Natália; Soares-Junior, José Maria; da Silva, Ismael Dale Cotrim Guerreiro; Maliqueo, Manuel; Baracat, Edmund Chada; Maciel, Gustavo Arantes Rosa

2017-08-01

Polycystic ovary syndrome is a heterogeneous endocrine disorder that affects reproductive-age women. The mechanisms underlying the endocrine heterogeneity and neuroendocrinology of polycystic ovary syndrome are still unclear. In this study, we investigated the expression of the kisspeptin system and gonadotropin-releasing hormone pulse regulators in the hypothalamus as well as factors related to luteinizing hormone secretion in the pituitary of polycystic ovary syndrome rat models induced by testosterone or estradiol. A single injection of testosterone propionate (1.25 mg) (n=10) or estradiol benzoate (0.5 mg) (n=10) was administered to female rats at 2 days of age to induce experimental polycystic ovary syndrome. Controls were injected with a vehicle (n=10). Animals were euthanized at 90-94 days of age, and the hypothalamus and pituitary gland were used for gene expression analysis. Rats exposed to testosterone exhibited increased transcriptional expression of the androgen receptor and estrogen receptor-β and reduced expression of kisspeptin in the hypothalamus. However, rats exposed to estradiol did not show any significant changes in hormone levels relative to controls but exhibited hypothalamic downregulation of kisspeptin, tachykinin 3 and estrogen receptor-α genes and upregulation of the gene that encodes the kisspeptin receptor. Testosterone- and estradiol-exposed rats with different endocrine phenotypes showed differential transcriptional expression of members of the kisspeptin system and sex steroid receptors in the hypothalamus. These differences might account for the different endocrine phenotypes found in testosterone- and estradiol-induced polycystic ovary syndrome rats.

Resistance to cycloxaprid in Laodelphax striatellus is associated with altered expression of nicotinic acetylcholine receptor subunits.

PubMed

Zhang, Yueliang; Han, Yangchun; Yang, Qiong; Wang, Lihua; He, Peng; Liu, Zewen; Li, Zhong; Guo, Huifang; Fang, Jichao

2018-04-01

Cycloxaprid is a new oxabridged cis-configuration neonicotinoid insecticide, the resistance development potential and underlying resistance mechanism of which were investigated in the small brown planthopper, Laodelphax striatellus (Fallén), an important agricultural pest of rice. A cycloxaprid-resistant strain (YN-CPD) only achieved 10-fold higher resistance, in contrast to 106-fold higher resistance to buprofezin and 332-fold higher resistance to chlorpyrifos achieved after exposure to similar selection pressure, and the cycloxaprid selected line showed no cross-resistance to the buprofezin and chlorpyrifos-selected resistance strains. Moreover, we identified 10 nicotinic acetylcholine receptor (nAChR) subunits from the transcriptome of L. striatellus, and six segments had open reading frames (ORFs). While we did not find mutations in the nAChR genes of L. striatellus, subunits Lsα1 and Lsβ1 exhibited, respectively, 9.60-fold and 3.36-fold higher expression in the resistant strain, while Lsα8 exhibited 0.44-fold lower expression. Suppression of Lsα1 through ingestion of dsLsα1 led to an increase in susceptibility to cycloxaprid. The findings indicate that resistance to cycloxaprid develops slowly compared with resistance to other chemicals and without cross-resistance to chlorpyrifos or buprofezin; over-expressed Lsα1 is associated with low cycloxaprid resistance levels, but the importance of over-expressed Lsβ1 and reduced expression of Lsα8 could not be excluded. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Human Mesenchymal Stem Cell Spheroids in Fibrin Hydrogels Exhibit Improved Cell Survival and Potential for Bone Healing

PubMed Central

Murphy, Kaitlin C.; Fang, Sophia Y.; Leach, J. Kent

2014-01-01

Mesenchymal stem cells (MSC) have great therapeutic potential for the repair of nonhealing bone defects due to their proliferative capacity, multilineage potential, trophic factor secretion, and lack of immunogenicity. However, a major barrier to the translation of cell-based therapies into clinical practice is ensuring their survival and function upon implantation into the defect site. We hypothesized that forming MSC into more physiologic 3-dimensional spheroids, rather than employing dissociated cells from 2-dimensional monolayer culture, would enhance their survival when exposed to a harsh microenvironment while maintaining their osteogenic potential. MSC spheroids were formed using the hanging drop method with increasing cell numbers. Compared to larger spheroids, the smallest spheroids which contained 15,000 cells exhibited increased metabolic activity, reduced apoptosis, and the most uniform distribution of proliferating cells. Spheroids were then entrapped in fibrin gels and cultured in serum-free media and 1% oxygen. Compared to identical numbers of dissociated MSC in fibrin gels, spheroids exhibited significantly reduced apoptosis and secreted up to 100-fold more VEGF. We also observed that fibrin gels containing spheroids and those containing an equivalent number of dissociated cells exhibited similar expression levels of early and late markers of osteogenic differentiation. These data demonstrate that MSC spheroids exhibit greater resistance to apoptosis and enhanced proangiogenic potential, while maintaining similar osteogenic potential to dissociated MSC entrapped in a clinically relevant biomaterial, supporting the use of MSC spheroids in cell-based approaches to bone repair. PMID:24781147

Vernonia DGATs can complement the disrupted oil and protein metabolism in epoxygenase-expressing soybean seeds.

PubMed

Li, Runzhi; Yu, Keshun; Wu, Yongmei; Tateno, Mizuki; Hatanaka, Tomoko; Hildebrand, David F

2012-01-01

Plant oils can be useful chemical feedstocks such as a source of epoxy fatty acids. High seed-specific expression of a Stokesia laevis epoxygenase (SlEPX) in soybeans only results in 3-7% epoxide levels. SlEPX-transgenic soybean seeds also exhibited other phenotypic alterations, such as altered seed fatty acid profiles, reduced oil accumulation, and variable protein levels. SlEPX-transgenic seeds showed a 2-5% reduction in total oil content and protein levels of 30.9-51.4%. To address these pleiotrophic effects of SlEPX expression on other traits, transgenic soybeans were developed to co-express SlEPX and DGAT (diacylglycerol acyltransferase) genes (VgDGAT1 & 2) isolated from Vernonia galamensis, a high accumulator of epoxy fatty acids. These side effects of SlEPX expression were largely overcome in the DGAT co-expressing soybeans. Total oil and protein contents were restored to the levels in non-transgenic soybeans, indicating that both VgDGAT1 and VgDGAT2 could complement the disrupted phenotypes caused by over-expression of an epoxygenase in soybean seeds. Copyright © 2011 Elsevier Inc. All rights reserved.

Resveratrol improves left ventricular diastolic relaxation in type 2 diabetes by inhibiting oxidative/nitrative stress: in vivo demonstration with magnetic resonance imaging

PubMed Central

Zhang, Hanrui; Morgan, Brandon; Potter, Barry J.; Ma, Lixin; Dellsperger, Kevin C.; Ungvari, Zoltan

2010-01-01

Resveratrol is a natural phytophenol that exhibits cardioprotective effects. This study was designed to elucidate the mechanisms by which resveratrol protects against diabetes-induced cardiac dysfunction. Normal control (m-Leprdb) mice and type 2 diabetic (Leprdb) mice were treated with resveratrol orally for 4 wk. In vivo MRI showed that resveratrol improved cardiac function by increasing the left ventricular diastolic peak filling rate in Leprdb mice. This protective role is partially explained by resveratrol's effects in improving nitric oxide (NO) production and inhibiting oxidative/nitrative stress in cardiac tissue. Resveratrol increased NO production by enhancing endothelial NO synthase (eNOS) expression and reduced O2·− production by inhibiting NAD(P)H oxidase activity and gp91phox mRNA and protein expression. The increased nitrotyrosine (N-Tyr) protein expression in Leprdb mice was prevented by the inducible NO synthase (iNOS) inhibitor 1400W. Resveratrol reduced both N-Tyr and iNOS expression in Leprdb mice. Furthermore, TNF-α mRNA and protein expression, as well as NF-κB activation, were reduced in resveratrol-treated Leprdb mice. Both Leprdb mice null for TNF-α (dbTNF−/dbTNF− mice) and Leprdb mice treated with the NF-κB inhibitor MG-132 showed decreased NAD(P)H oxidase activity and iNOS expression as well as elevated eNOS expression, whereas m-Leprdb mice treated with TNF-α showed the opposite effects. Thus, resveratrol protects against cardiac dysfunction by inhibiting oxidative/nitrative stress and improving NO availability. This improvement is due to the role of resveratrol in inhibiting TNF-α-induced NF-κB activation, therefore subsequently inhibiting the expression and activation of NAD(P)H oxidase and iNOS as well as increasing eNOS expression in type 2 diabetes. PMID:20675566

Zhx2 (zinc fingers and homeoboxes 2) regulates major urinary protein gene expression in the mouse liver

PubMed Central

Jiang, Jieyun; Creasy, Kate Townsend; Purnell, Justin; Peterson, Martha L.; Spear, Brett T.

2017-01-01

The mouse major urinary proteins (Mups) are encoded by a large family of highly related genes clustered on chromosome 4. Mups, synthesized primarily and abundantly in the liver and secreted through the kidneys, exhibit male-biased expression. Mups bind a variety of volatile ligands; these ligands, and Mup proteins themselves, influence numerous behavioral traits. Although urinary Mup protein levels vary between inbred mouse strains, this difference is most pronounced in BALB/cJ mice, which have dramatically low urinary Mup levels; this BALB/cJ trait had been mapped to a locus on chromosome 15. We previously identified Zhx2 (zinc fingers and homeoboxes 2) as a regulator of numerous liver-enriched genes. Zhx2 is located on chromosome 15, and a natural hypomorphic mutation in the BALB/cJ Zhx2 allele dramatically reduces Zhx2 expression. Based on these data, we hypothesized that reduced Zhx2 levels are responsible for lower Mup expression in BALB/cJ mice. Using both transgenic and knock-out mice along with in vitro assays, our data show that Zhx2 binds Mup promoters and is required for high levels of Mup expression in the adult liver. In contrast to previously identified Zhx2 targets that appear to be repressed by Zhx2, Mup genes are positively regulated by Zhx2. These data identify Zhx2 as a novel regulator of Mup expression and indicate that Zhx2 activates as well as represses expression of target genes. PMID:28258223

Uncoupling Lipid Metabolism from Inflammation through Fatty Acid Binding Protein-Dependent Expression of UCP2

PubMed Central

Xu, Hongliang; Hertzel, Ann V.; Steen, Kaylee A.; Wang, Qigui; Suttles, Jill

2015-01-01

Chronic inflammation in obese adipose tissue is linked to endoplasmic reticulum (ER) stress and systemic insulin resistance. Targeted deletion of the murine fatty acid binding protein (FABP4/aP2) uncouples obesity from inflammation although the mechanism underlying this finding has remained enigmatic. Here, we show that inhibition or deletion of FABP4/aP2 in macrophages results in increased intracellular free fatty acids (FFAs) and elevated expression of uncoupling protein 2 (UCP2) without concomitant increases in UCP1 or UCP3. Silencing of UCP2 mRNA in FABP4/aP2-deficient macrophages negated the protective effect of FABP loss and increased ER stress in response to palmitate or lipopolysaccharide (LPS). Pharmacologic inhibition of FABP4/aP2 with the FABP inhibitor HTS01037 also upregulated UCP2 and reduced expression of BiP, CHOP, and XBP-1s. Expression of native FABP4/aP2 (but not the non-fatty acid binding mutant R126Q) into FABP4/aP2 null cells reduced UCP2 expression, suggesting that the FABP-FFA equilibrium controls UCP2 expression. FABP4/aP2-deficient macrophages are resistant to LPS-induced mitochondrial dysfunction and exhibit decreased mitochondrial protein carbonylation and UCP2-dependent reduction in intracellular reactive oxygen species. These data demonstrate that FABP4/aP2 directly regulates intracellular FFA levels and indirectly controls macrophage inflammation and ER stress by regulating the expression of UCP2. PMID:25582199

Immunostimulation of Salmo salar L., and its effect on Lepeophtheirus salmonis (Krøyer) P-glycoprotein mRNA expression following subsequent emamectin benzoate exposure.

PubMed

Igboeli, O O; Purcell, S L; Wotton, H; Poley, J; Burka, J F; Fast, M D

2013-03-01

Control of sea lice, Lepeophtheirus salmonis, on farmed Atlantic salmon, Salmo salar, relies heavily on chemotherapeutants. However, reduced efficacy of many treatments and need for integrated sea lice management plans require innovative strategies. Resistance to emamectin benzoate (EMB), a major sea lice parasiticide, has been linked with P-glycoprotein (P-gp) expression. We hypothesized that host immunostimulation would complement EMB treatment outcome. Lepeophtheirus salmonis-infected Atlantic salmon were fed immunostimulatory or control feeds. Sea lice were collected for 24-h EMB bioassays 1 and 2 weeks prior to commencement of EMB treatment of the fish. Two weeks after cessation of immunostimulant-treated feed, EMB was administered at 150 μg kg(-1) fish biomass for 7 days. The bioassay revealed stage, gender and immunostimulant-related differences in EMB EC(50) . Sea lice attached to salmon with a history of immunostimulation exhibited significantly greater survival than those on control feeds, despite similar levels of EMB in host tissues. Lepeophtheirus salmonis from salmon with a history of immunostimulation also exhibited higher P-gp mRNA expression as well as greater survivability compared to controls. Administration of immunostimulants prior to EMB treatment caused increased expression of P-gp mRNA which could have consequently caused decreased efficacy of the parasiticide. © 2013 Blackwell Publishing Ltd.

The PHD Finger Protein MMD1/DUET Ensures the Progression of Male Meiotic Chromosome Condensation and Directly Regulates the Expression of the Condensin Gene CAP-D3.

PubMed

Wang, Jun; Niu, Baixiao; Huang, Jiyue; Wang, Hongkuan; Yang, Xiaohui; Dong, Aiwu; Makaroff, Christopher; Ma, Hong; Wang, Yingxiang

2016-08-01

Chromosome condensation, a process mediated by the condensin complex, is essential for proper chromosome segregation during cell division. Unlike rapid mitotic chromosome condensation, meiotic chromosome condensation occurs over a relatively long prophase I and is unusually complex due to the coordination with chromosome axis formation and homolog interaction. The molecular mechanisms that regulate meiotic chromosome condensation progression from prophase I to metaphase I are unclear. Here, we show that the Arabidopsis thaliana meiotic PHD-finger protein MMD1/DUET is required for progressive compaction of prophase I chromosomes to metaphase I bivalents. The MMD1 PHD domain is required for its function in chromosome condensation and binds to methylated histone tails. Transcriptome analysis and qRT-PCR showed that several condensin genes exhibit significantly reduced expression in mmd1 meiocytes. Furthermore, MMD1 specifically binds to the promoter region of the condensin subunit gene CAP-D3 to enhance its expression. Moreover, cap-d3 mutants exhibit similar chromosome condensation defects, revealing an MMD1-dependent mechanism for regulating meiotic chromosome condensation, which functions in part by promoting condensin gene expression. Together, these discoveries provide strong evidence that the histone reader MMD1/DUET defines an important step for regulating the progression of meiotic prophase I chromosome condensation. © 2016 American Society of Plant Biologists. All rights reserved.

The PHD Finger Protein MMD1/DUET Ensures the Progression of Male Meiotic Chromosome Condensation and Directly Regulates the Expression of the Condensin Gene CAP-D3[OPEN

PubMed Central

Wang, Jun; Niu, Baixiao; Huang, Jiyue; Wang, Hongkuan; Yang, Xiaohui; Dong, Aiwu

2016-01-01

Chromosome condensation, a process mediated by the condensin complex, is essential for proper chromosome segregation during cell division. Unlike rapid mitotic chromosome condensation, meiotic chromosome condensation occurs over a relatively long prophase I and is unusually complex due to the coordination with chromosome axis formation and homolog interaction. The molecular mechanisms that regulate meiotic chromosome condensation progression from prophase I to metaphase I are unclear. Here, we show that the Arabidopsis thaliana meiotic PHD-finger protein MMD1/DUET is required for progressive compaction of prophase I chromosomes to metaphase I bivalents. The MMD1 PHD domain is required for its function in chromosome condensation and binds to methylated histone tails. Transcriptome analysis and qRT-PCR showed that several condensin genes exhibit significantly reduced expression in mmd1 meiocytes. Furthermore, MMD1 specifically binds to the promoter region of the condensin subunit gene CAP-D3 to enhance its expression. Moreover, cap-d3 mutants exhibit similar chromosome condensation defects, revealing an MMD1-dependent mechanism for regulating meiotic chromosome condensation, which functions in part by promoting condensin gene expression. Together, these discoveries provide strong evidence that the histone reader MMD1/DUET defines an important step for regulating the progression of meiotic prophase I chromosome condensation. PMID:27385818

Myocardial Adiponectin Isoform Shift in Dogs with Congestive Heart Failure—A Comparison to Hibernating Brown Bears (Ursus arctos horribilis)

PubMed Central

Nelson, O. Lynne; Wood, Rachael M.; Häggström, Jens; Kvart, Clarence; Robbins, Charles T.

2017-01-01

Adiponectin is the most abundant plasma adipokine, and is well known for its role in energy homeostasis and cardiac protection. In humans with dilated cardiomyopathy, myocardial adiponectin protein expression is reduced compared to normal hearts and has been implicated in the pathology of cardiomyopathy. Serum adiponectin levels are often conflicting, with higher levels associated with poor survival in humans with congestive heart failure (CHF). We evaluated adiponectin serum concentrations and myocardial protein expression in dogs with naturally occurring myxomatous mitral valve disease and CHF. We compared the findings to active and hibernating brown bears as bears are adapted to endure an extreme period of low cardiac output during their annual hibernation. Bears exhibited largely the active high-molecular weight (HMW) versus the low-molecular weight isoforms of myocardial adiponectin (HMW:LMW = 6.3) during both the active period and hibernation, while healthy dogs exhibited a more balanced mix of isoforms. Dogs with CHF expressed predominately HMW isoforms of adiponectin (HMW:LMW = 12.5), appearing more similar to bears. In contrast to humans, serum adiponectin was significantly lower in dogs with CHF and lowest levels in the severest CHF class. In both dogs and bears, myocardial adiponectin was expressed independent of circulating adiponectin concentrations, suggesting a local regulatory mechanism within the heart. PMID:29056695

The C. elegans neural editome reveals an ADAR target mRNA required for proper chemotaxis

PubMed Central

Deffit, Sarah N; Yee, Brian A; Manning, Aidan C; Rajendren, Suba; Vadlamani, Pranathi; Wheeler, Emily C; Domissy, Alain; Washburn, Michael C

2017-01-01

ADAR proteins alter gene expression both by catalyzing adenosine (A) to inosine (I) RNA editing and binding to regulatory elements in target RNAs. Loss of ADARs affects neuronal function in all animals studied to date. Caenorhabditis elegans lacking ADARs exhibit reduced chemotaxis, but the targets responsible for this phenotype remain unknown. To identify critical neural ADAR targets in C. elegans, we performed an unbiased assessment of the effects of ADR-2, the only A-to-I editing enzyme in C. elegans, on the neural transcriptome. Development and implementation of publicly available software, SAILOR, identified 7361 A-to-I editing events across the neural transcriptome. Intersecting the neural editome with adr-2 associated gene expression changes, revealed an edited mRNA, clec-41, whose neural expression is dependent on deamination. Restoring clec-41 expression in adr-2 deficient neural cells rescued the chemotaxis defect, providing the first evidence that neuronal phenotypes of ADAR mutants can be caused by altered gene expression. PMID:28925356

Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue while maintaining potent anti-tumor activity

PubMed Central

Caruso, Hillary G.; Hurton, Lenka V.; Najjar, Amer; Rushworth, David; Ang, Sonny; Olivares, Simon; Mi, Tiejuan; Switzer, Kirsten; Singh, Harjeet; Huls, Helen; Lee, Dean A.; Heimberger, Amy B.; Champlin, Richard E.; Cooper, Laurence J. N.

2015-01-01

Many tumors over express tumor-associated antigens relative to normal tissue, such as epidermal growth factor receptor (EGFR). This limits targeting by human T cells modified to express chimeric antigen receptors (CARs) due to potential for deleterious recognition of normal cells. We sought to generate CAR+ T cells capable of distinguishing malignant from normal cells based on the disparate density of EGFR expression by generating two CARs from monoclonal antibodies which differ in affinity. T cells with low affinity Nimo-CAR selectively targeted cells over-expressing EGFR, but exhibited diminished effector function as the density of EGFR decreased. In contrast, the activation of T cells bearing high affinity Cetux-CAR was not impacted by the density of EGFR. In summary, we describe the generation of CARs able to tune T-cell activity to the level of EGFR expression in which a CAR with reduced affinity enabled T cells to distinguish malignant from non-malignant cells. PMID:26330164

Pilocarpine-Induced Status Epilepticus in Rats Involves Ischemic and Excitotoxic Mechanisms

PubMed Central

Fabene, Paolo Francesco; Merigo, Flavia; Galiè, Mirco; Benati, Donatella; Bernardi, Paolo; Farace, Paolo; Nicolato, Elena; Marzola, Pasquina; Sbarbati, Andrea

2007-01-01

The neuron loss characteristic of hippocampal sclerosis in temporal lobe epilepsy patients is thought to be the result of excitotoxic, rather than ischemic, injury. In this study, we assessed changes in vascular structure, gene expression, and the time course of neuronal degeneration in the cerebral cortex during the acute period after onset of pilocarpine-induced status epilepticus (SE). Immediately after 2 hr SE, the subgranular layers of somatosensory cortex exhibited a reduced vascular perfusion indicative of ischemia, whereas the immediately adjacent supragranular layers exhibited increased perfusion. Subgranular layers exhibited necrotic pathology, whereas the supergranular layers were characterized by a delayed (24 h after SE) degeneration apparently via programmed cell death. These results indicate that both excitotoxic and ischemic injuries occur during pilocarpine-induced SE. Both of these degenerative pathways, as well as the widespread and severe brain damage observed, should be considered when animal model-based data are compared to human pathology. PMID:17971868

Phosphorylation at Ser-181 of oncogenic KRAS is required for tumor growth.

PubMed

Barceló, Carles; Paco, Noelia; Morell, Mireia; Alvarez-Moya, Blanca; Bota-Rabassedas, Neus; Jaumot, Montserrat; Vilardell, Felip; Capella, Gabriel; Agell, Neus

2014-02-15

KRAS phosphorylation has been reported recently to modulate the activity of mutant KRAS protein in vitro. In this study, we defined S181 as a specific phosphorylation site required to license the oncogenic function of mutant KRAS in vivo. The phosphomutant S181A failed to induce tumors in mice, whereas the phosphomimetic mutant S181D exhibited an enhanced tumor formation capacity, compared with the wild-type KRAS protein. Reduced growth of tumors composed of cells expressing the nonphosphorylatable KRAS S181A mutant was correlated with increased apoptosis. Conversely, increased growth of tumors composed of cells expressing the phosphomimetic KRAS S181D mutant was correlated with increased activation of AKT and ERK, two major downstream effectors of KRAS. Pharmacologic treatment with PKC inhibitors impaired tumor growth associated with reduced levels of phosphorylated KRAS and reduced effector activation. In a panel of human tumor cell lines expressing various KRAS isoforms, we showed that KRAS phosphorylation was essential for survival and tumorigenic activity. Furthermore, we identified phosphorylated KRAS in a panel of primary human pancreatic tumors. Taken together, our findings establish that KRAS requires S181 phosphorylation to manifest its oncogenic properties, implying that its inhibition represents a relevant target to attack KRAS-driven tumors. ©2013 AACR.

The Agr quorum-sensing system regulates fibronectin binding but not hemolysis in the absence of a functional electron transport chain.

PubMed

Pader, Vera; James, Ellen H; Painter, Kimberley L; Wigneshweraraj, Sivaramesh; Edwards, Andrew M

2014-10-01

Staphylococcus aureus is responsible for numerous chronic and recurrent infections, which are frequently associated with the emergence of small-colony variants (SCVs) that lack a functional electron transport chain. SCVs exhibit enhanced expression of fibronectin-binding protein (FnBP) and greatly reduced hemolysin production, although the basis for this is unclear. One hypothesis is that these phenotypes are a consequence of the reduced Agr activity of SCVs, while an alternative is that the lack of a functional electron transport chain and the resulting reduction in ATP production are responsible. Disruption of the electron transport chain of S. aureus genetically (hemB and menD) or chemically, using 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibited both growth and Agr activity and conferred an SCV phenotype. Supplementation of the culture medium with synthetic autoinducing peptide (sAIP) significantly increased Agr expression in both hemB mutant strains and S. aureus grown with HQNO and significantly reduced staphylococcal adhesion to fibronectin. However, sAIP did not promote hemolysin expression in hemB mutant strains or S. aureus grown with HQNO. Therefore, while Agr regulates fibronectin binding in SCVs, it cannot promote hemolysin production in the absence of a functional electron transport chain. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The Agr Quorum-Sensing System Regulates Fibronectin Binding but Not Hemolysis in the Absence of a Functional Electron Transport Chain

PubMed Central

Pader, Vera; James, Ellen H.; Painter, Kimberley L.; Wigneshweraraj, Sivaramesh

2014-01-01

Staphylococcus aureus is responsible for numerous chronic and recurrent infections, which are frequently associated with the emergence of small-colony variants (SCVs) that lack a functional electron transport chain. SCVs exhibit enhanced expression of fibronectin-binding protein (FnBP) and greatly reduced hemolysin production, although the basis for this is unclear. One hypothesis is that these phenotypes are a consequence of the reduced Agr activity of SCVs, while an alternative is that the lack of a functional electron transport chain and the resulting reduction in ATP production are responsible. Disruption of the electron transport chain of S. aureus genetically (hemB and menD) or chemically, using 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibited both growth and Agr activity and conferred an SCV phenotype. Supplementation of the culture medium with synthetic autoinducing peptide (sAIP) significantly increased Agr expression in both hemB mutant strains and S. aureus grown with HQNO and significantly reduced staphylococcal adhesion to fibronectin. However, sAIP did not promote hemolysin expression in hemB mutant strains or S. aureus grown with HQNO. Therefore, while Agr regulates fibronectin binding in SCVs, it cannot promote hemolysin production in the absence of a functional electron transport chain. PMID:25092909

mRNA destabilization improves glycemic responsiveness of transcriptionally regulated hepatic insulin gene therapy in vitro and in vivo.

PubMed

Thulé, Peter M; Lin, Yulin; Jia, Dingwu; Olson, Darin E; Tang, Shiue-Cheng; Sambanis, Athanassios

2017-03-01

Hepatic insulin gene therapy (HIGT) employing a glucose and insulin sensitive promoter to direct insulin transcription can lower blood sugars within 2 h of an intraperitoneal glucose challenge. However, post-challenge blood sugars frequently decline to below baseline. We hypothesize that this 'over-shoot' hypoglycemia results from sustained translation of long-lived transgene message, and that reducing pro-insulin message half-life will ameliorate post-challenge hypoglycemia. We compared pro-insulin message content and insulin secretion from primary rat hepatocytes expressing insulin from either a standard construct (2xfur), or a construct producing a destabilized pro-insulin message (InsTail), following exposure to stimulating or inhibitory conditions. Hepatocytes transduced with a 2xfur construct accumulated pro-insulin message, and exhibited increased insulin secretion, under conditions that both inhibit or stimulate transcription. By contrast, pro-insulin message content remained stable in InsTail expressing cells, and insulin secretion increased less than 2xfur during prolonged stimulation. During transitions from stimulatory to inhibitory conditions, or vice versa, amounts of pro-insulin message changed more rapidly in InsTail expressing cells than 2xfur expressing cells. Importantly, insulin secretion increased during the transition from stimulation to inhibition in 2xfur expressing cells, although it remained unchanged in InsTail expressing cells. Use of the InsTail destabilized insulin message tended to more rapidly reduce glucose induced glycemic excursions, and limit post-load hypoglycemia in STZ-diabetic mice in vivo. The data obtained in the present study suggest that combining transcriptional and post-transcriptional regulatory strategies may reduce undesirable glycemic excursion in models of HIGT. Copyright © 2017 John Wiley & Sons, Ltd.

The small tellurium-based compound SAS suppresses inflammation in human retinal pigment epithelium

PubMed Central

Livnat, Tami; Halpert, Gilad; Jawad, Shayma; Nisgav, Yael; Azar-Avivi, Shirley; Liu, Baoying; Nussenblatt, Robert B.; Weinberger, Dov; Sredni, Benjamin

2016-01-01

Purpose Pathological angiogenesis and chronic inflammation greatly contribute to the development of choroidal neovascularization (CNV) in chorioretinal diseases involving abnormal contact between retinal pigment epithelial (RPE) and endothelial cells (ECs), associated with Bruch’s membrane rupture. We explored the ability of the small organotellurium compound octa-O-bis-(R,R)-tartarate ditellurane (SAS) to mitigate inflammatory processes in human RPE cells. Methods Cell adhesion assays and analyses of gene and protein expression were used to examine the effect of SAS on ARPE-19 cells or primary human RPE cells that were grown alone or in an RPE-EC co-culture. Results Adhesion assays showed that SAS inhibited αv integrins expressed on RPE cells. Co-cultures of RPE cells with ECs significantly reduced the gene expression of PEDF, as compared to RPE cells cultured alone. Both SAS and the anti-αvβ3 antibody LM609 significantly enhanced the production of PEDF at both mRNA and protein levels in RPE cells. RPE cells co-cultured with EC exhibited increased gene expression of CXCL5, COX1, MMP2, IGF1, and IL8, all of which are involved in both angiogenesis and inflammation. The enhanced expression of these genes was greatly suppressed by SAS, but interestingly, remained unaffected by LM609. Zymography assay showed that SAS reduced the level of MMP-2 activity in RPE cells. We also found that SAS significantly suppressed IL-1β-induced IL-6 expression and secretion from RPE cells by reducing the protein levels of phospho-IkappaBalpha (pIκBα). Conclusions Our results suggest that SAS is a promising anti-inflammatory agent in RPE cells, and may be an effective therapeutic approach for controlling chorioretinal diseases. PMID:27293373

The small tellurium-based compound SAS suppresses inflammation in human retinal pigment epithelium.

PubMed

Dardik, Rima; Livnat, Tami; Halpert, Gilad; Jawad, Shayma; Nisgav, Yael; Azar-Avivi, Shirley; Liu, Baoying; Nussenblatt, Robert B; Weinberger, Dov; Sredni, Benjamin

2016-01-01

Pathological angiogenesis and chronic inflammation greatly contribute to the development of choroidal neovascularization (CNV) in chorioretinal diseases involving abnormal contact between retinal pigment epithelial (RPE) and endothelial cells (ECs), associated with Bruch's membrane rupture. We explored the ability of the small organotellurium compound octa-O-bis-(R,R)-tartarate ditellurane (SAS) to mitigate inflammatory processes in human RPE cells. Cell adhesion assays and analyses of gene and protein expression were used to examine the effect of SAS on ARPE-19 cells or primary human RPE cells that were grown alone or in an RPE-EC co-culture. Adhesion assays showed that SAS inhibited αv integrins expressed on RPE cells. Co-cultures of RPE cells with ECs significantly reduced the gene expression of PEDF, as compared to RPE cells cultured alone. Both SAS and the anti-αvβ3 antibody LM609 significantly enhanced the production of PEDF at both mRNA and protein levels in RPE cells. RPE cells co-cultured with EC exhibited increased gene expression of CXCL5, COX1, MMP2, IGF1, and IL8, all of which are involved in both angiogenesis and inflammation. The enhanced expression of these genes was greatly suppressed by SAS, but interestingly, remained unaffected by LM609. Zymography assay showed that SAS reduced the level of MMP-2 activity in RPE cells. We also found that SAS significantly suppressed IL-1β-induced IL-6 expression and secretion from RPE cells by reducing the protein levels of phospho-IkappaBalpha (pIκBα). Our results suggest that SAS is a promising anti-inflammatory agent in RPE cells, and may be an effective therapeutic approach for controlling chorioretinal diseases.

Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency

DOE PAGES

Eudes, Aymerick; Sathitsuksanoh, Noppadon; Baidoo, Edward E. K.; ...

2015-01-13

Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3-dehydroshikimatemore » dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3-dehydroshikimate – an intermediate of the shikimate pathway – into protocatechuate. Compared to wild-type plants, lines expressing QsuB contain higher amounts of protocatechuate, p-coumarate, p-coumaraldehyde and p-coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D-NMR spectroscopy and pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS) reveal an increase of p-hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size-exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass.« less

Resveratrol Reduces Prostate Cancer Growth and Metastasis by Inhibiting the Akt/MicroRNA-21 Pathway

PubMed Central

Sheth, Sandeep; Jajoo, Sarvesh; Kaur, Tejbeer; Mukherjea, Debashree; Sheehan, Kelly; Rybak, Leonard P.; Ramkumar, Vickram

2012-01-01

The consumption of foods containing resveratrol produces significant health benefits. Resveratrol inhibits cancer by reducing cell proliferation and metastasis and by inducing apoptosis. These actions could be explained by its ability to inhibit (ERK-1/2), Akt and suppressing the levels of estrogen and insulin growth factor -1 (IGF-1) receptor. How these processes are manifested into the antitumor actions of resveratrol is not clear. Using microarray studies, we show that resveratrol reduced the expression of various prostate-tumor associated microRNAs (miRs) including miR-21 in androgen-receptor negative and highly aggressive human prostate cancer cells, PC-3M-MM2. This effect of resveratrol was associated with reduced cell viability, migration and invasiveness. Additionally, resveratrol increased the expression of tumor suppressors, PDCD4 and maspin, which are negatively regulated by miR-21. Short interfering (si) RNA against PDCD4 attenuated resveratrol’s effect on prostate cancer cells, and similar effects were observed following over expression of miR-21 with pre-miR-21 oligonucleotides. PC-3M-MM2 cells also exhibited high levels of phospho-Akt (pAkt), which were reduced by both resveratrol and LY294002 (a PI3-kinase inhibitor). MiR-21 expression in these cells appeared to be dependent on Akt, as LY294002 reduced the levels of miR-21 along with a concurrent increase in PDCD4 expression. These in vitro findings were further corroborated in a severe combined immunodeficient (SCID) mouse xenograft model of prostate cancer. Oral administration of resveratrol not only inhibited the tumor growth but also decreased the incidence and number of metastatic lung lesions. These tumor- and metastatic-suppressive effects of resveratrol were associated with reduced miR-21 and pAkt, and elevated PDCD4 levels. Similar anti-tumor effects of resveratrol were observed in DU145 and LNCaP prostate cancer cells which were associated with suppression of Akt and PDCD4, but independent of miR-21.These data suggest that resveratrol’s anti-tumor actions in prostate cancer could be explained, in part, through inhibition of Akt/miR-21 signaling pathway. PMID:23272133

The effects of laughter on post-prandial glucose levels and gene expression in type 2 diabetic patients.

PubMed

Hayashi, Takashi; Murakami, Kazuo

2009-07-31

This report mainly summarizes the results of our study in which the physiological effects of laughter--as a positive emotional expression--were analyzed with respect to gene expression changes to demonstrate the hypothesis that the mind and genes mutually influence each other. We observed that laughter suppressed 2-h postprandial blood glucose level increase in patients with type 2 diabetes and analyzed gene expression changes. Some genes showed specific changes in their expression. In addition, we revealed that laughter decreased the levels of prorenin in blood; prorenin is involved in the onset of diabetic complications. Further, laughter normalized the expression of the prorenin receptor gene on peripheral blood leukocytes, which had been reduced in diabetic patients; this demonstrated that the inhibitory effects of laughter on the onset/deterioration of diabetic complications at the gene-expression level. In a subsequent study, we demonstrated the effects of laughter by discriminating 14 genes, related to natural killer (NK) cell activity, to exhibit continuous increases in expression as a result of laughter. Our results supported NK cell-mediated improvement in glucose tolerance at the gene-expression level. In this report, we also review other previous studies on laughter.

FOXP2-positive diffuse large B-cell lymphomas exhibit a poor response to R-CHOP therapy and distinct biological signatures

PubMed Central

Wong, Kah Keng; Gascoyne, Duncan M.; Soilleux, Elizabeth J.; Lyne, Linden; Spearman, Hayley; Roncador, Giovanna; Pedersen, Lars M.; Møller, Michael B.; Green, Tina M.; Banham, Alison H.

2016-01-01

FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL. PMID:27224915

FOXP2-positive diffuse large B-cell lymphomas exhibit a poor response to R-CHOP therapy and distinct biological signatures.

PubMed

Wong, Kah Keng; Gascoyne, Duncan M; Soilleux, Elizabeth J; Lyne, Linden; Spearman, Hayley; Roncador, Giovanna; Pedersen, Lars M; Møller, Michael B; Green, Tina M; Banham, Alison H

2016-08-16

FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL.

Suppressing a Putative Sterol Carrier Gene Reduces Plasmodesmal Permeability and Activates Sucrose Transporter Genes during Cotton Fiber Elongation.

PubMed

Zhang, Zhiyuan; Ruan, Yong-Ling; Zhou, Na; Wang, Fang; Guan, Xueying; Fang, Lei; Shang, Xiaoguang; Guo, Wangzhen; Zhu, Shuijin; Zhang, Tianzhen

2017-08-01

Plasmodesmata (PDs) play vital roles in cell-to-cell communication and plant development. Emerging evidence suggests that sterols are involved in PD activity during cytokinesis. However, whether sterols contribute to PD gating between established cells remains unknown. Here, we isolated GhSCP2D , a putative sterol carrier protein gene from elongating cotton ( Gossypium hirsutum ) fibers. In contrast to wild-type fiber PDs, which opened at 5 to 10 d postanthesis (DPA) and closed only at 15 to 25 DPA, plants with suppressed GhSCP2D expression had reduced sterol contents and closed PDs at 5 through 25 DPA The GhSCP2D- suppressed fibers exhibited callose deposition at the PDs, likely due to reduced expression of GhPdBG3-2A/D , which encodes a PD-targeting β-1,3-glucanase. Both GhPdBG3-2A/D expression and callose deposition were sensitive to a sterol biosynthesis inhibitor. Moreover, suppressing GhSCP2D upregulated a cohort of SUT and SWEET sucrose transporter genes in fiber cells. Collectively, our results indicate that (1) GhSCP2D is required for GhPdBG3-2A/D expression to degrade callose at the PD, thereby contributing to the establishment of the symplasmic pathway; and (2) blocking the symplasmic pathway by downregulating GhSCP2D activates or increases the expression of SUTs and SWEETs , leading to the switch from symplasmic to apoplasmic pathways. © 2017 American Society of Plant Biologists. All rights reserved.

SASH1 mediates sensitivity of breast cancer cells to chloropyramine and is associated with prognosis in breast cancer

PubMed Central

Burgess, Joshua T.; Bolderson, Emma; Saunus, Jodi M.; Zhang, Shu-Dong; Reid, Lynne E.; McNicol, Anne Marie; Lakhani, Sunil R.; Cuff, Katharine; Richard, Kerry; Richard, Derek J.; O'Byrne, Kenneth J.

2016-01-01

Expression of the SASH1 protein is reduced in a range of human cancers and has been implicated in apoptotic cancer cell death. This study investigated whether increasing SASH1 expression could be a useful therapeutic strategy in breast cancer. Ectopic SASH1 expression increased apoptosis in 7/8 breast cancer cell lines. Subsequent in silico connectivity screening demonstrated that the clinically approved antihistamine drug, chloropyramine, increased SASH1 mRNA levels. Chloropyramine has previously been shown to have anti-tumour activity in breast cancer in part through modulation of FAK signalling, a pathway also regulated by SASH1. This study demonstrated that chloropyramine increased SASH1 protein levels in breast cancer cells. Consistent with this the agent reduced cell confluency in 7/8 cell lines treated irrespective of their ER status but not apoptosis incompetent MCF7 cells. In contrast SASH1 siRNA-transfected breast cancer cells exhibited reduced chloropyramine sensitivity. The prognostic significance of SASH1 expression was also investigated in two breast cancer cohorts. Expression was associated with favourable outcome in ER-positive cases, but only those of low histological grade/proliferative status. Conversely, we found a very strong inverse association in HER2+ disease irrespective of ER status, and in triple-negative, basal-like cases. Overall, the data suggest that SASH1 is prognostic in breast cancer and could have subtype-dependent effects on breast cancer progression. Pharmacologic induction of SASH1 by chloropyramine treatment of breast cancer warrants further preclinical and clinical investigation. PMID:27637080

SASH1 mediates sensitivity of breast cancer cells to chloropyramine and is associated with prognosis in breast cancer.

PubMed

Burgess, Joshua T; Bolderson, Emma; Saunus, Jodi M; Zhang, Shu-Dong; Reid, Lynne E; McNicol, Anne Marie; Lakhani, Sunil R; Cuff, Katharine; Richard, Kerry; Richard, Derek J; O'Byrne, Kenneth J

2016-11-08

Expression of the SASH1 protein is reduced in a range of human cancers and has been implicated in apoptotic cancer cell death. This study investigated whether increasing SASH1 expression could be a useful therapeutic strategy in breast cancer. Ectopic SASH1 expression increased apoptosis in 7/8 breast cancer cell lines. Subsequent in silico connectivity screening demonstrated that the clinically approved antihistamine drug, chloropyramine, increased SASH1 mRNA levels. Chloropyramine has previously been shown to have anti-tumour activity in breast cancer in part through modulation of FAK signalling, a pathway also regulated by SASH1. This study demonstrated that chloropyramine increased SASH1 protein levels in breast cancer cells. Consistent with this the agent reduced cell confluency in 7/8 cell lines treated irrespective of their ER status but not apoptosis incompetent MCF7 cells. In contrast SASH1 siRNA-transfected breast cancer cells exhibited reduced chloropyramine sensitivity. The prognostic significance of SASH1 expression was also investigated in two breast cancer cohorts. Expression was associated with favourable outcome in ER-positive cases, but only those of low histological grade/proliferative status. Conversely, we found a very strong inverse association in HER2+ disease irrespective of ER status, and in triple-negative, basal-like cases. Overall, the data suggest that SASH1 is prognostic in breast cancer and could have subtype-dependent effects on breast cancer progression. Pharmacologic induction of SASH1 by chloropyramine treatment of breast cancer warrants further preclinical and clinical investigation.

Mutations in CIC and IDH1 cooperatively regulate 2-hydroxyglutarate levels and cell clonogenicity

PubMed Central

Chittaranjan, Suganthi; Chan, Susanna; Yang, Cindy; Yang, Kevin C.; Chen, Vincent; Moradian, Annie; Firme, Marlo; Song, Jungeun; Go, Nancy E.; Blough, Michael D.; Chan, Jennifer A.; Cairncross, J. Gregory; Gorski, Sharon M.; Morin, Gregg B.; Yip, Stephen; Marra, Marco A.

2014-01-01

The majority of oligodendrogliomas (ODGs) exhibit combined losses of chromosomes 1p and 19q and mutations of isocitrate dehydrogenase (IDH1-R132H or IDH2-R172K). Approximately 70% of ODGs with 1p19q co-deletions harbor somatic mutations in the Capicua Transcriptional Repressor (CIC) gene on chromosome 19q13.2. Here we show that endogenous long (CIC-L) and short (CIC-S) CIC proteins are predominantly localized to the nucleus or cytoplasm, respectively. Cytoplasmic CIC-S is found in close proximity to the mitochondria. To study wild type and mutant CIC function and motivated by the paucity of 1p19q co-deleted ODG lines, we created HEK293 and HOG stable cell lines ectopically co-expressing CIC and IDH1. Non-mutant lines displayed increased clonogenicity, but cells co-expressing the mutant IDH1-R132H with either CIC-S-R201W or -R1515H showed reduced clonogenicity in an additive manner, demonstrating cooperative effects in our assays. Expression of mutant CIC-R1515H increased cellular 2-Hydroxyglutarate (2HG) levels compared to wild type CIC in IDH1-R132H background. Levels of phosphorylated ATP-citrate Lyase (ACLY) were lower in cell lines expressing mutant CIC-S proteins compared to cells expressing wild type CIC-S, supporting a cytosolic citrate metabolism-related mechanism of reduced clonogenicity in our in vitro model systems. ACLY or phospho-ACLY were similarly reduced in CIC-mutant 1p19q co-deleted oligodendroglioma patient samples. PMID:25277207

Delta-like ligand 4: A predictor of poor prognosis in clear cell renal cell carcinoma

PubMed Central

WANG, WEI; YU, YI; WANG, YA; LI, XIAOMING; BAO, JUNSHENG; WU, GONGJIN; CHANG, HONG; SHI, TINGKAI; YUE, ZHONGJIN

2014-01-01

Delta-like ligand 4 (Dll4)-Notch signaling is important in tumor angiogenesis; however, the prognostic value of D114 detection in patients with clear cell renal cell carcinoma (CCRCC) remains unclear. The present study aimed to determine whether the presence of high Dll4 expression levels was correlated with poor prognosis in CCRCC following curative resection. The D114 expression levels in four paired samples of CCRCC tissues and adjacent normal renal tissues were assayed by western blotting. Surgical specimens comprised 121 CCRCC tissue samples and 65 normal renal tissue samples, obtained from patients with CCRCC. The specimens were immunohistochemically assessed to determine Dll4 and vascular endothelial growth factor receptor 2 (VEGFR-2) expression levels. The prognostic significance of Dll4 expression levels was evaluated by the Kaplan-Meier method and Cox regression analysis. The correlation between Dll4 expression levels and VEGFR-2 expression levels, tumor stage, tumor grade and metastasis, was examined by χ2 test and multivariate logistic regression. As determined by the western blotting results, Dll4 protein expression levels were significantly increased in CCRCC tissues compared with those in adjacent non-cancerous tissues. From the analysis of the surgical specimens, 53 (43.8%) CCRCC patients exhibited immunohistochemically high Dll4 expression levels and 68 (56.2%) patients exhibited low Dll4 expression levels. The survival curves revealed that the patients with high Dll4 expression levels had significantly shorter survival times than the patients with low Dll4 expression levels (P<0.001). Multivariate survival analysis demonstrated that the presence of high Dll4 expression levels was independently associated with reduced overall survival and progression-free survival times (P=0.021 and 0.034, respectively). A positive correlation was also identified between Dll4 and VEGFR-2 expression levels (P=0.001). In conclusion, the results show that the presence of high Dll4 expression levels was clearly associated with high VEGFR-2 expression levels, tumor grade, tumor stage and poor prognosis in CCRCC patients. Therefore, inhibition of Dll4 may exert potent growth inhibitory effects on tumors resistant to anti-VEGF therapies for CCRCC. PMID:25364440

Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43

PubMed Central

1995-01-01

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin. PMID:7642714

Reduced Slc1a1 expression is associated with neuroinflammation and impaired sensorimotor gating and cognitive performance in mice: Implications for schizophrenia

PubMed Central

Afshari, Parisa; Yao, Wei-Dong

2017-01-01

We previously reported a 84-Kb hemi-deletion copy number variant at the SLC1A1 gene locus that reduces its expression and appeared causally linked to schizophrenia. In this report, we characterize the in vivo and in vitro consequences of reduced expression of Slc1a1 in mice. Heterozygous (HET) Slc1a1+/- mice, which more closely model the hemi-deletion we found in human subjects, were examined in a series of behavioral, anatomical and biochemical assays. Knockout (KO) mice were also included in the behavioral studies for comparative purposes. Both HET and KO mice exhibited evidence of increased anxiety-like behavior, impaired working memory, decreased exploratory activity and impaired sensorimotor gating, but no changes in overall locomotor activity. The magnitude of changes was approximately equivalent in the HET and KO mice suggesting a dominant effect of the haploinsufficiency. Behavioral changes in the HET mice were accompanied by reduced thickness of the dorsomedial prefrontal cortex. Whole transcriptome RNA-Seq analysis detected expression changes of genes and pathways involved in cytokine signaling and synaptic functions in both brain and blood. Moreover, the brains of Slc1a1+/- mice displayed elevated levels of oxidized glutathione, a trend for increased oxidative DNA damage, and significantly increased levels of cytokines. This latter finding was further supported by SLC1A1 knockdown and overexpression studies in differentiated human neuroblastoma cells, which led to decreased or increased cytokine expression, respectively. Taken together, our results suggest that partial loss of the Slc1a1 gene in mice causes haploinsufficiency associated with behavioral, histological and biochemical changes that reflect an altered redox state and may promote the expression of behavioral features and inflammatory states consistent with those observed in schizophrenia. PMID:28886095

A novel dissociative steroid VBP15 reduces MUC5AC gene expression in airway epithelial cells but lacks the GRE mediated transcriptional properties of dexamethasone.

