[Involvement of cellular immunity and humoral immunity in mixed allergy induced by trichloroethylene].

PubMed

Xu, Xinyun; Li, Xueyu; Liu, Yuefeng

2014-12-01

To investigate whether cellular immunity and humoral immunity are involved in trichlorethylene (TCE)-induced mixed allergy, then provide the scientific basis for the mechanism of this disease. Guinea pigs and rats were tested for this study by application of guinea pig maximization test (GPMT), the animals were randomly divided into negative control, positive control and TCE treatment groups. Animals of these groups were administrated with olive oil, 2, 4-dinitrochlorobenzene (DNCB), and TCE, respectively, by intradermal injection. After TCE administration, rat peripheral blood samples were collected by flow cytometry to detect lymphocytes CD3⁺, CD4⁺, CD8⁺. Guinea pig peripheral blood samples were collected to detect the levels of IgG, IgA, IgM, C3, C4, and the spleens were taken out from guinea pigs after various treatment, mRNA expression of GATA3, T-bet, CTLA4 and Foxp3 in lymphocytes of guinea pig spleen was detected by real-time fluorescent PCR assay. Additionally, TCE allergic dermatitis patients were selected for the study, the peripheral blood samples were collected from the TCE patients group and control group, quantitative PCR was applied to detect mRNA expression of immune-related genes Foxp3, GATA3, CTLA4, T-bet. TCE induced obvious skin allergic reaction in guinea pigs, the sensitization rate was 83.3%, IgG levels in TCE group and positive control increased significantly. Additionally, mRNA expression levels of GATA3, T-bet, CTLA4 significantly elevated in TCE group and positive control, but Foxp3 mRNA levels decreased. The lymphocytes CD3⁺ ratio in TCE group and positive control of rats was higher than that in negative control, we found that there was no statistical difference of CD4⁺, CD8⁺, CD4⁺/CD8⁺ between TCE group and negative control of rats. The mRNA expression levels of Foxp3, GATA3, CTLA4 in TCE patients increased by 115%, 97%, 241%, respectively as compared with the control, T-bet levels decreased by 47%when compared with the control. TCE could induce obvious changes of cellular immunity and humoral immunity in guinea pigs, rats, and TCE patients, these findings indicated that TCE-induced immunological disorder belongs to the mixed allergy with involvment of cellular immunity and humoral immunity, the mixed allergy might be type IV and type II allergy.

Exaptive origins of regulated mRNA decay in eukaryotes

PubMed Central

Hamid, Fursham M.

2016-01-01

Eukaryotic gene expression is extensively controlled at the level of mRNA stability and the mechanisms underlying this regulation are markedly different from their archaeal and bacterial counterparts. We propose that two such mechanisms, nonsense‐mediated decay (NMD) and motif‐specific transcript destabilization by CCCH‐type zinc finger RNA‐binding proteins, originated as a part of cellular defense against RNA pathogens. These branches of the mRNA turnover pathway might have been used by primeval eukaryotes alongside RNA interference to distinguish their own messages from those of RNA viruses and retrotransposable elements. We further hypothesize that the subsequent advent of “professional” innate and adaptive immunity systems allowed NMD and the motif‐triggered mechanisms to be efficiently repurposed for regulation of endogenous cellular transcripts. This scenario explains the rapid emergence of archetypical mRNA destabilization pathways in eukaryotes and argues that other aspects of post‐transcriptional gene regulation in this lineage might have been derived through a similar exaptation route. PMID:27438915

Exaptive origins of regulated mRNA decay in eukaryotes.

PubMed

Hamid, Fursham M; Makeyev, Eugene V

2016-09-01

Eukaryotic gene expression is extensively controlled at the level of mRNA stability and the mechanisms underlying this regulation are markedly different from their archaeal and bacterial counterparts. We propose that two such mechanisms, nonsense-mediated decay (NMD) and motif-specific transcript destabilization by CCCH-type zinc finger RNA-binding proteins, originated as a part of cellular defense against RNA pathogens. These branches of the mRNA turnover pathway might have been used by primeval eukaryotes alongside RNA interference to distinguish their own messages from those of RNA viruses and retrotransposable elements. We further hypothesize that the subsequent advent of "professional" innate and adaptive immunity systems allowed NMD and the motif-triggered mechanisms to be efficiently repurposed for regulation of endogenous cellular transcripts. This scenario explains the rapid emergence of archetypical mRNA destabilization pathways in eukaryotes and argues that other aspects of post-transcriptional gene regulation in this lineage might have been derived through a similar exaptation route. © 2016 The Authors BioEssays Published by WILEY Periodicals, Inc.

Understanding the cancer cell phenotype beyond the limitations of current omics analyses.