PubMed

Garvin, Lindsay M; Chen, Yajun; Damsker, Jesse M; Rose, Mary C

2016-06-01

Overproduction of secretory mucins contributes to morbidity/mortality in inflammatory lung diseases. Inflammatory mediators directly increase expression of mucin genes, but few drugs have been shown to directly repress mucin gene expression. IL-1β upregulates the MUC5AC mucin gene in part via the transcription factors NFκB while the glucocorticoid Dexamethasone (Dex) transcriptionally represses MUC5AC expression by Dex-activated GR binding to two GRE cis-sites in the MUC5AC promoter in lung epithelial cells. VBP compounds (ReveraGen BioPharma) maintain anti-inflammatory activity through inhibition of NFκB but exhibit reduced GRE-mediated transcriptional properties associated with adverse side-effects and thus have potential to minimize harmful side effects of long-term steroid therapy in inflammatory lung diseases. We investigated VBP15 efficacy as an anti-mucin agent in two types of airway epithelial cells and analyzed the transcription factor activity and promoter binding associated with VBP15-induced MUC5AC repression. VBP15 reduced MUC5AC mRNA abundance in a dose- and time-dependent manner similar to Dex in the presence or absence of IL-1β in A549 and differentiated human bronchial epithelial cells. Repression was abrogated in the presence of RU486, demonstrating a requirement for GR in the VBP15-induced repression of MUC5AC. Inhibition of NFκB activity resulted in reduced baseline expression of MUC5AC indicating that constitutive activity maintains MUC5AC production. Chromatin immunoprecipitation analysis demonstrated lack of GR and of p65 (NFκB) binding to composite GRE domains in the MUC5AC promoter following VBP15 exposure of cells, in contrast to Dex. These data demonstrate that VBP15 is a novel anti-mucin agent that mediates the reduction of MUC5AC gene expression differently than the classical glucocorticoid, Dex. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cysteine palmitoylation of the γ subunit has a dominant role in modulating activity of the epithelial sodium channel.

PubMed

Mukherjee, Anindit; Mueller, Gunhild M; Kinlough, Carol L; Sheng, Nan; Wang, Zhijian; Mustafa, S Atif; Kashlan, Ossama B; Kleyman, Thomas R; Hughey, Rebecca P

2014-05-16

The epithelial sodium channel (ENaC) is composed of three homologous subunits (α, β, and γ) with cytoplasmic N and C termini. Our previous work revealed that two cytoplasmic Cys residues in the β subunit, βCys-43 and βCys-557, are Cys-palmitoylated. ENaCs with mutant βC43A/C557A exhibit normal surface expression but enhanced Na(+) self-inhibition and reduced channel open probability. Although the α subunit is not palmitoylated, we now show that the two cytoplasmic Cys residues in the γ subunit are palmitoylated. ENaCs with mutant γC33A, γC41A, or γC33A/C41A exhibit reduced activity compared with wild type channels but normal surface expression and normal levels of α and γ subunit-activating cleavage. These mutant channels have significantly enhanced Na(+) self-inhibition and reduced open probability compared with wild type ENaCs. Channel activity was enhanced by co-expression with the palmitoyltransferase DHHC2 that also co-immunoprecipitates with ENaCs. Secondary structure prediction of the N terminus of the γ subunit places γCys-33 within an α-helix and γCys-44 on a coil before the first transmembrane domain within a short tract that includes a well conserved His-Gly motif, where mutations have been associated with altered channel gating. Our current and previous results suggest that palmitoylation of the β and γ subunits of ENaCs enhances interactions of their respective cytoplasmic domains with the plasma membrane and stabilizes the open state of the channel. Comparison of activities of channels lacking palmitoylation sites in individual or multiple subunits revealed that γ subunit palmitoylation has a dominant role over β subunit palmitoylation in modulating ENaC gating. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Contribution of programmed cell death receptor (PD)-1 to Kupffer cell dysfunction in murine polymicrobial sepsis.

PubMed

Wang, Fei; Huang, Xin; Chung, Chun-Shiang; Chen, Yaping; Hutchins, Noelle A; Ayala, Alfred

2016-08-01

Recent studies suggest that coinhibitory receptors appear to be important in contributing sepsis-induced immunosuppression. Our laboratory reported that mice deficient in programmed cell death receptor (PD)-1 have increased bacterial clearance and improved survival in experimental sepsis induced by cecal ligation and puncture (CLP). In response to infection, the liver clears the blood of bacteria and produces cytokines. Kupffer cells, the resident macrophages in the liver, are strategically situated to perform the above functions. However, it is not known if PD-1 expression on Kupffer cells is altered by septic stimuli, let alone if PD-1 ligation contributes to the altered microbial handling seen. Here we report that PD-1 is significantly upregulated on Kupffer cells during sepsis. PD-1-deficient septic mouse Kupffer cells displayed markedly enhanced phagocytosis and restoration of the expression of major histocompatibility complex II and CD86, but reduced CD80 expression compared with septic wild-type (WT) mouse Kupffer cells. In response to ex vivo LPS stimulation, the cytokine productive capacity of Kupffer cells derived from PD-1-/- CLP mice exhibited a marked, albeit partial, restoration of the release of IL-6, IL-12, IL-1β, monocyte chemoattractant protein-1, and IL-10 compared with septic WT mouse Kupffer cells. In addition, PD-1 gene deficiency decreased LPS-induced apoptosis of septic Kupffer cells, as indicated by decreased levels of cleaved caspase-3 and reduced terminal deoxynucleotidyl transferase dUTP nick end-labeling-positive cells. Exploring the signal pathways involved, we found that, after ex vivo LPS stimulation, septic PD-1-/- mouse Kupffer cells exhibited an increased Akt phosphorylation and a reduced p38 phosphorylation compared with septic WT mouse Kupffer cells. Together, these results indicate that PD-1 appears to play an important role in regulating the development of Kupffer cell dysfunction seen in sepsis. Copyright © 2016 the American Physiological Society.

Contribution of programmed cell death receptor (PD)-1 to Kupffer cell dysfunction in murine polymicrobial sepsis

PubMed Central

Wang, Fei; Huang, Xin; Chung, Chun-Shiang; Chen, Yaping; Hutchins, Noelle A.

2016-01-01

Recent studies suggest that coinhibitory receptors appear to be important in contributing sepsis-induced immunosuppression. Our laboratory reported that mice deficient in programmed cell death receptor (PD)-1 have increased bacterial clearance and improved survival in experimental sepsis induced by cecal ligation and puncture (CLP). In response to infection, the liver clears the blood of bacteria and produces cytokines. Kupffer cells, the resident macrophages in the liver, are strategically situated to perform the above functions. However, it is not known if PD-1 expression on Kupffer cells is altered by septic stimuli, let alone if PD-1 ligation contributes to the altered microbial handling seen. Here we report that PD-1 is significantly upregulated on Kupffer cells during sepsis. PD-1-deficient septic mouse Kupffer cells displayed markedly enhanced phagocytosis and restoration of the expression of major histocompatibility complex II and CD86, but reduced CD80 expression compared with septic wild-type (WT) mouse Kupffer cells. In response to ex vivo LPS stimulation, the cytokine productive capacity of Kupffer cells derived from PD-1−/− CLP mice exhibited a marked, albeit partial, restoration of the release of IL-6, IL-12, IL-1β, monocyte chemoattractant protein-1, and IL-10 compared with septic WT mouse Kupffer cells. In addition, PD-1 gene deficiency decreased LPS-induced apoptosis of septic Kupffer cells, as indicated by decreased levels of cleaved caspase-3 and reduced terminal deoxynucleotidyl transferase dUTP nick end-labeling-positive cells. Exploring the signal pathways involved, we found that, after ex vivo LPS stimulation, septic PD-1−/− mouse Kupffer cells exhibited an increased Akt phosphorylation and a reduced p38 phosphorylation compared with septic WT mouse Kupffer cells. Together, these results indicate that PD-1 appears to play an important role in regulating the development of Kupffer cell dysfunction seen in sepsis. PMID:27288425

Orbitofrontal participation in sign- and goal-tracking conditioned responses: Effects of nicotine.

PubMed

Stringfield, Sierra J; Palmatier, Matthew I; Boettiger, Charlotte A; Robinson, Donita L

2017-04-01

Pavlovian conditioned stimuli can acquire incentive motivational properties, and this phenomenon can be measured in animals using Pavlovian conditioned approach behavior. Drugs of abuse can influence the expression of this behavior, and nicotine in particular exhibits incentive amplifying effects. Both conditioned approach behavior and drug abuse rely on overlapping corticolimbic circuitry. We hypothesize that the orbitofrontal cortex (OFC) regulates conditioned approach, and that one site of nicotine action is in the OFC where it reduces cortical output. To test this, we repeatedly exposed rats to 0.4 mg/kg nicotine (s.c.) during training and then pharmacologically inactivated the lateral OFC or performed in vivo electrophysiological recordings of lateral OFC neurons in the presence or absence of nicotine. In Experiment 1, animals were trained in a Pavlovian conditioning paradigm and behavior was evaluated after inactivation of the OFC by microinfusion of the GABA agonists baclofen and muscimol. In Experiment 2, we monitored phasic firing of OFC neurons during Pavlovian conditioning sessions. Nicotine reliably enhanced conditioned responding to the conditioned cue, and inactivation of the OFC reduced conditioned responding, especially the sign-tracking response. OFC neurons exhibited phasic excitations to cue presentation and during goal tracking, and nicotine acutely blunted this phasic neuronal firing. When nicotine was withheld, both conditioned responding and phasic firing in the OFC returned to the level of controls. These results suggest that the OFC is recruited for the expression of conditioned responses, and that nicotine acutely influences this behavior by reducing phasic firing in the OFC. Copyright © 2016 Elsevier Ltd. All rights reserved.

Transgenic Suppression of AGAMOUS Genes in Apple Reduces Fertility and Increases Floral Attractiveness

PubMed Central

Klocko, Amy L.; Borejsza-Wysocka, Ewa; Brunner, Amy M.; Shevchenko, Olga; Aldwinckle, Herb; Strauss, Steven H.

2016-01-01

We investigated the ability of RNA interference (RNAi) directed against two co-orthologs of AGAMOUS (AG) from Malus domestica (domestic apple, MdAG) to reduce the risks of invasiveness and provide genetic containment of transgenes, while also promoting the attractiveness of flowers for ornamental usage. Suppression of two MdAG-like genes, MdMADS15 and MdMADS22, led to the production of trees with highly showy, polypetalous flowers. These “double-flowers” had strongly reduced expression of both MdAG-like genes. Members of the two other clades within in the MdAG subfamily showed mild to moderate differences in gene expression, or were unchanged, with the level of suppression approximately proportional to the level of sequence identity between the gene analyzed and the RNAi fragment. The double-flowers also exhibited reduced male and female fertility, had few viable pollen grains, a decreased number of stigmas, and produced few viable seeds after cross-pollination. Despite these floral alterations, RNAi-AG trees with double-flowers set full-sized fruit. Suppression or mutation of apple AG-like genes appears to be a promising method for combining genetic containment with improved floral attractiveness. PMID:27500731

Demonstration of Hadoop-GIS: A Spatial Data Warehousing System Over MapReduce.

PubMed

Aji, Ablimit; Sun, Xiling; Vo, Hoang; Liu, Qioaling; Lee, Rubao; Zhang, Xiaodong; Saltz, Joel; Wang, Fusheng

2013-11-01

The proliferation of GPS-enabled devices, and the rapid improvement of scientific instruments have resulted in massive amounts of spatial data in the last decade. Support of high performance spatial queries on large volumes data has become increasingly important in numerous fields, which requires a scalable and efficient spatial data warehousing solution as existing approaches exhibit scalability limitations and efficiency bottlenecks for large scale spatial applications. In this demonstration, we present Hadoop-GIS - a scalable and high performance spatial query system over MapReduce. Hadoop-GIS provides an efficient spatial query engine to process spatial queries, data and space based partitioning, and query pipelines that parallelize queries implicitly on MapReduce. Hadoop-GIS also provides an expressive, SQL-like spatial query language for workload specification. We will demonstrate how spatial queries are expressed in spatially extended SQL queries, and submitted through a command line/web interface for execution. Parallel to our system demonstration, we explain the system architecture and details on how queries are translated to MapReduce operators, optimized, and executed on Hadoop. In addition, we will showcase how the system can be used to support two representative real world use cases: large scale pathology analytical imaging, and geo-spatial data warehousing.

Human amniotic epithelial cells inhibit CD4+ T cell activation in acute kidney injury patients by influencing the miR-101-c-Rel-IL-2 pathway.

PubMed

Liu, Junfeng; Hua, Rong; Gong, Zhangbin; Shang, Bin; Huang, Yongyi; Guo, Lihe; Liu, Te; Xue, Jun

2017-01-01

In the pathogenesis of acute kidney injury (AKI), the release of multiple interleukins can lead to increased kidney damage. Human amniotic epithelial cells (HuAECs) can inhibit immune cell activation in vivo and in vitro. We hypothesized that HuAECs could weaken patient-derived peripheral blood CD4+ T-cell activation and decreasing the ability of these cells to express and release IL-2. -Cell proliferation assay revealed that under the same culture conditions, activated AKI patient-derived CD4+ T cells had a significantly reduced proliferation rate when were co-cultured with HuAECs. And the level of IL-2 released was also significantly reduced. Western blot and qRT-PCR assays showed that the expression of c-Rel in the CD4+ T cells was also significantly reduced. However, the expression level of endogenous miR-101 in the CD4+ T cells co-cultured with HuAECs was significantly increased. Luciferase reporter assay results suggested that miR-101 could bind to a specific site in the c-Rel 3' UTR and induce the post-transcriptional silencing of c-Rel. Subsequently, we over-expressed miR-101 in AKI patient-derived CD4+ T cells. The qRT-PCR and western blot assay results revealed that the expression of endogenous c-Rel was significantly reduced, while the ELISA results indicated that the level of IL-2 released was also significantly decreased. Finally, ChIP-PCR assay results showed that the miR-101-overexpressing CD4+ T-cell group and the HuAEC co-culture CD4+ T-cell group exhibited significantly decreased binding capacities between the 'c-Rel-NFκB' complex and the IL-2 gene promoter, and the transcriptional activity of IL-2 was also significantly decreased. Therefore, we confirmed that HuAECs can stimulate miR-101 expression in AKI patient-derived peripheral blood CD4+ T cells, thus inhibiting the expression of the miR-101 target gene c-Rel and leading to a reduction in IL-2 expression and release. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rheumatic Heart Disease and Myxomatous Degeneration: Differences and Similarities of Valve Damage Resulting from Autoimmune Reactions and Matrix Disorganization.

PubMed

Martins, Carlo de Oliveira; Demarchi, Lea; Ferreira, Frederico Moraes; Pomerantzeff, Pablo Maria Alberto; Brandao, Carlos; Sampaio, Roney Orismar; Spina, Guilherme Sobreira; Kalil, Jorge; Cunha-Neto, Edecio; Guilherme, Luiza

2017-01-01

Autoimmune inflammatory reactions leading to rheumatic fever (RF) and rheumatic heart disease (RHD) result from untreated Streptococcus pyogenes throat infections in individuals who exhibit genetic susceptibility. Immune effector mechanisms have been described that lead to heart tissue damage culminating in mitral and aortic valve dysfunctions. In myxomatous valve degeneration (MXD), the mitral valve is also damaged due to non-inflammatory mechanisms. Both diseases are characterized by structural valve disarray and a previous proteomic analysis of them has disclosed a distinct profile of matrix/structural proteins differentially expressed. Given their relevance in organizing valve tissue, we quantitatively evaluated the expression of vimentin, collagen VI, lumican, and vitronectin as well as performed immunohistochemical analysis of their distribution in valve tissue lesions of patients in both diseases. We identified abundant expression of two isoforms of vimentin (45 kDa, 42 kDa) with reduced expression of the full-size protein (54 kDa) in RHD valves. We also found increased vitronectin expression, reduced collagen VI expression and similar lumican expression between RHD and MXD valves. Immunohistochemical analysis indicated disrupted patterns of these proteins in myxomatous degeneration valves and disorganized distribution in rheumatic heart disease valves that correlated with clinical manifestations such as valve regurgitation or stenosis. Confocal microscopy analysis revealed a diverse pattern of distribution of collagen VI and lumican into RHD and MXD valves. Altogether, these results demonstrated distinct patterns of altered valve expression and tissue distribution/organization of structural/matrix proteins that play important pathophysiological roles in both valve diseases.

Myocilin Regulates Cell Proliferation and Survival*

PubMed Central

Joe, Myung Kuk; Kwon, Heung Sun; Cojocaru, Radu; Tomarev, Stanislav I.

2014-01-01

Myocilin, a causative gene for open angle glaucoma, encodes a secreted glycoprotein with poorly understood functions. To gain insight into its functions, we produced a stably transfected HEK293 cell line expressing myocilin under an inducible promoter and compared gene expression profiles between myocilin-expressing and vector control cell lines by a microarray analysis. A significant fraction of differentially expressed genes in myocilin-expressing cells was associated with cell growth and cell death, suggesting that myocilin may have a role in the regulation of cell growth and survival. Increased proliferation of myocilin-expressing cells was demonstrated by the WST-1 proliferation assay, direct cell counting, and immunostaining with antibodies against Ki-67, a cellular proliferation marker. Myocilin-containing conditioned medium also increased proliferation of unmodified HEK293 cells. Myocilin-expressing cells were more resistant to serum starvation-induced apoptosis than control cells. TUNEL-positive apoptotic cells were dramatically decreased, and two apoptotic marker proteins, cleaved caspase 7 and cleaved poly(ADP-ribose) polymerase, were significantly reduced in myocilin-expressing cells as compared with control cells under apoptotic conditions. In addition, myocilin-deficient mesenchymal stem cells exhibited reduced proliferation and enhanced susceptibility to serum starvation-induced apoptosis as compared with wild-type mesenchymal stem cells. Phosphorylation of ERK1/2 and its upstream kinases, c-Raf and MEK, was increased in myocilin-expressing cells compared with control cells. Elevated phosphorylation of ERK1/2 was also observed in the trabecular meshwork of transgenic mice expressing 6-fold higher levels of myocilin when compared with their wild-type littermates. These results suggest that myocilin promotes cell proliferation and resistance to apoptosis via the ERK1/2 MAPK signaling pathway. PMID:24563482

The gut microbiota regulates bone mass in mice

PubMed Central

Sjögren, Klara; Engdahl, Cecilia; Henning, Petra; Lerner, Ulf H; Tremaroli, Valentina; Lagerquist, Marie K; Bäckhed, Fredrik; Ohlsson, Claes

2012-01-01

The gut microbiota modulates host metabolism and development of immune status. Here we show that the gut microbiota is also a major regulator of bone mass in mice. Germ-free (GF) mice exhibit increased bone mass associated with reduced number of osteoclasts per bone surface compared with conventionally raised (CONV-R) mice. Colonization of GF mice with a normal gut microbiota normalizes bone mass. Furthermore, GF mice have decreased frequency of CD4+ T cells and CD11b+/GR 1 osteoclast precursor cells in bone marrow, which could be normalized by colonization. GF mice exhibited reduced expression of inflammatory cytokines in bone and bone marrow compared with CONV-R mice. In summary, the gut microbiota regulates bone mass in mice, and we provide evidence for a mechanism involving altered immune status in bone and thereby affected osteoclast-mediated bone resorption. Further studies are required to evaluate the gut microbiota as a novel therapeutic target for osteoporosis. © 2012 American Society for Bone and Mineral Research. PMID:22407806

PGL, encoding chlorophyllide a oxygenase 1, impacts leaf senescence and indirectly affects grain yield and quality in rice.

PubMed

Yang, Yaolong; Xu, Jie; Huang, Lichao; Leng, Yujia; Dai, Liping; Rao, Yuchun; Chen, Long; Wang, Yuqiong; Tu, Zhengjun; Hu, Jiang; Ren, Deyong; Zhang, Guangheng; Zhu, Li; Guo, Longbiao; Qian, Qian; Zeng, Dali

2016-03-01

Chlorophyll (Chl) b is a ubiquitous accessory pigment in land plants, green algae, and prochlorophytes. This pigment is synthesized from Chl a by chlorophyllide a oxygenase and plays a key role in adaptation to various environments. This study characterizes a rice mutant, pale green leaf (pgl), and isolates the gene PGL by using a map-based cloning approach. PGL, encoding chlorophyllide a oxygenase 1, is mainly expressed in the chlorenchyma and activated in the light-dependent Chl synthesis process. Compared with wild-type plants, pgl exhibits a lower Chl content with a reduced and disorderly thylakoid ultrastructure, which decreases the photosynthesis rate and results in reduced grain yield and quality. In addition, pgl exhibits premature senescence in both natural and dark-induced conditions and more severe Chl degradation and reactive oxygen species accumulation than does the wild-type. Moreover, pgl is sensitive to heat stress. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Polyploidization of murine mesenchymal cells is associated with suppression of the long noncoding RNA H19 and reduced tumorigenicity.

PubMed

Shoshani, Ofer; Massalha, Hassan; Shani, Nir; Kagan, Sivan; Ravid, Orly; Madar, Shalom; Trakhtenbrot, Luba; Leshkowitz, Dena; Rechavi, Gideon; Zipori, Dov

2012-12-15

Mesenchymal stromal cells (MSC) are used extensively in clinical trials; however, the possibility that MSCs have a potential for malignant transformation was raised. We examined the genomic stability versus the tumor-forming capacity of multiple mouse MSCs. Murine MSCs have been shown to be less stable and more prone to malignant transformation than their human counterparts. A large series of independently isolated MSC populations exhibited low tumorigenic potential under syngeneic conditions, which increased in immunocompromised animals. Unexpectedly, higher ploidy correlated with reduced tumor-forming capacity. Furthermore, in both cultured MSCs and primary hepatocytes, polyploidization was associated with a dramatic decrease in the expression of the long noncoding RNA H19. Direct knockdown of H19 expression in diploid cells resulted in acquisition of polyploid cell traits. Moreover, artificial tetraploidization of diploid cancer cells led to a reduction of H19 levels, as well as to an attenuation of the tumorigenic potential. Polyploidy might therefore serve as a protective mechanism aimed at reducing malignant transformation through the involvement of the H19 regulatory long noncoding RNA.

Neurotensin increases mortality and mast cells reduce neurotensin levels in a mouse model of sepsis

PubMed Central

Piliponsky, Adrian M.; Chen, Ching-Cheng; Nishimura, Toshihiko; Metz, Martin; Rios, Eon J.; Dobner, Paul R.; Wada, Etsuko; Wada, Keiji; Zacharias, Sherma; Mohanasundaram, Uma; Faix, James D.; Abrink, Magnus; Pejler, Gunnar; Pearl, Ronald; Tsai, Mindy; Galli, Stephen J.

2010-01-01

Sepsis is a complex, incompletely understood and often fatal disorder,1 typically accompanied by hypotension,2 that is considered to represent a dysregulated host response to infection.3,4,5 Neurotensin (NT) is a 13-amino-acid peptide that, among its multiple effects, induces hypotension.6 We find that intraperitoneal and plasma concentrations of NT are increased in mice after severe caecal ligation and puncture (CLP), a model of sepsis, and that mice treated with a pharmacological antagonist of NT, or NT-deficient mice, exhibit reduced mortality during severe CLP. In mice, mast cells can degrade NT and reduce NT-induced hypotension and CLP-associated mortality, and optimal expression of these effects requires mast cell expression of neurotensin receptor 1 and neurolysin. These findings show that NT contributes to sepsis-related mortality in mice during severe CLP and that mast cells can lower NT concentrations, and suggest that mast cell-dependent reduction in NT levels contributes to the ability of mast cells to enhance survival after CLP. PMID:18376408

To the Large Nucleolar Bodies in Apoptotic Leukaemic Granulocytic Progenitors without Further Differentiation. Are Large Nucleoli Always Present in Proliferating Cells?

PubMed

Smetana, K; Kuželová, K; Zápotocký, M; Hrkal, Z

2017-01-01

Large nucleoli have generally been believed to be present in less differentiated and proliferating cells including the malignant ones. Such nucleoli have also been considered to be active in the biosynthetic process and major cell developmental activities. In contrast, after cytostatic treatment, apoptotic leukaemic progenitors still containing nuclei did not exhibit substantial reduction of the nucleolar size but displayed decreased nucleolar biosynthetic activity. The present study was undertaken to provide more information on the large nucleoli in spontaneously occurring apoptotic leukaemic progenitors without further differentiation. Leukaemic progenitors of established cell lineages originating from leukaemic patients represented a very convenient model for such study. Some of them exhibit morphological signs of the spontaneously occurring apoptotic process. Since such signs are expressed by nuclear and cytoplasmic morphological variability, the present study dealt with spontaneously occurring apoptotic progenitors with preserved nuclei characterized by heavy chromatin condensation and occasional fragmentation. Based of nucleolar body and nuclear maximal diameter measurements it seems to be clear that the nucleolar size in these cells was not substantially reduced, contrary to that of the nucleus. However, large nucleolar bodies in spontaneously occurring apoptotic cells were characterized by markedly reduced biosynthetic activity, as expressed by the decreased number of nucleolar transcription markers such as nucleolar fibrillar centres. In conclusion, large nucleoli may be present not only in proliferating, but also in spontaneously occurring apoptotic cells.

Rapid loss of behavioral plasticity and immunocompetence under intense sexual selection.

PubMed

van Lieshout, Emile; McNamara, Kathryn B; Simmons, Leigh W

2014-09-01

Phenotypic plasticity allows animals to maximize fitness by conditionally expressing the phenotype best adapted to their environment. Although evidence for such adjustment in reproductive tactics is common, little is known about how phenotypic plasticity evolves in response to sexual selection. We examined the effect of sexual selection intensity on phenotypic plasticity in mating behavior using the beetle Callosobruchus maculatus. Male genital spines harm females during mating and females exhibit copulatory kicking, an apparent resistance trait aimed to dislodge mating males. After exposing individuals from male- and female-biased experimental evolution lines to male- and female-biased sociosexual environments, we examined behavioral plasticity in matings with standard partners. While females from female-biased lines kicked sooner after exposure to male-biased sociosexual contexts, in male-biased lines this plasticity was lost. Ejaculate size did not diverge in response to selection history, but males from both treatments exhibited plasticity consistent with sperm competition intensity models, reducing size as the number of competitors increased. Analysis of immunocompetence revealed reduced immunity in both sexes in male-biased lines, pointing to increased reproductive costs under high sexual selection. These results highlight how male and female reproductive strategies are shaped by interactions between phenotypically plastic and genetic mechanisms of sexual trait expression. © 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.

Steroid 5α-reductase 2 deficiency leads to reduced dominance-related and impulse-control behaviors.

PubMed

Mosher, Laura J; Godar, Sean C; Morissette, Marc; McFarlin, Kenneth M; Scheggi, Simona; Gambarana, Carla; Fowler, Stephen C; Di Paolo, Thérèse; Bortolato, Marco

2018-05-01

The enzyme steroid 5α-reductase 2 (5αR2) catalyzes the conversion of testosterone into the potent androgen 5α-dihydrotestosterone. Previous investigations showed that 5αR2 is expressed in key brain areas for emotional and socio-affective reactivity, yet the role of this enzyme in behavioral regulation remains mostly unknown. Here, we profiled the behavioral characteristics of 5αR2 heterozygous (HZ) and knockout (KO) mice, as compared with their wild-type (WT) littermates. While male 5αR2 KO mice displayed no overt alterations in motoric, sensory, information-processing and anxiety-related behaviors, they exhibited deficits in neurobehavioral correlates of dominance (including aggression against intruders, mating, and tube dominance) as well as novelty-seeking and risk-taking responses. Furthermore, male 5αR2 KO mice exhibited reduced D 2 -like dopamine receptor binding in the shell of the nucleus accumbens - a well-recognized molecular signature of social dominance. Collectively, these results suggest that 5αR2 is involved in the establishment of social dominance and its behavioral manifestations. Further studies are warranted to understand how the metabolic actions of 5αR2 on steroid profile may be implicated in social ranking, impulse control, and the modulation of dopamine receptor expression in the nucleus accumbens. Copyright © 2018 Elsevier Ltd. All rights reserved.

WNT5A enhances resistance of melanoma cells to targeted BRAF inhibitors

PubMed Central

Anastas, Jamie N.; Kulikauskas, Rima M.; Tamir, Tigist; Rizos, Helen; Long, Georgina V.; von Euw, Erika M.; Yang, Pei-Tzu; Chen, Hsiao-Wang; Haydu, Lauren; Toroni, Rachel A.; Lucero, Olivia M.; Chien, Andy J.; Moon, Randall T.

2014-01-01

About half of all melanomas harbor a mutation that results in a constitutively active BRAF kinase mutant (BRAFV600E/K) that can be selectively inhibited by targeted BRAF inhibitors (BRAFis). While patients treated with BRAFis initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. Here, we observed marked elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein were also elevated in BRAFi-resistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction of endogenous WNT5A in melanoma decreased cell growth, increased apoptosis in response to BRAFi challenge, and decreased the activity of prosurvival AKT signaling. Conversely, overexpression of WNT5A promoted melanoma growth, tumorigenesis, and activation of AKT signaling. Similarly to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibited growth, sensitized melanoma cells to BRAFi, and reduced AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which promotes AKT signaling through FZD7 and RYK, leading to increased growth and therapeutic resistance. Furthermore, increased WNT5A expression in BRAFi-resistant melanomas correlates with a specific transcriptional signature, which identifies potential therapeutic targets to reduce clinical BRAFi resistance. PMID:24865425

Oocyte quality in mice is affected by a mycotoxin-contaminated diet.

PubMed

Hou, Yan-Jun; Xiong, Bo; Zheng, Wei-Jiang; Duan, Xing; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Qiang; Xu, Yin-Xue; Sun, Shao-Chen

2014-05-01

Mycotoxins, such as deoxynivalenol (DON), zearalenone (ZEN), and aflatoxin (AF), are commonly found in many food commodities and may impair the growth and reproductive efficiency of animals and humans. We investigated the effects of a mycotoxin-contaminated diet on mouse oocyte quality. Maize contaminated with DON (3.875 mg/kg), ZEN (1,897 μg/kg), and AF (806 μg/kg) was incorporated into a mouse diet at three different levels (0, 15, and 30% w/w). After 4 weeks, ovarian and germinal vesicle oocyte indices decreased in mycotoxin-fed mice. Oocytes from these mice exhibited low developmental competence with reduced germinal vesicle breakdown and polar body extrusion rates. Embryo developmental competence also showed a similar pattern, and the majority of embryos could not develop to the morula stage. Actin expression was also reduced in both the oocyte cortex and cytoplasm, which was accompanied by decreased expression of the actin nucleation factors profilin-1 and mDia1. Moreover, a large percentage of oocytes derived from mice that were fed a mycotoxin-contaminated diet exhibited aberrant spindle morphology, a loss of the cortical granule-free domain, and abnormal mitochondrial distributions, which further supported the decreased oocyte quality. Thus, our results demonstrate that mycotoxins are toxic to the mouse reproductive system by affecting oocyte quality. Copyright © 2013 Wiley Periodicals, Inc.

Perturbation of auxin homeostasis by overexpression of wild-type IAA15 results in impaired stem cell differentiation and gravitropism in roots.

PubMed

Yan, Da-Wei; Wang, Jing; Yuan, Ting-Ting; Hong, Li-Wei; Gao, Xiang; Lu, Ying-Tang

2013-01-01

Aux/IAAs interact with auxin response factors (ARFs) to repress their transcriptional activity in the auxin signaling pathway. Previous studies have focused on gain-of-function mutations of domain II and little is known about whether the expression level of wild-type Aux/IAAs can modulate auxin homeostasis. Here we examined the perturbation of auxin homeostasis by ectopic expression of wild-type IAA15. Root gravitropism and stem cell differentiation were also analyzed. The transgenic lines were less sensitive to exogenous auxin and exhibited low-auxin phenotypes including failures in gravity response and defects in stem cell differentiation. Overexpression lines also showed an increase in auxin concentration and reduced polar auxin transport. These results demonstrate that an alteration in the expression of wild-type IAA15 can disrupt auxin homeostasis.

Anxiolytic effects of buspirone and MTEP in the Porsolt Forced Swim Test.

PubMed

Lee, Kaziya M; Coelho, Michal A; Sern, Kimberly R; Class, MacKayla A; Bocz, Mark D; Szumlinski, Karen K

2017-01-01

Traditionally, a reduction in floating behavior or immobility in the Porsolt forced swim test (FST) is employed as a predictor of antidepressant efficacy. However, over the past several years, our studies of alcohol withdrawal-induced negative affect consistently indicate the coincidence of increased anxiety-related behaviors on various behavioral tests with reduced immobility in the FST. Further, this behavioral profile correlates with increased mGlu5 protein expression within limbic brain regions. As the role for mGlu5 in anxiety is well established, we hypothesized that the reduced immobility exhibited by alcohol-withdrawn mice when tested in the FST might reflect anxiety, possibly a hyper-reactivity to the acute swim stressor. Herein, we evaluated whether or not the decreased FST immobility during alcohol withdrawal responds to systemic treatment with a behaviorally-effective dose of the prototypical anxiolytic, buspirone (5 mg/kg). We also determined the functional relevance of the withdrawal-induced increase in mGlu5 expression for FST behavior by comparing the effects of buspirone to a behaviorally effective dose of the mGlu5 negative allosteric modulator MTEP (3 mg/kg). Adult male C57BL/6J mice were subjected to a 14-day, multi-bottle, binge-drinking protocol that elicits hyper-anxiety and increases glutamate-related protein expression during early withdrawal. Control animals received only water. At 24hr withdrawal, animals from each drinking condition were subdivided into groups and treated with an IP injection of buspirone, MTEP, or vehicle, 30min prior to the FST. Drug effects on general locomotor activity were also assessed. As we reported previously, alcohol-withdrawn animals exhibited significantly reduced immobility in the FST compared to water controls. Both buspirone and MTEP significantly increased immobility in alcohol-withdrawn animals, with a modest increase also seen in water controls. No significant group differences were observed for locomotor activity, indicating that neither anxiolytic was sedating. These results provide predictive validity for increased swimming/reduced immobility in the FST as a model of anxiety and provide novel evidence in favor of mGlu5 inhibition as an effective therapeutic strategy for treating hyperanxiety during alcohol withdrawal.

Expression of an Arabidopsis molybdenum cofactor sulphurase gene in soybean enhances drought tolerance and increases yield under field conditions.

PubMed

Li, Yajun; Zhang, Jiachang; Zhang, Juan; Hao, Ling; Hua, Jinping; Duan, Liusheng; Zhang, Mingcai; Li, Zhaohu

2013-08-01

LOS5/ABA3 gene encoding molybdenum cofactor sulphurase is involved in aldehyde oxidase (AO) activity in Arabidopsis, which indirectly regulates ABA biosynthesis and increased stress tolerance. Here, we used a constitutive super promoter to drive LOS5/ABA3 overexpression in soybean (Glycine max L.) to enhance drought tolerance in growth chamber and field conditions. Expression of LOS5/ABA3 was up-regulated by drought stress, which led to increasing AO activity and then a notable increase in ABA accumulation. Transgenic soybean under drought stress had reduced water loss by decreased stomatal aperture size and transpiration rate, which alleviated leaf wilting and maintained higher relative water content. Exposed to drought stress, transgenic soybean exhibited reduced cell membrane damage by reducing electrolyte leakage and production of malondialdehyde and promoting proline accumulation and antioxidant enzyme activities. Also, overexpression of LOS5/ABA3 enhanced expression of stress-up-regulated genes. Furthermore, the seed yield of transgenic plants is at least 21% higher than that of wide-type plants under drought stress conditions in the field. These data suggest that overexpression of LOS5/ABA3 could improve drought tolerance in transgenic soybean via enhanced ABA accumulation, which could activate expression of stress-up-regulated genes and cause a series of physiological and biochemical resistant responses. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Arctigenin reduces blood pressure by modulation of nitric oxide synthase and NADPH oxidase expression in spontaneously hypertensive rats.

PubMed

Liu, Ying; Wang, Guoyuan; Yang, Mingguang; Chen, Haining; zhao, Yan; Yang, Shucai; Sun, Changhao

2015-12-25

Arctigenin is a bioactive constituent from dried seeds of Arctium lappa L., which was traditionally used as medicine. Arctigenin exhibits various bioactivities, but its effects on blood pressure regulation are still not widely studied. In this study, we investigated antihypertensive effects of arctigenin by long-term treatment in spontaneously hypertensive rats (SHRs). Arctigenin (50 mg/kg) or vehicle was administered to SHRs or Wistar rats as negative control by oral gavage once a day for total 8 weeks. Nifedipine (3 mg/kg) was used as a positive drug control. After treatment, hemodynamic and physical parameters, vascular reactivity in aorta, the concentration of plasma arctigenin and serum thromboxane B2, NO release and vascular p-eNOS, p-Akt, caveolin-1 protein expression, and vascular superoxide anion generation and p47phox protein expression were detected and analyzed. The results showed that arctigenin significantly reduced systolic blood pressure and ameliorated endothelial dysfunction of SHRs. Arctigenin reduced the levels of thromboxane B2 in plasma and superoxide anion in thoracic aorta of SHRs. Furthermore, arctigenin increased the NO production by enhancing the phosphorylation of Akt and eNOS (Ser 1177), and inhibiting the expression of NADPH oxidase in thoracic aorta of SHRs. Our data suggested that antihypertensive mechanisms of arctigenin were associated with enhanced eNOS phosphorylation and decreased NADPH oxidase-mediated superoxide anion generation. Copyright © 2015 Elsevier Inc. All rights reserved.

RNA interference-mediated NOTCH3 knockdown induces phenotype switching of vascular smooth muscle cells in vitro

PubMed Central

Liu, Nan; Li, Ying; Chen, Hui; Wei, Wei; An, Yulin; Zhu, Guangming

2015-01-01

Notch3 plays an important role in differentiation, migration and signal transduction of vascular smooth muscle cells (VSMCs). In this study, we used RNA interference (RNAi) technique to investigate the effect of knocking down the expression of the NOTCH3 gene in VSMCs on the phenotype determination under pathologic status. Real-time PCR and Western Blot experiments verified the expression levels of Notch3 mRNA and protein were reduced more than 40% and 50% in the NOTCH3 siRNA group. When the expression of Notch3 was decreased, the proliferation, apoptosis and immigration of VSMCs were enhanced compared to control groups (P < 0.01). NOTCH3 siRNA VSMCs observed using confocal microscopy showed abnormal nuclear configuration, a disorganized actin filament system, polygonal cell shapes, and decreasing cell sizes. Additionally, knocking down the expression of NOTCH3 may evoke the CASR and FAK expression. In Conclusion, interfering with the expression of NOTCH3 causes VSMCs to exhibit an intermediate phenotype. CaSR and FAK may be involved in the Notch3 signaling pathway. PMID:26550181

RNA interference-mediated NOTCH3 knockdown induces phenotype switching of vascular smooth muscle cells in vitro.

PubMed

Liu, Nan; Li, Ying; Chen, Hui; Wei, Wei; An, Yulin; Zhu, Guangming

2015-01-01

Notch3 plays an important role in differentiation, migration and signal transduction of vascular smooth muscle cells (VSMCs). In this study, we used RNA interference (RNAi) technique to investigate the effect of knocking down the expression of the NOTCH3 gene in VSMCs on the phenotype determination under pathologic status. Real-time PCR and Western Blot experiments verified the expression levels of Notch3 mRNA and protein were reduced more than 40% and 50% in the NOTCH3 siRNA group. When the expression of Notch3 was decreased, the proliferation, apoptosis and immigration of VSMCs were enhanced compared to control groups (P < 0.01). NOTCH3 siRNA VSMCs observed using confocal microscopy showed abnormal nuclear configuration, a disorganized actin filament system, polygonal cell shapes, and decreasing cell sizes. Additionally, knocking down the expression of NOTCH3 may evoke the CASR and FAK expression. In Conclusion, interfering with the expression of NOTCH3 causes VSMCs to exhibit an intermediate phenotype. CaSR and FAK may be involved in the Notch3 signaling pathway.

A Gata2-Dependent Transcription Network Regulates Uterine Progesterone Responsiveness and Endometrial Function.

PubMed

Rubel, Cory A; Wu, San-Pin; Lin, Lin; Wang, Tianyuan; Lanz, Rainer B; Li, Xilong; Kommagani, Ramakrishna; Franco, Heather L; Camper, Sally A; Tong, Qiang; Jeong, Jae-Wook; Lydon, John P; DeMayo, Francesco J

2016-10-25

Altered progesterone responsiveness leads to female infertility and cancer, but underlying mechanisms remain unclear. Mice with uterine-specific ablation of GATA binding protein 2 (Gata2) are infertile, showing failures in embryo implantation, endometrial decidualization, and uninhibited estrogen signaling. Gata2 deficiency results in reduced progesterone receptor (PGR) expression and attenuated progesterone signaling, as evidenced by genome-wide expression profiling and chromatin immunoprecipitation. GATA2 not only occupies at and promotes expression of the Pgr gene but also regulates downstream progesterone responsive genes in conjunction with the PGR. Additionally, Gata2 knockout uteri exhibit abnormal luminal epithelia with ectopic TRP63 expressing squamous cells and a cancer-related molecular profile in a progesterone-independent manner. Lastly, we found a conserved GATA2-PGR regulatory network in both human and mice based on gene signature and path analyses using gene expression profiles of human endometrial tissues. In conclusion, uterine Gata2 regulates a key regulatory network of gene expression for progesterone signaling at the early pregnancy stage. Published by Elsevier Inc.

Silencing is noisy: population and cell level noise in telomere-adjacent genes is dependent on telomere position and sir2.

PubMed

Anderson, Matthew Z; Gerstein, Aleeza C; Wigen, Lauren; Baller, Joshua A; Berman, Judith

2014-07-01

Cell-to-cell gene expression noise is thought to be an important mechanism for generating phenotypic diversity. Furthermore, telomeric regions are major sites for gene amplification, which is thought to drive genetic diversity. Here we found that individual subtelomeric TLO genes exhibit increased variation in transcript and protein levels at both the cell-to-cell level as well as at the population-level. The cell-to-cell variation, termed Telomere-Adjacent Gene Expression Noise (TAGEN) was largely intrinsic noise and was dependent upon genome position: noise was reduced when a TLO gene was expressed at an ectopic internal locus and noise was elevated when a non-telomeric gene was expressed at a telomere-adjacent locus. This position-dependent TAGEN also was dependent on Sir2p, an NAD+-dependent histone deacetylase. Finally, we found that telomere silencing and TAGEN are tightly linked and regulated in cis: selection for either silencing or activation of a TLO-adjacent URA3 gene resulted in reduced noise at the neighboring TLO but not at other TLO genes. This provides experimental support to computational predictions that the ability to shift between silent and active chromatin states has a major effect on cell-to-cell noise. Furthermore, it demonstrates that these shifts affect the degree of expression variation at each telomere individually.

The Nuclear Receptor Corepressor Has Organizational Effects within the Developing Amygdala on Juvenile Social Play and Anxiety-Like Behavior

PubMed Central

Jessen, Heather M.; Kolodkin, Mira H.; Bychowski, Meaghan E.; Auger, Catherine J.; Auger, Anthony P.

2010-01-01

Nuclear receptor function on DNA is regulated by the balanced recruitment of coregulatory complexes. Recruited proteins that increase gene expression are called coactivators, and those that decrease gene expression are called corepressors. Little is known about the role of corepressors, such as nuclear receptor corepressor (NCoR), on the organization of behavior. We used real-time PCR to show that NCoR mRNA levels are sexually dimorphic, that females express higher levels of NCoR mRNA within the developing amygdala and hypothalamus, and that NCoR mRNA levels are reduced by estradiol treatment. To investigate the functional role of NCoR on juvenile social behavior, we infused small interfering RNA targeted against NCoR within the developing rat amygdala and assessed the enduring impact on juvenile social play behavior, sociability, and anxiety-like behavior. As expected, control males exhibited higher levels of juvenile social play than control females. Reducing NCoR expression during development further increased juvenile play in males only. Interestingly, decreased NCoR expression within the developing amygdala had lasting effects on increasing juvenile anxiety-like behavior in males and females. These data suggest that the corepressor NCoR functions to blunt sex differences in juvenile play behavior, a sexually dimorphic and hormone-dependent behavior, and appears critical for appropriate anxiety-like behavior in juvenile males and females. PMID:20051490

The nuclear receptor corepressor has organizational effects within the developing amygdala on juvenile social play and anxiety-like behavior.

PubMed

Jessen, Heather M; Kolodkin, Mira H; Bychowski, Meaghan E; Auger, Catherine J; Auger, Anthony P

2010-03-01

Nuclear receptor function on DNA is regulated by the balanced recruitment of coregulatory complexes. Recruited proteins that increase gene expression are called coactivators, and those that decrease gene expression are called corepressors. Little is known about the role of corepressors, such as nuclear receptor corepressor (NCoR), on the organization of behavior. We used real-time PCR to show that NCoR mRNA levels are sexually dimorphic, that females express higher levels of NCoR mRNA within the developing amygdala and hypothalamus, and that NCoR mRNA levels are reduced by estradiol treatment. To investigate the functional role of NCoR on juvenile social behavior, we infused small interfering RNA targeted against NCoR within the developing rat amygdala and assessed the enduring impact on juvenile social play behavior, sociability, and anxiety-like behavior. As expected, control males exhibited higher levels of juvenile social play than control females. Reducing NCoR expression during development further increased juvenile play in males only. Interestingly, decreased NCoR expression within the developing amygdala had lasting effects on increasing juvenile anxiety-like behavior in males and females. These data suggest that the corepressor NCoR functions to blunt sex differences in juvenile play behavior, a sexually dimorphic and hormone-dependent behavior, and appears critical for appropriate anxiety-like behavior in juvenile males and females.

Social Alienation in Schizophrenia Patients: Association with Insula Responsiveness to Facial Expressions of Disgust

PubMed Central

Lindner, Christian; Dannlowski, Udo; Walhöfer, Kirsten; Rödiger, Maike; Maisch, Birgit; Bauer, Jochen; Ohrmann, Patricia; Lencer, Rebekka; Zwitserlood, Pienie; Kersting, Anette; Heindel, Walter; Arolt, Volker

2014-01-01

Introduction Among the functional neuroimaging studies on emotional face processing in schizophrenia, few have used paradigms with facial expressions of disgust. In this study, we investigated whether schizophrenia patients show less insula activation to macro-expressions (overt, clearly visible expressions) and micro-expressions (covert, very brief expressions) of disgust than healthy controls. Furthermore, departing from the assumption that disgust faces signal social rejection, we examined whether perceptual sensitivity to disgust is related to social alienation in patients and controls. We hypothesized that high insula responsiveness to facial disgust predicts social alienation. Methods We used functional magnetic resonance imaging to measure insula activation in 36 schizophrenia patients and 40 healthy controls. During scanning, subjects passively viewed covert and overt presentations of disgust and neutral faces. To measure social alienation, a social loneliness scale and an agreeableness scale were administered. Results Schizophrenia patients exhibited reduced insula activation in response to covert facial expressions of disgust. With respect to macro-expressions of disgust, no between-group differences emerged. In patients, insula responsiveness to covert faces of disgust was positively correlated with social loneliness. Furthermore, patients' insula responsiveness to covert and overt faces of disgust was negatively correlated with agreeableness. In controls, insula responsiveness to covert expressions of disgust correlated negatively with agreeableness. Discussion Schizophrenia patients show reduced insula responsiveness to micro-expressions but not macro-expressions of disgust compared to healthy controls. In patients, low agreeableness was associated with stronger insula response to micro- and macro-expressions of disgust. Patients with a strong tendency to feel uncomfortable with social interactions appear to be characterized by a high sensitivity for facial expression signaling social rejection. Given the associations of insula responsiveness to covert disgust expression with low agreeableness in healthy individuals, insula responsiveness to expressions of disgust might be in general a neural marker of the personality trait of agreeableness. PMID:24465469

Social alienation in schizophrenia patients: association with insula responsiveness to facial expressions of disgust.