PubMed

Moreno-Sánchez, Rafael; Saavedra, Emma; Gallardo-Pérez, Juan Carlos; Rumjanek, Franklin D; Rodríguez-Enríquez, Sara

2016-01-01

Efforts to understand the mechanistic principles driving cancer metabolism and proliferation have been lately governed by genomic, transcriptomic and proteomic studies. This paper analyzes the caveats of these approaches. As molecular biology's central dogma proposes a unidirectional flux of information from genes to mRNA to proteins, it has frequently been assumed that monitoring the changes in the gene sequences and in mRNA and protein contents is sufficient to explain complex cellular processes. Such a stance commonly disregards that post-translational modifications can alter the protein function/activity and also that regulatory mechanisms enter into action, to coordinate the protein activities of pathways/cellular processes, in order to keep the cellular homeostasis. Hence, the actual protein activities (as enzymes/transporters/receptors) and their regulatory mechanisms ultimately dictate the final outcomes of a pathway/cellular process. In this regard, it is here documented that the mRNA levels of many metabolic enzymes and transcriptional factors have no correlation with the respective protein contents and activities. The validity of current clinical mRNA-based tests and proposed metabolite biomarkers for cancer detection/prognosis is also discussed. Therefore, it is proposed that, to achieve a thorough understanding of the modifications undergone by proliferating cancer cells, it is mandatory to experimentally analyze the cellular processes at the functional level. This could be achieved (a) locally, by examining the actual protein activities in the cell and their kinetic properties (or at least kinetically characterize the most controlling steps of the pathway/cellular process); (b) systemically, by analyzing the main fluxes of the pathway/cellular process, and how they are modulated by metabolites, all which should contribute to comprehending the regulatory mechanisms that have been altered in cancer cells. By adopting a more holistic approach it may become possible to improve the design of therapeutic strategies that would target cancer cells more specifically. © 2015 FEBS.

Translational induction of heat shock transcription factor σ32: evidence for a built-in RNA thermosensor

PubMed Central

Morita, Miyo Terao; Tanaka, Yoshiyuki; Kodama, Takashi S.; Kyogoku, Yoshimasa; Yanagi, Hideki; Yura, Takashi

1999-01-01

Induction of heat shock proteins in Escherichia coli is primarily caused by increased cellular levels of the heat shock σ-factor σ32 encoded by the rpoH gene. Increased σ32 levels result from both enhanced synthesis and stabilization. Previous work indicated that σ32 synthesis is induced at the translational level and is mediated by the mRNA secondary structure formed within the 5′-coding sequence of rpoH, including the translation initiation region. To understand the mechanism of heat induction of σ32 synthesis further, we analyzed expression of rpoH–lacZ gene fusions with altered stability of mRNA structure before and after heat shock. A clear correlation was found between the stability and expression or the extent of heat induction. Temperature-melting profiles of mRNAs with or without mutations correlated well with the expression patterns of fusion genes carrying the corresponding mutations in vivo. Furthermore, temperature dependence of mRNA–30S ribosome–tRNAfMet complex formation with wild-type or mutant mRNAs in vitro agreed well with that of the expression of gene fusions in vivo. Our results support a novel mechanism in which partial melting of mRNA secondary structure at high temperature enhances ribosome entry and translational initiation without involvement of other cellular components, that is, intrinsic mRNA stability controls synthesis of a transcriptional regulator. PMID:10090722

Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells.

PubMed

Safina, Alfiya; Garcia, Henry; Commane, Mairead; Guryanova, Olga; Degan, Seamus; Kolesnikova, Kateryna; Gurova, Katerina V

2013-08-01

Facilitates chromatin transcription (FACT) is a chromatin remodeling complex with two subunits: SSRP1 and SPT16. Mechanisms controlling FACT levels are of interest, since the complex is not expressed in most differentiated cells, but is frequently upregulated in cancer, particularly in poorly differentiated, aggressive tumors. Moreover, inhibition of FACT expression or function in tumor cells interferes with their survival. Here we demonstrate that SSRP1 and SPT16 protein levels decline upon induction of cellular differentiation or senescence in vitro and that similar declines in protein levels for both SSRP1 and SPT16 occur upon RNAi-mediated knockdown of either SSRP1 or SPT16. The interdependence of SSRP1 and SPT16 protein levels was found to be due to their association with SSRP1 and SPT16 mRNAs, which stabilizes the proteins. In particular, presence of SSRP1 mRNA is critical for SPT16 protein stability. In addition, binding of SSRP1 and SPT16 mRNAs to the FACT complex increases the stability and efficiency of translation of the mRNAs. These data support a model in which the FACT complex is stable when SSRP1 mRNA is present, but quickly degrades when SSRP1 mRNA levels drop. In the absence of FACT complex, SSRP1 and SPT16 mRNAs are unstable and inefficiently translated, making reactivation of FACT function unlikely in normal cells. Thus, we have described a complex and unusual mode of regulation controlling cellular FACT levels that results in amplified and stringent control of FACT activity. The FACT dependence of tumor cells suggests that mechanisms controlling FACT levels could be targeted for anticancer therapy.