PubMed

Lindner, Christian; Dannlowski, Udo; Walhöfer, Kirsten; Rödiger, Maike; Maisch, Birgit; Bauer, Jochen; Ohrmann, Patricia; Lencer, Rebekka; Zwitserlood, Pienie; Kersting, Anette; Heindel, Walter; Arolt, Volker; Kugel, Harald; Suslow, Thomas

2014-01-01

Among the functional neuroimaging studies on emotional face processing in schizophrenia, few have used paradigms with facial expressions of disgust. In this study, we investigated whether schizophrenia patients show less insula activation to macro-expressions (overt, clearly visible expressions) and micro-expressions (covert, very brief expressions) of disgust than healthy controls. Furthermore, departing from the assumption that disgust faces signal social rejection, we examined whether perceptual sensitivity to disgust is related to social alienation in patients and controls. We hypothesized that high insula responsiveness to facial disgust predicts social alienation. We used functional magnetic resonance imaging to measure insula activation in 36 schizophrenia patients and 40 healthy controls. During scanning, subjects passively viewed covert and overt presentations of disgust and neutral faces. To measure social alienation, a social loneliness scale and an agreeableness scale were administered. Schizophrenia patients exhibited reduced insula activation in response to covert facial expressions of disgust. With respect to macro-expressions of disgust, no between-group differences emerged. In patients, insula responsiveness to covert faces of disgust was positively correlated with social loneliness. Furthermore, patients' insula responsiveness to covert and overt faces of disgust was negatively correlated with agreeableness. In controls, insula responsiveness to covert expressions of disgust correlated negatively with agreeableness. Schizophrenia patients show reduced insula responsiveness to micro-expressions but not macro-expressions of disgust compared to healthy controls. In patients, low agreeableness was associated with stronger insula response to micro- and macro-expressions of disgust. Patients with a strong tendency to feel uncomfortable with social interactions appear to be characterized by a high sensitivity for facial expression signaling social rejection. Given the associations of insula responsiveness to covert disgust expression with low agreeableness in healthy individuals, insula responsiveness to expressions of disgust might be in general a neural marker of the personality trait of agreeableness.

CD44 variant isoform 9 emerges in response to injury and contributes to the regeneration of the gastric epithelium

PubMed Central

Bertaux-Skeirik, Nina; Wunderlich, Mark; Teal, Emma; Chakrabarti, Jayati; Biesiada, Jacek; Mahe, Maxime; Sundaram, Nambirajan; Gabre, Joel; Hawkins, Jennifer; Jian, Gao; Engevik, Amy C.; Yang, Li; Wang, Jiang; Goldenring, James R.; Qualls, Joseph E.; Medvedovic, Mario; Helmrath, Michael A.; Diwan, Tayyab; Mulloy, James C.; Zavros, Yana

2017-01-01

The CD44 gene encodes several protein isoforms due to alternative splicing and post translational modifications. Given that CD44 variant isoform 9 (CD44v9) is expressed within Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) glands during repair, CD44v9 may be play a functional role during the process of regeneration of the gastric epithelium. Here we hypothesize that CD44v9 marks a regenerative cell lineage responsive to infiltrating macrophages during regeneration of the gastric epithelium. Ulcers were induced in CD44-decient (CD44KO) and C57BL/6 (BL6) mice by a localized application of acetic acid to the serosal surface of the stomach. Gastric organoids expressing CD44v9 were derived from mouse stomachs and transplanted at the ulcer site of CD44KO mice. Ulcers, CD44v9 expression, proliferation and histology were measured 1, 3, 5 and 7-days post-injury. Human-derived gastric organoids were generated from stomach tissue collected from elderly (>55 years) or young (14–20 years) patients. Organoids were transplanted into the stomachs of NOD scid gamma (NSG) mice at the site of injury. Gastric injury was induced in NRG-SGM3 (NRGS) mice harboring human-derived immune cells (hnNRGS) and the immune profile analyzed by CyTOF. CD44v9 expression emerged within regenerating glands the ulcer margin in response to injury. While ulcers in BL6 mice healed within 7-days post-injury, CD44KO mice exhibited loss of repair and epithelial regeneration. Ulcer healing was promoted in CD44KO mice by transplanted CD55v9-expressing gastric organoids. NSG mice exhibited loss of CD44v9 expression and gastric repair. Transplantation of human-derived gastric organoids from young, but not aged stomachs promoted repair in NSG mouse stomachs in response to injury. Finally, compared to NRGS mice, huNRGS animals exhibited reduced ulcer sizes, an infiltration of human CD162+ macrophages and an emergence of CD44v9 expression in SPEM. Thus, during repair of the gastric epithelium CD44v9 emerges within a regenerative cell lineage t hat coincides with macrophage infiltration within the injured mucosa. PMID:28497484

Sex-dependent alterations in motor and anxiety-like behavior of aged bacterial peptidoglycan sensing molecule 2 knockout mice.

PubMed

Arentsen, Tim; Khalid, Roksana; Qian, Yu; Diaz Heijtz, Rochellys

2018-01-01

Peptidoglycan recognition proteins (PGRPs) are key sensing-molecules of the innate immune system that specifically detect bacterial peptidoglycan (PGN) and its derivates. PGRPs have recently emerged as potential key regulators of normal brain development and behavior. To test the hypothesis that PGRPs play a role in motor control and anxiety-like behavior in later life, we used 15-month old male and female peptidoglycan recognition protein 2 (Pglyrp2) knockout (KO) mice. Pglyrp2 is an N-acetylmuramyl-l-alanine amidase that hydrolyzes PGN between the sugar backbone and the peptide chain (which is unique among the mammalian PGRPs). Using a battery of behavioral tests, we demonstrate that Pglyrp2 KO male mice display decreased levels of anxiety-like behavior compared with wild type (WT) males. In contrast, Pglyrp2 KO female mice show reduced rearing activity and increased anxiety-like behavior compared to WT females. In the accelerated rotarod test, however, Pglyrp2 KO female mice performed better compared to WT females (i.e., they had longer latency to fall off the rotarod). Further, Pglyrp2 KO male mice exhibited decreased expression levels of synaptophysin, gephyrin, and brain-derived neurotrophic factor in the frontal cortex, but not in the amygdala. Pglyrp2 KO female mice exhibited increased expression levels of spinophilin and alpha-synuclein in the frontal cortex, while exhibiting decreased expression levels of synaptophysin, gephyrin and spinophilin in the amygdala. Our findings suggest a novel role for Pglyrp2asa key regulator of motor and anxiety-like behavior in late life. Copyright © 2017. Published by Elsevier Inc.

Aging-associated inflammation promotes selection for adaptive oncogenic events in B cell progenitors.

PubMed

Henry, Curtis J; Casás-Selves, Matias; Kim, Jihye; Zaberezhnyy, Vadym; Aghili, Leila; Daniel, Ashley E; Jimenez, Linda; Azam, Tania; McNamee, Eoin N; Clambey, Eric T; Klawitter, Jelena; Serkova, Natalie J; Tan, Aik Choon; Dinarello, Charles A; DeGregori, James

2015-12-01

The incidence of cancer is higher in the elderly; however, many of the underlying mechanisms for this association remain unexplored. Here, we have shown that B cell progenitors in old mice exhibit marked signaling, gene expression, and metabolic defects. Moreover, B cell progenitors that developed from hematopoietic stem cells (HSCs) transferred from young mice into aged animals exhibited similar fitness defects. We further demonstrated that ectopic expression of the oncogenes BCR-ABL, NRAS(V12), or Myc restored B cell progenitor fitness, leading to selection for oncogenically initiated cells and leukemogenesis specifically in the context of an aged hematopoietic system. Aging was associated with increased inflammation in the BM microenvironment, and induction of inflammation in young mice phenocopied aging-associated B lymphopoiesis. Conversely, a reduction of inflammation in aged mice via transgenic expression of α-1-antitrypsin or IL-37 preserved the function of B cell progenitors and prevented NRAS(V12)-mediated oncogenesis. We conclude that chronic inflammatory microenvironments in old age lead to reductions in the fitness of B cell progenitor populations. This reduced progenitor pool fitness engenders selection for cells harboring oncogenic mutations, in part due to their ability to correct aging-associated functional defects. Thus, modulation of inflammation--a common feature of aging--has the potential to limit aging-associated oncogenesis.

Genetic Transformation and Hairy Root Induction Enhance the Antioxidant Potential of Lactuca serriola L.

PubMed

El-Esawi, Mohamed A; Elkelish, Amr; Elansary, Hosam O; Ali, Hayssam M; Elshikh, Mohamed; Witczak, Jacques; Ahmad, Margaret

2017-01-01

Lactuca serriola L. is a herbaceous species, used for human nutrition and medicinal purposes. The high antioxidant capacity of L. serriola indicates the possibility of enhancing its edible and health potential by increasing the flavonoid and phenolic contents. The present study aimed at enhancing the production of phenolics and flavonoids by hairy root cultures in Lactuca serriola transformed with Agrobacterium rhizogenes strain AR15834 harbouring the rolB gene. The genetic transformation of rolB in transformed roots was validated, and rolB expression level was evaluated using real-time qPCR analysis. Expression levels of flavonoid biosynthesis genes (CHI, PAL, FLS, and CHS) were assessed in the hairy and nontransformed roots. Results showed higher expression levels in the transgenic roots than in the nontransformed ones ( p < 0.01). Transgenic hairy roots exhibited a 54.8-96.7% increase in the total phenolic content, 38.1-76.2% increase in the total flavonoid content, and 56.7-96.7% increase in the total reducing power when compared with the nontransgenic roots ( p < 0.01). DPPH results also revealed that the transgenic hairy roots exhibited a 31.6-50% increase in antioxidant potential, when compared to normal roots. This study addressed the enhancement of secondary metabolite biosynthesis by hairy root induction in L. serriola .

Silencing and heterologous expression of ppo-2 indicate a specific function of a single polyphenol oxidase isoform in resistance of dandelion (Taraxacum officinale) against Pseudomonas syringae pv. tomato.

PubMed

Richter, Carolin; Dirks, Mareike E; Gronover, Christian Schulze; Prüfer, Dirk; Moerschbacher, Bruno M

2012-02-01

Dandelion (Taraxacum officinale) possesses an unusually high degree of disease resistance. As this plant exhibits high polyphenol oxidase (PPO) activity and PPO have been implicated in resistance against pests and pathogens, we analyzed the potential involvement of five PPO isoenzymes in the resistance of dandelion against Botrytis cinerea and Pseudomonas syringae pv. tomato. Only one PPO (ppo-2) was induced during infection, and ppo-2 promoter and β-glucuronidase marker gene fusions revealed strong induction of the gene surrounding lesions induced by B. cinerea. Specific RNAi silencing reduced ppo-2 expression only, and concomitantly increased plant susceptibility to P. syringae pv. tomato. At 4 days postinoculation, P. syringae pv. tomato populations were strongly increased in the ppo-2 RNAi lines compared with wild-type plants. When the dandelion ppo-2 gene was expressed in Arabidopsis thaliana, a plant having no PPO gene, active protein was formed and protein extracts of the transgenic plants exhibited substrate-dependent antimicrobial activity against P. syringae pv. tomato. These results clearly indicate a strong contribution of a specific, single PPO isoform to disease resistance. Therefore, we propose that specific PPO isoenzymes be included in a new family of pathogenesis-related (PR) proteins.

Modulation of Human Valve Interstitial Cell Phenotype and Function Using a Fibroblast Growth Factor 2 Formulation

PubMed Central

Latif, Najma; Quillon, Alfred; Sarathchandra, Padmini; McCormack, Ann; Lozanoski, Alec; Yacoub, Magdi H.; Chester, Adrian H.

2015-01-01

Valve interstitial cells (VICs) are fibroblastic in nature however in culture it is widely accepted that they differentiate into a myofibroblastic phenotype. This study assessed a fibroblast culture media formulation for its ability to maintain the phenotype and function of VICs as in the intact healthy valve. Normal human VICs were cultured separately in standard DMEM and in fibroblast media consisting of FGF2 (10ng/ml), insulin (50ng/ml) and 2% FCS for at least a week. Cell morphology, aspect ratio, size, levels and distribution of protein expression, proliferation, cell cycle, contraction and migration were assessed. Some VICs and some valve endothelial cells expressed FGF2 in valve tissue and this expression was increased in calcified valves. VICs in DMEM exhibited large, spread cells whereas VICs in fibroblast media were smaller, elongated and spindly. Aspect ratio and size were both significantly higher in DMEM (p<0.01). The level of expression of α-SMA was significantly reduced in fibroblast media at day 2 after isolation (p<0.01) and the expression of α-SMA, SM22 and EDA-fibronectin was significantly reduced in fibroblast media at days 7 and 12 post-isolation (p<0.01). Expression of cytoskeletal proteins, bone marker proteins and extracellular matrix proteins was reduced in fibroblast media. Proliferation of VICs in fibroblast media was significantly reduced at weeks 1 (p<0.05) and 2 (p<0.01). Collagen gel contraction was significantly reduced in fibroblast media (p<0.05). VICs were found to have significantly fewer and smaller focal adhesions in fibroblast media (p<0.01) with significantly fewer supermature focal adhesions in fibroblast media (p<0.001). Ultrastructurally, VICs in fibroblast media resembled native VICs from intact valves. VICs in fibroblast media demonstrated a slower migratory ability after wounding at 72 hours (p<0.01). Treatment of human VICs with this fibroblast media formulation has the ability to maintain and to dedifferentiate the VICs back to a fibroblastic phenotype with phenotypic and functional characteristics ascribed to cells in the intact valve. This methodology is fundamental in the study of normal valve biology, pathology and in the field of tissue engineering. PMID:26042674

Interleukin-33 treatment reduces secondary injury and improves functional recovery after contusion spinal cord injury.

PubMed

Pomeshchik, Yuriy; Kidin, Iurii; Korhonen, Paula; Savchenko, Ekaterina; Jaronen, Merja; Lehtonen, Sarka; Wojciechowski, Sara; Kanninen, Katja; Koistinaho, Jari; Malm, Tarja

2015-02-01

Interleukin-33 (IL-33) is a member of the interleukin-1 cytokine family and highly expressed in the naïve mouse brain and spinal cord. Despite the fact that IL-33 is known to be inducible by various inflammatory stimuli, its cellular localization in the central nervous system and role in pathological conditions is controversial. Administration of recombinant IL-33 has been shown to attenuate experimental autoimmune encephalomyelitis progression in one study, yet contradictory reports also exist. Here we investigated for the first time the pattern of IL-33 expression in the contused mouse spinal cord and demonstrated that after spinal cord injury (SCI) IL-33 was up-regulated and exhibited a nuclear localization predominantly in astrocytes. Importantly, we found that treatment with recombinant IL-33 alleviated secondary damage by significantly decreasing tissue loss, demyelination and astrogliosis in the contused mouse spinal cord, resulting in dramatically improved functional recovery. We identified both central and peripheral mechanisms of IL-33 action. In spinal cord, IL-33 treatment reduced the expression of pro-inflammatory tumor necrosis factor-alpha and promoted the activation of anti-inflammatory arginase-1 positive M2 microglia/macrophages, which chronically persisted in the injured spinal cord for up to at least 42 days after the treatment. In addition, IL-33 treatment showed a tendency towards reduced T-cell infiltration into the spinal cord. In the periphery, IL-33 treatment induced a shift towards the Th2 type cytokine profile and reduced the percentage and absolute number of cytotoxic, tumor necrosis factor-alpha expressing CD4+ cells in the spleen. Additionally, IL-33 treatment increased expression of T-regulatory cell marker FoxP3 and reduced expression of M1 marker iNOS in the spleen. Taken together, these results provide the first evidence that IL-33 administration is beneficial after CNS trauma. Treatment with IL33 may offer a novel therapeutic strategy for patients with acute contusion SCI. Copyright © 2014 Elsevier Inc. All rights reserved.

DLC1-dependent parathyroid hormone–like hormone inhibition suppresses breast cancer bone metastasis

PubMed Central

Wang, Yufeng; Lei, Rong; Zhuang, Xueqian; Zhang, Ning; Pan, Hong; Li, Gang; Hu, Jing; Pan, Xiaoqi; Tao, Qian; Fu, Da; Xiao, Jianru; Chin, Y. Eugene; Kang, Yibin; Yang, Qifeng; Hu, Guohong

2014-01-01

Bone metastasis is a frequent complication of breast cancer that is often accelerated by TGF-β signaling; however, little is known about how the TGF-β pathway is regulated during bone metastasis. Here we report that deleted in liver cancer 1 (DLC1) is an important regulator of TGF-β responses and osteolytic metastasis of breast cancer cells. In murine models, breast cancer cells lacking DLC1 expression exhibited enhanced capabilities of bone metastasis. Knockdown of DLC1 in cancer cells promoted bone metastasis, leading to manifested osteolysis and accelerated death in mice, while DLC1 overexpression suppressed bone metastasis. Activation of Rho-ROCK signaling in the absence of DLC1 mediated SMAD3 linker region phosphorylation and TGF-β–induced expression of parathyroid hormone–like hormone (PTHLH), leading to osteoclast maturation for osteolytic colonization. Furthermore, pharmacological inhibition of Rho-ROCK effectively reduced PTHLH production and breast cancer bone metastasis in vitro and in vivo. Evaluation of clinical breast tumor samples revealed that reduced DLC1 expression was linked to elevated PTHLH expression and organ-specific metastasis to bone. Overall, our findings define a stroma-dependent paradigm of Rho signaling in cancer and implicate Rho–TGF-β crosstalk in osteolytic bone metastasis. PMID:24590291

MicroRNA let-7b regulates neural stem cell proliferation and differentiation by targeting nuclear receptor TLX signaling

PubMed Central

Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Lang, Ming-Fei; Yang, Su; Li, Wendong; Shi, Yanhong

2010-01-01

Neural stem cell self-renewal and differentiation is orchestrated by precise control of gene expression involving nuclear receptor TLX. Let-7b, a member of the let-7 microRNA family, is expressed in mammalian brains and exhibits increased expression during neural differentiation. However, the role of let-7b in neural stem cell proliferation and differentiation remains unknown. Here we show that let-7b regulates neural stem cell proliferation and differentiation by targeting the stem cell regulator TLX and the cell cycle regulator cyclin D1. Overexpression of let-7b led to reduced neural stem cell proliferation and increased neural differentiation, whereas antisense knockdown of let-7b resulted in enhanced proliferation of neural stem cells. Moreover, in utero electroporation of let-7b to embryonic mouse brains led to reduced cell cycle progression in neural stem cells. Introducing an expression vector of Tlx or cyclin D1 that lacks the let-7b recognition site rescued let-7b-induced proliferation deficiency, suggesting that both TLX and cyclin D1 are important targets for let-7b-mediated regulation of neural stem cell proliferation. Let-7b, by targeting TLX and cyclin D1, establishes an efficient strategy to control neural stem cell proliferation and differentiation. PMID:20133835

MicroRNA let-7b regulates neural stem cell proliferation and differentiation by targeting nuclear receptor TLX signaling.

PubMed

Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Lang, Ming-Fei; Yang, Su; Li, Wendong; Shi, Yanhong

2010-02-02

Neural stem cell self-renewal and differentiation is orchestrated by precise control of gene expression involving nuclear receptor TLX. Let-7b, a member of the let-7 microRNA family, is expressed in mammalian brains and exhibits increased expression during neural differentiation. However, the role of let-7b in neural stem cell proliferation and differentiation remains unknown. Here we show that let-7b regulates neural stem cell proliferation and differentiation by targeting the stem cell regulator TLX and the cell cycle regulator cyclin D1. Overexpression of let-7b led to reduced neural stem cell proliferation and increased neural differentiation, whereas antisense knockdown of let-7b resulted in enhanced proliferation of neural stem cells. Moreover, in utero electroporation of let-7b to embryonic mouse brains led to reduced cell cycle progression in neural stem cells. Introducing an expression vector of Tlx or cyclin D1 that lacks the let-7b recognition site rescued let-7b-induced proliferation deficiency, suggesting that both TLX and cyclin D1 are important targets for let-7b-mediated regulation of neural stem cell proliferation. Let-7b, by targeting TLX and cyclin D1, establishes an efficient strategy to control neural stem cell proliferation and differentiation.

Claudin-1 induced sealing of blood-brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis.

PubMed

Pfeiffer, Friederike; Schäfer, Julia; Lyck, Ruth; Makrides, Victoria; Brunner, Sarah; Schaeren-Wiemers, Nicole; Deutsch, Urban; Engelhardt, Britta

2011-11-01

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood-brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have failed.

Indirect immobilized Jagged1 suppresses cell cycle progression and induces odonto/osteogenic differentiation in human dental pulp cells.

PubMed

Manokawinchoke, Jeeranan; Nattasit, Praphawi; Thongngam, Tanutchaporn; Pavasant, Prasit; Tompkins, Kevin A; Egusa, Hiroshi; Osathanon, Thanaphum

2017-08-31

Notch signaling regulates diverse biological processes in dental pulp tissue. The present study investigated the response of human dental pulp cells (hDPs) to the indirect immobilized Notch ligand Jagged1 in vitro. The indirect immobilized Jagged1 effectively activated Notch signaling in hDPs as confirmed by the upregulation of HES1 and HEY1 expression. Differential gene expression profiling using an RNA sequencing technique revealed that the indirect immobilized Jagged1 upregulated genes were mainly involved in extracellular matrix organization, disease, and signal transduction. Downregulated genes predominantly participated in the cell cycle, DNA replication, and DNA repair. Indirect immobilized Jagged1 significantly reduced cell proliferation, colony forming unit ability, and the number of cells in S phase. Jagged1 treated hDPs exhibited significantly higher ALP enzymatic activity, osteogenic marker gene expression, and mineralization compared with control. Pretreatment with a γ-secretase inhibitor attenuated the Jagged1-induced ALP activity and mineral deposition. NOTCH2 shRNA reduced the Jagged1-induced osteogenic marker gene expression, ALP enzymatic activity, and mineral deposition. In conclusion, indirect immobilized Jagged1 suppresses cell cycle progression and induces the odonto/osteogenic differentiation of hDPs via the canonical Notch signaling pathway.

Evidence that β7 Integrin Regulates Hematopoietic Stem Cell Homing and Engraftment Through Interaction with MAdCAM-1.

PubMed

Murakami, Jodi L; Xu, Baohui; Franco, Christopher B; Hu, Xingbin; Galli, Stephen J; Weissman, Irving L; Chen, Ching-Cheng

2016-01-01

α4β7 integrin is a cell adhesion receptor that is crucial for the migration of hematopoietic progenitors and mature effector cells in the periphery, but its role in adult hematopoiesis is controversial. We identified a subset of hematopoietic stem cells (HSCs) in the bone marrow (BM) that expressed β7 integrin. These β7(+) HSCs were capable of multilineage, long-term reconstitution and had an inherent competitive advantage over β7(-) HSCs. On the other hand, HSCs that lacked β7 integrin (β7KO) had reduced engraftment potential. Interestingly, quantitative RT-PCR and flow cytometry revealed that β7KO HSCs expressed lower levels of the chemokine receptor CXCR4. Accordingly, β7KO HSCs exhibited impaired migration abilities in vitro and BM homing capabilities in vivo. Lethal irradiation induced expression of the α4β7 integrin ligand-mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on BM endothelial cells. Moreover, blocking MAdCAM-1 reduced the homing of HSCs and impaired the survival of recipient mice. Altogether, these data indicate that β7 integrin, when expressed by HSCs, interacted with its endothelial ligand MAdCAM-1 in the BM microenvironment, thereby promoting HSC homing and engraftment.

Regulation of tissue factor and inflammatory mediators by Egr-1 in a mouse endotoxemia model.

PubMed

Pawlinski, Rafal; Pedersen, Brian; Kehrle, Bettina; Aird, William C; Frank, Rolf D; Guha, Mausumee; Mackman, Nigel

2003-05-15

In septic shock, tissue factor (TF) activates blood coagulation, and cytokines and chemokines orchestrate an inflammatory response. In this study, the role of Egr-1 in lipopolysaccharide (LPS) induction of TF and inflammatory mediators in vivo was evaluated using Egr-1(+/+) and Egr-1(-/-) mice. Administration of LPS transiently increased the steady-state levels of Egr-1 mRNA in the kidneys and lungs of Egr-1(+/+) mice with maximal induction at one hour. Egr-1 was expressed in epithelial cells in the kidneys and lungs in untreated and LPS-treated mice. LPS induction of monocyte chemoattractant protein mRNA in the kidneys and lungs of Egr-1(-/-) mice was not affected at 3 hours, but its expression was significantly reduced at 8 hours compared with the expression observed in Egr-1(+/+) mice. Similarly, LPS induction of TF mRNA expression in the kidneys and lungs at 8 hours was reduced in Egr-1(-/-) mice. However, Egr-1 deficiency did not affect plasma levels of tumor necrosis factor alpha in endotoxemic mice. Moreover, Egr-1(+/+) and Egr-1(-/-) mice exhibited similar survival times in a model of acute endotoxemia. These data indicate that Egr-1 does not contribute to the early inflammatory response in the kidneys and lungs or the early systemic inflammatory response in endotoxemic mice. However, Egr-1 does contribute to the sustained expression of inflammatory mediators and to the maximal expression of TF at 8 hours in the kidneys and lungs.

Signaling by Bone Morphogenetic Proteins directs formation of an ectodermal signaling center that regulates craniofacial development

PubMed Central

Foppiano, Silvia; Hu, Diane; Marcucio, Ralph S.

2008-01-01

We previously described a signaling center, the Frontonasal Ectodermal Zone (FEZ) that regulates growth and patterning of the frontonasal process (FNP). The FEZ is comprised of FNP ectoderm flanking a boundary between Sonic hedgehog (Shh) and Fibroblast growth factor 8 (Fgf8) expression domains. Our objective was to examine BMP signaling during formation of the FEZ. We blocked BMP signaling throughout the FNP prior to FEZ formation by infecting chick embryos at stage 10 (HH10) with a replication competent avian retrovirus encoding the BMP antagonist Noggin. We assessed gene expression patterns in the FNP 72 hours after infection (~HH22) and observed that Shh expression was reduced or absent. In the mesenchyme we observed that Bmp2 transcripts were absent while the Bmp4 expression domain was expanded proximally. In addition to the molecular changes, infected embryos also exhibited facial malformations at 72 and 96 hours after infection suggesting that the FEZ did not form. Our data indicate that reduced cell proliferation, but not apoptosis, in the mesenchyme contributed to the phenotype that we observed. Additionally, adding exogenous SHH into the mesenchyme of RCAS-Noggin infected embryos did not restore Bmp2 and Bmp4 to a normal pattern of expression. These data indicate that BMP signaling mediates interactions between tissues in the FNP that regulate FEZ formation; and that the correct pattern of Bmp2 and Bmp4, but not Bmp7, expression in the FNP mesenchyme requires signaling by the BMP pathway. PMID:18028903

Effects of an inducible aiiA gene on disease resistance in Eucalyptus urophylla × Eucalyptus grandis.

PubMed

Ouyang, L J; Li, L M

2016-08-01

N-acyl-homoserine lactones (AHLs) are metabolites of mostly gram-negative bacteria and are critical signaling molecules in bacterial quorum-sensing systems. At threshold concentrations, AHLs can activate the expression of pathogenic genes and induce diseases. Therefore, reducing AHL concentrations is a key point of disease control in plants. AHL-lactonase, which is expressed by aiiA, is widespread in Bacillus sp and can hydrolyze AHLs. In the present study, we cloned aiiA from Bacillus subtilis by PCR. A plant expression vector of aiiA was constructed and name Pcam-PPP3-aiiA, in which expression of aiiA was controlled by the pathogen-inducible plant promoter PPP3. The recombinant plasmid was transferred into Eucalyptus × urophylla × E. grandis by an Agrobacterium-mediated transformation. PCR and Southern blotting showed that aiiA was successfully integrated into the E. urophylla × E. grandis genome and its expression was induced by Ralstonia solanacearum 12 h after inoculation, as shown by reverse transcription-PCR. The transcription efficacy of aiiA increased 43.88-, 30.65-, and 18.95-fold after inoculation with R. solanacearum, Erwinia carotovora ssp. zeae (Sabet) and Cylindrocladium quinqueseptatum, respectively as shown by RT-real-time PCR. Transgenic E.urophylla × E.grandis expressing the AIIA protein exhibited significantly enhanced disease resistance compared to non-transgenic plants by delaying the onset of wilting and reducing the disease index.

Gene expression alterations associated with outcome in aromatase inhibitor-treated ER+ early-stage breast cancer patients.

PubMed

Thomsen, Karina G; Lyng, Maria B; Elias, Daniel; Vever, Henriette; Knoop, Ann S; Lykkesfeldt, Anne E; Lænkholm, Anne-Vibeke; Ditzel, Henrik J

2015-12-01

Aromatase inhibitors (AI), either alone or together with chemotherapy, have become the standard adjuvant treatment for postmenopausal, estrogen receptor-positive (ER+) breast cancer. Although AIs improve overall survival, resistance is still a major clinical problem, thus additional biomarkers predictive of outcome of ER+ breast cancer patients treated with AIs are needed. Global gene expression analysis was performed on ER+ primary breast cancers from patients treated with adjuvant AI monotherapy; half experienced recurrence (median follow-up 6.7 years). Gene expression alterations were validated by qRT-PCR, and functional studies evaluating the effect of siRNA-mediated gene knockdown on cell growth were performed. Twenty-six genes, including TFF3, DACH1, RGS5, and GHR, were shown to exhibit altered expression in tumors from patients with recurrence versus non-recurrent (fold change ≥1.5, p < 0.05), and the gene expression alterations were confirmed using qRT-PCR. Ten of these 26 genes could be linked in a network associated with cellular proliferation, growth, and development. TFF3, which encodes for trefoil factor 3 and is an estrogen-responsive oncogene shown to play a functional role in tamoxifen resistance and metastasis of ER+ breast cancer, was also shown to be upregulated in an AI-resistant cell line model, and reduction of TFF3 levels using TFF3-specific siRNAs decreased the growth of both the AI-resistant and -sensitive parental cell lines. Moreover, overexpression of TFF3 in parental AI-sensitive MCF-7/S0.5 cells resulted in reduced sensitivity to the AI exemestane, whereas TFF3 overexpression had no effect on growth in the absence of exemestane, indicating that TFF3 mediates growth and survival signals that abrogate the growth inhibitory effect of exemestane. We identified a panel of 26 genes exhibiting altered expression associated with disease recurrence in patients treated with adjuvant AI monotherapy, including TFF3, which was shown to exhibit a growth- and survival-promoting effect in the context of AI treatment.

Carboplatin resistant human laryngeal carcinoma cells are cross resistant to curcumin due to reduced curcumin accumulation.

PubMed

Rak, Sanjica; Cimbora-Zovko, Tamara; Gajski, Goran; Dubravčić, Klara; Domijan, Ana-Marija; Delaš, Ivančica; Garaj-Vrhovac, Verica; Batinić, Drago; Sorić, Jasna; Osmak, Maja

2013-03-01

Curcumin is a natural compound that exhibits a wide range of beneficial effects, among them the anti-tumor activity. Recently it was shown that curcumin may be efficient against drug resistant tumor cells. The goal of our investigation was to examine if human laryngeal carcinoma cells resistant to carboplatin display sensitivity to curcumin, as compared to parental cells, and if this sensitivity is altered, to determine the molecular mechanisms that are responsible for it. We found that carboplatin resistant 7T cells were also cross resistant to curcumin. After the treatment with equimolar concentration of curcumin, 7T cells exhibited lower intracellular accumulation of curcumin which coincided with reduced formation of reactive oxygen species (ROS), diminished lipid and DNA damage followed by reduced induction of apoptosis and expression of heat shock protein 70 (Hsp70), as compared to parental HEp-2 cells. However, after the treatment with equitoxic concentration of curcumin, intracellular accumulation and all the explored downstream effects were similar in both cell lines suggesting that resistance of 7T cells to curcumin was based on its reduced intracellular accumulation. Since curcumin accumulates mostly in the membranes, we explored the fatty acid composition of both cell lines, but we did not find any difference between them. Copyright © 2012 Elsevier Ltd. All rights reserved.

Nicotinic Receptor Alpha7 Expression during Tooth Morphogenesis Reveals Functional Pleiotropy

PubMed Central

Rogers, Scott W.; Gahring, Lorise C.

2012-01-01

The expression of nicotinic acetylcholine receptor (nAChR) subtype, alpha7, was investigated in the developing teeth of mice that were modified through homologous recombination to express a bi-cistronic IRES-driven tau-enhanced green fluorescent protein (GFP); alpha7GFP) or IRES-Cre (alpha7Cre). The expression of alpha7GFP was detected first in cells of the condensing mesenchyme at embryonic (E) day E13.5 where it intensifies through E14.5. This expression ends abruptly at E15.5, but was again observed in ameloblasts of incisors at E16.5 or molar ameloblasts by E17.5–E18.5. This expression remains detectable until molar enamel deposition is completed or throughout life as in the constantly erupting mouse incisors. The expression of alpha7GFP also identifies all stages of innervation of the tooth organ. Ablation of the alpha7-cell lineage using a conditional alpha7Cre×ROSA26-LoxP(diphtheria toxin A) strategy substantially reduced the mesenchyme and this corresponded with excessive epithelium overgrowth consistent with an instructive role by these cells during ectoderm patterning. However, alpha7knock-out (KO) mice exhibited normal tooth size and shape indicating that under normal conditions alpha7 expression is dispensable to this process. The function of ameloblasts in alpha7KO mice is altered relative to controls. High resolution micro-computed tomography analysis of adult mandibular incisors revealed enamel volume of the alpha7KO was significantly reduced and the organization of enamel rods was altered relative to controls. These results demonstrate distinct and varied spatiotemporal expression of alpha7 during tooth development, and they suggest that dysfunction of this receptor would have diverse impacts upon the adult organ. PMID:22666322

Systemic Sclerosis Patients Present Alterations in the Expression of Molecules Involved in B-Cell Regulation

PubMed Central

Soto, Lilian; Ferrier, Ashley; Aravena, Octavio; Fonseca, Elianet; Berendsen, Jorge; Biere, Andrea; Bueno, Daniel; Ramos, Verónica; Aguillón, Juan Carlos; Catalán, Diego

2015-01-01

The activation threshold of B cells is tightly regulated by an array of inhibitory and activator receptors in such a way that disturbances in their expression can lead to the appearance of autoimmunity. The aim of this study was to evaluate the expression of activating and inhibitory molecules involved in the modulation of B cell functions in transitional, naive, and memory B-cell subpopulations from systemic sclerosis patients. To achieve this, blood samples were drawn from 31 systemic sclerosis patients and 53 healthy individuals. Surface expression of CD86, MHC II, CD19, CD21, CD40, CD22, Siglec 10, CD35, and FcγRIIB was determined by flow cytometry. IL-10 production was evaluated by intracellular flow cytometry from isolated B cells. Soluble IL-6 and IL-10 levels were measured by ELISA from supernatants of stimulated B cells. Systemic sclerosis patients exhibit an increased frequency of transitional and naive B cells related to memory B cells compared with healthy controls. Transitional and naive B cells from patients express higher levels of CD86 and FcγRIIB than healthy donors. Also, B cells from patients show high expression of CD19 and CD40, whereas memory cells from systemic sclerosis patients show reduced expression of CD35. CD19 and CD35 expression levels associate with different autoantibody profiles. IL-10+ B cells and secreted levels of IL-10 were markedly reduced in patients. In conclusion, systemic sclerosis patients show alterations in the expression of molecules involved in B-cell regulation. These abnormalities may be determinant in the B-cell hyperactivation observed in systemic sclerosis. PMID:26483788

Hyperphenylalaninemia due to defects in tetrahydrobiopterin metabolism: Molecular characterization of mutations in 6-pyruvoyl-tetrahydropterin synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thoeny, B.; Leimbacher, W.; Blau, N.

1994-05-01

A variant type of hyperphenylalaninemia is caused by a deficiency of tetrahydrobiopterin (BH[sub 4]), the obligatory cofactor for phenylalanine hydroxylase. The most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (PTPS) activity, the second enzyme in the biosynthetic pathway for BH[sub 4]. The human liver cDNA for PTPS was previously isolated, and the recombinant protein was found to be active when expressed in Escherichia coli. The authors now have investigated two patients for their molecular nature of this autosomal recessive disorder. Both patients were diagnosed as PTPS deficient, one with the central and one withmore » the peripheral form, on the basis of an elevated serum phenylalanine concentration concomitant with lowered levels of urinary biopterin and PTPS activity in erythrocytes. Molecular analysis was performed on the patients' cultured primary skin fibroblasts. PTPS activities were found in vitro to be reduced to background activity. Direct cDNA sequence analysis using reverse transcriptase-PCR technology showed for the patient with the central form a homozygous G-to-A transition at codon 25, causing the replacement of an arginine by glutamine (R25Q). Expression of this mutant allele in E.coli revealed 14% activity when compared with the wild-type enzyme. The patient with the peripheral form exhibited compound heteroxygosity, having on one allele a C-to-T transition resulting in the substitution of arginine 16 for cysteine (R16C) in the enzyme and having on the second allele a 14-bp deletion ([Delta]14bp), leading to a frameshift at lysine 120 and a premature stop codon (K120[yields]Stop). Heterologous expression of the enzyme with the single-amino-acid exchange R16C revealed only 7% enzyme activity, whereas expression of the deletion allele [Delta]14bp exhibited no detectable activity. All three mutations result in reduced enzymatic activity when reconstituted in E. coli.« less

Preclinical evaluation of dasatinib alone and in combination with cabozantinib for the treatment of diffuse intrinsic pontine glioma

PubMed Central

Philippe, Cathy; Paulsson, Janna; Andreiuolo, Felipe; Guerrini-Rousseau, Léa; Cornilleau, Gaétan; Le Dret, Ludivine; Richon, Catherine; Lacroix, Ludovic; Puget, Stéphanie; Geoerger, Birgit; Vassal, Gilles; Östman, Arne; Grill, Jacques

2015-01-01

Abstract Background Platelet-derived growth factor receptor A is altered by amplification and/or mutation in diffuse intrinsic pontine glioma (DIPG). We explored in vitro on new DIPG models the efficacy of dasatinib, a multi-tyrosine kinase inhibitor targeting this receptor. Methods Gene expression profiles were generated from 41 DIPGs biopsied at diagnosis and compared with the signature associated with sensitivity/resistance to dasatinib. A panel of 12 new DIPG cell lines were established from biopsy at diagnosis, serially passaged, and characterized by gene expression analyses. Effects of dasatinib (1–10 μM) on proliferation, invasion, and cytotoxicity were determined on 4 of these cell lines using live-cell imaging and flow cytometry assays. Downstream signaling and receptor tyrosine kinases (RTKs) were assessed by western blot and phospho-RTK array. The effect of the combination with the c-Met inhibitor cabozantinib was studied on cellular growth and invasion analyzed by the Chou–Talaly method. Results DIPG primary tumors and cell lines exhibited the gene expression signature of sensitivity to dasatinib. Dasatinib reduced proliferation (half-maximal inhibitory concentration = 10–100 nM) and invasion (30%–60% reduction) at 100 nM in 4/4 cultures and induced apoptosis in 1 of 4 DIPG cell lines. Activity of downstream effectors of dasatinib targets including activin receptor 1 was strongly reduced. Since multiple RTKs were activated simultaneously in DIPG cell lines, including c-Met, which can be also amplified in DIPG, the benefit of the combination of dasatinib with cabozantinib was explored for its synergistic effects on proliferation and migration/invasion in these cell lines. Conclusion Dasatinib exhibits antitumor effects in vitro that could be increased by the combination with another RTK inhibitor targeting c-Met. PMID:25534822

The Arabidopsis vacuolar malate channel is a member of the ALMT family.

PubMed

Kovermann, Peter; Meyer, Stefan; Hörtensteiner, Stefan; Picco, Cristiana; Scholz-Starke, Joachim; Ravera, Silvia; Lee, Youngsook; Martinoia, Enrico

2007-12-01

In plants, malate is a central metabolite and fulfills a large number of functions. Vacuolar malate may reach very high concentrations and fluctuate rapidly, whereas cytosolic malate is kept at a constant level allowing optimal metabolism. Recently, a vacuolar malate transporter (Arabidopsis thaliana tonoplast dicarboxylate transporter, AttDT) was identified that did not correspond to the well-characterized vacuolar malate channel. We therefore hypothesized that a member of the aluminum-activated malate transporter (ALMT) gene family could code for a vacuolar malate channel. Using GFP fusion constructs, we could show that AtALMT9 (A. thaliana ALMT9) is targeted to the vacuole. Promoter-GUS fusion constructs demonstrated that this gene is expressed in all organs, but is cell-type specific as GUS activity in leaves was detected nearly exclusively in mesophyll cells. Patch-clamp analysis of an Atalmt9 T-DNA insertion mutant exhibited strongly reduced vacuolar malate channel activity. In order to functionally characterize AtALMT9 as a malate channel, we heterologously expressed this gene in tobacco and in oocytes. Overexpression of AtALMT9-GFP in Nicotiana benthamiana leaves strongly enhanced the malate current densities across the mesophyll tonoplasts. Functional expression of AtALMT9 in Xenopus oocytes induced anion currents, which were clearly distinguishable from endogenous oocyte currents. Our results demonstrate that AtALMT9 is a vacuolar malate channel. Deletion mutants for AtALMT9 exhibit only slightly reduced malate content in mesophyll protoplasts and no visible phenotype, indicating that AttDT and the residual malate channel activity are sufficient to sustain the transport activity necessary to regulate the cytosolic malate homeostasis.

Dysregulation of hepatic fatty acid metabolism in chronic kidney disease.

PubMed

Jin, Kyubok; Norris, Keith; Vaziri, Nosratola D

2013-02-01

Chronic kidney disease (CKD) results in hypertriglyceridemia which is largely due to impaired clearance of triglyceride-rich lipoproteins occasioned by downregulation of lipoprotein lipase and very low-density lipoprotein (LDL) receptor in the skeletal muscle and adipose tissue and of hepatic lipase and LDL receptor-related protein in the liver. However, data on the effect of CKD on fatty acid metabolism in the liver is limited and was investigated here. Male Sprague-Dawley rats were randomized to undergo 5/6 nephrectomy (CRF) or sham operation (control) and observed for 12 weeks. The animals were then euthanized and their liver tissue tested for nuclear translocation (activation) of carbohydrate-responsive element binding protein (ChREBP) and sterol-responsive element binding protein-1 (SREBP-1) which independently regulate the expression of key enzyme in fatty acid synthesis, i.e. fatty acid synthase (FAS) and acyl-CoA carboxylase (ACC) as well as nuclear Peroxisome proliferator-activated receptor alpha (PPARα) which regulates the expression of enzymes involved in fatty acid oxidation and transport, i.e. L-FABP and CPT1A. In addition, the expression of ATP synthase α, ATP synthase β, glycogen synthase and diglyceride acyltransferase 1 (DGAT1) and DGAT2 were determined. Compared with controls, the CKD rats exhibited hypertriglyceridemia, elevated plasma and liver tissue free fatty acids, increased nuclear ChREBP and reduced nuclear SREBP-1 and PPARα, upregulation of ACC and FAS and downregulation of L-FABP, CPT1A, ATP synthase α, glycogen synthase and DGAT in the liver tissue. Liver in animals with advanced CKD exhibits ChREBP-mediated upregulation of enzymes involved in fatty acid synthesis, downregulation of PPARα-regulated fatty acid oxidation system and reduction of DGAT resulting in reduced fatty acid incorporation in triglyceride.

6-Shogaol ameliorates diabetic nephropathy through anti-inflammatory, hyperlipidemic, anti-oxidative activity in db/db mice.

PubMed

Xu, Yun; Bai, Liwei; Chen, Xuehui; Li, Yan; Qin, Yan; Meng, Xiangyu; Zhang, Qinggui

2018-01-01

The prevalence of type 2 diabetes mellitus has been increasing worldwide and more than two thirds of the patients may develop diabetic nephropathy (DN). However, the efficiency of existing approaches on DN progression is limited. 6-Shogaol (6-SG), a major dehydrated derivative of gingerols, possesses various biological properties. The present study was designed to evaluate the possible effects of 6-SG on DN in db/db mice and to investigate the mechanisms. We revealed that 6-SG reduced the levels of fasting blood glucose, serum insulin, C-peptide, glycosylated hemoglobin A1c, and systolic blood pressure. 6-SG decreased the levels of blood urea nitrogen (BUN), serum creatinine, urinary albumin content and albumin/creatinine ratio (ACR), ameliorated the pathological injuries of kidneys, reduced the surface area of Bowman's capsule, Bowman's space, glomerular tuft, and decreased the expression of collagen IV and fibronectin in kidneys of db/db mice. The high levels of systemic and renal triglyceride and cholesterol were decreased by 6-SG. Moreover, 6-SG exhibited anti-inflammatory effects, as reflected by reduction of tumor necrosis factor ɑ (TNFɑ), monocyte chemotactic protein-1 (MCP-1), and IL-6 levels in circulation and kidneys, and decrease of NF-κB expression. Furthermore, 6-SG also inhibited oxidative stress and restored the expression of NF-E2-related factor 2 (Nrf2) in kidneys of db/db mice. In conclusion, we have demonstrated that 6-SG exhibits anti-diabetic and renal protective effects against DN, in which effect the anti-inflammatory, hyperlipidemic, anti-oxidative activities may be involved. Overall, 6-SG could be a promising therapeutic treatment to ameliorate diabetes and the development of DN. Copyright © 2017. Published by Elsevier Masson SAS.

Simvastatin Inhibits IL-5-Induced Chemotaxis and CCR3 Expression of HL-60-Derived and Human Primary Eosinophils.

PubMed

Fu, Chia-Hsiang; Tsai, Wan-Chun; Lee, Ta-Jen; Huang, Chi-Che; Chang, Po-Hung; Su Pang, Jong-Hwei

2016-01-01

IL-5-induced chemotaxis of eosinophils is an important feature of allergic airway inflammatory diseases. Simvastatin, a lipid lowering agent, has been shown to exhibit anti-inflammatory and anti-allergic effects. Our aim was to investigate the effect of simvastatin on IL-5-induced eosinophil chemotaxis and its regulatory mechanisms. Eosinophils were derived by treating HL-60 clone 15 (HC15) cells with butyric acid (BA) in an alkaline condition or through direct isolation from human peripheral blood. The expressions of CC chemokine receptor 3 (CCR3) and interleukin (IL)-5 receptors (IL5Rα and β) were analyzed using RT/real-time PCR. The granular proteins were stained using fast green. Eotaxin-induced chemotaxis was measured using a transwell migration assay. CCR3 protein expression was revealed by immunocytochemistry. An animal model of allergic rhinitis was established by challenging Sprague-Dawley® rats repeatedly with ovalbumin. Butyric acid significantly increased the expression of IL5Rα and IL5Rβ, CCR3 and granular proteins in HC15 cells, indicating the maturation of eosinophils (BA-E cells). IL-5 further enhanced the CCR3 expression at both the mRNA and protein levels and the eotaxin-induced chemotaxis of BA-E cells. Simvastatin inhibited the effects of IL-5 on BA-E cells, but not in the presence of mevalonate. Similar results were also exhibited in human primary eosinophils. In vivo animal studies further confirmed that oral simvastatin could significantly suppress the infiltration of eosinophils into turbinate tissues of allergic rats. Therefore, simvastatin was demonstrated to inhibit IL-5-induced CCR3 expression and chemotaxis of eosinophils mediated via the mevalonate pathway. We confirmed that simvastatin also reduced eosinophilic infiltration in allergic rhinitis.

Enhanced drought and heat stress tolerance of tobacco plants with ectopically enhanced cytokinin oxidase/dehydrogenase gene expression

PubMed Central

Macková, Hana; Hronková, Marie; Dobrá, Jana; Turečková, Veronika; Novák, Ondřej; Lubovská, Zuzana; Motyka, Václav; Haisel, Daniel; Hájek, Tomáš; Prášil, Ilja Tom; Gaudinová, Alena; Štorchová, Helena; Ge, Eva; Werner, Tomáš; Schmülling, Thomas; Vanková, Radomíra

2013-01-01

Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms. PMID:23669573

Reduction of methylviologen-mediated oxidative stress tolerance in antisense transgenic tobacco seedlings through restricted expression of StAPX.

PubMed

Sun, Wei-Hong; Wang, Yong; He, Hua-Gang; Li, Xue; Song, Wan; Du, Bin; Meng, Qing-Wei

2013-07-01

Ascorbate peroxidases are directly involved in reactive oxygen species (ROS) scavenging by reducing hydrogen peroxide to water. The tomato thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco. RNA gel blot analysis confirmed that StAPX in tomato leaves was induced by methylviologen-mediated oxidative stress. The sense transgenic seedlings exhibited higher tAPX activity than that of the wild type (WT) plants under oxidative stress conditions, while the antisense seedlings exhibited lower tAPX activity. Lower APX activities of antisense transgenic seedlings caused higher malondialdehyde contents and relative electrical conductivity. The sense transgenic seedlings with higher tAPX activity maintained higher chlorophyll content and showed the importance of tAPX in maintaining the optimal chloroplast development under methylviologen stress conditions, whereas the antisense lines maintained lower chlorophyll content than WT seedlings. Results indicated that the over-expression of StAPX enhanced tolerance to methylviologen-mediated oxidative stress in sense transgenic tobacco early seedlings, whereas the suppression of StAPX in antisense transgenic seedlings showed high sensitivity to oxidative stress.

Docosahexaenoic acid inhibits the growth of hormone-dependent prostate cancer cells by promoting the degradation of the androgen receptor.

PubMed

Hu, Zhimei; Qi, Haixia; Zhang, Ruixue; Zhang, Kun; Shi, Zhemin; Chang, Yanan; Chen, Linfeng; Esmaeili, Mohsen; Baniahmad, Aria; Hong, Wei

2015-09-01

Epidemiological and preclinical data have demonstrated the preventative effects of ω-3 polyunsaturated fatty acids, including docosahexaenoic acid (DHA), on prostate cancer. However, there are inconsistencies in these previous studies and the underlying mechanisms remain to be elucidated. In the present study, the androgen receptor (AR), which is a transcription factor involved in cell proliferation and prostate carcinogenesis, was identified as a target of DHA. It was revealed that DHA inhibited hormone‑dependent growth of LNCaP prostate cancer cells. Reverse transcription-quantitative polymerase chain reaction analysis revealed that treatment with DHA caused no alteration in the transcribed mRNA expression levels of the AR gene. However, immunoblotting revealed that this treatment reduces the protein expression level of the AR. The androgen‑induced genes were subsequently repressed by treatment with DHA. It was demonstrated that DHA exhibits no effect on the translation process of the AR, however, it promotes the proteasome‑mediated degradation of the AR. Therefore, the present study provided a novel mechanism by which DHA exhibits an inhibitory effect on growth of prostate cancer cells.

Perturbed desmosomal cadherin expression in grainy head-like 1-null mice.

PubMed

Wilanowski, Tomasz; Caddy, Jacinta; Ting, Stephen B; Hislop, Nikki R; Cerruti, Loretta; Auden, Alana; Zhao, Lin-Lin; Asquith, Stephen; Ellis, Sarah; Sinclair, Rodney; Cunningham, John M; Jane, Stephen M

2008-03-19

In Drosophila, the grainy head (grh) gene plays a range of key developmental roles through the regulation of members of the cadherin gene family. We now report that mice lacking the grh homologue grainy head-like 1 (Grhl1) exhibit hair and skin phenotypes consistent with a reduction in expression of the genes encoding the desmosomal cadherin, desmoglein 1 (Dsg1). Grhl1-null mice show an initial delay in coat growth, and older mice exhibit hair loss as a result of poor anchoring of the hair shaft in the follicle. The mice also develop palmoplantar keratoderma, analogous to humans with DSG1 mutations. Sequence analysis, DNA binding, and chromatin immunoprecipitation experiments demonstrate that the human and mouse Dsg1 promoters are direct targets of GRHL1. Ultrastructural analysis reveals reduced numbers of abnormal desmosomes in the interfollicular epidermis. These findings establish GRHL1 as an important regulator of the Dsg1 genes in the context of hair anchorage and epidermal differentiation, and suggest that cadherin family genes are key targets of the grainy head-like genes across 700 million years of evolution.

Perturbed desmosomal cadherin expression in grainy head-like 1-null mice

PubMed Central

Wilanowski, Tomasz; Caddy, Jacinta; Ting, Stephen B; Hislop, Nikki R; Cerruti, Loretta; Auden, Alana; Zhao, Lin-Lin; Asquith, Stephen; Ellis, Sarah; Sinclair, Rodney; Cunningham, John M; Jane, Stephen M

2008-01-01

In Drosophila, the grainy head (grh) gene plays a range of key developmental roles through the regulation of members of the cadherin gene family. We now report that mice lacking the grh homologue grainy head-like 1 (Grhl1) exhibit hair and skin phenotypes consistent with a reduction in expression of the genes encoding the desmosomal cadherin, desmoglein 1 (Dsg1). Grhl1-null mice show an initial delay in coat growth, and older mice exhibit hair loss as a result of poor anchoring of the hair shaft in the follicle. The mice also develop palmoplantar keratoderma, analogous to humans with DSG1 mutations. Sequence analysis, DNA binding, and chromatin immunoprecipitation experiments demonstrate that the human and mouse Dsg1 promoters are direct targets of GRHL1. Ultrastructural analysis reveals reduced numbers of abnormal desmosomes in the interfollicular epidermis. These findings establish GRHL1 as an important regulator of the Dsg1 genes in the context of hair anchorage and epidermal differentiation, and suggest that cadherin family genes are key targets of the grainy head-like genes across 700 million years of evolution. PMID:18288204

Orexin Regulates Bone Remodeling via a Dominant Positive Central Action and a Subordinate Negative Peripheral Action

PubMed Central

Wei, Wei; Motoike, Toshiyuki; Krzeszinski, Jing Y.; Jin, Zixue; Xie, Xian-Jin; Dechow, Paul C.; Yanagisawa, Masashi; Wan, Yihong

2014-01-01

SUMMARY Orexin neuropeptides promote arousal, appetite, reward, and energy expenditure. However, whether orexin affects bone mass accrual is unknown. Here we show that orexin functions centrally through orexin receptor 2 (OX2R) in the brain to enhance bone formation. OX2R-null mice exhibit low-bone-mass owing to elevated circulating leptin; whereas central administration of an OX2R-selective agonist augments bone mass. Conversely, orexin also functions peripherally through orexin receptor 1 (OX1R) in the bone to suppress bone formation. OX1R-null mice exhibit high-bone-mass owing to a mesenchymal stem cell differentiation shift from adipocyte to osteoblast that results from higher osseous ghrelin expression. The central action is dominant over the peripheral action because bone mass is reduced in orexin-null and OX1R2R-double-null mice but enhanced in orexin over-expressing transgenic mice. These findings reveal orexin as a critical rheostat of skeletal homeostasis that exerts a yin-yang dual regulation, and highlight orexin as a therapeutic target for osteoporosis. PMID:24794976

Weak vaccinia virus-induced NK cell regulation of CD4 T cells is associated with reduced NK cell differentiation and cytolytic activity.

PubMed

Hatfield, Steven D; Daniels, Keith A; O'Donnell, Carey L; Waggoner, Stephen N; Welsh, Raymond M

2018-06-01

Natural killer (NK) cells control antiviral adaptive immune responses in mice during some virus infections, but the universality of this phenomenon remains unknown. Lymphocytic choriomeningitis virus (LCMV) infection of mice triggered potent cytotoxic activity of NK cells (NK LCMV ) against activated CD4 T cells, tumor cells, and allogeneic lymphocytes. In contrast, NK cells activated by vaccinia virus (VACV) infection (NK VACV ) exhibited weaker cytolytic activity against each of these target cells. Relative to NK LCMV cells, NK VACV cells exhibited a more immature (CD11b - CD27 + ) phenotype, and lower expression levels of the activation marker CD69, cytotoxic effector molecules (perforin, granzyme B), and the transcription factor IRF4. NK VACV cells expressed higher levels of the inhibitory molecule NKG2A than NK LCMV cells. Consistent with this apparent lethargy, NK VACV cells only weakly constrained VACV-specific CD4 T-cell responses. This suggests that NK cell regulation of adaptive immunity, while universal, may be limited with viruses that poorly activate NK cells. Published by Elsevier Inc.

Sulfite Reductase Protects Plants against Sulfite Toxicity1[W][OA

PubMed Central

Yarmolinsky, Dmitry; Brychkova, Galina; Fluhr, Robert; Sagi, Moshe

2013-01-01

Plant sulfite reductase (SiR; Enzyme Commission 1.8.7.1) catalyzes the reduction of sulfite to sulfide in the reductive sulfate assimilation pathway. Comparison of SiR expression in tomato (Solanum lycopersicum ‘Rheinlands Ruhm’) and Arabidopsis (Arabidopsis thaliana) plants revealed that SiR is expressed in a different tissue-dependent manner that likely reflects dissimilarity in sulfur metabolism between the plant species. Using Arabidopsis and tomato SiR mutants with modified SiR expression, we show here that resistance to ectopically applied sulfur dioxide/sulfite is a function of SiR expression levels and that plants with reduced SiR expression exhibit higher sensitivity than the wild type, as manifested in pronounced leaf necrosis and chlorophyll bleaching. The sulfite-sensitive mutants accumulate applied sulfite and show a decline in glutathione levels. In contrast, mutants that overexpress SiR are more tolerant to sulfite toxicity, exhibiting little or no damage. Resistance to high sulfite application is manifested by fast sulfite disappearance and an increase in glutathione levels. The notion that SiR plays a role in the protection of plants against sulfite is supported by the rapid up-regulation of SiR transcript and activity within 30 min of sulfite injection into Arabidopsis and tomato leaves. Peroxisomal sulfite oxidase transcripts and activity levels are likewise promoted by sulfite application as compared with water injection controls. These results indicate that, in addition to participating in the sulfate assimilation reductive pathway, SiR also plays a role in protecting leaves against the toxicity of sulfite accumulation. PMID:23221833

Paternal epigenetic effects of population density on locust phase-related characteristics associated with heat-shock protein expression.

PubMed

Chen, Bing; Li, Shaoqin; Ren, Qiang; Tong, Xiwen; Zhang, Xia; Kang, Le

2015-02-01

Many species exhibit transgenerational plasticity by which environmental cues experienced by either parent can be transmitted to their offspring, resulting in phenotypic variants in offspring to match ancestral environments. However, the manner by which paternal experiences affect offspring plasticity through epigenetic inheritance in animals generally remains unclear. In this study, we examined the transgenerational effects of population density on phase-related traits in the migratory locust Locusta migratoria. Using an experimental design that explicitly controls genetic background, we found that the effects of crowd or isolation rearing on phase plasticity could be inherited to the offspring. The isolation of gregarious locusts resulted in reduced weight in offspring eggs and altered morphometric traits in hatchlings, whereas crowding of solitarious locusts exhibited opposite effects. The consequences of density changes were transmitted by both maternal and paternal inheritance, although the expression of paternal effects was not as pronounced as that of maternal effects. Prominent expression of heat-shock proteins (Hsps), such as Hsp90, Hsp70 and Hsp20.6, could be triggered by density changes. Hsps were significantly upregulated upon crowding but downregulated upon isolation. The variation in parental Hsp expression was also transmitted to the offspring, in which the pattern of inheritance was consistent with that of phase characteristics. These results revealed a paternal effect on phase polyphenism and Hsp expression induced by population density, and defined a model system that could be used to study the paternal epigenetic inheritance of environmental changes. © 2015 John Wiley & Sons Ltd.

Over-Expression of Arabidopsis EDT1 Gene Confers Drought Tolerance in Alfalfa (Medicago sativa L.)

PubMed Central

Zheng, Guangshun; Fan, Cunying; Di, Shaokang; Wang, Xuemin; Xiang, Chengbin; Pang, Yongzhen

2017-01-01

Alfalfa (Medicago sativa L.) is an important legume forage crop with great economic value. However, as the growth of alfalfa is seriously affected by an inadequate supply of water, drought is probably the major abiotic environmental factor that most severely affects alfalfa production worldwide. In an effort to enhance alfalfa drought tolerance, we transformed the Arabidopsis Enhanced Drought Tolerance 1 (AtEDT1) gene into alfalfa via Agrobacterium-mediated transformation. Compared with wild type plants, drought stress treatment resulted in higher survival rates and biomass, but reduced water loss rates in the transgenic plants. Furthermore, transgenic alfalfa plants had increased stomatal size, but reduced stomatal density, and these stomatal changes contributed greatly to reduced water loss from leaves. Importantly, transgenic alfalfa plants exhibited larger root systems with larger root lengths, root weight, and root diameters than wild type plants. The transgenic alfalfa plants had reduced membrane permeability and malondialdehyde content, but higher soluble sugar and proline content, higher superoxide dismutase activity, higher chlorophyll content, enhanced expression of drought-responsive genes, as compared with wild type plants. Notably, transgenic alfalfa plants grew better in a 2-year field trial and showed enhanced growth performance with increased biomass yield. All of our morphological, physiological, and molecular analyses demonstrated that the ectopic expression of AtEDT1 improved growth and enhanced drought tolerance in alfalfa. Our study provides alfalfa germplasm for use in forage improvement programs, and may help to increase alfalfa production in arid lands. PMID:29326737

Over-Expression of Arabidopsis EDT1 Gene Confers Drought Tolerance in Alfalfa (Medicago sativa L.).

PubMed

Zheng, Guangshun; Fan, Cunying; Di, Shaokang; Wang, Xuemin; Xiang, Chengbin; Pang, Yongzhen

2017-01-01

Alfalfa ( Medicago sativa L.) is an important legume forage crop with great economic value. However, as the growth of alfalfa is seriously affected by an inadequate supply of water, drought is probably the major abiotic environmental factor that most severely affects alfalfa production worldwide. In an effort to enhance alfalfa drought tolerance, we transformed the Arabidopsis Enhanced Drought Tolerance 1 ( AtEDT1 ) gene into alfalfa via Agrobacterium -mediated transformation. Compared with wild type plants, drought stress treatment resulted in higher survival rates and biomass, but reduced water loss rates in the transgenic plants. Furthermore, transgenic alfalfa plants had increased stomatal size, but reduced stomatal density, and these stomatal changes contributed greatly to reduced water loss from leaves. Importantly, transgenic alfalfa plants exhibited larger root systems with larger root lengths, root weight, and root diameters than wild type plants. The transgenic alfalfa plants had reduced membrane permeability and malondialdehyde content, but higher soluble sugar and proline content, higher superoxide dismutase activity, higher chlorophyll content, enhanced expression of drought-responsive genes, as compared with wild type plants. Notably, transgenic alfalfa plants grew better in a 2-year field trial and showed enhanced growth performance with increased biomass yield. All of our morphological, physiological, and molecular analyses demonstrated that the ectopic expression of AtEDT1 improved growth and enhanced drought tolerance in alfalfa. Our study provides alfalfa germplasm for use in forage improvement programs, and may help to increase alfalfa production in arid lands.

Proteasome inhibition blocks NF-κB and ERK1/2 pathways, restores antigen expression and sensitizes resistant human melanoma to TCR-engineered CTLs

PubMed Central

Jazirehi, Ali R.; Economou, James S.

2012-01-01

Adoptive cell transfer (ACT) of ex vivo engineered autologous lymphocytes encoding high-affinity MART-1/HLA-A*0201-specific T-cell receptor (TCR) α/β chains (F5 CTL), densely infiltrate into sites of metastatic disease, mediating dramatic but partial clinical responses in melanoma patients. We hypothesized that MART-1 down-modulation in addition to aberrant apoptotic/survival signaling could confer resistance to death signals delivered by transgenic CTLs. To explore this hypothesis, we established an in vitro model of resistant (R) lines from MART-1+/HLA-A*0201+ F5 CTL-sensitive parental (P) lines under serial F5 CTL-selective pressure. We have recently reported that several melanoma R lines, while retaining MART-1 expression, exhibited constitutive NF-κB activation and over-expression of NF-κB-dependent resistance factors. Another established melanoma cell line M244, otherwise sensitive to F5 CTL, yielded R lines after serial F5 CTL selective pressure which had both reduced MART-1 expression levels, thus, could not be recognized, and were resistant to CTL-delivered apoptotic death signals. The proteasome inhibitor bortezomib blocked NF-κB activity, decreased phopspho-ERK1/2, increased phospho-JNK levels, reduced expression of resistance-factors, restored MART-1 expression to sufficient levels, which in combination allowed M244R lines be sensitized to F5 CTL-killing. These findings suggest that proteasome inhibition in immune resistant tumors can restore proapoptotic signaling and improve tumor antigen expression. PMID:22532603

Reduced peripheral expression of the glucocorticoid receptor α isoform in individuals with posttraumatic stress disorder: a cumulative effect of trauma burden.

PubMed

Gola, Hannah; Engler, Andrea; Morath, Julia; Adenauer, Hannah; Elbert, Thomas; Kolassa, Iris-Tatjana; Engler, Harald

2014-01-01

Posttraumatic stress disorder (PTSD) is a serious psychiatric condition that was found to be associated with altered functioning of the hypothalamic-pituitary-adrenal (HPA) axis and changes in glucocorticoid (GC) responsiveness. The physiological actions of GCs are primarily mediated through GC receptors (GR) of which isoforms with different biological activities exist. This study aimed to investigate whether trauma-experience and/or PTSD are associated with altered expression of GR splice variants. GRα and GRβ mRNA expression levels were determined by real-time quantitative PCR in whole blood samples of individuals with chronic and severe forms of PTSD (n = 42) as well as in ethnically matched reference subjects (non-PTSD, n = 35). Individuals suffering from PTSD exhibited significantly lower expression of the predominant and functionally active GRα isoform compared to non-PTSD subjects. This effect remained significant when accounting for gender, smoking, psychotropic medication or comorbid depression. Moreover, the GRα expression level was significantly negatively correlated with the number of traumatic event types experienced, both in the whole sample and within the PTSD patient group. Expression of the less abundant and non-ligand binding GRβ isoform was comparable between patient and reference groups. Reduced expression of the functionally active GRα isoform in peripheral blood cells of individuals with PTSD seems to be a cumulative effect of trauma burden rather than a specific feature of PTSD since non-PTSD subjects with high trauma load showed an intermediate phenotype between PTSD patients and individuals with no or few traumatic experiences.

Halofuginone alleviates acute viral myocarditis in suckling BALB/c mice by inhibiting TGF-β1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Xiao-Hua; Fu, Jia; Sun, Da-Qing, E-mail: daqingsuncd@163.com

2016-04-29

Viral myocarditis (VMC) is an inflammation of heart muscle in infants and young adolescents. This study explored the function of halofuginone (HF) in Coxsackievirus B3 (CVB3) -treated suckling mice. HF-treated animal exhibited higher survival rate, lower heart/body weight, and more decreased blood sugar concentration than CVB3 group. HF also reduced the expressions of interleukin(IL)-17 and IL-23 and the numbers of Th17 cells. Moreover, HF downregulated pro-inflammatory cytokine levels and increased anti-inflammatory cytokine levels. The expressions of transforming growth factor(TGF-β1) and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) p65/ tumor necrosis factor-α (TNF-α) proteins were decreased by HF as well. Finally,more » the overexpression of TGF-β1 counteracted the protection effect of HF in CVB3-treated suckling mice. In summary, our study suggests HF increases the survival of CVB3 suckling mice, reduces the Th17 cells and pro-inflammatory cytokine levels, and may through downregulation of the TGF-β1-mediated expression of NF-κB p65/TNF-α pathway proteins. These results offer a potential therapeutic strategy for the treatment of VMC. - Highlights: • Halofuginone (HF) increases the survival of suckling BALB/c mice infected with acute CVB3. • HF reduces the expression of Th17 cell markers (IL-17 and IL-23) and the number of CD4{sup +} IL17{sup +} cells. • Pro-inflammatory cytokines levels associated with myocarditis were reduced by HF in CVB3-treated suckling mice. • HF alleviates VMC via inhibition of TGF-β1-mediated NF-κB p65/TNF-α pathway.« less

Inhibition of biphasic ethylene production enhances tolerance to abiotic stress by reducing the accumulation of reactive oxygen species in Nicotiana tabacum.

PubMed

Wi, Soo Jin; Jang, Su Jin; Park, Ky Young

2010-07-01

Reactive oxygen species (ROS), such as H(2)O(2), are important plant cell signaling molecules involved in responses to biotic and abiotic stresses and in developmental and physiological processes. Despite the well-known physiological functions of ethylene production and stress signaling via ROS during stresses, whether ethylene acts alone or in conjunction with ROS has not yet been fully elucidated. Therefore, we investigated the relationship between ethylene production and ROS accumulation during the response to abiotic stress. We used three independent transgenic tobacco lines, CAS-AS-2, -3 and -4, in which an antisense transcript of the senescence-related ACC synthase (ACS) gene from carnation flower (CARACC, Gen-Bank accession No. M66619) was expressed heterologously. Biphasic ethylene biosynthesis was reduced significantly in these transgenic plants, with or without H(2)O(2) treatment. These plants exhibited significantly reduced H(2)O(2)-induced gene-specific expression of ACS members, which were regulated in a time-dependent manner. The higher levels of NtACS1 expression in wild-type plants led to a second peak in ethylene production, which resulted in a more severe level of necrosis and cell death, as determined by trypan blue staining. In the transgenic lines, upregulated transcription of CAB, POR1 and RbcS resulted in increased photosynthetic performance following salt stress. This stress tolerance of H(2)O(2)-treated transgenic plants resulted from reduced ethylene biosynthesis, which decreased ROS accumulation via increased gene expression and activity of ROS-detoxifying enzymes, including MnSOD, CuZnSOD, and catalase. Therefore, it is suggested that ethylene plays a potentially critical role as an amplifier for ROS accumulation, implying a synergistic effect between biosynthesis of ROS and ethylene.

Hydrogen Sulfide Alleviates Aluminum Toxicity via Decreasing Apoplast and Symplast Al Contents in Rice

PubMed Central

Zhu, Chun Q.; Zhang, Jun H.; Sun, Li M.; Zhu, Lian F.; Abliz, Buhailiqem; Hu, Wen J.; Zhong, Chu; Bai, Zhi G.; Sajid, Hussain; Cao, Xiao C.; Jin, Qian Y.

2018-01-01

Hydrogen sulfide (H2S) plays a vital role in Al3+ stress resistance in plants, but the underlying mechanism is unclear. In the present study, pretreatment with 2 μM of the H2S donor NaHS significantly alleviated the inhibition of root elongation caused by Al toxicity in rice roots, which was accompanied by a decrease in Al contents in root tips under 50 μM Al3+ treatment. NaHS pretreatment decreased the negative charge in cell walls by reducing the activity of pectin methylesterase and decreasing the pectin and hemicellulose contents in rice roots. This treatment also masked Al-binding sites in the cell wall by upregulating the expression of OsSATR1 and OsSTAR2 in roots and reduced Al binding in the cell wall by stimulating the expression of the citrate acid exudation gene OsFRDL4 and increasing the secretion of citrate acid. In addition, NaHS pretreatment decreased the symplasmic Al content by downregulating the expression of OsNRAT1, and increasing the translocation of cytoplasmic Al to the vacuole via upregulating the expression of OsALS1. The increment of antioxidant enzyme [superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD)] activity with NaHS pretreatment significantly decreased the MDA and H2O2 content in rice roots, thereby reducing the damage of Al3+ toxicity on membrane integrity in rice. H2S exhibits crosstalk with nitric oxide (NO) in response to Al toxicity, and through reducing NO content in root tips to alleviate Al toxicity. Together, this study establishes that H2S alleviates Al toxicity by decreasing the Al content in the apoplast and symplast of rice roots. PMID:29559992

A topical treatment containing heat-treated Lactobacillus johnsonii NCC 533 reduces Staphylococcus aureus adhesion and induces antimicrobial peptide expression in an in vitro reconstructed human epidermis model.

PubMed

Rosignoli, Carine; Thibaut de Ménonville, Séverine; Orfila, Danielle; Béal, Méline; Bertino, Béatrice; Aubert, Jérôme; Mercenier, Annick; Piwnica, David

2018-04-01

Staphylococcus aureus colonization is thought to contribute to the pathophysiology of atopic dermatitis (AD). AD patients exhibit reduced levels of cutaneous antimicrobial peptides (AMPs), which may explain their increased susceptibility to infections. Using an in vitro reconstructed human epidermis (RHE) model, we sought to determine whether topical application of a non-replicating probiotic, heat-treated Lactobacillus johnsonii NCC 533 (HT La1), could inhibit S. aureus adhesion to skin and boost cutaneous innate immunity. We found that application of HT La1 suspension to RHE samples reduced the binding of radiolabelled S. aureus by up to 74%. To investigate a potential effect of HT La1 on innate immunity, we analysed the expression of nine AMP genes, including those encoding beta defensins and S100 proteins, following topical application of HT La1 in suspension or in a daily moisturizer lotion. Analysed genes were induced by up to fourfold in a dose-dependent manner by HT La1 in suspension and by up to 2.4-fold by HT La1 in the moisturizer lotion. Finally, using ELISA and immunohistochemical detection, we evaluated the expression and secretion of the AMPs hBD-2 and psoriasin and determined that both proteins were induced by topical HT La1, particularly in the stratum corneum of the RHE. These findings demonstrate that a topically applied, non-replicating probiotic can modulate endogenous AMP expression and inhibit binding of S. aureus to an RHE model in vitro. Moreover, they suggest that a topical formulation containing HT La1 could benefit atopic skin by enhancing cutaneous innate immunity and reducing S. aureus colonization. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Hydrogen Sulfide Alleviates Aluminum Toxicity via Decreasing Apoplast and Symplast Al Contents in Rice.

PubMed

Zhu, Chun Q; Zhang, Jun H; Sun, Li M; Zhu, Lian F; Abliz, Buhailiqem; Hu, Wen J; Zhong, Chu; Bai, Zhi G; Sajid, Hussain; Cao, Xiao C; Jin, Qian Y

2018-01-01

Hydrogen sulfide (H 2 S) plays a vital role in Al 3+ stress resistance in plants, but the underlying mechanism is unclear. In the present study, pretreatment with 2 μM of the H 2 S donor NaHS significantly alleviated the inhibition of root elongation caused by Al toxicity in rice roots, which was accompanied by a decrease in Al contents in root tips under 50 μM Al 3+ treatment. NaHS pretreatment decreased the negative charge in cell walls by reducing the activity of pectin methylesterase and decreasing the pectin and hemicellulose contents in rice roots. This treatment also masked Al-binding sites in the cell wall by upregulating the expression of OsSATR1 and OsSTAR2 in roots and reduced Al binding in the cell wall by stimulating the expression of the citrate acid exudation gene OsFRDL4 and increasing the secretion of citrate acid. In addition, NaHS pretreatment decreased the symplasmic Al content by downregulating the expression of OsNRAT1 , and increasing the translocation of cytoplasmic Al to the vacuole via upregulating the expression of OsALS1 . The increment of antioxidant enzyme [superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD)] activity with NaHS pretreatment significantly decreased the MDA and H 2 O 2 content in rice roots, thereby reducing the damage of Al 3+ toxicity on membrane integrity in rice. H 2 S exhibits crosstalk with nitric oxide (NO) in response to Al toxicity, and through reducing NO content in root tips to alleviate Al toxicity. Together, this study establishes that H 2 S alleviates Al toxicity by decreasing the Al content in the apoplast and symplast of rice roots.

The NAv1.7 blocker protoxin II reduces burn injury-induced spinal nociceptive processing.

PubMed

Torres-Pérez, Jose Vicente; Adamek, Pavel; Palecek, Jiri; Vizcaychipi, Marcela; Nagy, Istvan; Varga, Angelika

2018-01-01

Controlling pain in burn-injured patients poses a major clinical challenge. Recent findings suggest that reducing the activity of the voltage-gated sodium channel Na v 1.7 in primary sensory neurons could provide improved pain control in burn-injured patients. Here, we report that partial thickness scalding-type burn injury on the rat paw upregulates Na v 1.7 expression in primary sensory neurons 3 h following injury. The injury also induces upregulation in phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB), a marker for nociceptive activation in primary sensory neurons. The upregulation in p-CREB occurs mainly in Na v 1.7-immunopositive neurons and exhibits a peak at 5 min and, following a decline at 30 min, a gradual increase from 1 h post-injury. The Na v 1.7 blocker protoxin II (ProTxII) or morphine injected intraperitoneally 15 min before or after the injury significantly reduces burn injury-induced spinal upregulation in phosphorylated serine 10 in histone H3 and phosphorylated extracellular signal-regulated kinase 1/2, which are both markers for spinal nociceptive processing. Further, ProTxII significantly reduces the frequency of spontaneous excitatory post-synaptic currents in spinal dorsal horn neurons following burn injury. Together, these findings indicate that using Na v 1.7 blockers should be considered to control pain in burn injury. • Burn injury upregulates Na v 1.7 expression in primary sensory neurons. • Burn injury results in increased activity of Na v 1.7-expressing primary sensory neurons. • Inhibiting Na v 1.7 by protoxin II reduces spinal nociceptive processing. • Na v 1.7 represents a potential target to reduce pain in burn injury.

Hyperphagia, lower body temperature, and reduced running wheel activity precede development of morbid obesity in New Zealand obese mice.

PubMed

Jürgens, Hella S; Schürmann, Annette; Kluge, Reinhart; Ortmann, Sylvia; Klaus, Susanne; Joost, Hans-Georg; Tschöp, Matthias H

2006-04-13

Among polygenic mouse models of obesity, the New Zealand obese (NZO) mouse exhibits the most severe phenotype, with fat depots exceeding 40% of total body weight at the age of 6 mo. Here we dissected the components of energy balance including feeding behavior, locomotor activity, energy expenditure, and thermogenesis compared with the related lean New Zealand black (NZB) and obese B6.V-Lep(ob)/J (ob/ob) strains (11% and 65% fat at 23 wk, respectively). NZO mice exhibited a significant hyperphagia that, when food intake was expressed per metabolic body mass, was less pronounced than that of the ob/ob strain. Compared with NZB, NZO mice exhibited increased meal frequency, meal duration, and meal size. Body temperature as determined by telemetry with implanted sensors was reduced in NZO mice, but again to a lesser extent than in the ob/ob strain. In striking contrast to ob/ob mice, NZO mice were able to maintain a constant body temperature during a 20-h cold exposure, thus exhibiting a functioning cold-induced thermogenesis. No significant differences in spontaneous home cage activity were observed among NZO, NZB, and ob/ob strains. When mice had access to voluntary running wheels, however, running activity was significantly lower in NZO than NZB mice and even lower in ob/ob mice. These data indicate that obesity in NZO mice, just as in humans, is due to a combination of hyperphagia, reduced energy expenditure, and insufficient physical activity. Because NZO mice differ strikingly from the ob/ob strain in their resistance to cold stress, we suggest that the molecular defects causing hyperphagia in NZO mice are located distal from leptin and its receptor.

Lack of effect of the alpha2C-adrenoceptor Del322-325 polymorphism on inhibition of cyclic AMP production in HEK293 cells.

PubMed

Montgomery, M D; Bylund, D B

2010-02-01

The alpha(2C)-adrenoceptor has multiple functions, including inhibiting release of noradrenaline from presynaptic nerve terminals. A human alpha(2C) polymorphism, Del322-325, a potential risk factor for heart failure, has been reported to exhibit reduced signalling in CHO cells. To further understand the role of the Del322-325 polymorphism on receptor signalling, we attempted to replicate and further study the reduced signalling in HEK293 cells. Human alpha(2C) wild-type (WT) and Del322-325 adrenoceptors were stably transfected into HEK293 cells. Radioligand binding was performed to determine affinities for both receptors. In intact cells, inhibition of forskolin-stimulated cyclic AMP production by WT and Del322-325 clones with a range of receptor densities (200-2320 fmol.mg(-1) protein) was measured following agonist treatment. Noradrenaline, brimonidine and clonidine exhibited similar binding affinities for WT and Del322-325. Brimonidine and clonidine also had similar efficacies and potencies for both receptors for the inhibition of cyclic AMP production at all receptor densities tested. A linear regression analysis comparing efficacy and potency with receptor expression levels showed no differences in slopes between WT and Del322-325. The alpha(2C) WT and Del322-325 adrenoceptors exhibited similar binding properties. Additionally, inhibition of cyclic AMP production by Del322-325 was similar to that of WT over a range of receptor densities. Therefore, in intact HEK293 cells, the alpha(2C)-Del322-325 polymorphism does not exhibit reduced signalling to adenylyl cyclase and may not represent a clinically important phenotype.

Emerging Hawking-Like Radiation from Gravitational Bremsstrahlung Beyond the Planck Scale.

PubMed

Ciafaloni, Marcello; Colferai, Dimitri; Veneziano, Gabriele

2015-10-23

We argue that, as a consequence of the graviton's spin-2, its bremsstrahlung in trans-Planckian-energy (E≫M(P)) gravitational scattering at small deflection angle can be nicely expressed in terms of helicity-transformation phases and their transfer within the scattering process. The resulting spectrum exhibits deeply sub-Planckian characteristic energies of order M(P)(2)/E≪M(P) (reminiscent of Hawking radiation), a suppressed fragmentation region, and a reduced rapidity plateau, in broad agreement with recent classical estimates.

Over-expression of NADH-dependent oxidoreductase (fucO) for increasing furfural or 5-hydroxymethylfurfural tolerance

DOEpatents

Miller, Elliot N.; Zhang, Xueli; Yomano, Lorraine P.; Wang, Xuan; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

2015-10-13

The subject invention pertains to the discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce furfural. This allows for a new approach to improve furfural tolerance in bacterial and/or yeast cells used to produce desired products. Thus, novel biocatalysts (bacterial, fungal or yeast cells) exhibiting increased tolerance to furfural and 5-hydroxymethylfurfural (5-HMF) are provided as are methods of making and using such biocatalysts for the production of a desired product.

Synergistic and Antagonistic Interplay between Myostatin Gene Expression and Physical Activity Levels on Gene Expression Patterns in Triceps Brachii Muscles of C57/BL6 Mice

PubMed Central

Caetano-Anollés, Kelsey; Mishra, Sanjibita; Rodriguez-Zas, Sandra L.

2015-01-01

Levels of myostatin expression and physical activity have both been associated with transcriptome dysregulation and skeletal muscle hypertrophy. The transcriptome of triceps brachii muscles from male C57/BL6 mice corresponding to two genotypes (wild-type and myostatin-reduced) under two conditions (high and low physical activity) was characterized using RNA-Seq. Synergistic and antagonistic interaction and ortholog modes of action of myostatin genotype and activity level on genes and gene pathways in this skeletal muscle were uncovered; 1,836, 238, and 399 genes exhibited significant (FDR-adjusted P-value < 0.005) activity-by-genotype interaction, genotype and activity effects, respectively. The most common differentially expressed profiles were (i) inactive myostatin-reduced relative to active and inactive wild-type, (ii) inactive myostatin-reduced and active wild-type, and (iii) inactive myostatin-reduced and inactive wild-type. Several remarkable genes and gene pathways were identified. The expression profile of nascent polypeptide-associated complex alpha subunit (Naca) supports a synergistic interaction between activity level and myostatin genotype, while Gremlin 2 (Grem2) displayed an antagonistic interaction. Comparison between activity levels revealed expression changes in genes encoding for structural proteins important for muscle function (including troponin, tropomyosin and myoglobin) and for fatty acid metabolism (some linked to diabetes and obesity, DNA-repair, stem cell renewal, and various forms of cancer). Conversely, comparison between genotype groups revealed changes in genes associated with G1-to-S-phase transition of the cell cycle of myoblasts and the expression of Grem2 proteins that modulate the cleavage of the myostatin propeptide. A number of myostatin-feedback regulated gene products that are primarily regulatory were uncovered, including microRNA impacting central functions and Piezo proteins that make cationic current-controlling mechanosensitive ion channels. These important findings extend hypotheses of myostatin and physical activity master regulation of genes and gene pathways, impacting medical practices and therapies associated with muscle atrophy in humans and companion animal species and genome-enabled selection practices applied to food-production animal species. PMID:25710176

Glutathione S-transferase Mu 2-transduced mesenchymal stem cells ameliorated anti-glomerular basement membrane antibody-induced glomerulonephritis by inhibiting oxidation and inflammation

PubMed Central

2014-01-01

Introduction Oxidative stress is implicated in tissue inflammation, and plays an important role in the pathogenesis of immune-mediated nephritis. Using the anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM-GN) mouse model, we found that increased expression of glutathione S-transferase Mu 2 (GSTM2) was related to reduced renal damage caused by anti-GBM antibodies. Furthermore, mesenchymal stem cell (MSC)-based therapy has shed light on the treatment of immune-mediated kidney diseases. The aim of this study was to investigate if MSCs could be utilized as vehicles to deliver the GSTM2 gene product into the kidney and to evaluate its potential therapeutic effect on anti-GBM-GN. Methods The human GSTM2 gene (hGSTM2) was transduced into mouse bone marrow-derived MSCs via a lentivirus vector to create a stable cell line (hGSTM2-MSC). The cultured hGSTM2-MSCs were treated with 0.5mM H2O2, and apoptotic cells were measured by terminal dUTP nick-end labeling (TUNEL) assay. The 129/svj mice, which were challenged with anti-GBM antibodies, were injected with 106 hGSTM2-MSCs via the tail vein. Expression of hGSTM2 and inflammatory cytokines in the kidney was assayed by quantitative PCR and western blotting. Renal function of mice was evaluated by monitoring proteinuria and levels of blood urea nitrogen (BUN), and renal pathological changes were analyzed by histochemistry. Immunohistochemical analysis was performed to measure inflammatory cell infiltration and renal cell apoptosis. Results MSCs transduced with hGSTM2 exhibited similar growth and differentiation properties to MSCs. hGSTM2-MSCs persistently expressed hGSTM2 and resisted H2O2-induced apoptosis. Upon injection into 129/svj mice, hGSTM2-MSCs migrated to the kidney and expressed hGSTM2. The anti-GBM-GN mice treated with hGSTM2-MSCs exhibited reduced proteinuria and BUN (58% and 59% reduction, respectively) and ameliorated renal pathological damage, compared with control mice. Mice injected with hGSTM2-MSCs showed alleviated renal inflammatory cell infiltration and reduced expression of chemokine (C-C motif) ligand 2 (CCL2), interleukin (IL)-1β and IL-6 (53%, 46% and 52% reduction, respectively), compared with controls. Moreover, hGSTM2-MSCs increased expression of renal superoxide dismutase and catalase, which may associate with detoxifying reactive oxygen species to prevent oxidative renal damage. Conclusions Our data suggest that the enhanced protective effect of GSTM2-transduced MSCs against anti-GBM-GN might be associated with inhibition of oxidative stress-induced renal cell apoptosis and inflammation, through over-expression of hGSTM2 in mouse kidneys. PMID:24480247

Glutathione S-transferase Mu 2-transduced mesenchymal stem cells ameliorated anti-glomerular basement membrane antibody-induced glomerulonephritis by inhibiting oxidation and inflammation.

PubMed

Li, Yajuan; Yan, Mei; Yang, Jichen; Raman, Indu; Du, Yong; Min, Soyoun; Fang, Xiangdong; Mohan, Chandra; Li, Quan-Zhen

2014-01-30

Oxidative stress is implicated in tissue inflammation, and plays an important role in the pathogenesis of immune-mediated nephritis. Using the anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM-GN) mouse model, we found that increased expression of glutathione S-transferase Mu 2 (GSTM2) was related to reduced renal damage caused by anti-GBM antibodies. Furthermore, mesenchymal stem cell (MSC)-based therapy has shed light on the treatment of immune-mediated kidney diseases. The aim of this study was to investigate if MSCs could be utilized as vehicles to deliver the GSTM2 gene product into the kidney and to evaluate its potential therapeutic effect on anti-GBM-GN. The human GSTM2 gene (hGSTM2) was transduced into mouse bone marrow-derived MSCs via a lentivirus vector to create a stable cell line (hGSTM2-MSC). The cultured hGSTM2-MSCs were treated with 0.5 mM H2O2, and apoptotic cells were measured by terminal dUTP nick-end labeling (TUNEL) assay. The 129/svj mice, which were challenged with anti-GBM antibodies, were injected with 10⁶ hGSTM2-MSCs via the tail vein. Expression of hGSTM2 and inflammatory cytokines in the kidney was assayed by quantitative PCR and western blotting. Renal function of mice was evaluated by monitoring proteinuria and levels of blood urea nitrogen (BUN), and renal pathological changes were analyzed by histochemistry. Immunohistochemical analysis was performed to measure inflammatory cell infiltration and renal cell apoptosis. MSCs transduced with hGSTM2 exhibited similar growth and differentiation properties to MSCs. hGSTM2-MSCs persistently expressed hGSTM2 and resisted H2O2-induced apoptosis. Upon injection into 129/svj mice, hGSTM2-MSCs migrated to the kidney and expressed hGSTM2. The anti-GBM-GN mice treated with hGSTM2-MSCs exhibited reduced proteinuria and BUN (58% and 59% reduction, respectively) and ameliorated renal pathological damage, compared with control mice. Mice injected with hGSTM2-MSCs showed alleviated renal inflammatory cell infiltration and reduced expression of chemokine (C-C motif) ligand 2 (CCL2), interleukin (IL)-1β and IL-6 (53%, 46% and 52% reduction, respectively), compared with controls. Moreover, hGSTM2-MSCs increased expression of renal superoxide dismutase and catalase, which may associate with detoxifying reactive oxygen species to prevent oxidative renal damage. Our data suggest that the enhanced protective effect of GSTM2-transduced MSCs against anti-GBM-GN might be associated with inhibition of oxidative stress-induced renal cell apoptosis and inflammation, through over-expression of hGSTM2 in mouse kidneys.

Reinforcement of wheel running in BALB/c mice: role of motor activity and endogenous opioids.

PubMed

Vargas-Pérez, Héctor; Sellings, Laurie H L; Paredes, Raúl G; Prado-Alcalá, Roberto A; Díaz, José-Luis

2008-11-01

The authors investigated the effect of the opioid antagonist naloxone on wheel-running behavior in Balb/c mice. Naloxone delayed the acquisition of wheel-running behavior, but did not reduce the expression of this behavior once acquired. Delayed acquisition was not likely a result of reduced locomotor activity, as naloxone-treated mice did not exhibit reduced wheel running after the behavior was acquired, and they performed normally on the rotarod test. However, naloxone-blocked conditioned place preference for a novel compartment paired previously with wheel running, suggesting that naloxone may delay wheel-running acquisition by blocking the rewarding or reinforcing effects of the behavior. These results suggest that the endogenous opioid system mediates the initial reinforcing effects of wheel running that are important in acquisition of the behavior.

Correlation between p65 and TNF-α in patients with acute myelocytic leukemia.

PubMed

Dong, Qiao-Mei; Ling, Chun; Zhu, Jun-Fang; Chen, Xuan; Tang, Yan; Zhao, L I

2015-11-01

The correlation between the expression levels of p65 and TNF-α in patients with acute myelocytic leukemia (AML) and AML cell lines were investigated. The bone marrow samples of 30 AML patients and 10 non-leukemia controls were studied. The mRNA expression levels of p65 and TNF-α were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Pearson's Correlation test was used to demonstrate the correlation between TNF-α and p65 expression levels in AML specimens. Receiver operating characteristic (ROC) curves were plotted to determine whether TNF-α and p65 expression levels could be used to differentiate AML samples from non-leukemia samples. MG132 and anti-TNF-α antibody were used to inhibit the expression of p65 and TNF-α in the AML cell line, HL-60. The expression of p65 and TNF-α were detected by RT-qPCR and western blot analysis. The mRNA expression levels of p65 and TNF-α were significantly increased in AML patients compared with non-leukemia control bone marrow samples by RT-qPCR, and the two molecules expression pattern's exhibited sufficient predictive power to distinguish AML patients from non-leukemia control samples. Pearson's correlation analysis demonstrated that TNF-α expression was strongly correlated with p65 expression in AML bone marrow samples. In HL-60 cells, inhibition of TNF-α reduced the expression of p65; in addition, inhibition of p65 reduced the expression of TNF-α as assessed by RT-qPCR and western blot analysis. p65 and TNF-α were highly expressed in AML patients, and these 2 molecules were strongly correlated. The present study indicates that p65 and TNF-α have potential as molecular markers to distinguish AML patients from non-leukemia control samples, and that these 2 molecules may be useful prognostic factor for patients with AML.

Altered Behavioral Performance and Live Imaging of Circuit-Specific Neural Deficiencies in a Zebrafish Model for Psychomotor Retardation

PubMed Central

Lerer-Goldshtein, Tali; Vatine, Gad David; Appelbaum, Lior

2014-01-01

The mechanisms and treatment of psychomotor retardation, which includes motor and cognitive impairment, are indefinite. The Allan-Herndon-Dudley syndrome (AHDS) is an X-linked psychomotor retardation characterized by delayed development, severe intellectual disability, muscle hypotonia, and spastic paraplegia, in combination with disturbed thyroid hormone (TH) parameters. AHDS has been associated with mutations in the monocarboxylate transporter 8 (mct8/slc16a2) gene, which is a TH transporter. In order to determine the pathophysiological mechanisms of AHDS, MCT8 knockout mice were intensively studied. Although these mice faithfully replicated the abnormal serum TH levels, they failed to exhibit the neurological and behavioral symptoms of AHDS patients. Here, we generated an mct8 mutant (mct8−/−) zebrafish using zinc-finger nuclease (ZFN)-mediated targeted gene editing system. The elimination of MCT8 decreased the expression levels of TH receptors; however, it did not affect the expression of other TH-related genes. Similar to human patients, mct8−/− larvae exhibited neurological and behavioral deficiencies. High-throughput behavioral assays demonstrated that mct8−/− larvae exhibited reduced locomotor activity, altered response to external light and dark transitions and an increase in sleep time. These deficiencies in behavioral performance were associated with altered expression of myelin-related genes and neuron-specific deficiencies in circuit formation. Time-lapse imaging of single-axon arbors and synapses in live mct8−/− larvae revealed a reduction in filopodia dynamics and axon branching in sensory neurons and decreased synaptic density in motor neurons. These phenotypes enable assessment of the therapeutic potential of three TH analogs that can enter the cells in the absence of MCT8. The TH analogs restored the myelin and axon outgrowth deficiencies in mct8−/− larvae. These findings suggest a mechanism by which MCT8 regulates neural circuit assembly, ultimately mediating sensory and motor control of behavioral performance. We also propose that the administration of TH analogs early during embryo development can specifically reduce neurological damage in AHDS patients. PMID:25255244

Hepcidin suppression in β-thalassemia is associated with the down-regulation of atonal homolog 8.

PubMed

Upanan, Supranee; McKie, Andrew T; Latunde-Dada, Gladys O; Roytrakul, Sittiruk; Uthaipibull, Chairat; Pothacharoen, Peraphan; Kongtawelert, Prachya; Fucharoen, Suthat; Srichairatanakool, Somdet

2017-08-01

Atonal homolog 8 (ATOH8) is defined as a positive regulator of hepcidin transcription, which links erythropoietic activity with iron-sensing molecules. In the present study, we investigated the association between hepcidin and ATOH8 expression in β-thalassemia. We found that inhibition of hepcidin expression in β-thalassemia is correlated with reduced ATOH8 expression. Hepatic hepcidin 1 (Hamp1) and Atoh8 mRNA expression were down-regulated in β-thalassemic mice. Hepcidin (HAMP) and ATOH8 mRNA expression were consistently suppressed in Huh7 cells cultured in medium supplemented with β-thalassemia patient serum. The Huh7 cells, which were transfected with ATOH8-FLAG expression plasmid and cultured in the supplemented medium, exhibited increased levels of ATOH8 mRNA, ATOH8-FLAG protein, pSMAD1,5,8, and HAMP mRNA. Interestingly, over-expression of ATOH8 reversed the effects of hepcidin suppression induced by the β-thalassemia patient sera. In conclusion, hepcidin suppression in β-thalassemia is associated with the down-regulation of ATOH8 in response to anemia. We, therefore, suggest that ATOH8 is an important transcriptional regulator of hepcidin in β-thalassemia.

Effects of increased Kindlin-2 expression in bladder cancer stromal fibroblasts.