Mechanisms of information decoding in a cascade system of gene expression

NASA Astrophysics Data System (ADS)

Wang, Haohua; Yuan, Zhanjiang; Liu, Peijiang; Zhou, Tianshou

2016-05-01

Biotechnology advances have allowed investigation of heterogeneity of cellular responses to stimuli on the single-cell level. Functionally, this heterogeneity can compromise cellular responses to environmental signals, and it can also enlarge the repertoire of possible cellular responses and hence increase the adaptive nature of cellular behaviors. However, the mechanism of how this response heterogeneity is generated remains elusive. Here, by systematically analyzing a representative cellular signaling system, we show that (1) the upstream activator always amplifies the downstream burst frequency (BF) but the noiseless activator performs better than the noisy one, remarkably for small or moderate input signal strengths, and the repressor always reduces the downstream BF but the difference in the reducing effect between noiseless and noise repressors is very small; (2) both the downstream burst size and mRNA mean are a monotonically increasing function of the activator strength but a monotonically decreasing function of the repressor strength; (3) for repressor-type input, there is a noisy signal strength such that the downstream mRNA noise arrives at an optimal level, but for activator-type input, the output noise intensity is fundamentally a monotonically decreasing function of the input strength. Our results reveal the essential mechanisms of both signal information decoding and cellular response heterogeneity, whereas our analysis provides a paradigm for analyzing dynamics of noisy biochemical signaling systems.

Biomolecular engineering of intracellular switches in eukaryotes

PubMed Central

Pastuszka, M.K.; Mackay, J.A.

2010-01-01

Tools to selectively and reversibly control gene expression are useful to study and model cellular functions. When optimized, these cellular switches can turn a protein's function “on” and “off” based on cues designated by the researcher. These cues include small molecules, drugs, hormones, and even temperature variations. Here we review three distinct areas in gene expression that are commonly targeted when designing cellular switches. Transcriptional switches target gene expression at the level of mRNA polymerization, with examples including the tetracycline gene induction system as well as nuclear receptors. Translational switches target the process of turning the mRNA signal into protein, with examples including riboswitches and RNA interference. Post-translational switches control how proteins interact with one another to attenuate or relay signals. Examples of post-translational modification include dimerization and intein splicing. In general, the delay times between switch and effect decreases from transcription to translation to post-translation; furthermore, the fastest switches may offer the most elegant opportunities to influence and study cell behavior. We discuss the pros and cons of these strategies, which directly influence their usefulness to study and implement drug targeting at the tissue and cellular level. PMID:21209849

Cytosolic increased labile Zn2+ contributes to arrhythmogenic action potentials in left ventricular cardiomyocytes through protein thiol oxidation and cellular ATP depletion.

PubMed

Degirmenci, Sinan; Olgar, Yusuf; Durak, Aysegul; Tuncay, Erkan; Turan, Belma

2018-07-01

Intracellular labile (free) Zn 2+ -level ([Zn 2+ ] i ) is low and increases markedly under pathophysiological conditions in cardiomyocytes. High [Zn 2+ ] i is associated with alterations in excitability and ionic-conductances while exact mechanisms are not clarified yet. Therefore, we examined the elevated-[Zn 2+ ] i on some sarcolemmal ionic-mechanisms, which can mediate cardiomyocyte dysfunction. High-[Zn 2+ ] i induced significant changes in action potential (AP) parameters, including depolarization in resting membrane-potential and prolongations in AP-repolarizing phases. We detected also the time-dependent effects such as induction of spontaneous APs at the time of ≥ 3 min following [Zn 2+ ] i increases, a manner of cellular ATP dependent and reversible with disulfide-reducing agent dithiothreitol, DTT. High-[Zn 2+ ] i induced inhibitions in voltage-dependent K + -channel currents, such as transient outward K + -currents, I to , steady-state currents, I ss and inward-rectifier K + -currents, I K1 , reversible with DTT seemed to be responsible from the prolongations in APs. We, for the first time, demonstrated that lowering cellular ATP level induced significant decreaeses in both I ss and I K1 , while no effect on I to . However, the increased-[Zn 2+ ] i could induce marked activation in ATP-sensitive K + -channel currents, I KATP , depending on low cellular ATP and thiol-oxidation levels of these channels. The mRNA levels of Kv4.3, Kv1.4 and Kv2.1 were depressed markedly with increased-[Zn 2+ ] i with no change in mRNA level of Kv4.2, while the mRNA level of I KATP subunit, SUR2A was increased significantly with increased-[Zn 2+ ] i , being reversible with DTT. Overall we demonstrated that high-[Zn 2+ ] i, even if nanomolar levels, alters cardiac function via prolonged APs of cardiomyocytes, at most, due to inhibitions in voltage-dependent K + -currents, although activation of I KATP is playing cardioprotective role, through some biochemical changes in cellular ATP- and thiol-oxidation levels. It seems, a well-controlled [Zn 2+ ] i can be novel therapeutic target for cardiac complications under pathological conditions including oxidative stress. Copyright © 2018 Elsevier GmbH. All rights reserved.