PubMed

Wu, Jitao; Yu, Cuicui; Cai, Li; Lu, Youyi; Jiang, Lei; Liu, Chu; Li, Yongwei; Feng, Fan; Gao, Zhenli; Zhu, Zhe; Yu, Shengqiang; Yuan, Hejia; Cui, Yuanshan

2017-08-01

Kindlin-2 is a focal adhesion protein highly expressed in bladder cancer stromal fibroblasts. We investigated the prognostic significance of Kindlin-2 in bladder cancer stromal fibroblasts and evaluated the effects of Kindlin-2 on the malignant behaviors of tumor cells. Immunohistochemical staining of 203 paraffin-embedded bladder cancer tissues showed that Kindlin-2 expression correlated with advanced stage, high grade, and relapse of bladder cancer. Kaplan-Meier survival analysis demonstrated that patients exhibiting high Kindlin-2 expression had shorter survival times than those with low Kindlin-2 expression ( p < 0.01). Multivariate analysis revealed that high Kindlin-2 expression leads to poor prognosis in bladder cancer. Using cancer-associated fibroblasts (CAFs) isolated from human bladder cancer tissue, we observed that Kindlin-2 knockdown decreased CAFs activation, resulting in decreased expression of α-smooth muscle actin (α-SMA) and the extracellular matrix protein fibronectin. Kindlin-2 suppression also reduced CAF-induced bladder cancer cell migration and invasion. Moreover, we found that Kindlin-2 activates CAFs and promotes the invasiveness of bladder cancer cells by stimulating TGF-β-induced epithelial-mesenchymal transition. These results support targeting Kindlin-2 and the corresponding activated CAFs in bladder cancer therapy.

Differential gene expression in porcine SK6 cells infected with wild-type and SAP domain-mutant foot-and-mouth disease virus.

PubMed

Ni, Zixin; Yang, Fan; Cao, Weijun; Zhang, Xiangle; Jin, Ye; Mao, Ruoqing; Du, Xiaoli; Li, Weiwei; Guo, Jianhong; Liu, Xiangtao; Zhu, Zixiang; Zheng, Haixue

2016-06-01

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious disease in livestock. The viral proteinase L(pro) of FMDV is involved in pathogenicity, and mutation of the L(pro) SAP domain reduces FMDV pathogenicity in pigs. To determine the gene expression profiles associated with decreased pathogenicity in porcine cells, we performed transcriptome analysis using next-generation sequencing technology and compared differentially expressed genes in SK6 cells infected with FMDV containing L(pro) with either a wild-type or mutated version of the SAP domain. This analysis yielded 1,853 genes that exhibited a ≥ 2-fold change in expression and was validated by real-time quantitative PCR detection of several differentially expressed genes. Many of the differentially expressed genes correlated with antiviral responses corresponded to genes associated with transcription factors, immune regulation, cytokine production, inflammatory response, and apoptosis. Alterations in gene expression profiles may be responsible for the variations in pathogenicity observed between the two FMDV variants. Our results provided genes of interest for the further study of antiviral pathways and pathogenic mechanisms related to FMDV L(pro).

Increased expression of high mobility group box protein 1 and vascular endothelial growth factor in placenta previa.

PubMed

Xie, Han; Qiao, Ping; Lu, Yi; Li, Ying; Tang, Yuping; Huang, Yiying; Bao, Yirong; Ying, Hao

2017-12-01

Placenta previa is often associated with preterm delivery, reduced birth weight, a higher frequency of placental accreta and postpartum haemorrhage, and increased likelihood of blood transfusion. The present study aimed to examine the expression of high mobility group box protein 1 (HMGB1) in the placenta of women with or without placenta previa. The study group consisted of placental tissues obtained from women with or without placenta previa. The expression levels of HMGB1 and vascular endothelial growth factor (VEGF) were evaluated in the placental tissues using reverse transcription‑quantitative polymerase chain reaction, western blotting and immunohistochemistry. The mRNA expression levels of HMGB1 and VEGF were significantly increased in the placenta previa group compared with in the normal group. In addition, the placenta previa group exhibited increased HMGB1 and VEGF staining in vascular endothelial cells and trophoblasts. There were no significant differences in the expression of HMGB1 or VEGF between groups with or without placenta accreta or postpartum haemorrhage. The present study hypothesised that the increased expression of HMGB1 in the placenta may be associated with the pathogenesis of placenta previa by regulating the expression of the proangiogenic factor VEGF.

Analysis of Protein Kinase C Delta (PKCδ) Expression in Endometrial Tumors

PubMed Central

Reno, Elaine M.; Haughian, James M.; Dimitrova, Irina K.; Jackson, Twila A.; Shroyer, Kenneth R; Bradford., Andrew P.

2007-01-01

Endometrial cancer is the most common gynecological malignancy in the US, however, its underlying molecular mechanisms are poorly understood and few prognostic indicators have been identified. The Protein Kinase C (PKC) family have been shown to regulate pathways critical to malignant transformation, and in endometrial tumors, changes in PKC expression and activity have been linked to a more aggressive phenotype and poor prognosis. We have recently shown that PKCδ is a critical regulator of apoptosis and cell survival in endometrial cancer cells; however, PKCδ levels in endometrial tumors had not been determined. We used immunohistochemistry to examine PKCδ protein levels in normal endometrium and endometrioid carcinomas of increasing grade. Normal endometrium exhibited abundant nuclear and cytoplasmic staining of PKCδ, confined to glandular epithelium. In endometrial tumors, decreased PKCδ expression, both in intensity and fraction of epithelial cells stained, was observed with increasing tumor grade, with PKCδ being preferentially lost from the nucleus. Consistent with these observations, endometrial cancer cell lines derived from poorly differentiated tumors exhibited reduced PKCδ levels relative to well-differentiated lines. Treatment of endometrial cancer cells with etoposide resulted in a translocation of PKCδ from cytoplasm to nucleus concomitant with induction of apoptosis. Decreased PKCδ expression, particularly in the nucleus, may compromise the ability of cells to undergo apoptosis, perhaps conferring resistance to chemotherapy. Our results indicate that loss of PKCδ is an indicator of endometrial malignancy and increasing grade of cancer. Thus, PKCδ may function as a tumor suppressor in endometrial cancer. PMID:17959229

Tubular Epithelial NF-κB Activity Regulates Ischemic AKI

PubMed Central

Vigolo, Emilia; Hinze, Christian; Park, Joon-Keun; Roël, Giulietta; Balogh, András; Choi, Mira; Wübken, Anne; Cording, Jimmi; Blasig, Ingolf E.; Luft, Friedrich C.; Scheidereit, Claus; Schmidt-Ott, Kai M.; Schmidt-Ullrich, Ruth; Müller, Dominik N.

2016-01-01

NF-κB is a key regulator of innate and adaptive immunity and is implicated in the pathogenesis of AKI. The cell type–specific functions of NF-κB in the kidney are unknown; however, the pathway serves distinct functions in immune and tissue parenchymal cells. We analyzed tubular epithelial-specific NF-κB signaling in a mouse model of ischemia-reperfusion injury (IRI)–induced AKI. NF-κB reporter activity and nuclear localization of phosphorylated NF-κB subunit p65 analyses in mice revealed that IRI induced widespread NF-κB activation in renal tubular epithelia and in interstitial cells that peaked 2–3 days after injury. To genetically antagonize tubular epithelial NF-κB activity, we generated mice expressing the human NF-κB super-repressor IκBαΔN in renal proximal, distal, and collecting duct epithelial cells. Compared with control mice, these mice exhibited improved renal function, reduced tubular apoptosis, and attenuated neutrophil and macrophage infiltration after IRI-induced AKI. Furthermore, tubular NF-κB–dependent gene expression profiles revealed temporally distinct functional gene clusters for apoptosis, chemotaxis, and morphogenesis. Primary proximal tubular cells isolated from IκBαΔN-expressing mice and exposed to hypoxia-mimetic agent cobalt chloride exhibited less apoptosis and expressed lower levels of chemokines than cells from control mice did. Our results indicate that postischemic NF-κB activation in renal tubular epithelia aggravates tubular injury and exacerbates a maladaptive inflammatory response. PMID:26823548

Downregulation of mucosal mast cell activation and immune response in diarrhoea-irritable bowel syndrome by oral disodium cromoglycate: A pilot study

PubMed Central

Lobo, Beatriz; Ramos, Laura; Martínez, Cristina; Guilarte, Mar; González-Castro, Ana M; Alonso-Cotoner, Carmen; Pigrau, Marc; de Torres, Inés; Rodiño-Janeiro, Bruno K; Salvo-Romero, Eloisa; Fortea, Marina; Pardo-Camacho, Cristina; Guagnozzi, Danila; Azpiroz, Fernando

2017-01-01

Background and goal Diarrhoea-predominant irritable bowel syndrome (IBS-D) exhibits intestinal innate immune and mucosal mast cell (MC) activation. MC stabilisers have been shown to improve IBS symptoms but the mechanism is unclear. Our primary aim was to investigate the effect of oral disodium cromoglycate (DSCG) on jejunal MC activation and specific innate immune signalling pathways in IBS-D, and secondarily, its potential clinical benefit. Study Mucosal MC activation (by ultrastructural changes, tryptase release and gene expression) and innate immune signalling (by protein and gene expression) were quantified in jejunal biopsies from healthy (HS; n = 16) and IBS-D subjects after six months of either treatment with DSCG (600 mg/day, IBS-D-DSCG group; n = 18) or without treatment (IBS-D-NT group; n = 25). All IBS-D patients recorded abdominal pain and bowel habits at baseline and in the last 10 days prior to jejunal sampling. Results IBS-D-NT exhibited significant MC activation and over-expression of immune-related genes as compared to HS, whereas in IBS-D-DSCG MC activity and gene expression were similar to HS. Furthermore, DSCG significantly reduced abdominal pain and improved stool consistency. Conclusion Oral DSCG modulates mucosal immune activity and improves gut symptoms in IBS-D patients. Future placebo-controlled clinical trials are needed for confirmation of clinical benefit of DSCG for IBS-D. PMID:29026603

Adhesion of Epstein–Barr virus-positive natural killer cell lines to cultured endothelial cells stimulated with inflammatory cytokines

PubMed Central

Kanno, H; Watabe, D; Shimizu, N; Sawai, T

2008-01-01

Chronic active Epstein–Barr virus (EBV) infection (CAEBV) is characterized by chronic recurrent infectious mononucleosis-like symptoms. Approximately one-fourth of CAEBV patients develop vascular lesions with infiltration of EBV-positive lymphoid cells. Furthermore, EBV-positive natural killer (NK)/T cell lymphomas often exhibit angiocentric or angiodestructive lesions. These suggest an affinity of EBV-positive NK/T cells to vascular components. In this study, we evaluated the expression of adhesion molecules and cytokines in EBV-positive NK lymphoma cell lines, SNK1 and SNK6, and examined the role of cytokines in the interaction between NK cell lines and endothelial cells. SNKs expressed intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) at much higher levels than those in EBV-negative T cell lines. SNKs produced the larger amount of tumour necrosis factor (TNF)-α, which caused increased expression of ICAM-1 and VCAM-1 in cultured human endothelial cells, than that from EBV-negative T cell lines. Furthermore, SNKs exhibited increased adhesion to cultured endothelial cells stimulated with TNF-α or interleukin (IL)-1β, and the pretreatment of cytokine-stimulated endothelial cells with anti-VCAM-1-antibodies reduced cell adhesion. These indicate that the up-regulated expression of VCAM-1 on cytokine-stimulated endothelial cells would be important for the adhesion of EBV-positive NK cells and might initiate the vascular lesions. PMID:18190605

Protein kinase C βII and TGFβRII in ω-3 fatty acid–mediated inhibition of colon carcinogenesis

PubMed Central

Murray, Nicole R.; Weems, Capella; Chen, Lu; Leon, Jessica; Yu, Wangsheng; Davidson, Laurie A.; Jamieson, Lee; Chapkin, Robert S.; Thompson, E. Aubrey; Fields, Alan P.

2002-01-01

Încreasing evidence demonstrates that protein kinase C βII (PKCβII) promotes colon carcinogenesis. We previously reported that colonic PKCβII is induced during colon carcinogenesis in rodents and humans, and that elevated expression of PKCβII in the colon of transgenic mice enhances colon carcinogenesis. Here, we demonstrate that PKCβII represses transforming growth factor β receptor type II (TGFβRII) expression and reduces sensitivity to TGF-β–mediated growth inhibition in intestinal epithelial cells. Transgenic PKCβII mice exhibit hyperproliferation, enhanced colon carcinogenesis, and marked repression of TGFβRII expression. Chemopreventive dietary ω-3 fatty acids inhibit colonic PKCβII activity in vivo and block PKCβII-mediated hyperproliferation, enhanced carcinogenesis, and repression of TGFβRII expression in the colonic epithelium of transgenic PKCβII mice. These data indicate that dietary ω-3 fatty acids prevent colon cancer, at least in part, through inhibition of colonic PKCβII signaling and restoration of TGF-β responsiveness. PMID:12058013

Deletion of Monoglyceride Lipase in Astrocytes Attenuates Lipopolysaccharide-induced Neuroinflammation*

PubMed Central

Grabner, Gernot F.; Eichmann, Thomas O.; Wagner, Bernhard; Gao, Yuanqing; Farzi, Aitak; Taschler, Ulrike; Radner, Franz P. W.; Schweiger, Martina; Lass, Achim; Holzer, Peter; Zinser, Erwin; Tschöp, Matthias H.; Yi, Chun-Xia; Zimmermann, Robert

2016-01-01

Monoglyceride lipase (MGL) is required for efficient hydrolysis of the endocannabinoid 2-arachidonoylglyerol (2-AG) in the brain generating arachidonic acid (AA) and glycerol. This metabolic function makes MGL an interesting target for the treatment of neuroinflammation, since 2-AG exhibits anti-inflammatory properties and AA is a precursor for pro-inflammatory prostaglandins. Astrocytes are an important source of AA and 2-AG, and highly express MGL. In the present study, we dissected the distinct contribution of MGL in astrocytes on brain 2-AG and AA metabolism by generating a mouse model with genetic deletion of MGL specifically in astrocytes (MKOGFAP). MKOGFAP mice exhibit moderately increased 2-AG and reduced AA levels in brain. Minor accumulation of 2-AG in the brain of MKOGFAP mice does not cause cannabinoid receptor desensitization as previously observed in mice globally lacking MGL. Importantly, MKOGFAP mice exhibit reduced brain prostaglandin E2 and pro-inflammatory cytokine levels upon peripheral lipopolysaccharide (LPS) administration. These observations indicate that MGL-mediated degradation of 2-AG in astrocytes provides AA for prostaglandin synthesis promoting LPS-induced neuroinflammation. The beneficial effect of astrocyte-specific MGL-deficiency is not fully abrogated by the inverse cannabinoid receptor 1 agonist SR141716 (Rimonabant) suggesting that the anti-inflammatory effects are rather caused by reduced prostaglandin synthesis than by activation of cannabinoid receptors. In conclusion, our data demonstrate that MGL in astrocytes is an important regulator of 2-AG levels, AA availability, and neuroinflammation. PMID:26565024

Age-Related Changes in Mouse Taste Bud Morphology, Hormone Expression, and Taste Responsivity

PubMed Central

Shin, Yu-Kyong; Cong, Wei-na; Cai, Huan; Kim, Wook; Maudsley, Stuart; Martin, Bronwen

2012-01-01

Normal aging is a complex process that affects every organ system in the body, including the taste system. Thus, we investigated the effects of the normal aging process on taste bud morphology, function, and taste responsivity in male mice at 2, 10, and 18 months of age. The 18-month-old animals demonstrated a significant reduction in taste bud size and number of taste cells per bud compared with the 2- and 10-month-old animals. The 18-month-old animals exhibited a significant reduction of protein gene product 9.5 and sonic hedgehog immunoreactivity (taste cell markers). The number of taste cells expressing the sweet taste receptor subunit, T1R3, and the sweet taste modulating hormone, glucagon-like peptide-1, were reduced in the 18-month-old mice. Concordant with taste cell alterations, the 18-month-old animals demonstrated reduced sweet taste responsivity compared with the younger animals and the other major taste modalities (salty, sour, and bitter) remained intact. PMID:22056740

Inhibitory effect of corn silk on skin pigmentation.

PubMed

Choi, Sang Yoon; Lee, Yeonmi; Kim, Sung Soo; Ju, Hyun Min; Baek, Ji Hwoon; Park, Chul-Soo; Lee, Dong-Hyuk

2014-03-03

In this study, the inhibitory effect of corn silk on melanin production was evaluated. This study was performed to investigate the inhibitory effect of corn silk on melanin production in Melan-A cells by measuring melanin production and protein expression. The corn silk extract applied on Melan-A cells at a concentration of 100 ppm decreased melanin production by 37.2% without cytotoxicity. This was a better result than arbutin, a positive whitening agent, which exhibited a 26.8% melanin production inhibitory effect at the same concentration. The corn silk extract did not suppress tyrosinase activity but greatly reduced the expression of tyrosinase in Melan-A cells. In addition, corn silk extract was applied to the human face with hyperpigmentation, and skin color was measured to examine the degree of skin pigment reduction. The application of corn silk extract on faces with hyperpigmentation significantly reduced skin pigmentation without abnormal reactions. Based on the results above, corn silk has good prospects for use as a material for suppressing skin pigmentation.

Age-related changes in mouse taste bud morphology, hormone expression, and taste responsivity.

PubMed

Shin, Yu-Kyong; Cong, Wei-na; Cai, Huan; Kim, Wook; Maudsley, Stuart; Egan, Josephine M; Martin, Bronwen

2012-04-01

Normal aging is a complex process that affects every organ system in the body, including the taste system. Thus, we investigated the effects of the normal aging process on taste bud morphology, function, and taste responsivity in male mice at 2, 10, and 18 months of age. The 18-month-old animals demonstrated a significant reduction in taste bud size and number of taste cells per bud compared with the 2- and 10-month-old animals. The 18-month-old animals exhibited a significant reduction of protein gene product 9.5 and sonic hedgehog immunoreactivity (taste cell markers). The number of taste cells expressing the sweet taste receptor subunit, T1R3, and the sweet taste modulating hormone, glucagon-like peptide-1, were reduced in the 18-month-old mice. Concordant with taste cell alterations, the 18-month-old animals demonstrated reduced sweet taste responsivity compared with the younger animals and the other major taste modalities (salty, sour, and bitter) remained intact.

NADPH oxidase 4 limits bone mass by promoting osteoclastogenesis

PubMed Central

Goettsch, Claudia; Babelova, Andrea; Trummer, Olivia; Erben, Reinhold G.; Rauner, Martina; Rammelt, Stefan; Weissmann, Norbert; Weinberger, Valeska; Benkhoff, Sebastian; Kampschulte, Marian; Obermayer-Pietsch, Barbara; Hofbauer, Lorenz C.; Brandes, Ralf P.; Schröder, Katrin

2013-01-01

ROS are implicated in bone diseases. NADPH oxidase 4 (NOX4), a constitutively active enzymatic source of ROS, may contribute to the development of such disorders. Therefore, we studied the role of NOX4 in bone homeostasis. Nox4–/– mice displayed higher bone density and reduced numbers and markers of osteoclasts. Ex vivo, differentiation of monocytes into osteoclasts with RANKL and M-CSF induced Nox4 expression. Loss of NOX4 activity attenuated osteoclastogenesis, which was accompanied by impaired activation of RANKL-induced NFATc1 and c-JUN. In an in vivo model of murine ovariectomy–induced osteoporosis, pharmacological inhibition or acute genetic knockdown of Nox4 mitigated loss of trabecular bone. Human bone obtained from patients with increased osteoclast activity exhibited increased NOX4 expression. Moreover, a SNP of NOX4 was associated with elevated circulating markers of bone turnover and reduced bone density in women. Thus, NOX4 is involved in bone loss and represents a potential therapeutic target for the treatment of osteoporosis. PMID:24216508

Antioxidative characteristics and inhibition of alpha-melanocyte-stimulating hormone-stimulated melanogenesis of vanillin and vanillic acid from Origanum vulgare.

PubMed

Chou, Tzung-Han; Ding, Hsiou-Yu; Hung, Wei Jing; Liang, Chia-Hua

2010-08-01

The antioxidant activities of vanillin and vanillic acid isolated from Origanum vulgare are investigated. These compounds may serve as agents for antimelanogenesis. Vanillic acid is a stronger antioxidant than vanillin, in terms of free radical scavenging activity, reducing power and inhibition of lipid peroxidation. The inhibition of cellular reactive oxygen species (ROS) in H(2)O(2)-treated BNLCL2 cells by vanillic acid exceeds that of ascorbic acid (AA) or trolox. In B16F0 cells stimulated with alpha-melanocyte-stimulating hormone (alpha-MSH), vanillic acid reduced cellular tyrosinase activity, DOPA oxidase and melanin contents, as well as down-regulated expressions of melanocortin-1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related proteins 2 (TRP-2) and TRP-1. Vanillin did not express inhibition of tyrosinase activity. These results supported that vanillic acid is a significantly stronger antioxidant than vanillin and exhibited stronger antimelanogenesis performance because of the structural presence of the carboxyl group.

Hyperglycemia Promotes Schwann Cell De-differentiation and De-myelination via Sorbitol Accumulation and Igf1 Protein Down-regulation*

PubMed Central

Hao, Wu; Tashiro, Syoichi; Hasegawa, Tomoka; Sato, Yuiko; Kobayashi, Tami; Tando, Toshimi; Katsuyama, Eri; Fujie, Atsuhiro; Watanabe, Ryuichi; Morita, Mayu; Miyamoto, Kana; Morioka, Hideo; Nakamura, Masaya; Matsumoto, Morio; Amizuka, Norio; Toyama, Yoshiaki; Miyamoto, Takeshi

2015-01-01

Diabetes mellitus (DM) is frequently accompanied by complications, such as peripheral nerve neuropathy. Schwann cells play a pivotal role in regulating peripheral nerve function and conduction velocity; however, changes in Schwann cell differentiation status in DM are not fully understood. Here, we report that Schwann cells de-differentiate into immature cells under hyperglycemic conditions as a result of sorbitol accumulation and decreased Igf1 expression in those cells. We found that de-differentiated Schwann cells could be re-differentiated in vitro into mature cells by treatment with an aldose reductase inhibitor, to reduce sorbitol levels, or with vitamin D3, to elevate Igf1 expression. In vivo DM models exhibited significantly reduced nerve function and conduction, Schwann cell de-differentiation, peripheral nerve de-myelination, and all conditions were significantly rescued by aldose reductase inhibitor or vitamin D3 administration. These findings reveal mechanisms underlying pathological changes in Schwann cells seen in DM and suggest ways to treat neurological conditions associated with this condition. PMID:25998127

Snapin-regulated late endosomal transport is critical for efficient autophagy-lysosomal function in neurons.

PubMed

Cai, Qian; Lu, Li; Tian, Jin-Hua; Zhu, Yi-Bing; Qiao, Haifa; Sheng, Zu-Hang

2010-10-06

Neuron maintenance and survival require late endocytic transport from distal processes to the soma where lysosomes are predominantly localized. Here, we report a role for Snapin in attaching dynein to late endosomes through its intermediate chain (DIC). snapin(-/-) neurons exhibit aberrant accumulation of immature lysosomes, clustering and impaired retrograde transport of late endosomes along processes, reduced lysosomal proteolysis due to impaired delivery of internalized proteins and hydrolase precursors from late endosomes to lysosomes, and impaired clearance of autolysosomes, combined with reduced neuron viability and neurodegeneration. The phenotypes are rescued by expressing the snapin transgene, but not the DIC-binding-defective Snapin-L99K mutant. Snapin overexpression in wild-type neurons enhances late endocytic transport and lysosomal function, whereas expressing the mutant defective in Snapin-DIC coupling shows a dominant-negative effect. Altogether, our study highlights new mechanistic insights into how Snapin-DIC coordinates retrograde transport and late endosomal-lysosomal trafficking critical for autophagy-lysosomal function, and thus neuronal homeostasis. Copyright © 2010 Elsevier Inc. All rights reserved.

Hemoglobin genetics: recent contributions of GWAS and gene editing

PubMed Central

Smith, Elenoe C.; Orkin, Stuart H.

2016-01-01

The β-hemoglobinopathies are inherited disorders resulting from altered coding potential or expression of the adult β-globin gene. Impaired expression of β-globin reduces adult hemoglobin (α2β2) production, the hallmark of β-thalassemia. A single-base mutation at codon 6 leads to formation of HbS (α2βS2) and sickle cell disease. While the basis of these diseases is known, therapy remains largely supportive. Bone marrow transplantation is the only curative therapy. Patients with elevated levels of fetal hemoglobin (HbF, α2γ2) as adults exhibit reduced symptoms and enhanced survival. The β-globin gene locus is a paradigm of cell- and developmental stage-specific regulation. Although the principal erythroid cell transcription factors are known, mechanisms responsible for silencing of the γ-globin gene were obscure until application of genome-wide association studies (GWAS). Here, we review findings in the field. GWAS identified BCL11A as a candidate negative regulator of γ-globin expression. Subsequent studies have established BCL11A as a quantitative repressor. GWAS-related single-nucleotide polymorphisms lie within an essential erythroid enhancer of the BCL11A gene. Disruption of a discrete region within the enhancer reduces BCL11A expression and induces HbF expression, providing the basis for gene therapy using gene editing tools. A recently identified, second silencing factor, leukemia/lymphoma-related factor/Pokemon, shares features with BCL11A, including interaction with the nucleosome remodeling deacetylase repressive complex. These findings suggest involvement of a common pathway for HbF silencing. In addition, we discuss other factors that may be involved in γ-globin gene silencing and their potential manipulation for therapeutic benefit in treating the β-hemoglobinopathies. PMID:27340226

Transient and Prolonged Response of Chicken Cecum Mucosa to Colonization with Different Gut Microbiota

PubMed Central

Volf, Jiri; Polansky, Ondrej; Varmuzova, Karolina; Gerzova, Lenka; Sekelova, Zuzana; Faldynova, Marcela; Babak, Vladimir; Medvecky, Matej; Smith, Adrian L.; Kaspers, Bernd; Velge, Philippe; Rychlik, Ivan

2016-01-01

In this study we determined protein and gene expression in the caeca of newly hatched chickens inoculated with cecal contents sourced from hens of different ages. Over 250 proteins exhibited modified expression levels in response to microbiota inoculation. The most significant inductions were observed for ISG12-2, OASL, ES1, LYG2, DMBT1-L, CDD, ANGPTL6, B2M, CUZD1, IgM and Ig lambda chain. Of these, ISG12-2, ES1 and both immunoglobulins were expressed at lower levels in germ-free chickens compared to conventional chickens. In contrast, CELA2A, BRT-2, ALDH1A1, ADH1C, AKR1B1L, HEXB, ALDH2, ALDOB, CALB1 and TTR were expressed at lower levels following inoculation of microbiota. When chicks were given microbiota preparations from different age donors, the recipients mounted differential responses to the inoculation which also differed from the response profile in naturally colonised birds. For example, B2M, CUZD1 and CELA2A responded differently to the inoculation with microbiota of 4- or 40-week-old hens. The increased or decreased gene expression could be recorded 6 weeks after the inoculation of newly hatched chickens. To characterise the proteins that may directly interact with the microbiota we characterised chicken proteins that co-purified with the microbiota and identified a range of host proteins including CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda. We propose that induction of ISG12-2 results in reduced apoptosis of host cells exposed to the colonizing commensal microbiota and that CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda reduce contact of luminal microbiota with the gut epithelium thereby reducing the inflammatory response. PMID:27685470

Exposure to welding fumes and lower airway infection with Streptococcus pneumoniae.

PubMed

Suri, Reetika; Periselneris, Jimstan; Lanone, Sophie; Zeidler-Erdely, Patti C; Melton, Geoffrey; Palmer, Keith T; Andujar, Pascal; Antonini, James M; Cohignac, Vanessa; Erdely, Aaron; Jose, Ricardo J; Mudway, Ian; Brown, Jeremy; Grigg, Jonathan

2016-02-01

Welders are at increased risk of pneumococcal pneumonia. The mechanism for this association is not known. The capacity of pneumococci to adhere to and infect lower airway cells is mediated by host-expressed platelet-activating factor receptor (PAFR). We sought to assess the effect of mild steel welding fumes (MS-WF) on PAFR-dependent pneumococcal adhesion and infection to human airway cells in vitro and on pneumococcal airway infection in a mouse model. The oxidative potential of MS-WF was assessed by their capacity to reduce antioxidants in vitro. Pneumococcal adhesion and infection of A549, BEAS-2B, and primary human bronchial airway cells were assessed by means of quantitative bacterial culture and expressed as colony-forming units (CFU). After intranasal instillation of MS-WF, mice were infected with Streptococcus pneumoniae, and bronchoalveolar lavage fluid (BALF) and lung CFU values were determined. PAFR protein levels were assessed by using immunofluorescence and immunohistochemistry, and PAFR mRNA expression was assessed by using quantitative PCR. PAFR was blocked by CV-3988, and oxidative stress was attenuated by N-acetylcysteine. MS-WF exhibited high oxidative potential. In A549 and BEAS-2B cells MS-WF increased pneumococcal adhesion and infection and PAFR protein expression. Both CV-3988 and N-acetylcysteine reduced MS-WF-stimulated pneumococcal adhesion and infection of airway cells. MS-WF increased mouse lung PAFR mRNA expression and increased BALF and lung pneumococcal CFU values. In MS-WF-exposed mice CV-3988 reduced BALF CFU values. Hypersusceptibility of welders to pneumococcal pneumonia is in part mediated by the capacity of welding fumes to increase PAFR-dependent pneumococcal adhesion and infection of lower airway cells. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. All rights reserved.

Exposure to welding fumes and lower airway infection with Streptococcus pneumoniae

PubMed Central

Suri, Reetika; Periselneris, Jimstan; Lanone, Sophie; Zeidler-Erdely, Patti C.; Melton, Geoffrey; Palmer, Keith T.; Andujar, Pascal; Antonini, James M.; Cohignac, Vanessa; Erdely, Aaron; Jose, Ricardo J.; Mudway, Ian; Brown, Jeremy; Grigg, Jonathan

2015-01-01

Background Welders are at increased risk of pneumococcal pneumonia. The mechanism for this association is not known. The capacity of pneumococci to adhere to and infect lower airway cells is mediated by host-expressed platelet-activating factor receptor (PAFR). Objective We sought to assess the effect of mild steel welding fumes (MS-WF) on PAFR-dependent pneumococcal adhesion and infection to human airway cells in vitro and on pneumococcal airway infection in a mouse model. Methods The oxidative potential of MS-WF was assessed by their capacity to reduce antioxidants in vitro. Pneumococcal adhesion and infection of A549, BEAS-2B, and primary human bronchial airway cells were assessed by means of quantitative bacterial culture and expressed as colony-forming units (CFU). After intranasal instillation of MS-WF, mice were infected with Streptococcus pneumoniae, and bronchoalveolar lavage fluid (BALF) and lung CFU values were determined. PAFR protein levels were assessed by using immunofluorescence and immunohistochemistry, and PAFR mRNA expression was assessed by using quantitative PCR. PAFR was blocked by CV-3988, and oxidative stress was attenuated by N-acetylcysteine. Results: MS-WF exhibited high oxidative potential. In A549 and BEAS-2B cells MS-WF increased pneumococcal adhesion and infection and PAFR protein expression. Both CV-3988 and N-acetylcysteine reduced MS-WF–stimulated pneumococcal adhesion and infection of airway cells. MS-WF increased mouse lung PAFR mRNA expression and increased BALF and lung pneumococcal CFU values. In MS-WF–exposed mice CV-3988 reduced BALF CFU values. Conclusions Hypersusceptibility of welders to pneumococcal pneumonia is in part mediated by the capacity of welding fumes to increase PAFR-dependent pneumococcal adhesion and infection of lower airway cells. PMID:26277596

Peripheral insulin resistance in ILK-depleted mice by reduction of GLUT4 expression.

PubMed

Hatem-Vaquero, Marco; Griera, Mercedes; García-Jerez, Andrea; Luengo, Alicia; Álvarez, Julia; Rubio, José A; Calleros, Laura; Rodríguez-Puyol, Diego; Rodríguez-Puyol, Manuel; De Frutos, Sergio

2017-08-01

The development of insulin resistance is characterized by the impairment of glucose uptake mediated by glucose transporter 4 (GLUT4). Extracellular matrix changes are induced when the metabolic dysregulation is sustained. The present work was devoted to analyze the possible link between the extracellular-to-intracellular mediator integrin-linked kinase (ILK) and the peripheral tissue modification that leads to glucose homeostasis impairment. Mice with general depletion of ILK in adulthood (cKD-ILK) maintained in a chow diet exhibited increased glycemia and insulinemia concurrently with a reduction of the expression and membrane presence of GLUT4 in the insulin-sensitive peripheral tissues compared with their wild-type littermates (WT). Tolerance tests and insulin sensitivity indexes confirmed the insulin resistance in cKD-ILK, suggesting a similar stage to prediabetes in humans. Under randomly fed conditions, no differences between cKD-ILK and WT were observed in the expression of insulin receptor (IR-B) and its substrate IRS-1 expressions. The IR-B isoform phosphorylated at tyrosines 1150/1151 was increased, but the AKT phosphorylation in serine 473 was reduced in cKD-ILK tissues. Similarly, ILK-blocked myotubes reduced their GLUT4 promoter activity and GLUT4 expression levels. On the other hand, the glucose uptake capacity in response to exogenous insulin was impaired when ILK was blocked in vivo and in vitro , although IR/IRS/AKT phosphorylation states were increased but not different between groups. We conclude that ILK depletion modifies the transcription of GLUT4, which results in reduced peripheral insulin sensitivity and glucose uptake, suggesting ILK as a molecular target and a prognostic biomarker of insulin resistance. © 2017 Society for Endocrinology.

Activation of ER stress and mTORC1 suppresses hepatic sortilin-1 levels in obese mice

PubMed Central

Ai, Ding; Baez, Juan M.; Jiang, Hongfeng; Conlon, Donna M.; Hernandez-Ono, Antonio; Frank-Kamenetsky, Maria; Milstein, Stuart; Fitzgerald, Kevin; Murphy, Andrew J.; Woo, Connie W.; Strong, Alanna; Ginsberg, Henry N.; Tabas, Ira; Rader, Daniel J.; Tall, Alan R.

2012-01-01

Recent GWAS have identified SNPs at a human chromosom1 locus associated with coronary artery disease risk and LDL cholesterol levels. The SNPs are also associated with altered expression of hepatic sortilin-1 (SORT1), which encodes a protein thought to be involved in apoB trafficking and degradation. Here, we investigated the regulation of Sort1 expression in mouse models of obesity. Sort1 expression was markedly repressed in both genetic (ob/ob) and high-fat diet models of obesity; restoration of hepatic sortilin-1 levels resulted in reduced triglyceride and apoB secretion. Mouse models of obesity also exhibit increased hepatic activity of mammalian target of rapamycin complex 1 (mTORC1) and ER stress, and we found that administration of the mTOR inhibitor rapamycin to ob/ob mice reduced ER stress and increased hepatic sortilin-1 levels. Conversely, genetically increased hepatic mTORC1 activity was associated with repressed Sort1 and increased apoB secretion. Treating WT mice with the ER stressor tunicamycin led to marked repression of hepatic sortilin-1 expression, while administration of the chemical chaperone PBA to ob/ob mice led to amelioration of ER stress, increased sortilin-1 expression, and reduced apoB and triglyceride secretion. Moreover, the ER stress target Atf3 acted at the SORT1 promoter region as a transcriptional repressor, whereas knockdown of Atf3 mRNA in ob/ob mice led to increased hepatic sortilin-1 levels and decreased apoB and triglyceride secretion. Thus, in mouse models of obesity, induction of mTORC1 and ER stress led to repression of hepatic Sort1 and increased VLDL secretion via Atf3. This pathway may contribute to dyslipidemia in metabolic disease. PMID:22466652

The atypical chemokine receptor ACKR2 drives pulmonary fibrosis by tuning influx of CCR2+ and CCR5+ IFNγ-producing γδT cells in mice.

PubMed

Russo, Remo Castro; Savino, Benedetta; Mirolo, Massimiliano; Buracchi, Chiara; Germano, Giovanni; Anselmo, Achille; Zammataro, Luca; Pasqualini, Fabio; Mantovani, Alberto; Locati, Massimo; Teixeira, Mauro M

2018-02-22

Chemokines coordinate lung inflammation and fibrosis by acting on chemokine receptors expressed on leukocytes and other cell types. Atypical chemokine receptors (ACKRs) bind, internalize and degrade chemokines, tuning homeostasis and immune responses. ACKR2 recognizes and decreases levels of inflammatory CC chemokines. The role of ACKR2 in fibrogenesis is unknown. Investigate the role of ACKR2 in the context of pulmonary fibrosis. The effects of ACKR2 expression and deficiency during inflammation and fibrosis were analyzed using a bleomycin-model of fibrosis, ACKR2-deficient mice, bone marrow chimeras and antibody-mediated leukocyte depletion. ACKR2 was up-regulated acutely in response to bleomycin and normalized over time. ACKR2-/- mice showed reduced lethality and lung fibrosis. Bone marrow chimeras showed that lethality and fibrosis depended on ACKR2 expression in pulmonary resident (non-hematopoietic) cells but not on leukocytes. ACKR2-/- mice exhibited decreased expression of tissue remodeling genes, reduced leukocyte influx, pulmonary injury, and dysfunction. ACKR2-/- mice had early-increased levels of CCL5, CCL12, CCL17 and IFNγ, and increased number of CCR2+ and CCR5+ IFNγ-producing γδT cells in the airways counterbalanced by low Th17 lymphocyte influx. There was reduced accumulation of IFNγ-producing γδT cells in CCR2-/- and CCR5-/- mice. Moreover, depletion of γδT cells worsened the clinical symptoms induced by bleomycin and reversed the phenotype of ACKR2-/- mice exposed to bleomycin. ACKR2 controls the CC chemokine expression that drives the influx of CCR2+ and CCR5+ IFNγ-producing γδT cells tuning the Th17 response that mediate pulmonary fibrosis triggered by bleomycin instillation.

Histone deacetylase 3 supports endochondral bone formation by controlling cytokine signaling and matrix remodeling

PubMed Central

Carpio, Lomeli R.; Bradley, Elizabeth W.; McGee-Lawrence, Meghan E.; Weivoda, Megan M.; Poston, Daniel D.; Dudakovic, Amel; Xu, Ming; Tchkonia, Tamar; Kirkland, James L.; van Wijnen, Andre J.; Oursler, Merry Jo; Westendorf, Jennifer J.

2017-01-01

Histone deacetylase (HDAC) inhibitors are efficacious epigenetic-based therapies for some cancers and neurological disorders; however, each of these drugs inhibits multiple HDACs and has detrimental effects on the skeleton. To better understand how HDAC inhibitors affect endochondral bone formation, we conditionally deleted one of their targets, Hdac3, pre- and postnatally in type II collagen α1 (Col2α1)–expressing chondrocytes. Embryonic deletion was lethal, but postnatal deletion of Hdac3 delayed secondary ossification center formation, altered maturation of growth plate chondrocytes, and increased osteoclast activity in the primary spongiosa. HDAC3-deficient chondrocytes exhibited increased expression of cytokine and matrix-degrading genes (Il-6, Mmp3, Mmp13, and Saa3) and a reduced abundance of genes related to extracellular matrix production, bone development, and ossification (Acan, Col2a1, Ihh, and Col10a1). Histone acetylation increased at and near genes that had increased expression. The acetylation and activation of nuclear factor κB (NF-κB) were also increased in HDAC3-deficient chondrocytes. Increased cytokine signaling promoted autocrine activation of Janus kinase (JAK)–signal transducer and activator of transcription (STAT) and NF-κB pathways to suppress chondrocyte maturation, as well as paracrine activation of osteoclasts and bone resorption. Blockade of interleukin-6 (IL-6)–JAK–STAT signaling, NF-κB signaling, and bromodomain extraterminal proteins, which recognize acetylated lysines and promote transcriptional elongation, significantly reduced Il-6 and Mmp13 expression in HDAC3-deficient chondrocytes and secondary activation in osteoclasts. The JAK inhibitor ruxolitinib also reduced osteoclast activity in Hdac3 conditional knockout mice. Thus, HDAC3 controls the temporal and spatial expression of tissue-remodeling genes and inflammatory responses in chondrocytes to ensure proper endochondral ossification during development. PMID:27507649

Tyrosine kinase receptor c-ros-oncogene 1 inhibition alleviates aberrant bone formation of TWIST-1 haploinsufficient calvarial cells from Saethre-Chotzen syndrome patients.

PubMed

Camp, Esther; Anderson, Peter J; Zannettino, Andrew C W; Glackin, Carlotta A; Gronthos, Stan

2018-09-01

Saethre-Chotzen syndrome (SCS), associated with TWIST-1 mutations, is characterized by premature fusion of cranial sutures. TWIST-1 haploinsufficiency, leads to alterations in suture mesenchyme cellular gene expression patterns, resulting in aberrant osteogenesis and craniosynostosis. We analyzed the expression of the TWIST-1 target, Tyrosine kinase receptor c-ros-oncogene 1 (C-ROS-1) in TWIST-1 haploinsufficient calvarial cells derived from SCS patients and calvaria of Twist-1 del/+ mutant mice and found it to be highly expressed when compared to TWIST-1 wild-type controls. Knock-down of C-ROS-1 expression in TWIST-1 haploinsufficient calvarial cells derived from SCS patients was associated with decreased capacity for osteogenic differentiation in vitro. Furthermore, treatment of human SCS calvarial cells with the tyrosine kinase chemical inhibitor, Crizotinib, resulted in reduced C-ROS-1 activity and the osteogenic potential of human SCS calvarial cells with minor effects on cell viability or proliferation. Cultured human SCS calvarial cells treated with Crizotinib exhibited a dose-dependent decrease in alkaline phosphatase activity and mineral deposition, with an associated decrease in expression levels of Runt-related transcription factor 2 and OSTEOPONTIN, with reduced PI3K/Akt signalling in vitro. Furthermore, Crizotinib treatment resulted in reduced BMP-2 mediated bone formation potential of whole Twist-1 del/+ mutant mouse calvaria organotypic cultures. Collectively, these results suggest that C-ROS-1 promotes osteogenic differentiation of TWIST-1 haploinsufficient calvarial osteogenic progenitor cells. Furthermore, the aberrant osteogenic potential of these cells is inhibited by the reduction of C-ROS-1. Therefore, targeting C-ROS-1 with a pharmacological agent, such as Crizotinib, may serve as a novel therapeutic strategy to alleviate craniosynostosis associated with aberrant TWIST-1 function. © 2018 Wiley Periodicals, Inc.

Changes in Leptin Signaling by SOCS3 Modulate Fasting-Induced Hyperphagia and Weight Regain in Mice.

PubMed

Pedroso, João A B; Silveira, Marina A; Lima, Leandro B; Furigo, Isadora C; Zampieri, Thais T; Ramos-Lobo, Angela M; Buonfiglio, Daniella C; Teixeira, Pryscila D S; Frazão, Renata; Donato, Jose

2016-10-01

Weight regain frequently follows interventions that reduce body weight, leading to a failure in long-term obesity treatment. Inhibitory proteins of the leptin signaling pathway, such as the suppressor of cytokine signaling 3 (SOCS3), have been studied in conditions that predispose animals to obesity. However, whether SOCS3 modulates postrestriction hyperphagia and weight regain remains unknown. Mice lacking SOCS3 protein specifically in leptin receptor (LepR)-expressing cells (LepR SOCS3 knockout [KO]) were generated and studied in fasting and refeeding conditions. LepR SOCS3 KO mice exhibited increased leptin sensitivity in the hypothalamus. Notably, LepR SOCS3 KO males and females showed attenuated food intake and weight regain after 48 hours of fasting. Postrestriction hyperleptinemia was also prevented in LepR SOCS3 KO mice. Next, we studied possible mechanisms and neural circuits involved in the SOCS3 effects. SOCS3 deletion did not prevent fasting- or refeeding-induced c-Fos expression in the arcuate nucleus of the hypothalamus (ARH) nor fasting-induced increased excitability of ARH LepR-expressing cells. On the other hand, SOCS3 ablation reduced the mRNA levels of hypothalamic orexigenic neuropeptides during fasting (neuropeptide Y, agouti-related protein, orexin, and melanin-concentrating hormone). In summary, our findings suggest that increased leptin sensitivity contributes to the maintenance of a reduced body weight after food deprivation. In addition, the attenuated postrestriction food intake observed in mutant mice was not explained by fasting-induced changes in the activity of ARH neurons but exclusively by a lower transcription of orexigenic neuropeptides during fasting. These results indicate a partial dissociation between the regulation of neuronal activity and gene expression in ARH LepR-expressing cells.

Peroxisome proliferator-activated receptor-β/δ inhibits human neuroblastoma cell tumorigenesis by inducing p53- and SOX2-mediated cell differentiation.

PubMed

Yao, Pei-Li; Chen, Liping; Dobrzański, Tomasz P; Zhu, Bokai; Kang, Boo-Hyon; Müller, Rolf; Gonzalez, Frank J; Peters, Jeffrey M

2017-05-01

Neuroblastoma is a common childhood cancer typically treated by inducing differentiation with retinoic acid (RA). Peroxisome proliferator-activated receptor-β/δ, (PPARβ/δ) is known to promote terminal differentiation of many cell types. In the present study, PPARβ/δ was over-expressed in three human neuroblastoma cell lines, NGP, SK-N-BE(2), and IMR-32, that exhibit high, medium, and low sensitivity, respectively, to retinoic acid-induced differentiation to determine if PPARβ/δ and retinoic acid receptors (RARs) could be jointly targeted to increase the efficacy of treatment. All-trans-RA (atRA) decreased expression of SRY (sex determining region Y)-box 2 (SOX2), a stem cell regulator and marker of de-differentiation, in NGP and SK-N-BE(2) cells with inactive or mutant tumor suppressor p53, respectively. However, atRA did not suppress SOX2 expression in IMR-32 cells carrying wild-type p53. Over-expression and/or ligand activation of PPARβ/δ reduced the average volume and weight of ectopic tumor xenografts from NGP, SK-N-BE(2), or IMR-32 cells compared to controls. Compared with that found with atRA, PPARβ/δ suppressed SOX2 expression in NGP and SK-N-BE(2) cells and ectopic xenografts, and was also effective in suppressing SOX2 expression in IMR-32 cells that exhibit higher p53 expression compared to the former cell lines. Combined, these observations demonstrate that activating or over-expressing PPARβ/δ induces cell differentiation through p53- and SOX2-dependent signaling pathways in neuroblastoma cells and tumors. This suggests that combinatorial activation of both RARα and PPARβ/δ may be suitable as an alternative therapeutic approach for RA-resistant neuroblastoma patients. Published [2016]. This article is a U.S. Government work and is in the public domain in the USA.

Effect of hyperbaric oxygen preconditioning on peri-hemorrhagic focal edema and aquaporin-4 expression.

PubMed

Fang, Jinyong; Li, Hongling; Li, Guanglei; Wang, Lichun

2015-08-01

The aim of the present study was to investigate the effect of hyperbaric oxygen preconditioning (HBO-PC) on peri-hemorrhagic focal edema and aquaporin-4 (AQP-4) expression in an experimental intracerebral hemorrhage (ICH) rat model. Sixty-six Sprague Dawley® rats were divided into three groups: The sham-surgery group (SHG; n=6); the control group (A-ICH; n=30), in which the rats were injected with autologous blood; and the experimental HBO-PC group (P-HBO; n=30). The rats underwent brain edema and AQP-4 detection at 5 postoperative time-points (24, 48 and 72 h and 5 and 7 days). The water content in the brain tissues of the A-ICH animals was higher than that in the brain tissues of the SHG rats at each time-point (P<0.05), and the edema in the P-HBO was significantly more severe 24 and 48 h postoperatively than that at 7 days postoperatively (P<0.05). The difference between the P-HBO and A-ICH was significant at 48 and 72 h postoperatively (P<0.05). AQP-4 was expressed in the post-hemorrhagic rat brains of all groups; the SHG animals exhibited low expression, while the A-ICH animals exhibited an increased expression 24 h postoperatively. In the A-ICH, expression peaked at 48 h postoperatively and began to decrease gradually after 72 h. At the 7-day time-point, the expression level in the A-ICH was closer to but still higher than that of the SHG animals (P<0.05). The differences between the P-HBO and A-ICH animals at the postoperative 24-h, 48-h and 7-day time-points were statistically significant (P<0.05). In conclusion, HBO-PC may downregulate AQP-4 expression to reduce the intracerebral edema, thus strengthening tolerance to ICH and protecting the nerves.

Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells.

PubMed

Ko, Jen-Chung; Chen, Jyh-Cheng; Wang, Tai-Jing; Zheng, Hao-Yu; Chen, Wen-Ching; Chang, Po-Yuan; Lin, Yun-Wei

2016-04-01

Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20 μM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with MMC for the treatment of NSCLC. Copyright © 2016 Elsevier Inc. All rights reserved.

Elevated Mirc1/Mir17-92 cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages.

PubMed

Tazi, Mia F; Dakhlallah, Duaa A; Caution, Kyle; Gerber, Madelyn M; Chang, Sheng-Wei; Khalil, Hany; Kopp, Benjamin T; Ahmed, Amr E; Krause, Kathrin; Davis, Ian; Marsh, Clay; Lovett-Racke, Amy E; Schlesinger, Larry S; Cormet-Boyaka, Estelle; Amer, Amal O

2016-11-01

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (Mirs), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of Mirs in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the Mirc1/Mir17-92 cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between Mirc1/Mir17-92 and autophagy gene expression. An in silico study for targets of Mirs that comprise the cluster suggested that the majority of the Mirs target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated Mirs, via restoration of autophagy. In vivo, downregulation of Mir17 and Mir20a partially restored autophagy expression and hence improved the clearance of Burkholderia cenocepacia. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.

Aquaporin 4 is a Ubiquitously Expressed Isoform in the Dogfish (Squalus acanthias) Shark.

PubMed

Cutler, Christopher P; Maciver, Bryce; Cramb, Gordon; Zeidel, Mark

2011-01-01

The dogfish ortholog of aquaporin 4 (AQP4) was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5' and 3' RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3%) of homology with higher vertebrate sequences but lower levels of homology to Agnathan (38.2%) or teleost (57.5%) fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ). Semi-quantitative PCR further indicates that AQP4 is ubiquitous, being expressed in all tissues measured but at low levels in certain tissues, where the level in liver > gill >  intestine. Manipulation of the external environmental salinity of groups of dogfish showed that when fish were acclimated in stages to 120% seawater (SW) or 75% SW, there was no change in AQP4 mRNA expression in either rectal gland, kidney, or esophagus/cardiac stomach. Whereas quantitative PCR experiments using the RNA samples from the same experiment, showed a significant 63.1% lower abundance of gill AQP4 mRNA expression in 120% SW-acclimated dogfish. The function of dogfish AQP4 was also determined by measuring the effect of the AQP4 expression in Xenopus laevis oocytes. Dogfish AQP4 expressing-oocytes, exhibited significantly increased osmotic water permeability (P(f)) compared to controls, and this was invariant with pH. Permeability was not significantly reduced by treatment of oocytes with mercury chloride, as is also the case with AQP4 in other species. Similarly AQP4 expressing-oocytes did not exhibit enhanced urea or glycerol permeability, which is also consistent with the water-selective property of AQP4 in other species.

Aquaporin 4 is a Ubiquitously Expressed Isoform in the Dogfish (Squalus acanthias) Shark

PubMed Central

Cutler, Christopher P; MacIver, Bryce; Cramb, Gordon; Zeidel, Mark

2012-01-01

The dogfish ortholog of aquaporin 4 (AQP4) was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5′ and 3′ RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3%) of homology with higher vertebrate sequences but lower levels of homology to Agnathan (38.2%) or teleost (57.5%) fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ). Semi-quantitative PCR further indicates that AQP4 is ubiquitous, being expressed in all tissues measured but at low levels in certain tissues, where the level in liver > gill >  intestine. Manipulation of the external environmental salinity of groups of dogfish showed that when fish were acclimated in stages to 120% seawater (SW) or 75% SW, there was no change in AQP4 mRNA expression in either rectal gland, kidney, or esophagus/cardiac stomach. Whereas quantitative PCR experiments using the RNA samples from the same experiment, showed a significant 63.1% lower abundance of gill AQP4 mRNA expression in 120% SW-acclimated dogfish. The function of dogfish AQP4 was also determined by measuring the effect of the AQP4 expression in Xenopus laevis oocytes. Dogfish AQP4 expressing-oocytes, exhibited significantly increased osmotic water permeability (Pf) compared to controls, and this was invariant with pH. Permeability was not significantly reduced by treatment of oocytes with mercury chloride, as is also the case with AQP4 in other species. Similarly AQP4 expressing-oocytes did not exhibit enhanced urea or glycerol permeability, which is also consistent with the water-selective property of AQP4 in other species. PMID:22291652

Haploinsufficiency of the Myc regulator Mtbp extends survival and delays tumor development in aging mice.

PubMed

Grieb, Brian C; Boyd, Kelli; Mitra, Ramkrishna; Eischen, Christine M

2016-10-30

Alterations of specific genes can modulate aging. Myc, a transcription factor that regulates the expression of many genes involved in critical cellular functions was shown to have a role in controlling longevity. Decreased expression of Myc inhibited many of the deleterious effects of aging and increased lifespan in mice. Without altering Myc expression, reduced levels of Mtbp, a recently identified regulator of Myc, limit Myc transcriptional activity and proliferation, while increased levels promote Myc-mediated effects. To determine the contribution of Mtbp to the effects of Myc on aging, we studied a large cohort of Mtbp heterozygous mice and littermate matched wild-type controls. Mtbp haploinsufficiency significantly increased longevity and maximal survival in mice. Reduced levels of Mtbp did not alter locomotor activity, litter size, or body size, but Mtbp heterozygous mice did exhibit elevated markers of metabolism, particularly in the liver. Mtbp +/- mice also had a significant delay in spontaneous cancer development, which was most prominent in the hematopoietic system, and an altered tumor spectrum compared to Mtbp +/+ mice. Therefore, the data suggest Mtbp is a regulator of longevity in mice that mimics some, but not all, of the properties of Myc in aging.

MORIN MITIGATES OXIDATIVE STRESS, APOPTOSIS AND INFLAMMATION IN CEREBRAL ISCHEMIC RATS

PubMed Central

Chen, Yanrong; Li, Yanke; Xu, Huali; Li, Gang; Ma, Yunxia; Pang, Yu Jun

2017-01-01

Background: Morin is a flavanoid which exhibits potent antioxidant activity in various oxidative stress related diseases. The current study was attempted to scrutinize the preclinical bio-efficacy of morin on focal ischemia. Methods: The animal model of focal cerebral ischemic injury was done by midbrain carotid artery occlusion (MCAO) method, followed by Morin (30mg/kg) administration for seven days. Results: The outcome of the study showed that treatment with morin displayed positive effects in reducing the focal cerebral ischemia. This effect was evident with the improvements in neurological deficits, reduction in MDA content and elevation of antioxidant levels (SOD, GSH and Gpx). Furthermore, protein expression of Bax and caspase-3 were effectively down-regulated, whilst the expression of Bcl-2 was significantly elevated. On the other hand, the mRNA expression of proinflammatory cytokines was significantly reduced in focal cerebral ischemic rats upon morin intervention. Conclusion: Thus, the beneficial effects of morin on cerebral ischemia assault may result from the reduction of oxidative stress, inhibition of apoptosis and inflammation. The neuroprotective effects of morin supplement may serve as potent adjuvant in the amelioration of ischemic stroke. PMID:28573251

Optimized expression in Pichia pastoris eliminates common protein contaminants from subsequent His-tag purification.

PubMed

Chen, Yong; Li, Yang; Liu, Peng; Sun, Qun; Liu, Zhu

2014-04-01

A weakness of using immobilized metal affinity chromatography (IMAC) to purify recombinant proteins expressed in Pichia pastoris is the co-purification of native proteins that exhibit high affinities for Ni-IMAC. We have determined the elution profiles of P. pastoris proteins and have examined the native proteins that co-purify when eluting with 100 mM imidazole. Four major contaminants were identified: mitochondrial alcohol dehydrogenase isozyme III (mADH), nucleotide excision repair endonuclease, and the hypothetical proteins TPHA_0L01390 and TDEL_0B02190 which are homologous proteins derived from Tetrapisispora phaffii and Torulaspora delbrueckii, respectively. A new P. pastoris expression strain was engineered that eliminated the predominant contaminant, mADH, by gene disruption. The total amount of protein contaminants was reduced by 55 % without effecting cell growth. The present study demonstrates the feasibility of using a proteomic approach to facilitate bioprocess optimization.

Oxygen partial pressure modulates 67-kDa laminin receptor expression, leading to altered activity of the green tea polyphenol, EGCG.

PubMed

Tsukamoto, Shuntaro; Yamashita, Shuya; Kim, Yoon Hee; Kumazoe, Motofumi; Huang, Yuhui; Yamada, Koji; Tachibana, Hirofumi

2012-09-21

(-)-Epigallocatechin-3-O-gallate (EGCG) exhibits anti-tumor activity mediated via the 67-kDa laminin receptor (67LR). In this study, we found that 67LR protein levels are reduced by exposure to low O(2) levels (5%), without affecting the expression of HIF-1α. We also found that EGCG-induced anti-cancer activity is abrogated under low O(2) levels (5%) in various cancer cells. Notably, treatment with the proteasome inhibitor, prevented down-regulation of 67LR and restored sensitivity to EGCG under 5% O(2). In summary, 67LR expression is highly sensitive to O(2) partial pressure, and the activity of EGCG can be regulated in cancer cells by O(2) partial pressure. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

An HMGA2-IGF2BP2 Axis Regulates Myoblast Proliferation and Myogenesis

PubMed Central

Li, Zhizhong; Gilbert, Jason A.; Zhang, Yunyu; Zhang, Minsi; Qiu, Qiong; Ramanujan, Krishnan; Shavlakadze, Tea; Eash, John K.; Scaramozza, Annarita; Goddeeris, Matthew M.; Kirsch, David G.; Campbell, Kevin P.; Brack, Andrew S.; Glass, David J.

2013-01-01

Summary A group of genes that are highly and specifically expressed in proliferating skeletal myoblasts during myogenesis was identified. Expression of one of these genes, Hmga2, increases coincident with satellite cell activation, and later its expression significantly declines correlating with fusion of myoblasts into myotubes. Hmga2 knockout mice exhibit impaired muscle development and reduced myoblast proliferation, while overexpression of HMGA2 promotes myoblast growth. This perturbation in proliferation can be explained by the finding that HMGA2 directly regulates the RNA-binding protein IGF2BP2. Add-back of IGF2BP2 rescues the phenotype. IGF2BP2 in turn binds to and controls the translation of a set of mRNAs, including c-myc, Sp1, and Igf1r. These data demonstrate that the HMGA2-IGF2BP2 axis functions as a key regulator of satellite cell activation and therefore skeletal muscle development. PMID:23177649

Follistatin-like 1 expression is decreased in the alveolar epithelium of hypoplastic rat lungs with nitrofen-induced congenital diaphragmatic hernia.

PubMed

Takahashi, Toshiaki; Zimmer, Julia; Friedmacher, Florian; Puri, Prem

2017-05-01

Pulmonary hypoplasia (PH), characterized by incomplete alveolar development, remains a major therapeutic challenge associated with congenital diaphragmatic hernia (CDH). Follistatin-like 1 (Fstl1) is a crucial regulator of alveolar formation and maturation, which is strongly expressed in distal airway epithelium. Fstl1-deficient mice exhibit reduced airspaces, impaired alveolar epithelial cell differentiation, and insufficient production of surfactant proteins similar to PH in human CDH. We hypothesized that pulmonary Fstl1 expression is decreased during alveolarization in the nitrofen-induced CDH model. Timed-pregnant rats received nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D18 and D21 and divided into control-/nitrofen-exposed specimens. Alveolarization was assessed using morphometric analysis techniques. Pulmonary gene expression of Fstl1 was determined by qRT-PCR. Immunofluorescence-double-staining for Fstl1 and alveolar epithelial marker surfactant protein C (SP-C) was performed to evaluate protein expression/localization. Radial alveolar count was significantly reduced in hypoplastic lungs of nitrofen-exposed fetuses with significant down regulation of Fstl1 mRNA expression on D18 and D21 compared to controls. Confocal-laser-scanning-microscopy revealed strikingly diminished Fstl1 immunofluorescence and SP-C expression in distal alveolar epithelium of nitrofen-exposed fetuses with CDH-associated PH on D18 and D21 compared to controls. Decreased expression of Fstl1 in alveolar epithelium may disrupt alveolarization and pulmonary surfactant production, thus contributing to the development of PH in the nitrofen-induced CDH model. 2b (Centre for Evidence-Based Medicine, Oxford). Copyright © 2017 Elsevier Inc. All rights reserved.

TMEM106B expression is reduced in Alzheimer’s disease brains

PubMed Central

2014-01-01

Introduction TMEM106B is a transmembrane glycoprotein of unknown function located within endosome/lysosome compartments expressed ubiquitously in various cell types. Previously, the genome-wide association study (GWAS) identified a significant association of TMEM106B single nucleotide polymorphisms (SNPs) with development of frontotemporal lobar degeneration with ubiquitinated TAR DNA-binding protein-43 (TDP-43)-positive inclusions (FTLD-TDP), particularly in the patients exhibiting the progranulin (PGRN) gene (GRN) mutations. Recent studies indicate that TMEM106B plays a pathological role in various neurodegenerative diseases, including Alzheimer’s disease (AD). However, at present, the precise levels of TMEM106B expression in AD brains remain unknown. Methods By quantitative reverse transcription (RT)-PCR (qPCR), western blot and immunohistochemistry, we studied TMEM106B and PGRN expression levels in a series of AD and control brains, including amyotrophic lateral sclerosis, Parkinson’s disease, multiple system atrophy and non-neurological cases. Results In AD brains, TMEM106B mRNA and protein levels were significantly reduced, whereas PGRN mRNA levels were elevated, compared with the levels in non-AD brains. In all brains, TMEM106B was expressed in the majority of cortical neurons, hippocampal neurons, and some populations of oligodendrocytes, reactive astrocytes and microglia with the location in the cytoplasm. In AD brains, surviving neurons expressed intense TMEM106B immunoreactivity, while senile plaques, neurofibrillary tangles and the perivascular neuropil, almost devoid of TMEM106B, intensely expressed PGRN. Conclusions We found an inverse relationship between TMEM106B (downregulation) and PGRN (upregulation) expression levels in AD brains, suggesting a key role of TMEM106B in the pathological processes of AD. PMID:24684749

Cortisol increases CXCR4 expression but does not affect CD62L and CCR7 levels on specific T cell subsets in humans.

PubMed

Besedovsky, Luciana; Linz, Barbara; Dimitrov, Stoyan; Groch, Sabine; Born, Jan; Lange, Tanja

2014-06-01

Glucocorticoids are well known to affect T cell migration, leading to a redistribution of the cells from blood to the bone marrow, accompanied by a concurrent suppression of lymph node homing. Despite numerous studies in this context, with most of them employing synthetic glucocorticoids in nonphysiological doses, the mechanisms of this redistribution are not well understood. Here, we investigated in healthy men the impact of cortisol at physiological concentrations on the expression of different migration molecules on eight T cell subpopulations in vivo and in vitro. Hydrocortisone (cortisol, 22 mg) infused during nocturnal rest when endogenous cortisol levels are low, compared with placebo, differentially reduced numbers of T cell subsets, with naive CD4(+) and CD8(+) subsets exhibiting the strongest reduction. Hydrocortisone in vivo and in vitro increased CXCR4 expression, which presumably mediates the recruitment of T cells to the bone marrow. Expression of the lymph node homing receptor CD62L on total CD3(+) and CD8(+) T cells appeared reduced following hydrocortisone infusion. However, this was due to a selective extravasation of CD62L(+) T cell subsets, as hydrocortisone affected neither CD62L expression on a subpopulation level nor CD62L expression in vitro. Corresponding results in the opposite direction were observed after blocking of endogenous cortisol synthesis by metyrapone. CCR7, another lymph node homing receptor, was also unaffected by hydrocortisone in vitro. Thus, cortisol seems to redirect T cells to the bone marrow by upregulating their CXCR4 expression, whereas its inhibiting effect on T cell homing to lymph nodes is apparently regulated independently of the expression of classical homing receptors. Copyright © 2014 the American Physiological Society.

Amyloid precursor protein regulates migration and metalloproteinase gene expression in prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyazaki, Toshiaki; Ikeda, Kazuhiro; Horie-Inoue, Kuniko

Highlights: • APP knockdown reduced proliferation and migration of prostate cancer cells. • APP knockdown reduced expression of metalloproteinase and EMT-related genes. • APP overexpression promoted LNCaP cell migration. • APP overexpression increased expression of metalloproteinase and EMT-related genes. - Abstract: Amyloid precursor protein (APP) is a type I transmembrane protein, and one of its processed forms, β-amyloid, is considered to play a central role in the development of Alzheimer’s disease. We previously showed that APP is a primary androgen-responsive gene in prostate cancer and that its increased expression is correlated with poor prognosis for patients with prostate cancer. APPmore » has also been implicated in several human malignancies. Nevertheless, the mechanism underlying the pro-proliferative effects of APP on cancers is still not well-understood. In the present study, we explored a pathophysiological role for APP in prostate cancer cells using siRNA targeting APP (siAPP). The proliferation and migration of LNCaP and DU145 prostate cancer cells were significantly suppressed by siAPP. Differentially expressed genes in siAPP-treated cells compared to control siRNA-treated cells were identified by microarray analysis. Notably, several metalloproteinase genes, such as ADAM10 and ADAM17, and epithelial–mesenchymal transition (EMT)-related genes, such as VIM, and SNAI2, were downregulated in siAPP-treated cells as compared to control cells. The expression of these genes was upregulated in LNCaP cells stably expressing APP when compared with control cells. APP-overexpressing LNCaP cells exhibited enhanced migration in comparison to control cells. These results suggest that APP may contribute to the proliferation and migration of prostate cancer cells by modulating the expression of metalloproteinase and EMT-related genes.« less

Omega-3 fatty acid deficient male rats exhibit abnormal behavioral activation in the forced swim test following chronic fluoxetine treatment: association with altered 5-HT1A and alpha2A adrenergic receptor expression.

PubMed

Able, Jessica A; Liu, Yanhong; Jandacek, Ronald; Rider, Therese; Tso, Patrick; McNamara, Robert K

2014-03-01

Omega-3 fatty acid deficiency during development leads to enduing alterations in central monoamine neurotransmission in rat brain. Here we investigated the effects of omega-3 fatty acid deficiency on behavioral and neurochemical responses to chronic fluoxetine (FLX) treatment. Male rats were fed diets with (CON, n = 34) or without (DEF, n = 30) the omega-3 fatty acid precursor alpha-linolenic acid (ALA) during peri-adolescent development (P21-P90). A subset of CON (n = 14) and DEF (n = 12) rats were administered FLX (10 mg/kg/d) through their drinking water for 30 d beginning on P60. The forced swimming test (FST) was initiated on P90, and regional brain mRNA markers of serotonin and noradrenaline neurotransmission were determined. Dietary ALA depletion led to significant reductions in frontal cortex docosahexaenoic acid (DHA, 22:6n-3) composition in DEF (-26%, p = 0.0001) and DEF + FLX (-32%, p = 0.0001) rats. Plasma FLX and norfluoxetine concentrations did not different between FLX-treated DEF and CON rats. During the 15-min FST pretest, DEF + FLX rats exhibited significantly greater climbing behavior compared with CON + FLX rats. During the 5-min test trial, FLX treatment reduced immobility and increased swimming in CON and DEF rats, and only DEF + FLX rats exhibited significant elevations in climbing behavior. DEF + FLX rats exhibited greater midbrain, and lower frontal cortex, 5-HT1A mRNA expression compared with all groups including CON + FLX rats. DEF + FLX rats also exhibited greater midbrain alpha2A adrenergic receptor mRNA expression which was positively correlated with climbing behavior in the FST. These preclinical data demonstrate that low omega-3 fatty acid status leads to abnormal behavioral and neurochemical responses to chronic FLX treatment in male rats. Copyright © 2013 Elsevier Ltd. All rights reserved.

Jasmonates act positively in adventitious root formation in petunia cuttings.

PubMed

Lischweski, Sandra; Muchow, Anne; Guthörl, Daniela; Hause, Bettina

2015-09-22

Petunia is a model to study the process of adventitious root (AR) formation on leafy cuttings. Excision of cuttings leads to a transient increase in jasmonates, which is regarded as an early, transient and critical event for rooting. Here, the role of jasmonates in AR formation on petunia cuttings has been studied by a reverse genetic approach. To reduce the endogenous levels of jasmonates, transgenic plants were generated expressing a Petunia hybrida ALLENE OXIDE CYCLASE (PhAOC)-RNAi construct. The transgenic plants exhibited strongly reduced PhAOC transcript and protein levels as well as diminished accumulation of cis-12-oxo-phytodienoic acid, jasmonic acid and jasmonoyl-isoleucine after wounding in comparison to wild type and empty vector expressing plants. Reduced levels of endogenous jasmonates resulted in formation of lower numbers of ARs. However, this effect was not accompanied by altered levels of auxin and aminocyclopropane carboxylate (ACC, precursor of ethylene) or by impaired auxin and ethylene-induced gene expression. Neither activity of cell-wall invertases nor accumulation of soluble sugars was altered by jasmonate deficiency. Diminished numbers of AR in JA-deficient cuttings suggest that jasmonates act as positive regulators of AR formation in petunia wild type. However, wound-induced rise in jasmonate levels in petunia wild type cuttings seems not to be causal for increased auxin and ethylene levels and for sink establishment.

Cetuximab Resistance in Head and Neck Cancer Is Mediated by EGFR-K521 Polymorphism.

PubMed

Braig, Friederike; Kriegs, Malte; Voigtlaender, Minna; Habel, Beate; Grob, Tobias; Biskup, Karina; Blanchard, Veronique; Sack, Markus; Thalhammer, Anja; Ben Batalla, Isabel; Braren, Ingke; Laban, Simon; Danielczyk, Antje; Goletz, Steffen; Jakubowicz, Elzbieta; Märkl, Bruno; Trepel, Martin; Knecht, Rainald; Riecken, Kristoffer; Fehse, Boris; Loges, Sonja; Bokemeyer, Carsten; Binder, Mascha

2017-03-01

Head and neck squamous cell carcinomas (HNSCC) exhibiting resistance to the EGFR-targeting drug cetuximab poses a challenge to their effective clinical management. Here, we report a specific mechanism of resistance in this setting based upon the presence of a single nucleotide polymorphism encoding EGFR-K 521 (K-allele), which is expressed in >40% of HNSCC cases. Patients expressing the K-allele showed significantly shorter progression-free survival upon palliative treatment with cetuximab plus chemotherapy or radiation. In several EGFR-mediated cancer models, cetuximab failed to inhibit downstream signaling or to kill cells harboring a high K-allele frequency. Cetuximab affinity for EGFR-K 521 was reduced slightly, but ligand-mediated EGFR activation was intact. We found a lack of glycan sialyation on EGFR-K 521 that associated with reduced protein stability, suggesting a structural basis for reduced cetuximab efficacy. CetuGEX, an antibody with optimized Fc glycosylation targeting the same epitope as cetuximab, restored HNSCC sensitivity in a manner associated with antibody-dependent cellular cytotoxicity rather than EGFR pathway inhibition. Overall, our results highlight EGFR-K 521 expression as a key mechanism of cetuximab resistance to evaluate prospectively as a predictive biomarker in HNSCC patients. Further, they offer a preclinical rationale for the use of ADCC-optimized antibodies to treat tumors harboring this EGFR isoform. Cancer Res; 77(5); 1188-99. ©2016 AACR . ©2016 American Association for Cancer Research.

Reduced Notch signalling leads to postnatal skeletal muscle hypertrophy in Pofut1cax/cax mice.

PubMed

Al Jaam, Bilal; Heu, Katy; Pennarubia, Florian; Segelle, Alexandre; Magnol, Laetitia; Germot, Agnès; Legardinier, Sébastien; Blanquet, Véronique; Maftah, Abderrahman

2016-09-01

Postnatal skeletal muscle growth results from the activation of satellite cells and/or an increase in protein synthesis. The Notch signalling pathway maintains satellite cells in a quiescent state, and once activated, sustains their proliferation and commitment towards differentiation. In mammals, POFUT1-mediated O-fucosylation regulates the interactions between NOTCH receptors and ligands of the DELTA/JAGGED family, thus initiating the activation of canonical Notch signalling. Here, we analysed the consequences of downregulated expression of the Pofut1 gene on postnatal muscle growth in mutant Pofut1(cax/cax) (cax, compact axial skeleton) mice and differentiation of their satellite cell-derived myoblasts (SCDMs). Pofut1(cax/cax) mice exhibited muscle hypertrophy, no hyperplasia and a decrease in satellite cell numbers compared with wild-type C3H mice. In agreement with these observations, Pofut1(cax/cax) SCDMs differentiated earlier concomitant with reduced Pax7 expression and decrease in PAX7(+)/MYOD(-) progenitor cells. In vitro binding assays showed a reduced interaction of DELTA-LIKE 1 ligand (DLL1) with NOTCH receptors expressed at the cell surface of SCDMs, leading to a decreased Notch signalling as seen by the quantification of cleaved NICD and Notch target genes. These results demonstrated that POFUT1-mediated O-fucosylation of NOTCH receptors regulates myogenic cell differentiation and affects postnatal muscle growth in mice. © 2016 The Authors.

Reduced Notch signalling leads to postnatal skeletal muscle hypertrophy in Pofut1cax/cax mice

PubMed Central

Al Jaam, Bilal; Heu, Katy; Pennarubia, Florian; Segelle, Alexandre; Magnol, Laetitia; Germot, Agnès; Blanquet, Véronique; Maftah, Abderrahman

2016-01-01

Postnatal skeletal muscle growth results from the activation of satellite cells and/or an increase in protein synthesis. The Notch signalling pathway maintains satellite cells in a quiescent state, and once activated, sustains their proliferation and commitment towards differentiation. In mammals, POFUT1-mediated O-fucosylation regulates the interactions between NOTCH receptors and ligands of the DELTA/JAGGED family, thus initiating the activation of canonical Notch signalling. Here, we analysed the consequences of downregulated expression of the Pofut1 gene on postnatal muscle growth in mutant Pofut1cax/cax (cax, compact axial skeleton) mice and differentiation of their satellite cell-derived myoblasts (SCDMs). Pofut1cax/cax mice exhibited muscle hypertrophy, no hyperplasia and a decrease in satellite cell numbers compared with wild-type C3H mice. In agreement with these observations, Pofut1cax/cax SCDMs differentiated earlier concomitant with reduced Pax7 expression and decrease in PAX7+/MYOD− progenitor cells. In vitro binding assays showed a reduced interaction of DELTA-LIKE 1 ligand (DLL1) with NOTCH receptors expressed at the cell surface of SCDMs, leading to a decreased Notch signalling as seen by the quantification of cleaved NICD and Notch target genes. These results demonstrated that POFUT1-mediated O-fucosylation of NOTCH receptors regulates myogenic cell differentiation and affects postnatal muscle growth in mice. PMID:27628322

The bHLH transcription factor GmPIB1 facilitates resistance to Phytophthora sojae in Glycine max

PubMed Central

Cheng, Qun; Dong, Lidong; Gao, Tianjiao; Liu, Tengfei; Li, Ninghui; Wang, Le; Chang, Xin; Wu, Junjiang; Xu, Pengfei

2018-01-01

Abstract Phytophthora sojae Kaufmann and Gerdemann causes Phytophthora root rot, a destructive soybean disease worldwide. A basic helix–loop–helix (bHLH) transcription factor is thought to be involved in the response to P. sojae infection in soybean, as revealed by RNA sequencing (RNA-seq). However, the molecular mechanism underlying this response is currently unclear. Here, we explored the function and underlying mechanisms of a bHLH transcription factor in soybean, designated GmPIB1 (P. sojae-inducible bHLH transcription factor), during host responses to P. sojae. GmPIB1 was significantly induced by P. sojae in the resistant soybean cultivar ‘L77-1863’. Analysis of transgenic soybean hairy roots with elevated or reduced expression of GmPIB1 demonstrated that GmPIB1 enhances resistance to P. sojae and reduces reactive oxygen species (ROS) accumulation. Quantitative reverse transcription PCR and chromatin immunoprecipitation–quantitative PCR assays revealed that GmPIB1 binds directly to the promoter of GmSPOD1 and represses its expression; this gene encodes a key enzyme in ROS production. Moreover, transgenic soybean hairy roots with GmSPOD1 silencing through RNA interference exhibited improved resistance to P. sojae and reduced ROS generation. These findings suggest that GmPIB1 enhances resistance to P. sojae by repressing the expression of GmSPOD1. PMID:29579245

Prader-Willi Critical Region, a Non-Translated, Imprinted Central Regulator of Bone Mass: Possible Role in Skeletal Abnormalities in Prader-Willi Syndrome.

PubMed

Khor, Ee-Cheng; Fanshawe, Bruce; Qi, Yue; Zolotukhin, Sergei; Kulkarni, Rishikesh N; Enriquez, Ronaldo F; Purtell, Louise; Lee, Nicola J; Wee, Natalie K; Croucher, Peter I; Campbell, Lesley; Herzog, Herbert; Baldock, Paul A

2016-01-01

Prader-Willi Syndrome (PWS), a maternally imprinted disorder and leading cause of obesity, is characterised by insatiable appetite, poor muscle development, cognitive impairment, endocrine disturbance, short stature and osteoporosis. A number of causative loci have been located within the imprinted Prader-Willi Critical Region (PWCR), including a set of small non-translated nucleolar RNA's (snoRNA). Recently, micro-deletions in humans identified the snoRNA Snord116 as a critical contributor to the development of PWS exhibiting many of the classical symptoms of PWS. Here we show that loss of the PWCR which includes Snord116 in mice leads to a reduced bone mass phenotype, similar to that observed in humans. Consistent with reduced stature in PWS, PWCR KO mice showed delayed skeletal development, with shorter femurs and vertebrae, reduced bone size and mass in both sexes. The reduction in bone mass in PWCR KO mice was associated with deficiencies in cortical bone volume and cortical mineral apposition rate, with no change in cancellous bone. Importantly, while the length difference was corrected in aged mice, consistent with continued growth in rodents, reduced cortical bone formation was still evident, indicating continued osteoblastic suppression by loss of PWCR expression in skeletally mature mice. Interestingly, deletion of this region included deletion of the exclusively brain expressed Snord116 cluster and resulted in an upregulation in expression of both NPY and POMC mRNA in the arcuate nucleus. Importantly, the selective deletion of the PWCR only in NPY expressing neurons replicated the bone phenotype of PWCR KO mice. Taken together, PWCR deletion in mice, and specifically in NPY neurons, recapitulates the short stature and low BMD and aspects of the hormonal imbalance of PWS individuals. Moreover, it demonstrates for the first time, that a region encoding non-translated RNAs, expressed solely within the brain, can regulate bone mass in health and disease.

Expression of bioactive recombinant human fibroblast growth factor 10 in Carthamus tinctorius L. seeds.

PubMed

Huang, Jian; Yang, Jing; Guan, Lili; Yi, Shanyong; Du, Linna; Tian, Haishan; Guo, Yongxin; Zhai, Feng; Lu, Zhen; Li, Haiyan; Li, Xiaokun; Jiang, Chao

2017-10-01

Fibroblast growth factor 10 (FGF10) is a member of the FGF superfamily. It exhibits diverse biological functions, and is extensively used for fundamental research and clinical applications involving hair growth, tissue repair, and burn wounds. Oil bodies, obtained from oil seeds, have been exploited for a variety of biotechnology applications. The use of oil bodies reduces purification steps and costs associated with the production of heterogonous proteins. Here, recombinant human FGF10 (rhFGF10) was expressed in safflower (Carthamus tinctorius L.) seeds using oilbody-oleosin technology. A plant expression vector, pOTBar-oleosin-rhFGF10, was constructed and introduced into safflower using Agrobacterium tumefaciens transformation, and mature safflower plants were obtained by grafting. Oleosin-rhFGF10 was successfully transformed and expressed in safflower seeds and inherited to the T 3 generation. Moreover, MTT assays demonstrated that oil bodies expressed oleosin-FGF10 had a dose-dependent effect on cellular proliferation. In conclusion, this may provide a method of producing oleosin-rhFGF10, and help us meet the increasing pharmacological demands for the protein. Copyright © 2016. Published by Elsevier Inc.

Induction of expression and co-localization of heat shock polypeptides with the polyalanine expansion mutant of poly(A)-binding protein N1 after chemical stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Qishan; Bag, Jnanankur

Formation of nuclear inclusions consisting of aggregates of a polyalanine expansion mutant of nuclear poly(A)-binding protein (PABPN1) is the hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a late onset autosomal dominant disease. Patients with this disorder exhibit progressive swallowing difficulty and drooping of their eye lids, which starts around the age of 50. Previously we have shown that treatment of cells expressing the mutant PABPN1 with a number of chemicals such as ibuprofen, indomethacin, ZnSO{sub 4}, and 8-hydroxy-quinoline induces HSP70 expression and reduces PABPN1 aggregation. In these studies we have shown that expression of additional HSPs including HSP27, HSP40,more » and HSP105 were induced in mutant PABPN1 expressing cells following exposure to the chemicals mentioned above. Furthermore, all three additional HSPs were translocated to the nucleus and probably helped to properly fold the mutant PABPN1 by co-localizing with this protein.« less

Glycine Betaine Catabolism Contributes to Pseudomonas syringae Tolerance to Hyperosmotic Stress by Relieving Betaine-Mediated Suppression of Compatible Solute Synthesis

PubMed Central

Li, Shanshan; Yu, Xilan

2013-01-01

Many bacteria can accumulate glycine betaine for osmoprotection and catabolize it as a growth substrate, but how they regulate these opposing roles is poorly understood. In Pseudomonas syringae B728a, expression of the betaine catabolism genes was reduced by an osmotic upshift to an intermediate stress level, consistent with betaine accumulation, but was increased by an upshift to a high stress level, as confirmed by an accompanying increase in degradation of radiolabeled betaine. Deletion of the gbcAB betaine catabolism genes reduced osmotolerance at a high osmolarity, and this reduction was due to the relief of betaine-mediated suppression of compatible solute synthesis. This conclusion was supported by the findings that, at high osmolarity, the ΔgbcAB mutant accumulated high betaine levels and low endogenous solutes and exhibited reduced expression of the solute synthesis genes. Moreover, the ΔgbcAB mutant and a mutant deficient in the synthesis of the compatible solutes NAGGN and trehalose exhibited similar reductions in osmotolerance and also in fitness on bean leaves. Activation of betaine catabolism at high osmotic stress resulted, in part, from induction of gbdR, which encodes the transcriptional activator GbdR. Betaine catabolism was subject to partial repression by succinate under hyperosmotic stress conditions, in contrast to strong repression in the absence of stress, suggesting that betaine functions both in nutrition and as an intracellular signal modulating solute synthesis under hyperosmotic stress conditions. Collectively, these results begin to provide a detailed mechanistic understanding of how P. syringae transitions from reliance on exogenously derived betaine to the use of endogenous solutes during adaptation to hyperosmotic conditions. PMID:23524610

Dietary HMB and β-alanine co-supplementation does not improve in situ muscle function in sedentary, aged male rats.

PubMed

Russ, David W; Acksel, Cara; Boyd, Iva M; Maynard, John; McCorkle, Katherine W; Edens, Neile K; Garvey, Sean M

2015-12-01

This study evaluated the effects of dietary β-hydroxy-β-methylbutyrate (HMB) combined with β-alanine (β-Ala) in sedentary, aged male rats. It has been suggested that dietary HMB or β-Ala supplementation may mitigate age-related declines in muscle strength and fatigue resistance. A total of 20 aged Sprague-Dawley rats were studied. At age 20 months, 10 rats were administered a control, purified diet and 10 rats were administered a purified diet supplemented with both HMB and β-Ala (HMB+β-Ala) for 8 weeks (approximately equivalent to 3 and 2.4 g per day human dose). We measured medial gastrocnemius (MG) size, force, fatigability, and myosin composition. We also evaluated an array of protein markers related to muscle mitochondria, protein synthesis and breakdown, and autophagy. HMB+β-Ala had no significant effects on body weight, MG mass, force or fatigability, myosin composition, or muscle quality. Compared with control rats, those fed HMB+β-Ala exhibited a reduced (41%, P = 0.039) expression of muscle RING-finger protein 1 (MURF1), a common marker of protein degradation. Muscle from rats fed HMB+β-Ala also exhibited a 45% reduction (P = 0.023) in p70s6K phosphorylation following fatiguing stimulation. These data suggest that HMB+β-Ala at the dose studied may reduce muscle protein breakdown by reducing MURF1 expression, but has minimal effects on muscle function in this model of uncomplicated aging. They do not, however, rule out potential benefits of HMB+β-Ala co-supplementation at other doses or durations of supplementation in combination with exercise or in situations where extreme muscle protein breakdown and loss of mass occur (e.g., bedrest, cachexia, failure-to-thrive).

RNAi silenced Dd-grp94 (Dictyostelium discoideum glucose-regulated protein 94 kDa) cell lines in Dictyostelium exhibit marked reduction in growth rate and delay in development.

PubMed

Baviskar, Sandhya N; Shields, Malcolm S

2010-01-01

Glucose-regulated 94 kDa protein (Grp94) is a resident of the endoplasmic reticulum (ER) of multicellular eukaryotes. It is a constitutively expressed protein that is overexpressed in certain abnormal conditions of the cell such as depletion of glucose and calcium, and low oxygen and pH. The protein is also implicated in diseased conditions like cancer and Alzheimer's disease. In this study, the consequences of downregulation of Grp94 were investigated at both unicellular and multicellular stages of Dictyostelium discoideum. Previous studies have shown the expression of Dd-Grp94 (Dictyostelium discoideum glucose-regulated 94 kDa protein) in wild-type cells varies during development, and overexpression of Dd-Grp94 leads to abnormal cell shape and inhibition of development (i.e., formation of fruiting bodies). Grp94 is a known calcium binding protein and an efficient calcium buffer. Therefore, in the present study we hypothesized that downregulation of Dd-Grp94 protein would affect Dictyostelium cell structure, growth, and development. We found that Dd-grp94 RNAi recombinants exhibited reduced growth rate, cell size, and a subtle change in cell motility compared to the parental cells. The recombinants also exhibited a delay in development and small fruiting bodies. These results establish that Dd-grp94 plays a crucial role in determining normal cell structure, growth and differentiation.

A combination of sorafenib and nilotinib reduces the growth of castrate-resistant prostate cancer

PubMed Central

Archibald, Monica; Pritchard, Tara; Nehoff, Hayley; Rosengren, Rhonda J; Greish, Khaled; Taurin, Sebastien

2016-01-01

Castrate-resistant prostate cancer (CRPC) remains incurable due to the lack of effective therapies. Several tyrosine kinases have been implicated in the development and growth of CRPC, as such targeting these kinases may offer an alternative therapeutic strategy. We established the combination of two tyrosine kinase inhibitors (TKIs), sorafenib and nilotinib, as the most cytotoxic. In addtion, to improve their bioavailability and reduce their metabolism, we encapsulated sorafenib and nilotinib into styrene-co-maleic acid micelles. The micelles’ charge, size, and release rate were characterized. We assessed the effect of the combination on the cytotoxicity, cell cycle, apoptosis, protein expression, tumor spheroid integrity, migration, and invasion. The micelles exhibited a mean diameter of 100 nm, a neutral charge, and appeared highly stable. The micellar TKIs promoted greater cytotoxicity, decreased cell proliferation, and increased apoptosis relative to the free TKIs. In addition, the combination reduced the expression and activity of several tyrosine kinases and reduced tumor spheroid integrity and metastatic potential of CRPC cell lines more efficiently than the single treatments. The combination increased the therapeutic potential and demonstrated the relevance of a targeted combination therapy for the treatment of CRPC. In addition, the efficacy of the encapsulated drugs provides the basis for an in vivo preclinical testing. PMID:26811677

Demonstration of Hadoop-GIS: A Spatial Data Warehousing System Over MapReduce

PubMed Central

Aji, Ablimit; Sun, Xiling; Vo, Hoang; Liu, Qioaling; Lee, Rubao; Zhang, Xiaodong; Saltz, Joel; Wang, Fusheng

2016-01-01

The proliferation of GPS-enabled devices, and the rapid improvement of scientific instruments have resulted in massive amounts of spatial data in the last decade. Support of high performance spatial queries on large volumes data has become increasingly important in numerous fields, which requires a scalable and efficient spatial data warehousing solution as existing approaches exhibit scalability limitations and efficiency bottlenecks for large scale spatial applications. In this demonstration, we present Hadoop-GIS – a scalable and high performance spatial query system over MapReduce. Hadoop-GIS provides an efficient spatial query engine to process spatial queries, data and space based partitioning, and query pipelines that parallelize queries implicitly on MapReduce. Hadoop-GIS also provides an expressive, SQL-like spatial query language for workload specification. We will demonstrate how spatial queries are expressed in spatially extended SQL queries, and submitted through a command line/web interface for execution. Parallel to our system demonstration, we explain the system architecture and details on how queries are translated to MapReduce operators, optimized, and executed on Hadoop. In addition, we will showcase how the system can be used to support two representative real world use cases: large scale pathology analytical imaging, and geo-spatial data warehousing. PMID:27617325

The RhoGEF Net1 Is Required for Normal Mammary Gland Development

PubMed Central

Zuo, Yan; Berdeaux, Rebecca

2014-01-01

Neuroepithelial transforming gene 1 (Net1) is a RhoA subfamily-specific guanine nucleotide exchange factor that is overexpressed in human breast cancer and is required for breast cancer cell migration and invasion. However, the role of Net1 in normal mammary gland development or function has never been assessed. To understand the role of Net1 in the mammary gland, we have created a conditional Net1 knockout mouse model. Whole-body deletion of Net1 results in delayed mammary gland development during puberty characterized by slowed of ductal extension and reduced ductal branching. Epithelial cells within the developing ducts show reduced proliferation that is accompanied by diminished estrogen receptor-α expression and activity. Net1-deficient mammary glands also exhibit reduced phosphorylation of regulatory subunits of myosin light chain and myosin light-chain phosphatase, indicating that RhoA-dependent actomyosin contraction is compromised. Net1 deficiency also leads to disorganization of myoepithelial and ductal epithelial cells and increased periductal collagen deposition. Mammary epithelial cell transplantation experiments indicate that reduced ductal branching and disorganization are cell autonomous. These data identify for the first time a role for NET1 in vivo and indicate that NET1 expression is essential for the proliferation and differentiation of mammary epithelial cells in the developing mammary gland. PMID:25321414

Molecular Prerequisites for Diminished Cold Sensitivity in Ground Squirrels and Hamsters.

PubMed

Matos-Cruz, Vanessa; Schneider, Eve R; Mastrotto, Marco; Merriman, Dana K; Bagriantsev, Sviatoslav N; Gracheva, Elena O

2017-12-19

Thirteen-lined ground squirrels and Syrian hamsters are known for their ability to withstand cold during hibernation. We found that hibernators exhibit cold tolerance even in the active state. Imaging and electrophysiology of squirrel somatosensory neurons reveal a decrease in cold sensitivity of TRPM8-expressing cells. Characterization of squirrel and hamster TRPM8 showed that the channels are chemically activated but exhibit poor activation by cold. Cold sensitivity can be re-introduced into squirrel and hamster TRPM8 by transferring the transmembrane domain from the cold sensitive rat ortholog. The same can be achieved in squirrel TRPM8 by mutating only six amino acids. Reciprocal mutations suppress cold sensitivity of the rat ortholog, supporting functional significance of these residues. Our results suggest that ground squirrels and hamsters exhibit reduced cold sensitivity, partially due to modifications in the transmembrane domain of TRPM8. Our study reveals molecular adaptations that accompany cold tolerance in two species of mammalian hibernators. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Transformation of Epithelial Ovarian Cancer Stemlike Cells into Mesenchymal Lineage via EMT Results in Cellular Heterogeneity and Supports Tumor Engraftment

PubMed Central

Jiang, Hua; Lin, Xiaolong; Liu, Yingtao; Gong, Wenjia; Ma, Xiaoling; Yu, Yinhua; Xie, Yi; Sun, Xiaoxi; Feng, Youji; Janzen, Viktor; Chen, Tong

2012-01-01

Ovarian cancers are heterogeneous and contain stemlike cells that are able to self-renew and are responsible for sustained tumor growth. Metastasis in the peritoneal cavity occurs more frequently in ovarian cancer than in other malignancies, but the underlying mechanism remains largely unknown. We have identified that ovarian cancer stemlike cells (CSCs), which were defined as side population (SP) cells, were present in patients’ ascitic fluid and mesenchymally transformed cell lines, ES-2 and HO-8910PM. SP cells, which were sorted from both cell lines and implanted into immunocompromised mice, were localized to the xenografted tumor boundary. In addition, SP cells exhibited an epithelial phenotype and showed a distinct gene expression profile with reduced expression of cell adhesion molecules (CAMs), indicating that SP cells exert an important role in ovarian cancer progression on the basis of their delicate interaction with the surrounding microenvironment and anatomical localization in tumors. In contrast, non-SP cells exhibited a more mesenchymal phenotype and showed more increased invasive potential than SP cells. This heterogeneity was observed as an endogenous transformation via the epithelial–mesenchymal transition (EMT) process. Inhibition of the EMT process by Snail1 silencing reduced the SP cell frequency, and affected their invasive capacity and engraftment. These findings illustrate the interplay between epithelial ovarian CSCs and the EMT, and exert a link to explain tumor heterogeneity and its necessity for ovarian cancer maintenance, metastasis and progression. PMID:22801793

Empathy and aversion: the neural signature of mentalizing in Tourette syndrome.

PubMed

Eddy, C M; Cavanna, A E; Hansen, P C

2017-02-01

Previous studies suggest that adults with Tourette syndrome (TS) can respond unconventionally on tasks involving social cognition. We therefore hypothesized that these patients would exhibit different neural responses to healthy controls in response to emotionally salient expressions of human eyes. Twenty-five adults with TS and 25 matched healthy controls were scanned using fMRI during the standard version of the Reading the Mind in the Eyes Task which requires mental state judgements, and a novel comparison version requiring judgements about age. During prompted mental state recognition, greater activity was apparent in TS within left orbitofrontal cortex, posterior cingulate, right amygdala and right temporo-parietal junction (TPJ), while reduced activity was apparent in regions including left inferior parietal cortex. Age judgement elicited greater activity in TS within precuneus, medial prefrontal and temporal regions involved in mentalizing. The interaction between group and task revealed differential activity in areas including right inferior frontal gyrus. Task-related activity in the TPJ covaried with global ratings of the urge to tic. While recognizing mental states, adults with TS exhibit greater activity than controls in brain areas involved in the processing of negative emotion, in addition to reduced activity in regions associated with the attribution of agency. In addition, increased recruitment of areas involved in mental state reasoning is apparent in these patients when mentalizing is not a task requirement. Our findings highlight differential neural reactivity in response to emotive social cues in TS, which may interact with tic expression.

Flightless I interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling

PubMed Central

Arora, Pamma D.; Wang, Yongqiang; Bresnick, Anne; Janmey, Paul A.; McCulloch, Christopher A.

2015-01-01

We examined the role of the actin-capping protein flightless I (FliI) in collagen remodeling by mouse fibroblasts. FliI-overexpressing cells exhibited reduced spreading on collagen but formed elongated protrusions that stained for myosin10 and fascin and penetrated pores of collagen-coated membranes. Inhibition of Cdc42 blocked formation of cell protrusions. In FliI-knockdown cells, transfection with constitutively active Cdc42 did not enable protrusion formation. FliI-overexpressing cells displayed increased uptake and degradation of exogenous collagen and strongly compacted collagen fibrils, which was blocked by blebbistatin. Mass spectrometry analysis of FliI immunoprecipitates showed that FliI associated with nonmuscle myosin IIA (NMMIIA), which was confirmed by immunoprecipitation. GFP-FliI colocalized with NMMIIA at cell protrusions. Purified FliI containing gelsolin-like domains (GLDs) 1–6 capped actin filaments efficiently, whereas FliI GLD 2–6 did not. Binding assays showed strong interaction of purified FliI protein (GLD 1–6) with the rod domain of NMMIIA (kD = 0.146 μM), whereas FliI GLD 2–6 showed lower binding affinity (kD = 0.8584 μM). Cells expressing FliI GLD 2–6 exhibited fewer cell extensions, did not colocalize with NMMIIA, and showed reduced collagen uptake compared with cells expressing FliI GLD 1–6. We conclude that FliI interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling in fibroblasts. PMID:25877872

All-trans retinoic acid results in irregular repair of septa and fails to inhibit proinflammatory macrophages.

PubMed

Seifart, C; Muyal, J P; Plagens, A; Yildirim, A Ö; Kohse, K; Grau, V; Sandu, S; Reinke, C; Tschernig, T; Vogelmeier, C; Fehrenbach, H

2011-08-01

All-trans retinoic acid (ATRA) is controversially discussed in emphysema therapy. We re-evaluated ATRA in the elastase model and hypothesised that beneficial effects should be reflected by increased alveolar surface area, elastin expression and downregulation of inflammatory mediators and matrix metalloproteinases (MMPs). Emphysema was induced by porcine pancreatic elastase versus saline in Sprague-Dawley rats. On days 26-37, rats received daily intraperitoneal injections with ATRA (500 μg · kg(-1) body weight) versus olive oil. Lungs were removed at day 38. Rat alveolar epithelial L2 cells were incubated with/without elastase followed by ATRA- or vehicle-treatment, respectively. ATRA only partially ameliorated structural defects. Alveolar walls exhibited irregular architecture: increased arithmetic mean thickness, reduction in surface coverage by alveolar epithelial cells type II. ATRA only partially restored reduced soluble elastin. It tended to increase the ratio of ED1(+):ED2(+) macrophages. Bronchoalveolar lavage (BAL) cells exhibited a proinflammatory state and high expression of interleukin-1β, cytokine-induced neutrophil chemoattractant-1, tumour necrosis factor-α, nuclear factor-κB, MMP-2, MMP-9, MMP-12, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in emphysema, with ATRA exerting only few effects. MMP-7 was highly induced by ATRA in healthy but not in emphysematous lungs. ATRA reduced both MMP-2 and TIMP-1 activity in BAL fluid of emphysematous lungs. ATRA-therapy may bear the risk of unwanted side-effects on alveolar septal architecture in emphysematous lungs.

RNASeq-derived transcriptome comparisons reveal neuromodulatory deficiency in the CO2 insensitive brown Norway rat

PubMed Central

Puissant, Madeleine M; Echert, Ashley E; Yang, Chun; Mouradian, Gary C; Novotny, Tyler; Liu, Pengyuan; Liang, Mingyu; Hodges, Matthew R

2015-01-01

Raphé-derived serotonin (5-HT) and thyrotropin-releasing hormone (TRH) play important roles in fundamental, homeostatic control systems such as breathing and specifically the ventilatory CO2 chemoreflex. Brown Norway (BN) rats exhibit an inherent and severe ventilatory insensitivity to hypercapnia but also exhibit relatively normal ventilation at rest and during other conditions, similar to multiple genetic models of 5-HT system dysfunction in mice. Herein, we tested the hypothesis that the ventilatory insensitivity to hypercapnia in BN rats is due to altered raphé gene expression and the consequent deficiencies in raphé-derived neuromodulators such as TRH. Medullary raphé transcriptome comparisons revealed lower expression of multiple 5-HT neuron-specific genes in BN compared to control Dahl salt-sensitive rats, predictive of reduced central nervous system monoamines by bioinformatics analyses and confirmed by high-performance liquid chromatography measurements. In particular, raphé Trh mRNA and peptide levels were significantly reduced in BN rats, and injections of the stable TRH analogue Taltirelin (TAL) stimulated breathing dose-dependently, with greater effects in BN versus control Sprague–Dawley rats. Importantly, TAL also effectively normalized the ventilatory CO2 chemoreflex in BN rats, but TAL did not affect CO2 sensitivity in control Sprague–Dawley rats. These data establish a molecular basis of the neuromodulatory deficiency in BN rats, and further suggest an important functional role for TRH signalling in the mammalian CO2 chemoreflex. PMID:25630262

Variable salinity responses of 12 alfalfa genotypes and comparative expression analyses of salt-response genes

PubMed Central

Sandhu, Devinder; Cornacchione, Monica V.; Ferreira, Jorge F. S.; Suarez, Donald L.

2017-01-01

Twelve alfalfa genotypes that were selected for biomass under salinity, differences in Na and Cl concentrations in shoots and K/Na ratio were evaluated in this long-term salinity experiment. The selected plants were cloned to reduce genetic variability within each genotype. Salt tolerance (ST) index of the genotypes ranged from 0.39 to 1. The most salt-tolerant genotypes SISA14-1 (G03) and AZ-90ST (G10), the top performers for biomass, exhibited the least effect on shoot number and height. SISA14-1 (G03) accumulated low Na and Cl under salinity. Most genotypes exhibited a net reduction in shoot Ca, Mg, P, Fe, and Cu, while Mn and Zn increased under salinity. Salinity reduced foliar area and stomatal conductance; while net photosynthetic rate and transpiration were not affected. Interestingly, salinity increased chlorophyll and antioxidant capacity in most genotypes; however neither parameter correlated well to ST index. Salt-tolerant genotypes showed upregulation of the SOS1, SOS2, SOS3, HKT1, AKT1, NHX1, P5CS1, HSP90.7, HSP81.2, HSP71.1, HSPC025, OTS1, SGF29 and SAL1 genes. Gene expression analyses allowed us to classify genotypes based on their ability to regulate different components of the salt tolerance mechanism. Pyramiding different components of the salt tolerance mechanism may lead to superior salt-tolerant alfalfa genotypes. PMID:28225027

Effects of intrauterine growth restriction during late pregnancy on the cell apoptosis and related gene expression in ovine fetal liver.

PubMed

Liu, Yingchun; Ma, Chi; Li, Hui; Li, Lingyao; Gao, Feng; Ao, Changjin

2017-03-01

This study investigated the effect of intrauterine growth restriction (IUGR) during late pregnancy on the cell apoptosis and related gene expression in ovine fetal liver. Eighteen time-mated Mongolian ewes with singleton fetuses were allocated to three groups at d 90 of pregnancy: Restricted Group 1 (RG1, 0.18 MJ ME kg BW -0.75  d -1 , n = 6), Restricted Group 2 (RG2, 0.33 MJ ME kg BW -0.75  d -1 , n = 6) and a Control Group (CG, ad libitum, 0.67 MJ ME kg BW -0.75  d -1 , n = 6). Fetuses were recovered at slaughter on d 140. Fetal liver weight, DNA content and protein/DNA ratio, proliferation index, cytochrome c, activities of Caspase-3, 8, and 9 were examined, along with relative expression of genes related to apoptosis. Fetuses in both restricted groups exhibited decreased BW, hepatic weight, DNA content, and protein/DNA ratio when compared to CG (P < 0.05), as well as reduced proliferation index (P < 0.05). However, the increased numbers of apoptotic cells in fetal liver were observed in both restricted groups (P < 0.05). Fetuses with severe IUGR (RG1) exhibited increased (P < 0.05) activities of Caspase-3, 8, 9, as higher levels of mitochondrial cytochrome c in fetal liver; intermediate changes were found in RG2 fetuses, but the difference were not significant (P > 0.05). Hepatic expression of gene related to apoptosis showed reduced protein 21 (P21), B-cell lymphoma 2 (Bcl-2) and apoptosis antigen 1 ligand (FasL) expression in RG1 and RG2 (P < 0.05). In contrast, the increased hepatic expression of protein 53 (P53), Bcl-2 associated X protein (Bax) and apoptosis antigen 1 (Fas) in both IUGR fetuses were found (P < 0.05). These results indicate that the fetal hepatocyte proliferation were arrested in G1 cell cycle, and the fetal hepatocyte apoptosis was sensitive to the IUGR resulted from maternal undernutrition. The cell apoptosis in IUGR fetal liver were the potential mechanisms for its retarded proliferation and impaired development. Copyright © 2016 Elsevier Inc. All rights reserved.

Developmental Stage-Specific Manifestations of Absent TPO/c-MPL Signalling in Newborn Mice.

PubMed

Lorenz, Viola; Ramsey, Haley; Liu, Zhi-Jian; Italiano, Joseph; Hoffmeister, Karin; Bihorel, Sihem; Mager, Donald; Hu, Zhongbo; Slayton, William B; Kile, Benjamin T; Sola-Visner, Martha; Ferrer-Marin, Francisca

2017-12-01

Congenital amegakaryocytic thrombocytopaenia (CAMT) is a disorder caused by c-MPL mutations that impair thrombopoietin (TPO) signalling, resulting in a near absence of megakaryocytes (MKs). While this phenotype is consistent in adults, neonates with CAMT can present with severe thrombocytopaenia despite normal MK numbers. To investigate this, we characterized MKs and platelets in newborn c-MPL –/– mice. Liver MKs in c-MPL –/– neonates were reduced in number and size compared with wild-type (WT) age-matched MKs, and exhibited ultrastructural abnormalities not found in adult c-MPL –/– MKs. Platelet counts were lower in c-MPL –/– compared with WT mice at birth and did not increase over the first 2 weeks of life. In vivo biotinylation revealed a significant reduction in the platelet half-life of c-MPL –/– newborn mice (P2) compared with age-matched WT pups, which was not associated with ultrastructural abnormalities. Genetic deletion of the pro-apoptotic Bak did not rescue the severely reduced platelet half-life of c-MPL –/– newborn mice, suggesting that it was due to factors other than platelets entering apoptosis early. Indeed, adult GFP+ (green fluorescent protein transgenic) platelets transfused into thrombocytopenic c-MPL –/– P2 pups also had a shortened lifespan, indicating the importance of cell-extrinsic factors. In addition, neonatal platelets from WT and c-MPL –/– mice exhibited reduced P-selectin surface expression following stimulation compared with adult platelets of either genotype, and platelets from c-MPL –/– neonates exhibited reduced glycoprotein IIb/IIIa (GPIIb/IIIa) activation in response to thrombin compared with age-matched WT platelets. Taken together, our findings indicate that c-MPL deficiency is associated with abnormal maturation of neonatal MKs and developmental stage-specific defects in platelet function.

The Proprotein Convertase Subtilisin/Kexin FurinA Regulates Zebrafish Host Response against Mycobacterium marinum

PubMed Central

Ojanen, Markus J. T.; Turpeinen, Hannu; Cordova, Zuzet M.; Hammarén, Milka M.; Harjula, Sanna-Kaisa E.; Parikka, Mataleena; Rämet, Mika

2015-01-01

Tuberculosis is a chronic bacterial disease with a complex pathogenesis. An effective immunity against Mycobacterium tuberculosis requires both the innate and adaptive immune responses, including proper T helper (Th) type 1 cell function. FURIN is a proprotein convertase subtilisin/kexin (PCSK) enzyme, which is highly expressed in Th1 type cells. FURIN expression in T cells is essential for maintaining peripheral immune tolerance, but its role in the innate immunity and infections has remained elusive. Here, we utilized Mycobacterium marinum infection models in zebrafish (Danio rerio) to investigate how furin regulates host responses against mycobacteria. In steady-state furinAtd204e/+ fish reduced furinA mRNA levels associated with low granulocyte counts and elevated Th cell transcription factor expressions. Silencing furin genes reduced the survival of M. marinum-infected zebrafish embryos. A mycobacterial infection upregulated furinA in adult zebrafish, and infected furinAtd204e/+ mutants exhibited a proinflammatory phenotype characterized by elevated tumor necrosis factor a (tnfa), lymphotoxin alpha (lta) and interleukin 17a/f3 (il17a/f3) expression levels. The enhanced innate immune response in the furinAtd204e/+ mutants correlated with a significantly decreased bacterial burden in a chronic M. marinum infection model. Our data show that upregulated furinA expression can serve as a marker for mycobacterial disease, since it inhibits early host responses and consequently promotes bacterial growth in a chronic infection. PMID:25624351

SigEMD: A powerful method for differential gene expression analysis in single-cell RNA sequencing data.

PubMed

Wang, Tianyu; Nabavi, Sheida

2018-04-24

Differential gene expression analysis is one of the significant efforts in single cell RNA sequencing (scRNAseq) analysis to discover the specific changes in expression levels of individual cell types. Since scRNAseq exhibits multimodality, large amounts of zero counts, and sparsity, it is different from the traditional bulk RNA sequencing (RNAseq) data. The new challenges of scRNAseq data promote the development of new methods for identifying differentially expressed (DE) genes. In this study, we proposed a new method, SigEMD, that combines a data imputation approach, a logistic regression model and a nonparametric method based on the Earth Mover's Distance, to precisely and efficiently identify DE genes in scRNAseq data. The regression model and data imputation are used to reduce the impact of large amounts of zero counts, and the nonparametric method is used to improve the sensitivity of detecting DE genes from multimodal scRNAseq data. By additionally employing gene interaction network information to adjust the final states of DE genes, we further reduce the false positives of calling DE genes. We used simulated datasets and real datasets to evaluate the detection accuracy of the proposed method and to compare its performance with those of other differential expression analysis methods. Results indicate that the proposed method has an overall powerful performance in terms of precision in detection, sensitivity, and specificity. Copyright © 2018 Elsevier Inc. All rights reserved.

Sensitivity of Lipid Metabolism and Insulin Signaling to Genetic Alterations in Hepatic Peroxisome Proliferator–Activated Receptor-γ Coactivator-1α Expression

PubMed Central

Estall, Jennifer L.; Kahn, Mario; Cooper, Marcus P.; Fisher, ffolliott Martin; Wu, Michele K.; Laznik, Dina; Qu, Lishu; Cohen, David E.; Shulman, Gerald I.; Spiegelman, Bruce M.

2009-01-01

OBJECTIVE The peroxisome proliferator–activated receptor-γ coactivator (PGC)-1 family of transcriptional coactivators controls hepatic function by modulating the expression of key metabolic enzymes. Hepatic gain of function and complete genetic ablation of PGC-1α show that this coactivator is important for activating the programs of gluconeogenesis, fatty acid oxidation, oxidative phosphorylation, and lipid secretion during times of nutrient deprivation. However, how moderate changes in PGC-1α activity affect metabolism and energy homeostasis has yet to be determined. RESEARCH DESIGN AND METHODS To identify key metabolic pathways that may be physiologically relevant in the context of reduced hepatic PGC-1α levels, we used the Cre/Lox system to create mice heterozygous for PGC-1α specifically within the liver (LH mice). RESULTS These mice showed fasting hepatic steatosis and diminished ketogenesis associated with decreased expression of genes involved in mitochondrial β-oxidation. LH mice also exhibited high circulating levels of triglyceride that correlated with increased expression of genes involved in triglyceride-rich lipoprotein assembly. Concomitant with defects in lipid metabolism, hepatic insulin resistance was observed both in LH mice fed a high-fat diet as well as in primary hepatocytes. CONCLUSIONS These data highlight both the dose-dependent and long-term effects of reducing hepatic PGC-1α levels, underlining the importance of tightly regulated PGC-1α expression in the maintenance of lipid homeostasis and glucose metabolism. PMID:19366863

The Essential Role of the Deinococcus radiodurans ssb Gene in Cell Survival and Radiation Tolerance

PubMed Central

Lockhart, J. Scott; DeVeaux, Linda C.

2013-01-01

Recent evidence has implicated single-stranded DNA-binding protein (SSB) expression level as an important factor in microbial radiation resistance. The genome of the extremely radiation resistant bacterium Deinococcus radiodurans contains genes for two SSB homologs: the homodimeric, canonical Ssb, encoded by the gene ssb, and a novel pentameric protein encoded by the gene ddrB. ddrB is highly induced upon exposure to radiation, and deletions result in decreased radiation-resistance, suggesting an integral role of the protein in the extreme resistance exhibited by this organism. Although expression of ssb is also induced after irradiation, Ssb is thought to be involved primarily in replication. In this study, we demonstrate that Ssb in D. radiodurans is essential for cell survival. The lethality of an ssb deletion cannot be complemented by providing ddrB in trans. In addition, the radiation-sensitive phenotype conferred by a ddrB deletion is not alleviated by providing ssb in trans. By altering expression of the ssb gene, we also show that lower levels of transcription are required for optimal growth than are necessary for high radiation resistance. When expression is reduced to that of E. coli, ionizing radiation resistance is similarly reduced. UV resistance is also decreased under low ssb transcript levels where growth is unimpaired. These results indicate that the expression of ssb is a key component of both normal cellular metabolism as well as pathways responsible for the high radiation tolerance of D. radiodurans. PMID:23951213

Prefrontal cortical-specific differences in behavior and synaptic plasticity between adolescent and adult mice.

PubMed

Konstantoudaki, Xanthippi; Chalkiadaki, Kleanthi; Vasileiou, Elisabeth; Kalemaki, Katerina; Karagogeos, Domna; Sidiropoulou, Kyriaki

2018-03-01

Adolescence is a highly vulnerable period for the emergence of major neuropsychological disorders and is characterized by decreased cognitive control and increased risk-taking behavior and novelty-seeking. The prefrontal cortex (PFC) is involved in the cognitive control of impulsive and risky behavior. Although the PFC is known to reach maturation later than other cortical areas, little information is available regarding the functional changes from adolescence to adulthood in PFC, particularly compared with other primary cortical areas. This study aims to understand the development of PFC-mediated, compared with non-PFC-mediated, cognitive functions. Toward this aim, we performed cognitive behavioral tasks in adolescent and adult mice and subsequently investigated synaptic plasticity in two different cortical areas. Our results showed that adolescent mice exhibit impaired performance in PFC-dependent cognitive tasks compared with adult mice, whereas their performance in non-PFC-dependent tasks is similar to that of adults. Furthermore, adolescent mice exhibited decreased long-term potentiation (LTP) within upper-layer synapses of the PFC but not the barrel cortex. Blocking GABA A receptor function significantly augments LTP in both the adolescent and adult PFC. No change in intrinsic excitability of PFC pyramidal neurons was observed between adolescent and adult mice. Finally, increased expression of the NR2A subunit of the N-methyl-d-aspartate receptors is found only in the adult PFC, a change that could underlie the emergence of LTP. In conclusion, our results demonstrate physiological and behavioral changes during adolescence that are specific to the PFC and could underlie the reduced cognitive control in adolescents. NEW & NOTEWORTHY This study reports that adolescent mice exhibit impaired performance in cognitive functions dependent on the prefrontal cortex but not in cognitive functions dependent on other cortical regions. The current results propose reduced synaptic plasticity in the upper layers of the prefrontal cortex as a cellular correlate of this weakened cognitive function. This decreased synaptic plasticity is due to reduced N-methyl-d-aspartate receptor expression but not due to dampened intrinsic excitability or enhanced GABAergic signaling during adolescence.

Quantitative analysis of lentiviral transgene expression in mice over seven generations.

PubMed

Wang, Yong; Song, Yong-tao; Liu, Qin; Liu, Cang'e; Wang, Lu-lu; Liu, Yu; Zhou, Xiao-yang; Wu, Jun; Wei, Hong

2010-10-01

Lentiviral transgenesis is now recognized as an extremely efficient and cost-effective method to produce transgenic animals. Transgenes delivered by lentiviral vectors exhibited inheritable expression in many species including those which are refractory to genetic modification such as non-human primates. However, epigenetic modification was frequently observed in lentiviral integrants, and transgene expression found to be inversely correlated with methylation density. Recent data showed that about one-third lentiviral integrants exhibited hypermethylation and low expression, but did not demonstrate whether those integrants with high expression could remain constant expression and hypomethylated during long term germline transmission. In this study, using lentiviral eGFP transgenic mice as the experimental animals, lentiviral eGFP expression levels and its integrant numbers in genome were quantitatively analyzed by fluorescent quantitative polymerase-chain reaction (FQ-PCR), using the house-keeping gene ribosomal protein S18 (Rps18) and the single copy gene fatty acid binding protein of the intestine (Fabpi) as the internal controls respectively. The methylation densities of the integrants were quantitatively analyzed by bisulfite sequencing. We found that the lentiviral integrants with high expression exhibited a relative constant expression level per integrant over at least seven generations. Besides, the individuals containing these integrants exhibited eGFP expression levels which were positively and almost linearly correlated with the integrant numbers in their genomes, suggesting that no remarkable position effect on transgene expression of the integrants analyzed was observed. In addition, over seven generations the methylation density of these integrants did not increase, but rather decreased remarkably, indicating that these high expressing integrants were not subjected to de novo methylation during at least seven generations of germline transmission. Taken together, these data suggested that transgenic lines with long term stable expression and no position effect can be established by lentiviral transgenesis.

(Z)-3,5,4'-Trimethoxystilbene Limits Hepatitis C and Cancer Pathophysiology by Blocking Microtubule Dynamics and Cell Cycle Progression

PubMed Central

Nguyen, Charles B.; Kotturi, Hari; Waris, Gulam; Mohammed, Altaf; Chandrakesan, Parthasarathy; May, Randal; Sureban, Sripathi; Weygant, Nathaniel; Qu, Dongfeng; Rao, Chinthalapally V.; Dhanasekaran, Danny N.; Bronze, Michael S.; Houchen, Courtney W.; Ali, Naushad

2016-01-01

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. Chronic hepatitis C virus (HCV) infection causes induction of several tumor/cancer stem cell (CSC) markers and is known to be a major risk factor for development of HCC. Therefore, drugs that simultaneously target viral replication and CSC properties are needed for a risk-free treatment of advanced stage liver diseases including HCC. Here, we demonstrated that (Z)-3,5,4’-trimethoxystilbene (Z-TMS) exhibits potent anti-tumor and anti-HCV activities without exhibiting cytotoxicity to human hepatocytes in vitro or in mice livers. Diethylnitrosamine (DEN)/carbon tetrachloride (CCl4) extensively induced expression of DCLK1 (a CSC marker) in the livers of C57BL/6 mice following hepatic injury. Z-TMS exhibited hepatoprotective effects against DEN/CCl4-induced injury by reducing DCLK1 expression and improving histological outcomes. The drug caused bundling of DCLK1 with microtubules and blocked cell cycle progression at G2/M phase in hepatoma cells via downregulation of CDK1, induction of p21cip1/waf1 expression, and inhibition of Akt (Ser473) phosphorylation. Z-TMS also inhibited proliferation of erlotinib-resistant lung adenocarcinoma cells (H1975) bearing the T790M EGFR mutation most likely by promoting autophagy and nuclear fragmentation. In conclusion, Z-TMS appears to be a unique therapeutic agent targeting HCV and concurrently eliminating cells with neoplastic potential during chronic liver diseases including HCC. It may also be a valuable drug for targeting drug-resistant carcinomas and cancers of the lungs, pancreas, colon, and intestine in which DCLK1 is involved in tumorigenesis. PMID:27287718

Patients with metabolic syndrome exhibit higher platelet activity than those with conventional risk factors for vascular disease.

PubMed

Serebruany, Victor L; Malinin, Alex; Ong, Stephen; Atar, Dan

2008-04-01

The metabolic syndrome is a matter of ongoing debate with regard to its existence, classification, clinical meaningfulness, and associated risks for vessel occlusion. Considering that persistent platelet activation is a cornerstone for the development of acute vascular events, and that patients with type 2 diabetes consistently exhibit high platelet activity, these characteristics may be critical for distinguishing and triageing specific features of metabolic syndrome among established risk factors for vascular disease. We assessed the platelet activity by conventional aggregation, expression of major surface receptors by flow cytometry, and quantitatively by rapid bedside analyzers in 20 aspirin-naïve patients with documented metabolic syndrome, and compared these with 20 untreated subjects with multiple cardiovascular risk factors. Closure time by the PFA-100 analyzer was significantly (P = 0.002) shorter in patients with metabolic syndrome indicating platelet inhibition under high shear conditions. Ultegra analyzer readings revealed increased fibrinogen binding (P = 0.0003) what in combination with the increased expression of PAC-1 (P = 0.32) strongly suggest activation of platelet glycoprotein IIb/IIIa receptor. Surface expression of CD107a (P = 0.014), and SPAN-12 (P = 0.003) were also higher in patients with metabolic syndrome. In contrast, platelet aggregation induced by collagen or ADP, CD31, CD41, CD42b, CD51/61, CD62p, CD63, CD154, CD165, so as formation of platelet-monocyte aggregates, PAR-1 thrombin receptor, and thrombospondin did not differ between groups. Patients with metabolic syndrome exhibited a higher degree of platelet activation than subjects with conventional risk factors for vascular disease. Conceptually, applying adequate antiplatelet strategies may reduce the risk of acute thrombotic events in these patients. Further prospective studies exploring this notion are encouraged.

Secreted Frizzled-related protein 2 as a target in antifibrotic therapeutic intervention.

PubMed

Mastri, Michalis; Shah, Zaeem; Hsieh, Karin; Wang, Xiaowen; Wooldridge, Bailey; Martin, Sean; Suzuki, Gen; Lee, Techung

2014-03-15

Progressive fibrosis is a pathological hallmark of many chronic diseases responsible for organ failure. Although there is currently no therapy on the market that specifically targets fibrosis, the dynamic fibrogenic process is known to be regulated by multiple soluble mediators that may be therapeutically intervened. The failing hamster heart exhibits marked fibrosis and increased expression of secreted Frizzled-related protein 2 (sFRP2) amenable to reversal by mesenchymal stem cell (MSC) therapy. Given the previous demonstration that sFRP2-null mice subjected to myocardial infarction exhibited reduced fibrosis and improved function, we tested whether antibody-based sFRP2 blockade might counteract the fibrogenic pathway and repair cardiac injury. Cardiomyopathic hamsters were injected intraperitoneally twice a week each with 20 μg of sFRP2 antibody. Echocardiography, histology, and biochemical analyses were performed after 1 mo. sFRP2 antibody increased left ventricular ejection fraction from 40 ± 1.2 to 49 ± 6.5%, whereas saline and IgG control exhibited a further decline to 37 ± 0.9 and 31 ± 3.2%, respectively. Functional improvement is associated with a ∼ 50% reduction in myocardial fibrosis, ∼ 65% decrease in apoptosis, and ∼ 75% increase in wall thickness. Consistent with attenuated fibrosis, both MSC therapy and sFRP2 antibody administration significantly increased the activity of myocardial matrix metalloproteinase-2. Gene expression analysis of the hamster heart and cultured fibroblasts identified Axin2 as a downstream target, the expression of which was activated by sFRP2 but inhibited by therapeutic intervention. sFRP2 blockade also increased myocardial levels of VEGF and hepatocyte growth factor (HGF) along with increased angiogenesis. These findings highlight the pathogenic effect of dysregulated sFRP2, which may be specifically targeted for antifibrotic therapy.

Immortalization and characterization of osteoblast cell lines generated from wild-type and Nmp4-null mouse bone marrow stromal cells using murine telomerase reverse transcriptase (mTERT).

PubMed

Alvarez, Marta B; Childress, Paul; Philip, Binu K; Gerard-O'Riley, Rita; Hanlon, Michael; Herbert, Brittney-Shea; Robling, Alexander G; Pavalko, Fredrick M; Bidwell, Joseph P

2012-05-01

Intermittent parathyroid hormone (PTH) adds new bone to the osteoporotic skeleton; the transcription factor Nmp4/CIZ represses PTH-induced bone formation in mice and as a consequence is a potential drug target for improving hormone clinical efficacy. To explore the impact of Nmp4/CIZ on osteoblast phenotype, we immortalized bone marrow stromal cells from wildtype (WT) and Nmp4-knockout (KO) mice using murine telomerase reverse transcriptase. Clonal lines were initially chosen based on their positive staining for alkaline phosphatase and capacity for mineralization. Disabling Nmp4/CIZ had no gross impact on osteoblast phenotype development. WT and KO clones exhibited identical sustained growth, reduced population doubling times, extended maintenance of the mature osteoblast phenotype, and competency for differentiating toward the osteoblast and adipocyte lineages. Additional screening of the immortalized cells for PTH-responsiveness permitted further studies with single WT and KO clones. We recently demonstrated that PTH-induced c-fos femoral mRNA expression is enhanced in Nmp4-KO mice and in the present study we observed that hormone stimulated either an equivalent or modestly enhanced increase in c-fos mRNA expression in both primary null and KO clone cells depending on PTH concentration. The null primary osteoblasts and KO clone cells exhibited a transiently enhanced response to bone morphogenetic protein 2 (BMP2). The clones exhibited lower and higher expressions of the PTH receptor (Pthr1) and the BMP2 receptor (Bmpr1a, Alk3), respectively, as compared to primary cells. These immortalized cell lines will provide a valuable tool for disentangling the complex functional roles underlying Nmp4/CIZ regulation of bone anabolism. Copyright © 2011 Wiley Periodicals, Inc.

Role of CB2 receptors in social and aggressive behavior in male mice.

PubMed

Rodríguez-Arias, Marta; Navarrete, Francisco; Blanco-Gandia, M Carmen; Arenas, M Carmen; Aguilar, María A; Bartoll-Andrés, Adrián; Valverde, Olga; Miñarro, José; Manzanares, Jorge

2015-08-01

Male CB1KO mice exhibit stronger aggressive responses than wild-type mice. This study was designed to examine the role of cannabinoid CB2r in social and aggressive behavior. The social interaction test and resident-intruder paradigm were performed in mice lacking CB2r (CB2KO) and in wild-type (WT) littermates. The effects of the CB2r selective agonist JWH133 (1 and 2 mg/kg) on aggression were also evaluated in Oncins France 1 (OF1) mice. Gene expression analyses of monoamine oxidase-A (MAO-A), catechol-o-methyltransferase (COMT), 5-hydroxytryptamine transporter (5-HTT), and 5-HT1B receptor (5HT1Br) in the dorsal raphe nuclei (DR) and the amygdala (AMY) were carried out using real-time PCR. Group-housed CB2KO mice exhibited higher levels of aggression in the social interaction test and displayed more aggression than resident WT mice. Isolation increased aggressive behavior in WT mice but did not affect CB2KO animals; however, the latter mice exhibited higher levels of social interaction with their WT counterparts. MAO-A and 5-HTT gene expression was significantly higher in grouped CB2KO mice. The expression of 5HT1Br, COMT, and MAO-A in the AMY was more pronounced in CB2KO mice than in WT counterparts. Acute administration of the CB2 agonist JWH133 significantly reduced the level of aggression in aggressive isolated OF1 mice, an effect that decreased after pretreatment with the CB2 receptor antagonist AM630. Our results suggest that CB2r is implicated in social interaction and aggressive behavior and deserves further consideration as a potential new target for the management of aggression.

Cadm1-Expressing Synapses on Purkinje Cell Dendrites Are Involved in Mouse Ultrasonic Vocalization Activity

PubMed Central

Fujita, Eriko; Tanabe, Yuko; Imhof, Beat A.; Momoi, Mariko Y.; Momoi, Takashi

2012-01-01

Foxp2(R552H) knock-in (KI) mouse pups with a mutation related to human speech–language disorders exhibit poor development of cerebellar Purkinje cells and impaired ultrasonic vocalization (USV), a communication tool for mother-offspring interactions. Thus, human speech and mouse USV appear to have a Foxp2-mediated common molecular basis in the cerebellum. Mutations in the gene encoding the synaptic adhesion molecule CADM1 (RA175/Necl2/SynCAM1/Cadm1) have been identified in people with autism spectrum disorder (ASD) who have impaired speech and language. In the present study, we show that both Cadm1-deficient knockout (KO) pups and Foxp2(R552H) KI pups exhibit impaired USV and smaller cerebellums. Cadm1 was preferentially localized to the apical–distal portion of the dendritic arbor of Purkinje cells in the molecular layer of wild-type pups, and VGluT1 level decreased in the cerebellum of Cadm1 KO mice. In addition, we detected reduced immunoreactivity of Cadm1 and VGluT1 on the poorly developed dendritic arbor of Purkinje cells in the Foxp2(R552H) KI pups. However, Cadm1 mRNA expression was not altered in the Foxp2(R552H) KI pups. These results suggest that although the Foxp2 transcription factor does not target Cadm1, Cadm1 at the synapses of Purkinje cells and parallel fibers is necessary for USV function. The loss of Cadm1-expressing synapses on the dendrites of Purkinje cells may be associated with the USV impairment that Cadm1 KO and Foxp2(R552H) KI mice exhibit. PMID:22272290

Kaiso overexpression promotes intestinal inflammation and potentiates intestinal tumorigenesis in Apc(Min/+) mice.

PubMed

Pierre, Christina C; Longo, Joseph; Mavor, Meaghan; Milosavljevic, Snezana B; Chaudhary, Roopali; Gilbreath, Ebony; Yates, Clayton; Daniel, Juliet M

2015-09-01

Constitutive Wnt/β-catenin signaling is a key contributor to colorectal cancer (CRC). Although inactivation of the tumor suppressor adenomatous polyposis coli (APC) is recognized as an early event in CRC development, it is the accumulation of multiple subsequent oncogenic insults facilitates malignant transformation. One potential contributor to colorectal carcinogenesis is the POZ-ZF transcription factor Kaiso, whose depletion extends lifespan and delays polyp onset in the widely used Apc(Min/+) mouse model of intestinal cancer. These findings suggested that Kaiso potentiates intestinal tumorigenesis, but this was paradoxical as Kaiso was previously implicated as a negative regulator of Wnt/β-catenin signaling. To resolve Kaiso's role in intestinal tumorigenesis and canonical Wnt signaling, we generated a transgenic mouse model (Kaiso(Tg/+)) expressing an intestinal-specific myc-tagged Kaiso transgene. We then mated Kaiso(Tg/+) and Apc(Min/+) mice to generate Kaiso(Tg/+):Apc(Min/+) mice for further characterization. Kaiso(Tg/+):Apc(Min/+) mice exhibited reduced lifespan and increased polyp multiplicity compared to Apc(Min/+) mice. Consistent with this murine phenotype, we found increased Kaiso expression in human CRC tissue, supporting a role for Kaiso in human CRC. Interestingly, Wnt target gene expression was increased in Kaiso(Tg/+):Apc(Min/+) mice, suggesting that Kaiso's function as a negative regulator of canonical Wnt signaling, as seen in Xenopus, is not maintained in this context. Notably, Kaiso(Tg/+):Apc(Min/+) mice exhibited increased inflammation and activation of NFκB signaling compared to their Apc(Min/+) counterparts. This phenotype was consistent with our previous report that Kaiso(Tg/+) mice exhibit chronic intestinal inflammation. Together our findings highlight a role for Kaiso in promoting Wnt signaling, inflammation and tumorigenesis in the mammalian intestine. Copyright © 2015 Elsevier B.V. All rights reserved.

Transection of Preganglionic Axons Leads to CNS Neuronal Plasticity Followed by Survival and Target Reinnervation

PubMed Central

Coulibaly, Aminata P.; Gannon, Sean M.; Hawk, Kiel; Walsh, Brian F.; Isaacson, Lori G.

2013-01-01

The goals of the present study were to investigate the changes in sympathetic preganglionic neurons following transection of distal axons in the cervical sympathetic trunk (CST) that innervate the superior cervical ganglion (SCG) and to assess changes in the protein expression of brain derived neurotrophic factor (BDNF) and its receptor TrkB in the thoracic spinal cord. . At 1 week, a significant decrease in soma volume and reduced soma expression of choline acetyltransferase (ChAT) in the intermediolateral cell column (IML) of T1 spinal cord were observed, with both ChAT-ir and non-immunoreactive neurons expressing the injury marker activating transcription factor 3. . These changes were transient, and at later time points, ChAT expression and soma volume returned to control values and the number of ATF3 neurons declined. No evidence for cell loss or neuronal apoptosis was detected at any time point. Protein levels of BDNF and/or full length TrkB in the spinal cord were increased throughout the survival period. In the SCG, both ChAT-ir axons and ChAT protein remained decreased at 16 weeks, but were increased compared to the 10 week time point. These results suggest that though IML neurons show reduced ChAT expression and cell volume at 1 week following CST transection, at later time points, the neurons recovered and exhibited no significant signs of neurodegeneration. The alterations in BDNF and/or TrkB may have contributed to the survival of the IML neurons and the recovery of ChAT expression, as well as to the reinnervation of the SCG. PMID:23891533

Reduced facial reactivity as a contributor to preserved emotion regulation in older adults.

PubMed

Pedder, David J; Terrett, Gill; Bailey, Phoebe E; Henry, Julie D; Ruffman, Ted; Rendell, Peter G

2016-02-01

This study investigated whether differences in the type of strategy used, or age-related differences in intensity of facial reactivity, might contribute to preserved emotion regulation ability in older adults. Young (n = 35) and older (n = 33) adults were instructed to regulate their emotion to positive and negative pictures under 3 conditions (watch, expressive suppression, cognitive 'detached' reappraisal). Participants were objectively monitored using facial electromyography (EMG) and assessed on memory performance. Both age groups were effectively, and equivalently, able to reduce their facial expressions. In relation to facial reactivity, the percentage increase of older adults' facial muscle EMG activity in the watch condition was significantly reduced relative to young adults. Recall of pictures following regulation was similar to the watch condition, and there was no difference in memory performance between the 2 regulation strategies for both groups. These findings do not support the proposal that the type of strategy used explains preserved emotion regulation ability in older adults. Coupled with the lack of memory costs following regulation, these data instead are more consistent with the suggestion that older adults may retain emotion regulation capacity partly because they exhibit less facial reactivity to begin with. (c) 2016 APA, all rights reserved).

Dairy Cows Naturally Infected with Bovine Leukemia Virus Exhibit Abnormal B- and T-Cell Phenotypes after Primary and Secondary Exposures to Keyhole Limpet Hemocyanin

PubMed Central

Frie, Meredith C.; Sporer, Kelly R. B.; Benitez, Oscar J.; Wallace, Joseph C.; Droscha, Casey J.; Bartlett, Paul C.; Coussens, Paul M.

2017-01-01

Bovine leukemia virus (BLV) is a retrovirus that is highly prevalent in US dairy herds: over 83% are BLV infected and the within-herd infection rate can be almost 50% on average. While BLV is known to cause lymphosarcomas, only 5% or fewer infected cattle will develop lymphoma; this low prevalence of cancer has historically not been a concern to dairy producers. However, more recent research has found that BLV+ cows without lymphoma produce less milk and have shorter lifespans than uninfected herdmates. It has been hypothesized that BLV infection interferes with normal immune function in infected cattle, and this could lead to reduced dairy production. To assess how naturally infected BLV+ cows responded to a primary and secondary immune challenge, 10 BLV+ and 10 BLV− cows were injected subcutaneously with keyhole limpet hemocyanin (KLH) and dimethyldioctadecylammonium bromide. B- and T-cell responses were characterized over the following 28 days. A total of 56 days after primary KLH exposure, cows were re-injected with KLH and B- and T-cell responses were characterized again over the following 28 days. BLV+ cows produced less KLH-specific IgM after primary immune stimulation; demonstrated fewer CD45R0+ B cells, altered proportions of CD5+ B cells, altered expression of CD5 on CD5+ B cells, and reduced MHCII surface expression on B cells ex vivo; exhibited reduced B-cell activation in vitro; and displayed an increase in BLV proviral load after KLH exposure. In addition, BLV+ cows had a reduced CD45R0+γδ+ T-cell population in the periphery and demonstrated a greater prevalence of IL4-producing T cells in vitro. All together, our results demonstrate that both B- and T-cell immunities are disrupted in BLV+ cows and that antigen-specific deficiencies can be detected in BLV+ cows even after a primary immune exposure. PMID:28770217

Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression

PubMed Central

Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

2014-01-01

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division. PMID:24714560

Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression.

PubMed

Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

2014-05-02

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division.

Reducing FLI1 Levels in the MRL/lpr Lupus Mouse Model Impacts T Cell Function by Modulating Glycosphingolipid Metabolism

PubMed Central

Richard, Erin Morris; Thiyagarajan, Thirumagal; Bunni, Marlene A.; Basher, Fahmin; Roddy, Patrick O.; Siskind, Leah J.; Nietert, Paul J.; Nowling, Tamara K.

2013-01-01

Systemic Lupus erythematosus (SLE) is an autoimmune disease caused, in part, by abnormalities in cells of the immune system including B and T cells. Genetically reducing globally the expression of the ETS transcription factor FLI1 by 50% in two lupus mouse models significantly improves disease measures and survival through an unknown mechanism. In this study we analyze the effects of reducing FLI1 in the MRL/lpr lupus prone model on T cell function. We demonstrate that adoptive transfer of MRL/lpr Fli1 +/+ or Fli1 +/- T cells and B cells into Rag1-deficient mice results in significantly decreased serum immunoglobulin levels in animals receiving Fli1 +/- lupus T cells compared to animals receiving Fli1 +/+ lupus T cells regardless of the genotype of co-transferred lupus B cells. Ex vivo analyses of MRL/lpr T cells demonstrated that Fli1 +/- T cells produce significantly less IL-4 during early and late disease and exhibited significantly decreased TCR-specific activation during early disease compared to Fli1 +/+ T cells. Moreover, the Fli1 +/- T cells expressed significantly less neuraminidase 1 (Neu1) message and decreased NEU activity during early disease and significantly decreased levels of glycosphingolipids during late disease compared to Fli1 +/+ T cells. FLI1 dose-dependently activated the Neu1 promoter in mouse and human T cell lines. Together, our results suggest reducing FLI1 in lupus decreases the pathogenicity of T cells by decreasing TCR-specific activation and IL-4 production in part through the modulation of glycosphingolipid metabolism. Reducing the expression of FLI1 or targeting the glycosphingolipid metabolic pathway in lupus may serve as a therapeutic approach to treating lupus. PMID:24040398

Reducing FLI1 levels in the MRL/lpr lupus mouse model impacts T cell function by modulating glycosphingolipid metabolism.

PubMed

Richard, Erin Morris; Thiyagarajan, Thirumagal; Bunni, Marlene A; Basher, Fahmin; Roddy, Patrick O; Siskind, Leah J; Nietert, Paul J; Nowling, Tamara K

2013-01-01

Systemic Lupus erythematosus (SLE) is an autoimmune disease caused, in part, by abnormalities in cells of the immune system including B and T cells. Genetically reducing globally the expression of the ETS transcription factor FLI1 by 50% in two lupus mouse models significantly improves disease measures and survival through an unknown mechanism. In this study we analyze the effects of reducing FLI1 in the MRL/lpr lupus prone model on T cell function. We demonstrate that adoptive transfer of MRL/lpr Fli1(+/+) or Fli1(+/-) T cells and B cells into Rag1-deficient mice results in significantly decreased serum immunoglobulin levels in animals receiving Fli1(+/-) lupus T cells compared to animals receiving Fli1(+/+) lupus T cells regardless of the genotype of co-transferred lupus B cells. Ex vivo analyses of MRL/lpr T cells demonstrated that Fli1(+/-) T cells produce significantly less IL-4 during early and late disease and exhibited significantly decreased TCR-specific activation during early disease compared to Fli1(+/+) T cells. Moreover, the Fli1(+/-) T cells expressed significantly less neuraminidase 1 (Neu1) message and decreased NEU activity during early disease and significantly decreased levels of glycosphingolipids during late disease compared to Fli1(+/+) T cells. FLI1 dose-dependently activated the Neu1 promoter in mouse and human T cell lines. Together, our results suggest reducing FLI1 in lupus decreases the pathogenicity of T cells by decreasing TCR-specific activation and IL-4 production in part through the modulation of glycosphingolipid metabolism. Reducing the expression of FLI1 or targeting the glycosphingolipid metabolic pathway in lupus may serve as a therapeutic approach to treating lupus.

Altered entrainment to the day/night cycle attenuates the daily rise in circulating corticosterone in the mouse.

PubMed

Sollars, Patricia J; Weiser, Michael J; Kudwa, Andrea E; Bramley, Jayne R; Ogilvie, Malcolm D; Spencer, Robert L; Handa, Robert J; Pickard, Gary E

2014-01-01

The suprachiasmatic nucleus (SCN) is a circadian oscillator entrained to the day/night cycle via input from the retina. Serotonin (5-HT) afferents to the SCN modulate retinal signals via activation of 5-HT1B receptors, decreasing responsiveness to light. Consequently, 5-HT1B receptor knockout (KO) mice entrain to the day/night cycle with delayed activity onsets. Since circulating corticosterone levels exhibit a robust daily rhythm peaking around activity onset, we asked whether delayed entrainment of activity onsets affects rhythmic corticosterone secretion. Wheel-running activity and plasma corticosterone were monitored in mice housed under several different lighting regimens. Both duration of the light:dark cycle (T cycle) and the duration of light within that cycle was altered. 5-HT1B KO mice that entrained to a 9.5L:13.5D (short day in a T = 23 h) cycle with activity onsets delayed more than 4 h after light offset exhibited a corticosterone rhythm in phase with activity rhythms but reduced 50% in amplitude compared to animals that initiated daily activity <4 h after light offset. Wild type mice in 8L:14D (short day in a T = 22 h) conditions with highly delayed activity onsets also exhibited a 50% reduction in peak plasma corticosterone levels. Exogenous adrenocorticotropin (ACTH) stimulation in animals exhibiting highly delayed entrainment suggested that the endogenous rhythm of adrenal responsiveness to ACTH remained aligned with SCN-driven behavioral activity. Circadian clock gene expression in the adrenal cortex of these same animals suggested that the adrenal circadian clock was also aligned with SCN-driven behavior. Under T cycles <24 h, altered circadian entrainment to short day (winter-like) conditions, manifest as long delays in activity onset after light offset, severely reduces the amplitude of the diurnal rhythm of plasma corticosterone. Such a pronounced reduction in the glucocorticoid rhythm may alter rhythmic gene expression in the central nervous system and in peripheral organs contributing to an array of potential pathophysiologies.

Altered Entrainment to the Day/Night Cycle Attenuates the Daily Rise in Circulating Corticosterone in the Mouse

PubMed Central

Sollars, Patricia J.; Weiser, Michael J.; Kudwa, Andrea E.; Bramley, Jayne R.; Ogilvie, Malcolm D.; Spencer, Robert L.; Handa, Robert J.; Pickard, Gary E.

2014-01-01

The suprachiasmatic nucleus (SCN) is a circadian oscillator entrained to the day/night cycle via input from the retina. Serotonin (5-HT) afferents to the SCN modulate retinal signals via activation of 5-HT1B receptors, decreasing responsiveness to light. Consequently, 5-HT1B receptor knockout (KO) mice entrain to the day/night cycle with delayed activity onsets. Since circulating corticosterone levels exhibit a robust daily rhythm peaking around activity onset, we asked whether delayed entrainment of activity onsets affects rhythmic corticosterone secretion. Wheel-running activity and plasma corticosterone were monitored in mice housed under several different lighting regimens. Both duration of the light∶dark cycle (T cycle) and the duration of light within that cycle was altered. 5-HT1B KO mice that entrained to a 9.5L:13.5D (short day in a T = 23 h) cycle with activity onsets delayed more than 4 h after light offset exhibited a corticosterone rhythm in phase with activity rhythms but reduced 50% in amplitude compared to animals that initiated daily activity <4 h after light offset. Wild type mice in 8L:14D (short day in a T = 22 h) conditions with highly delayed activity onsets also exhibited a 50% reduction in peak plasma corticosterone levels. Exogenous adrenocorticotropin (ACTH) stimulation in animals exhibiting highly delayed entrainment suggested that the endogenous rhythm of adrenal responsiveness to ACTH remained aligned with SCN-driven behavioral activity. Circadian clock gene expression in the adrenal cortex of these same animals suggested that the adrenal circadian clock was also aligned with SCN-driven behavior. Under T cycles <24 h, altered circadian entrainment to short day (winter-like) conditions, manifest as long delays in activity onset after light offset, severely reduces the amplitude of the diurnal rhythm of plasma corticosterone. Such a pronounced reduction in the glucocorticoid rhythm may alter rhythmic gene expression in the central nervous system and in peripheral organs contributing to an array of potential pathophysiologies. PMID:25365210

Long term effects of fetal undernutrition on rat heart. Role of hypertension and oxidative stress

PubMed Central

Rodríguez-Rodríguez, Pilar; López de Pablo, Angel L.; García-Prieto, Concha F.; Somoza, Beatriz; Quintana-Villamandos, Begoña; Gómez de Diego, José J.; Gutierrez-Arzapalo, Perla Y.; Ramiro-Cortijo, David; González, M. Carmen

2017-01-01

Background and aims Fetal undernutrition is a risk factor for heart disease in both genders, despite the protection of women against hypertension development. Using a rat model of maternal undernutrition (MUN) we aimed to assess possible sex differences in the development of cardiac alterations and the implication of hypertension and cardiac oxidative stress. Methods Male and female offspring from rats fed ad libitum (control) or with 50% of the normal daily intake during the second half of gestation (MUN) were used. Heart weight/body weight ratio (HW/BW), hemodynamic parameters (anaesthetized rats) and plasma brain natriuretic peptide (BNP, ELISA) were assessed in 21-day, 6-month and 22-month old rats. Plasma testosterone (ELISA) and cardiac protein expression of enzymes related to reactive oxygen species synthesis (p22phox, xanthine-oxidase) and degradation (catalase, Cu/Zn-SOD, Mn-SOD, Ec-SOD) were evaluated in 21-day and 6-month old rats (Western Blot). Heart structure and function was studied at the age of 22 months (echocardiography). Results At the age of 21 days MUN males exhibited significantly larger HW/BW and cardiac p22phox expression while females had reduced p22phox expression, compared to their respective sex-matched controls. At the age of 6-months, MUN males showed significantly larger blood pressure and cardiac xanthine-oxidase expression; MUN females were normotensive and had a lower cardiac expression of antioxidant enzymes, compared to their respective sex-matched controls. At the age of 22 months, both MUN males and females showed larger HW/BW and left ventricular mass and lower ejection fraction compared to sex-matched controls; only MUN males exhibited hypertension and a larger plasma BNP compared to aged male controls. Conclusions 1) During perinatal life females exposed to fetal undernutrition are protected from cardiac alterations, but in ageing they exhibit ventricular hypertrophy and functional loss, like MUN males; 2) cardiac oxidative stress might be implicated in the observed heart alterations in both sexes and 3) the severity of cardiac damage might be greater in males due to hypertension. PMID:28212445

Aag Hypoxanthine-DNA Glycosylase Is Synthesized in the Forespore Compartment and Involved in Counteracting the Genotoxic and Mutagenic Effects of Hypoxanthine and Alkylated Bases in DNA during Bacillus subtilis Sporulation.

PubMed

Ayala-García, Víctor M; Valenzuela-García, Luz I; Setlow, Peter; Pedraza-Reyes, Mario

2016-12-15

Aag from Bacillus subtilis has been implicated in in vitro removal of hypoxanthine and alkylated bases from DNA. The regulation of expression of aag in B. subtilis and the resistance to genotoxic agents and mutagenic properties of an Aag-deficient strain were studied here. A strain with a transcriptional aag-lacZ fusion expressed low levels of β-galactosidase during growth and early sporulation but exhibited increased transcription during late stages of this developmental process. Notably, aag-lacZ expression was higher inside the forespore than in the mother cell compartment, and this expression was abolished in a sigG-deficient background, suggesting a forespore-specific mechanism of aag transcription. Two additional findings supported this suggestion: (i) expression of an aag-yfp fusion was observed in the forespore, and (ii) in vivo mapping of the aag transcription start site revealed the existence of upstream regulatory sequences possessing homology to σ G -dependent promoters. In comparison with the wild-type strain, disruption of aag significantly reduced survival of sporulating B. subtilis cells following nitrous acid or methyl methanesulfonate treatments, and the Rif r mutation frequency was significantly increased in an aag strain. These results suggest that Aag protects the genome of developing B. subtilis sporangia from the cytotoxic and genotoxic effects of base deamination and alkylation. In this study, evidence is presented revealing that aag, encoding a DNA glycosylase implicated in processing of hypoxanthine and alkylated DNA bases, exhibits a forespore-specific pattern of gene expression during B. subtilis sporulation. Consistent with this spatiotemporal mode of expression, Aag was found to protect the sporulating cells of this microorganism from the noxious and mutagenic effects of base deamination and alkylation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The chromatin remodeler chd5 is necessary for proper head development during embryogenesis of Danio rerio.

PubMed

Bishop, Brett; Ho, Kwok Ki; Tyler, Kim; Smith, Amanda; Bonilla, Sylvia; Leung, Yuk Fai; Ogas, Joe

2015-08-01

The chromatin remodeler CHD5 plays a critical role in tumor suppression and neurogenesis in mammals. CHD5 contributes to gene expression during neurogenesis, but there is still much to learn regarding how this class of remodelers contributes to differentiation and development. CHD5 remodelers are vertebrate-specific, raising the prospect that CHD5 plays one or more conserved roles in this phylum. Expression of chd5 in adult fish closely mirrors expression of CHD5 in adult mammals. Knockdown of Chd5 during embryogenesis suggests new roles for CHD5 remodelers based on resulting defects in craniofacial development including reduced head and eye size as well as reduced cartilage formation in the head. In addition, knockdown of Chd5 results in altered expression of neural markers in the developing brain and eye as well as a profound defect in differentiation of dopaminergic amacrine cells. Recombinant zebrafish Chd5 protein exhibits nucleosome remodeling activity in vitro, suggesting that it is the loss of this activity that contributes to the observed phenotypes. Our studies indicate that zebrafish is an appropriate model for functional characterization of CHD5 remodelers in vertebrates and highlight the potential of this model for generating novel insights into the role of this vital class of remodelers. Copyright © 2015. Published by Elsevier B.V.

Inhibitory effects of Pycnogenol® (French maritime pine bark extract) on airway inflammation in ovalbumin-induced allergic asthma.

PubMed

Shin, In-Sik; Shin, Na-Rae; Jeon, Chan-Mi; Hong, Ju-Mi; Kwon, Ok-Kyoung; Kim, Jong-Choon; Oh, Sei-Ryang; Hahn, Kyu-Woung; Ahn, Kyung-Seop

2013-12-01

Pycnogenol® (PYC) is a standardized extracts from the bark of the French maritime pine (Pinus maritime) and used as a herbal remedy for various diseases. In this study, we evaluated the effects of PYC on airway inflammation using a model of ovalbumin (OVA)-induced allergic asthma and RAW264.7 cells. PYC decreased nitric oxide production and reduced the interleukine (IL)-1β and IL-6 levels in LPS-stimulated RAW264.7 cells. PYC also reduced the expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase (MMP)-9 and enhanced the expression of hemeoxygenase (HO)-1. In the in vivo experiment, PYC decreased the inflammatory cell count and the levels of IL-4, IL-5, IL-13, and immunoglobulin (Ig) E in BALF or serum. These results are consistent with the histological analysis findings, which showed that PYC attenuated the airway inflammation and mucus hypersecretion induced by OVA challenge. In addition, PYC enhanced the expression of HO-1. In contrast, PYC inhibited the elevated expression of iNOS and MMP-9 proteins induced by OVA challenge. In conclusion, PYC exhibits protective effects against OVA-induced asthma and LPS-stimulated RAW264.7 cells. These results suggest that PYC has potential as a therapeutic agent for the treatment of allergic asthma. Copyright © 2013 Elsevier Ltd. All rights reserved.

Histone posttranslational modifications and cell fate determination: lens induction requires the lysine acetyltransferases CBP and p300

PubMed Central

Wolf, Louise; Harrison, Wilbur; Huang, Jie; Xie, Qing; Xiao, Ningna; Sun, Jian; Kong, Lingkun; Lachke, Salil A.; Kuracha, Murali R.; Govindarajan, Venkatesh; Brindle, Paul K.; Ashery-Padan, Ruth; Beebe, David C.; Overbeek, Paul A.; Cvekl, Ales

2013-01-01

Lens induction is a classical embryologic model to study cell fate determination. It has been proposed earlier that specific changes in core histone modifications accompany the process of cell fate specification and determination. The lysine acetyltransferases CBP and p300 function as principal enzymes that modify core histones to facilitate specific gene expression. Herein, we performed conditional inactivation of both CBP and p300 in the ectodermal cells that give rise to the lens placode. Inactivation of both CBP and p300 resulted in the dramatic discontinuation of all aspects of lens specification and organogenesis, resulting in aphakia. The CBP/p300−/− ectodermal cells are viable and not prone to apoptosis. These cells showed reduced expression of Six3 and Sox2, while expression of Pax6 was not upregulated, indicating discontinuation of lens induction. Consequently, expression of αB- and αA-crystallins was not initiated. Mutant ectoderm exhibited markedly reduced levels of histone H3 K18 and K27 acetylation, subtly increased H3 K27me3 and unaltered overall levels of H3 K9ac and H3 K4me3. Our data demonstrate that CBP and p300 are required to establish lens cell-type identity during lens induction, and suggest that posttranslational histone modifications are integral to normal cell fate determination in the mammalian lens. PMID:24038357

Nerve growth factor reduces apoptotic cell death in rat facial motor neurons after facial nerve injury.

PubMed

Hui, Lian; Yuan, Jing; Ren, Zhong; Jiang, Xuejun

2015-01-01

To assess the effects of nerve growth factor (NGF) on motor neurons after induction of a facial nerve lesion, and to compare the effects of different routes of NGF injection on motor neuron survival. This study was carried out in the Department of Otolaryngology Head & Neck Surgery, China Medical University, Liaoning, China from October 2012 to March 2013. Male Wistar rats (n = 65) were randomly assigned into 4 groups: A) healthy controls; B) facial nerve lesion model + normal saline injection; C) facial nerve lesion model + NGF injection through the stylomastoid foramen; D) facial nerve lesion model + intraperitoneal injection of NGF. Apoptotic cell death was detected using the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. Expression of caspase-3 and p53 up-regulated modulator of apoptosis (PUMA) was determined by immunohistochemistry. Injection of NGF significantly reduced cell apoptosis, and also greatly decreased caspase-3 and PUMA expression in injured motor neurons. Group C exhibited better efficacy for preventing cellular apoptosis and decreasing caspase-3 and PUMA expression compared with group D (p<0.05). Our findings suggest that injections of NGF may prevent apoptosis of motor neurons by decreasing caspase-3 and PUMA expression after facial nerve injury in rats. The NGF injected through the stylomastoid foramen demonstrated better protective efficacy than when injected intraperitoneally.

Secreted HoxA3 Promotes Epidermal Proliferation and Angiogenesis in Genetically Modified Three-Dimensional Composite Skin Constructs

PubMed Central

Kuo, Jennifer H.; Cuevas, Ileana; Chen, Amy; Dunn, Ashley; Kuri, Mauricio; Boudreau, Nancy

2014-01-01

Objective: Homeobox (HOX) transcription factors coordinate gene expression in wound repair and angiogenesis. Previous studies have shown that gene transfer of HoxA3 to wounds of diabetic mice accelerates wound healing, increasing angiogenesis and keratinocyte migration. In this study, we examined whether HoxA3 can also improve angiogenesis, epidermal integrity, and viability of composite skin grafts. Approach: To determine the effects of HoxA3 on composite skin grafts, we constructed bilayered composite grafts incorporating fibroblasts engineered to constitutively secrete HoxA3. We then transplanted these composite grafts in vivo. Results: The composite grafts produced a stratified epidermal layer after seventeen days in culture and following transplantation in vivo, these grafts exhibit normal epidermal differentiation and reduced contraction compared to controls. In addition, HoxA3 grafts showed increased angiogenesis. Quantitative polymerase chain reaction (PCR) analyses of HoxA3 graft tissue reveal an increase in the downstream HoxA3 target genes MMP-14 and uPAR expression, as well as a reduction in CCL-2 and CxCl-12. Innovation: Expression of secreted HoxA3 in composite grafts represents a comprehensive approach that targets both keratinocytes and endothelial cells to promote epidermal proliferation and angiogenesis. Conclusion: Secreted HoxA3 improves angiogenesis, reduces expression of inflammatory mediators, and prolongs composite skin graft integrity. PMID:25302136

Autism-like behavior caused by deletion of vaccinia-related kinase 3 is improved by TrkB stimulation

PubMed Central

Kang, Myung-Su; Lee, Dohyun; Lee, Seung-Hyun

2017-01-01

Vaccinia-related kinases (VRKs) are multifaceted serine/threonine kinases that play essential roles in various aspects of cell signaling, cell cycle progression, apoptosis, and neuronal development and differentiation. However, the neuronal function of VRK3 is still unknown despite its etiological potential in human autism spectrum disorder (ASD). Here, we report that VRK3-deficient mice exhibit typical symptoms of autism-like behavior, including hyperactivity, stereotyped behaviors, reduced social interaction, and impaired context-dependent spatial memory. A significant decrease in dendritic spine number and arborization were identified in the hippocampus CA1 of VRK3-deficient mice. These mice also exhibited a reduced rectification of AMPA receptor–mediated current and changes in expression of synaptic and signaling proteins, including tyrosine receptor kinase B (TrkB), Arc, and CaMKIIα. Notably, TrkB stimulation with 7,8-dihydroxyflavone reversed the altered synaptic structure and function and successfully restored autism-like behavior in VRK3-deficient mice. These results reveal that VRK3 plays a critical role in neurodevelopmental disorders and suggest a potential therapeutic strategy for ASD. PMID:28899869

Autism-like behavior caused by deletion of vaccinia-related kinase 3 is improved by TrkB stimulation.

PubMed

Kang, Myung-Su; Choi, Tae-Yong; Ryu, Hye Guk; Lee, Dohyun; Lee, Seung-Hyun; Choi, Se-Young; Kim, Kyong-Tai

2017-10-02

Vaccinia-related kinases (VRKs) are multifaceted serine/threonine kinases that play essential roles in various aspects of cell signaling, cell cycle progression, apoptosis, and neuronal development and differentiation. However, the neuronal function of VRK3 is still unknown despite its etiological potential in human autism spectrum disorder (ASD). Here, we report that VRK3 -deficient mice exhibit typical symptoms of autism-like behavior, including hyperactivity, stereotyped behaviors, reduced social interaction, and impaired context-dependent spatial memory. A significant decrease in dendritic spine number and arborization were identified in the hippocampus CA1 of VRK3 -deficient mice. These mice also exhibited a reduced rectification of AMPA receptor-mediated current and changes in expression of synaptic and signaling proteins, including tyrosine receptor kinase B (TrkB), Arc, and CaMKIIα. Notably, TrkB stimulation with 7,8-dihydroxyflavone reversed the altered synaptic structure and function and successfully restored autism-like behavior in VRK3 -deficient mice. These results reveal that VRK3 plays a critical role in neurodevelopmental disorders and suggest a potential therapeutic strategy for ASD. © 2017 Kang et al.

The Cytoplasmic Tail of the T Cell Receptor CD3 ε Subunit Contains a Phospholipid-Binding Motif that Regulates T Cell Functions1

PubMed Central

DeFord-Watts, Laura M.; Tassin, Tara C.; Becker, Amy M.; Medeiros, Jennifer J.; Albanesi, Joseph P.; Love, Paul E.; Wülfing, Christoph; van Oers, Nicolai S. C.

2010-01-01

The CD3 ε subunit of the TCR complex contains two defined signaling domains, a proline-rich sequence and an ITAM. We identified a third signaling sequence in CD3 ε, termed the basic-rich stretch (BRS). Herein, we show that the positively charged residues of the BRS enable this region of CD3 ε to complex a subset of acidic phospholipids, including PI(3)P, PI(4)P, PI(5)P, PI(3,4,5)P3, and PI(4,5)P2. Transgenic mice containing mutations of the BRS exhibited varying developmental defects, ranging from reduced thymic cellularity to a complete block in T cell development. Peripheral T cells from BRS-modified mice also exhibited several defects, including decreased TCR surface expression, reduced TCR-mediated signaling responses to agonist peptide-loaded APCs, and delayed CD3 ε localization to the immunological synapse. Overall, these findings demonstrate a functional role for the CD3 ε lipid-binding domain in T cell biology. PMID:19542373

Overexpression of suppressor of cytokine signaling 3 in the arcuate nucleus of juvenile Phodopus sungorus alters seasonal body weight changes.

PubMed

Ganjam, Goutham K; Benzler, Jonas; Pinkenburg, Olaf; Boucsein, Alisa; Stöhr, Sigrid; Steger, Juliane; Culmsee, Carsten; Barrett, Perry; Tups, Alexander

2013-12-01

The profound seasonal cycle in body weight exhibited by the Djungarian hamster (Phodopus sungorus) is associated with the development of hypothalamic leptin resistance during long day photoperiod (LD, 16:8 h light dark cycle), when body weight is elevated relative to short day photoperiod (SD, 8:16 h light dark cycle). We previously have shown that this seasonal change in physiology is associated with higher levels of mRNA for the potent inhibitor of leptin signaling, suppressor of cytokine signaling-3 (SOCS3), in the arcuate nucleus (ARC) of LD hamsters relative to hamsters in SD. The alteration in SOCS3 gene expression preceded the body weight change suggesting that SOCS3 might be the molecular switch of seasonal body weight changes. To functionally characterize the role of SOCS3 in seasonal body weight regulation, we injected SOCS3 expressing recombinant adeno-associated virus type-2 (rAAV2-SOCS3) constructs into the ARC of leptin sensitive SD hamsters immediately after weaning. Hamsters that received rAAV2 expressing enhanced green fluorescent protein (rAAV2-EGFP) served as controls. ARC-directed SOCS3 overexpression led to a significant increase in body weight over a period of 12 weeks without fully restoring the LD phenotype. This increase was partially due to elevated brown and white adipose tissue mass. Gene expression of pro-opiomelanocortin was increased while thyroid hormone converting enzyme DIO3 mRNA levels were reduced in SD hamsters with SOCS3 overexpression. In conclusion, our data suggest that ARC-directed SOCS3 overexpression partially overcomes the profound seasonal body weight cycle exhibited by the hamster which is associated with altered pro-opiomelanocortin and DIO3 gene expression.

PEST-containing nuclear protein mediates the proliferation, migration, and invasion of human neuroblastoma cells through MAPK and PI3K/AKT/mTOR signaling pathways.

PubMed

Wu, Dong-Dong; Gao, Ying-Ran; Li, Tao; Wang, Da-Yong; Lu, Dan; Liu, Shi-Yu; Hong, Ya; Ning, Hui-Bin; Liu, Jun-Ping; Shang, Jia; Shi, Jun-Feng; Wei, Jian-She; Ji, Xin-Ying

2018-05-02

PEST-containing nuclear protein (PCNP), a novel nuclear protein, is involved in cell proliferation and tumorigenesis. However, the precise mechanism of action of PCNP in the process of tumor growth has not yet been fully elucidated. ShRNA knockdown and overexpression of PCNP were performed in human neuroblastoma cells. Tumorigenic and metastatic effects of PCNP were examined by tumor growth, migration, and invasion assays in vitro, as well as xenograft tumor assay in vivo. PCNP over-expression decreased the proliferation, migration, and invasion of human neuroblastoma cells and down-regulation of PCNP showed reverse effects. PCNP over-expression increased protein expressions of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, and cleaved poly adenosine diphosphate-ribose polymerase, as well as ratios of B-cell lymphoma-2 (Bcl-2)-associated X protein/Bcl-2 and Bcl-2-associated death promoter/B-cell lymphoma-extra large in human neuroblastoma cells, however PCNP knockdown exhibited reverse trends. PCNP over-expression increased phosphorylations of extracellular signal-regulated protein kinase 1/2, p38, c-Jun N-terminal kinase, as well as decreased phosphorylations of phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR), nevertheless PCNP knockdown exhibited opposite effects. Furthermore, PCNP over-expression significantly reduced the growth of human neuroblastoma xenograft tumors by down-regulating angiogenesis, whereas PCNP knockdown markedly promoted the growth of human neuroblastoma xenograft tumors through up-regulation of angiogenesis. PCNP mediates the proliferation, migration, and invasion of human neuroblastoma cells through mitogen-activated protein kinase and PI3K/AKT/mTOR signaling pathways, implying that PCNP is a therapeutic target for patients with neuroblastoma.

Formononetin inhibits enterovirus 71 replication by regulating COX- 2/PGE₂ expression.

PubMed

Wang, Huiqiang; Zhang, Dajun; Ge, Miao; Li, Zhuorong; Jiang, Jiandong; Li, Yuhuan

2015-03-01

The activation of ERK, p38 and JNK signal cascade in host cells has been demonstrated to up-regulate of enterovirus 71 (EV71)-induced cyclooxygenase-2 (COX-2)/ prostaglandins E2 (PGE₂) expression which is essential for viral replication. So, we want to know whether a compound can inhibit EV71 infection by suppressing COX-2/PGE₂ expression. The antiviral effect of formononetin was determined by cytopathic effect (CPE) assay and the time course assays. The influence of formononetin for EV71 replication was determined by immunofluorescence assay, western blotting assay and qRT-PCR assay. The mechanism of the antiviral activity of formononetin was determined by western blotting assay and ELISA assay. Formononetin could reduce EV71 RNA and protein synthesis in a dose-dependent manner. The time course assays showed that formononetin displayed significant antiviral activity both before (24 or 12 h) and after (0-6 h) EV71 inoculation in SK-N-SH cells. Formononetin was also able to prevent EV71-induced cytopathic effect (CPE) and suppress the activation of ERK, p38 and JNK signal pathways. Furthermore, formononetin could suppress the EV71-induced COX-2/PGE₂ expression. Also, formononetin exhibited similar antiviral activities against other members of Picornaviridae including coxsackievirus B2 (CVB2), coxsackievirus B3 (CVB3) and coxsackievirus B6 (CVB6). Formononetin could inhibit EV71-induced COX-2 expression and PGE₂ production via MAPKs pathway including ERK, p38 and JNK. Formononetin exhibited antiviral activities against some members of Picornaviridae. These findings suggest that formononetin could be a potential lead or supplement for the development of new anti-EV71 agents in the future.

Fibroblast Growth Factor Receptors Are Components of Autocrine Signaling Networks in Head and Neck Squamous Cell Carcinoma Cells

PubMed Central

Marshall, Marianne E.; Hinz, Trista K.; Kono, Scott A.; Singleton, Katherine R.; Bichon, Brady; Ware, Kathryn E.; Marek, Lindsay; Frederick, Barbara A.; Raben, David; Heasley, Lynn E.

2011-01-01

Purpose We previously reported that a fibroblast growth factor (FGF) receptor (FGFR) signaling pathway drives growth of lung cancer cell lines of squamous and large cell histologies. Herein, we explored FGFR dependency in cell lines derived from the tobacco-related malignancy, head and neck squamous cell carcinoma (HNSCC). Experimental Design FGF and FGFR mRNA and protein expression was assessed in nine HNSCC cell lines. Dependence on secreted FGF2 for cell growth was tested with FP-1039, an FGFR1-Fc fusion protein. FGFR and EGFR-dependence was defined by sensitivity to multiple inhibitors selective for FGFRs or EGFR. Results FGF2 was expressed in eight of the nine HNSCC cell lines examined. Also, FGFR2 and FGFR3 were frequently expressed while only two lines expressed FGFR1. FP-1039 inhibited growth of HNSCC cell lines expressing FGF2, identifying FGF2 as an autocrine growth factor. FGFR inhibitors selectively reduced in vitro growth and ERK signaling in three HNSCC cell lines while three distinct lines exhibited responsiveness to both EGFR and FGFR inhibitors. Combinations of these drugs yielded additive growth inhibition. Finally, three cell lines were highly sensitive to EGFR TKIs with no contribution from FGFR pathways. Conclusions FGFR signaling was dominant or co-dominant with EGFR in six HNSCC lines while three lines exhibited little or no role for FGFRs and were highly EGFR-dependent. Thus, the HNSCC cell lines can be divided into subsets defined by sensitivity to EGFR and FGFR-specific TKIs. FGFR inhibitors may represent novel therapeutics to deploy alone or in combination with EGFR inhibitors in HNSCC. PMID:21673064

Light irradiation helps magnetotactic bacteria eliminate intracellular reactive oxygen species.

PubMed

Li, Kefeng; Wang, Pingping; Chen, Chuanfang; Chen, Changyou; Li, Lulu; Song, Tao

2017-09-01

Magnetotactic bacteria (MTB) demonstrate photoresponse. However, little is known about the biological significance of this behaviour. Magnetosomes exhibit peroxidase-like activity and can scavenge reactive oxygen species (ROS). Magnetosomes extracted from the Magnetospirillum magneticum strain AMB-1 show enhanced peroxidase-like activity under illumination. The present study investigated the effects of light irradiation on nonmagnetic (without magnetosomes) and magnetic (with magnetosomes) AMB-1 cells. Results showed that light irradiation did not affect the growth of nonmagnetic and magnetic cells but significantly increased magnetosome synthesis and reduced intracellular ROS level in magnetic cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to analyse the expression level of magnetosome formation-associated genes (mamA, mms6, mms13 and mmsF) and stress-related genes (recA, oxyR, SOD, amb0664 and amb2684). Results showed that light irradiation upregulated the expression of mms6, mms13 and mmsF. Furthermore, light irradiation upregulated the expression of stress-related genes in nonmagnetic cells but downregulated them in magnetic cells. Additionally, magnetic cells exhibited stronger phototactic behaviour than nonmagnetic ones. These results suggested that light irradiation could heighten the ability of MTB to eliminate intracellular ROS and help them adapt to lighted environments. This phenomenon may be related to the enhanced peroxidase-like activity of magnetosomes under light irradiation. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

NASA Astrophysics Data System (ADS)

Ibrahim, Ahmed M. A.; Kim, Yonggyun

2008-01-01

Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

Precursor feeding studies and molecular characterization of geraniol synthase establish the limiting role of geraniol in monoterpene indole alkaloid biosynthesis in Catharanthus roseus leaves.

PubMed

Kumar, Krishna; Kumar, Sarma Rajeev; Dwivedi, Varun; Rai, Avanish; Shukla, Ashutosh K; Shanker, Karuna; Nagegowda, Dinesh A

2015-10-01

The monoterpene indole alkaloids (MIAs) are generally derived from strictosidine, which is formed by condensation of the terpene moiety secologanin and the indole moiety tryptamine. There are conflicting reports on the limitation of either terpene or indole moiety in the production of MIAs in Catharanthus roseus cell cultures. Formation of geraniol by geraniol synthase (GES) is the first step in secologanin biosynthesis. In this study, feeding of C. roseus leaves with geraniol, but not tryptophan (precursor for tryptamine), increased the accumulation of the MIAs catharanthine and vindoline, indicating the limitation of geraniol in MIA biosynthesis. This was further validated by molecular and in planta characterization of C. roseus GES (CrGES). CrGES transcripts exhibited leaf and shoot specific expression and were induced by methyl jasmonate. Virus-induced gene silencing (VIGS) of CrGES significantly reduced the MIA content, which was restored to near-WT levels upon geraniol feeding. Moreover, over-expression of CrGES in C. roseus leaves increased MIA content. Further, CrGES exhibited correlation with MIA levels in leaves of different C. roseus cultivars and has significantly lower expression relative to other pathway genes. These results demonstrated that the transcriptional regulation of CrGES and thus, the in planta geraniol availability plays crucial role in MIA biosynthesis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Genetic Transformation and Hairy Root Induction Enhance the Antioxidant Potential of Lactuca serriola L.

PubMed Central

Elkelish, Amr; Elansary, Hosam O.; Ali, Hayssam M.; Elshikh, Mohamed; Witczak, Jacques; Ahmad, Margaret

2017-01-01

Lactuca serriola L. is a herbaceous species, used for human nutrition and medicinal purposes. The high antioxidant capacity of L. serriola indicates the possibility of enhancing its edible and health potential by increasing the flavonoid and phenolic contents. The present study aimed at enhancing the production of phenolics and flavonoids by hairy root cultures in Lactuca serriola transformed with Agrobacterium rhizogenes strain AR15834 harbouring the rolB gene. The genetic transformation of rolB in transformed roots was validated, and rolB expression level was evaluated using real-time qPCR analysis. Expression levels of flavonoid biosynthesis genes (CHI, PAL, FLS, and CHS) were assessed in the hairy and nontransformed roots. Results showed higher expression levels in the transgenic roots than in the nontransformed ones (p < 0.01). Transgenic hairy roots exhibited a 54.8–96.7% increase in the total phenolic content, 38.1–76.2% increase in the total flavonoid content, and 56.7–96.7% increase in the total reducing power when compared with the nontransgenic roots (p < 0.01). DPPH results also revealed that the transgenic hairy roots exhibited a 31.6–50% increase in antioxidant potential, when compared to normal roots. This study addressed the enhancement of secondary metabolite biosynthesis by hairy root induction in L. serriola. PMID:28835782

Polydimethylsiloxane (PDMS) modulates CD38 expression, absorbs retinoic acid and may perturb retinoid signalling.

PubMed

Futrega, Kathryn; Yu, Jianshi; Jones, Jace W; Kane, Maureen A; Lott, William B; Atkinson, Kerry; Doran, Michael R

2016-04-21

Polydimethylsiloxane (PDMS) is the most commonly used material in the manufacture of customized cell culture devices. While there is concern that uncured PDMS oligomers may leach into culture medium and/or hydrophobic molecules may be absorbed into PDMS structures, there is no consensus on how or if PDMS influences cell behaviour. We observed that human umbilical cord blood (CB)-derived CD34(+) cells expanded in standard culture medium on PDMS exhibit reduced CD38 surface expression, relative to cells cultured on tissue culture polystyrene (TCP). All-trans retinoic acid (ATRA) induces CD38 expression, and we reasoned that this hydrophobic molecule might be absorbed by PDMS. Through a series of experiments we demonstrated that ATRA-mediated CD38 expression was attenuated when cultures were maintained on PDMS. Medium pre-incubated on PDMS for extended durations resulted in a time-dependant reduction of ATRA in the medium and increasingly attenuated CD38 expression. This indicated a time-dependent absorption of ATRA into the PDMS. To better understand how PDMS might generally influence cell behaviour, Ingenuity Pathway Analysis (IPA) was used to identify potential upstream regulators. This analysis was performed for differentially expressed genes in primary cells including CD34(+) haematopoietic progenitor cells, mesenchymal stromal cells (MSC), and keratinocytes, and cell lines including prostate cancer epithelial cells (LNCaP), breast cancer epithelial cells (MCF-7), and myeloid leukaemia cells (KG1a). IPA predicted that the most likely common upstream regulator of perturbed pathways was ATRA. We demonstrate here that ATRA is absorbed by PDMS in a time-dependent manner and results in the concomitant reduced expression of CD38 on the cell surface of CB-derived CD34(+) cells.

Cbl downregulation increases RBP4 expression in adipocytes of female mice

PubMed Central

Ameen, Gulizar Issa

2018-01-01

Obesity leads to adipose tissue dysfunction, insulin resistance and diabetes. Adipose tissue produces adipokines that contribute to regulate insulin sensitivity. In turn, insulin stimulates the production and release of some adipokines. Casitas-b-lymphoma proteins (c-Cbl, Cbl-b and Cbl3) are intracellular adaptor signalling proteins that are rapidly phosphorylated by activation of tyrosine kinase receptors. c-Cbl is rapidly phosphorylated by insulin in adipocytes. Here, we tested the hypothesis that Cbl signalling regulates adipokine expression in adipose tissue. We determined the adipokine profile of WAT of Cbl−/− and Cbl+/+ mice in the C57BL6 background. Female Cbl−/− mice exhibited altered expression of adiponectin, leptin and RBP4 in visceral adipose tissue, while no significant changes were seen in male mice. TNFα and IL6 levels were unaffected by Cbl depletion. RBP4 expression was unchanged in liver. Adipose tissue of Cbl−/− animals showed increased basal activation of extracellular regulated kinases (ERK1/2) compared to Cbl+/+. c-Cbl knockdown in 3T3L1 adipocytes also increased basal ERK phosphorylation and RBP4 expression. Inhibition of ERK1/2 phosphorylation in Cbl-depleted 3T3L1 adipocytes or in adipose tissue explants of Cbl−/− mice reduced RBP4 mRNA. 17β-Estradiol increased RBP4 mRNA in adipocytes. Cbl depletion did not change ER expression but increased phosphorylation of ERα at S118, a target site for ERK1/2. ERK1/2 inhibition reduced phosphoER and RBP4 levels. These findings suggest that Cbl contributes to regulate RBP4 expression in adipose of female mice through ERK1/2-mediated activation of ERα. Since Cbl signalling is compromised in diabetes, these data highlight a novel mechanism that upregulates RBP4 locally. PMID:29114012

Cardiac Development in Zebrafish and Human Embryonic Stem Cells Is Inhibited by Exposure to Tobacco Cigarettes and E-Cigarettes

PubMed Central

Palpant, Nathan J.; Hofsteen, Peter; Pabon, Lil; Reinecke, Hans; Murry, Charles E.

2015-01-01

Background Maternal smoking is a risk factor for low birth weight and other adverse developmental outcomes. Objective We sought to determine the impact of standard tobacco cigarettes and e-cigarettes on heart development in vitro and in vivo. Methods Zebrafish (Danio rerio) were used to assess developmental effects in vivo and cardiac differentiation of human embryonic stem cells (hESCs) was used as a model for in vitro cardiac development. Results In zebrafish, exposure to both types of cigarettes results in broad, dose-dependent developmental defects coupled with severe heart malformation, pericardial edema and reduced heart function. Tobacco cigarettes are more toxic than e-cigarettes at comparable nicotine concentrations. During cardiac differentiation of hESCs, tobacco smoke exposure results in a delayed transition through mesoderm. Both types of cigarettes decrease expression of cardiac transcription factors in cardiac progenitor cells, suggesting a persistent delay in differentiation. In definitive human cardiomyocytes, both e-cigarette- and tobacco cigarette-treated samples showed reduced expression of sarcomeric genes such as MLC2v and MYL6. Furthermore, tobacco cigarette-treated samples had delayed onset of beating and showed low levels and aberrant localization of N-cadherin, reduced myofilament content with significantly reduced sarcomere length, and increased expression of the immature cardiac marker smooth muscle alpha-actin. Conclusion These data indicate a negative effect of both tobacco cigarettes and e-cigarettes on heart development in vitro and in vivo. Tobacco cigarettes are more toxic than E-cigarettes and exhibit a broader spectrum of cardiac developmental defects. PMID:25978043

Withaferin-A Reduces Type I Collagen Expression In Vitro and Inhibits Development of Myocardial Fibrosis In Vivo

PubMed Central

Challa, Azariyas A.; Vukmirovic, Milica; Blackmon, John; Stefanovic, Branko

2012-01-01

Type I collagen is the most abundant protein in the human body. Its excessive synthesis results in fibrosis of various organs. Fibrosis is a major medical problem without an existing cure. Excessive synthesis of type I collagen in fibrosis is primarily due to stabilization of collagen mRNAs. We recently reported that intermediate filaments composed of vimentin regulate collagen synthesis by stabilizing collagen mRNAs. Vimentin is a primary target of Withaferin-A (WF-A). Therefore, we hypothesized that WF-A may reduce type I collagen production by disrupting vimentin filaments and decreasing the stability of collagen mRNAs. This study is to determine if WF-A exhibits anti-fibrotic properties in vitro and in vivo and to elucidate the molecular mechanisms of its action. In lung, skin and heart fibroblasts WF-A disrupted vimentin filaments at concentrations of 0.5–1.5 µM and reduced 3 fold the half-lives of collagen α1(I) and α2(I) mRNAs and protein expression. In addition, WF-A inhibited TGF-β1 induced phosphorylation of TGF-β1 receptor I, Smad3 phosphorylation and transcription of collagen genes. WF-A also inhibited in vitro activation of primary hepatic stellate cells and decreased their type I collagen expression. In mice, administration of 4 mg/kg WF-A daily for 2 weeks reduced isoproterenol-induced myocardial fibrosis by 50%. Our findings provide strong evidence that Withaferin-A could act as an anti-fibrotic compound against fibroproliferative diseases, including, but not limited to, cardiac interstitial fibrosis. PMID:22900077

Downregulation of Lysyl Oxidase Protects Retinal Endothelial Cells From High Glucose-Induced Apoptosis.

PubMed

Kim, Dongjoon; Mecham, Robert P; Trackman, Philip C; Roy, Sayon

2017-05-01

To investigate the effect of reducing high glucose (HG)-induced lysyl oxidase (LOX) overexpression and increased activity on retinal endothelial cell apoptosis. Rat retinal endothelial cells (RRECs) were grown in normal (N) or HG (30 mM glucose) medium for 7 days. In parallel, RRECs were grown in HG medium and transfected with LOX small interfering RNA (siRNA), scrambled siRNA as control, or exposed to β-aminopropionitrile (BAPN), a LOX inhibitor. LOX expression, AKT activation, and caspase-3 activity were determined by Western blot (WB) analysis and apoptosis by differential dye staining assay. Moreover, to determine whether diabetes-induced LOX overexpression alters AKT activation and promotes apoptosis, changes in LOX expression, AKT phosphorylation, caspase-3 activation, and Bax expression were assessed in retinas of streptozotocin (STZ)-induced diabetic mice and LOX heterozygous knockout (LOX+/-) mice. WB analysis indicated significant LOX overexpression and reduced AKT activation under HG condition in RRECs. Interestingly, when cells grown in HG were transfected with LOX siRNA or exposed to BAPN, the number of apoptotic cells was significantly decreased concomitant with increased AKT phosphorylation. Diabetic mouse retinas exhibited LOX overexpression, decreased AKT phosphorylation, and increased Bax and caspase-3 activation compared to values in nondiabetic mice. In LOX+/- mice, reduced LOX levels were observed with increased AKT activity, and reduced Bax and caspase-3 activity. Furthermore, decreased levels of LOX in the LOX+/- mice was protective against diabetes-induced apoptosis. Findings from this study indicate that preventing LOX overexpression may be protective against HG-induced apoptosis in retinal vascular cells associated with diabetic retinopathy.

Characterization of an ntrX mutant of Neisseria gonorrhoeae reveals a response regulator that controls expression of respiratory enzymes in oxidase-positive proteobacteria.

PubMed

Atack, John M; Srikhanta, Yogitha N; Djoko, Karrera Y; Welch, Jessica P; Hasri, Norain H M; Steichen, Christopher T; Vanden Hoven, Rachel N; Grimmond, Sean M; Othman, Dk Seti Maimonah Pg; Kappler, Ulrike; Apicella, Michael A; Jennings, Michael P; Edwards, Jennifer L; McEwan, Alastair G

2013-06-01

NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria. Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA), and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.

A role for anterior thalamic nuclei in affective cognition: interaction with environmental conditions.

PubMed

Dupire, Alexandra; Kant, Patricia; Mons, Nicole; Marchand, Alain R; Coutureau, Etienne; Dalrymple-Alford, John; Wolff, Mathieu

2013-05-01

Damage to anterior thalamic nuclei (ATN) is a well-known cause of diencephalic pathology that produces a range of cognitive deficits reminiscent of a hippocampal syndrome. Anatomical connections of the ATN also extend to cerebral areas that support affective cognition. Enriched environments promote recovery of declarative/relational memory after ATN lesions and are known to downregulate emotional behaviors. Hence, the performance of standard-housed and enriched ATN rats in a range of behavioral tasks engaging affective cognition was compared. ATN rats exhibited reduced anxiety responses in the elevated plus maze, increased activity and reduced corticosterone responses when exploring an open field, and delayed acquisition of a conditioned contextual fear response. ATN rats also exhibited reduced c-Fos and phosphorylated cAMP response element-binding protein (pCREB) immunoreactivity in the hippocampal formation and the amygdala after completion of the contextual fear test. Marked c-Fos hypoactivity and reduced pCREB levels were also evident in the granular retrosplenial cortex and, to a lesser extent, in the anterior cingulate cortex. Unlike standard-housed ATN rats, enriched ATN rats expressed virtually no fear of the conditioned context. These results show that the ATN regulate affective cognition and that damage to this region may produce markedly different behavioral effects as a function of environmental housing conditions. Copyright © 2013 Wiley Periodicals, Inc.

Retinoic Acid Improves Morphology of Cultured Peritoneal Mesothelial Cells from Patients Undergoing Dialysis

PubMed Central

Retana, Carmen; Sanchez, Elsa I.; Gonzalez, Sirenia; Perez-Lopez, Alejandro; Cruz, Armando; Lagunas-Munoz, Jesus; Alfaro-Cruz, Carmen; Vital-Flores, Socorro; Reyes, José L.

2013-01-01

Patients undergoing continuous ambulatory peritoneal dialysis are classified according to their peritoneal permeability as low transporter (low solute permeability) or High transporter (high solute permeability). Factors that determine the differences in permeability between them have not been fully disclosed. We investigated morphological features of cultured human peritoneal mesothelial cells from low or high transporter patients and its response to All trans retinoic Acid (ATRA, vitamin A active metabolite), as compared to non-uremic human peritoneal mesothelial cells. Control cells were isolated from human omentum. High or low transporter cells were obtained from dialysis effluents. Cells were cultured in media containing ATRA (0, 50, 100 or 200 nM). We studied length and distribution of microvilli and cilia (scanning electron microscopy), epithelial (cytokeratin, claudin-1, ZO-1 and occludin) and mesenchymal (vimentin and α-smooth muscle actin) transition markers by immunofluorescence and Western blot, and transforming growth factor β1 expression by Western blot. Low and high transporter exhibited hypertrophic cells, reduction in claudin-1, occludin and ZO-1 expression, cytokeratin and vimentin disorganization and positive α-smooth muscle actin label. Vimentin, α-smooth muscle actin and transforming growth factor- β1 were overexpressed in low transporter. Ciliated cells were diminished in low and high transporters. Microvilli number and length were severely reduced in high transporter. ATRA reduced hypertrophic cells number in low transporter. It also improved cytokeratin and vimentin organization, decreased vimentin and α-smooth muscle actin expression, and increased claudin 1, occludin and ZO-1 expression, in low and high transporter. In low transporter, ATRA reduced transforming growth factor-β1 expression. ATRA augmented percentage of ciliated cells in low and high transporter. It also augmented cilia length in high transporter. Alterations in structure, epithelial mesenchymal markers and transforming growth factor-β1expression were differential between low and high transporter. Beneficial effects of ATRA were improved human peritoneal mesothelial cells morphology tending to normalize structures. PMID:24223992

Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency.

PubMed

Eudes, Aymerick; Sathitsuksanoh, Noppadon; Baidoo, Edward E K; George, Anthe; Liang, Yan; Yang, Fan; Singh, Seema; Keasling, Jay D; Simmons, Blake A; Loqué, Dominique

2015-12-01

Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3-dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3-dehydroshikimate - an intermediate of the shikimate pathway - into protocatechuate. Compared to wild-type plants, lines expressing QsuB contain higher amounts of protocatechuate, p-coumarate, p-coumaraldehyde and p-coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D-NMR spectroscopy and pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS) reveal an increase of p-hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size-exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Cationic star-shaped polymer as an siRNA carrier for reducing MMP-9 expression in skin fibroblast cells and promoting wound healing in diabetic rats.

PubMed

Li, Na; Luo, Heng-Cong; Yang, Chuan; Deng, Jun-Jie; Ren, Meng; Xie, Xiao-Ying; Lin, Diao-Zhu; Yan, Li; Zhang, Li-Ming

2014-01-01

Excessive expression of matrix metalloproteinase-9 (MMP-9) is deleterious to the cutaneous wound-healing process in the context of diabetes. The aim of the present study was to explore whether a cationic star-shaped polymer consisting of β-cyclodextrin (β-CD) core and poly(amidoamine) dendron arms (β-CD-[D₃]₇) could be used as the gene carrier of small interfering RNA (siRNA) to reduce MMP-9 expression for enhanced diabetic wound healing. The cytotoxicity of β-CD-(D₃)₇ was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MMT) method in the rat CRL1213 skin fibroblast cell line. The transfection efficiency of β-CD-(D₃)₇/MMP-9-small interfering RNA (siRNA) complexes was determined by confocal microscopy and flow cytometry. Quantitative real time (RT) polymerase chain reaction was performed to measure the gene expression of MMP-9 after the transfection by β-CD-(D₃)₇/MMP-9-siRNA complexes. The β-CD-(D₃)₇/MMP-9-siRNA complexes were injected on the wounds of streptozocin-induced diabetic rats. Wound closure was measured on days 4 and 7 post-wounding. β-CD-(D₃)₇ exhibited low cytotoxicity in fibroblast cells, and easily formed the complexes with MMP-9-siRNA. The β-CD-(D₃)₇/MMP-9-siRNA complexes were readily taken up by fibroblast cells, resulting in the downregulation of MMP-9 gene expression (P<0.01). Animal experiments revealed that the treatment by β-CD-(D₃)₇/MMP-9-siRNA complexes enhanced wound closure in diabetic rats on day 7 post-wounding (P<0.05). β-CD-(D₃)₇ may be used as an efficient carrier for the delivery of MMP-9-siRNA to reduce MMP-9 expression in skin fibroblast cells and promote wound healing in diabetic rats.

Dioxin-induced retardation of development through a reduction in the expression of pituitary hormones and possible involvement of an aryl hydrocarbon receptor in this defect: A comparative study using two strains of mice with different sensitivities to dioxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takeda, Tomoki; Taura, Junki; Hattori, Yukiko

We have previously revealed that treating pregnant rats with 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) reduces the expression of gonadotropins and growth hormone (GH) in the fetal and neonatal pituitary. A change in gonadotropin expression impairs the testicular expression of steroidogenic proteins in perinatal pups, and imprint defects in sexual behavior after reaching maturity. In this study, we examined whether TCDD also affects the expression of gonadotropin and GH in mice using C57BL/6J and DBA/2J strains which express the aryl hydrocarbon receptor (Ahr) exhibiting a different affinity for TCDD. When pregnant C57BL/6J mice at gestational day (GD) 12 were given oral TCDD (0.2–20 μg/kg),more » all doses significantly attenuated the pituitary expression of gonadotropin mRNAs in fetuses at GD18. On the other hand, in DBA/2J mice, a much higher dose of TCDD (20 μg/kg) was needed to produce a significant attenuation. Such reduction in the C57BL/6J strain continued until at least postnatal day (PND) 4. In agreement with this, TCDD reduced the testicular expression of steroidogenic proteins in C57BL/6J neonates at PND2 and 4, although the same did not occur in the fetal testis and ovary. Furthermore, TCDD reduced the perinatal expression of GH, litter size and the body weight of newborn pups only in the C57BL/6J strain. These results suggest that 1) also in mice, maternal exposure to TCDD attenuates gonadotropin-regulated steroidogenesis and GH expression leading to the impairment of pup development and sexual immaturity; and 2) Ahr activation during the late fetal and early postnatal stages is required for these defects. - Highlights: • The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on mouse growth was studied. • TCDD reduced the levels of luteinizing hormone and growth hormone in perinatal pups. • Maternal exposure to TCDD also attenuated testicular steroidogenesis in pups. • The above effects of TCDD were more pronounced in C57BL/6J than in DBA/2J strain. • TCDD seems to disturb pup maturation by activating aryl hydrocarbon receptor.« less

Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging.

PubMed

Davis, Hannah M; Pacheco-Costa, Rafael; Atkinson, Emily G; Brun, Lucas R; Gortazar, Arancha R; Harris, Julia; Hiasa, Masahiro; Bolarinwa, Surajudeen A; Yoneda, Toshiyuki; Ivan, Mircea; Bruzzaniti, Angela; Bellido, Teresita; Plotkin, Lilian I

2017-06-01

Skeletal aging results in apoptosis of osteocytes, cells embedded in bone that control the generation/function of bone forming and resorbing cells. Aging also decreases connexin43 (Cx43) expression in bone; and osteocytic Cx43 deletion partially mimics the skeletal phenotype of old mice. Particularly, aging and Cx43 deletion increase osteocyte apoptosis, and osteoclast number and bone resorption on endocortical bone surfaces. We examined herein the molecular signaling events responsible for osteocyte apoptosis and osteoclast recruitment triggered by aging and Cx43 deficiency. Cx43-silenced MLO-Y4 osteocytic (Cx43 def ) cells undergo spontaneous cell death in culture through caspase-3 activation and exhibit increased levels of apoptosis-related genes, and only transfection of Cx43 constructs able to form gap junction channels reverses Cx43 def cell death. Cx43 def cells and bones from old mice exhibit reduced levels of the pro-survival microRNA miR21 and, consistently, increased levels of the miR21 target phosphatase and tensin homolog (PTEN) and reduced phosphorylated Akt, whereas PTEN inhibition reduces Cx43 def cell apoptosis. miR21 reduction is sufficient to induce apoptosis of Cx43-expressing cells and miR21 deletion in miR21 fl/fl bones increases apoptosis-related gene expression, whereas a miR21 mimic prevents Cx43 def cell apoptosis, demonstrating that miR21 lies downstream of Cx43. Cx43 def cells release more osteoclastogenic cytokines [receptor activator of NFκB ligand (RANKL)/high-mobility group box-1 (HMGB1)], and caspase-3 inhibition prevents RANKL/HMGB1 release and the increased osteoclastogenesis induced by conditioned media from Cx43 def cells, which is blocked by antagonizing HMGB1-RAGE interaction. These findings identify a novel Cx43/miR21/HMGB1/RANKL pathway involved in preventing osteocyte apoptosis that also controls osteoclast formation/recruitment and is impaired with aging. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.