Characterization of the African Swine Fever Virus Decapping Enzyme during Infection

PubMed Central

Quintas, Ana; Pérez-Núñez, Daniel; Sánchez, Elena G.; Nogal, Maria L.; Hentze, Matthias W.; Castelló, Alfredo

2017-01-01

ABSTRACT African swine fever virus (ASFV) infection is characterized by a progressive decrease in cellular protein synthesis with a concomitant increase in viral protein synthesis, though the mechanism by which the virus achieves this is still unknown. Decrease of cellular mRNA is observed during ASFV infection, suggesting that inhibition of cellular proteins is due to an active mRNA degradation process. ASFV carries a gene (Ba71V D250R/Malawi g5R) that encodes a decapping protein (ASFV-DP) that has a Nudix hydrolase motif and decapping activity in vitro. Here, we show that ASFV-DP was expressed from early times and accumulated throughout the infection with a subcellular localization typical of the endoplasmic reticulum, colocalizing with the cap structure and interacting with the ribosomal protein L23a. ASFV-DP was capable of interaction with poly(A) RNA in cultured cells, primarily mediated by the N-terminal region of the protein. ASFV-DP also interacted with viral and cellular RNAs in the context of infection, and its overexpression in infected cells resulted in decreased levels of both types of transcripts. This study points to ASFV-DP as a viral decapping enzyme involved in both the degradation of cellular mRNA and the regulation of viral transcripts. IMPORTANCE Virulent ASFV strains cause a highly infectious and lethal disease in domestic pigs for which there is no vaccine. Since 2007, an outbreak in the Caucasus region has spread to Russia, jeopardizing the European pig population and making it essential to deepen knowledge about the virus. Here, we demonstrate that ASFV-DP is a novel RNA-binding protein implicated in the regulation of mRNA metabolism during infection, making it a good target for vaccine development. PMID:29021398

Characterization of the African Swine Fever Virus Decapping Enzyme during Infection.

PubMed

Quintas, Ana; Pérez-Núñez, Daniel; Sánchez, Elena G; Nogal, Maria L; Hentze, Matthias W; Castelló, Alfredo; Revilla, Yolanda

2017-12-15

African swine fever virus (ASFV) infection is characterized by a progressive decrease in cellular protein synthesis with a concomitant increase in viral protein synthesis, though the mechanism by which the virus achieves this is still unknown. Decrease of cellular mRNA is observed during ASFV infection, suggesting that inhibition of cellular proteins is due to an active mRNA degradation process. ASFV carries a gene (Ba71V D250R/Malawi g5R) that encodes a decapping protein (ASFV-DP) that has a Nudix hydrolase motif and decapping activity in vitro Here, we show that ASFV-DP was expressed from early times and accumulated throughout the infection with a subcellular localization typical of the endoplasmic reticulum, colocalizing with the cap structure and interacting with the ribosomal protein L23a. ASFV-DP was capable of interaction with poly(A) RNA in cultured cells, primarily mediated by the N-terminal region of the protein. ASFV-DP also interacted with viral and cellular RNAs in the context of infection, and its overexpression in infected cells resulted in decreased levels of both types of transcripts. This study points to ASFV-DP as a viral decapping enzyme involved in both the degradation of cellular mRNA and the regulation of viral transcripts. IMPORTANCE Virulent ASFV strains cause a highly infectious and lethal disease in domestic pigs for which there is no vaccine. Since 2007, an outbreak in the Caucasus region has spread to Russia, jeopardizing the European pig population and making it essential to deepen knowledge about the virus. Here, we demonstrate that ASFV-DP is a novel RNA-binding protein implicated in the regulation of mRNA metabolism during infection, making it a good target for vaccine development. Copyright © 2017 Quintas et al.

A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export.

PubMed

Gong, Danyang; Kim, Yong Hoon; Xiao, Yuchen; Du, Yushen; Xie, Yafang; Lee, Kevin K; Feng, Jun; Farhat, Nisar; Zhao, Dawei; Shu, Sara; Dai, Xinghong; Chanda, Sumit K; Rana, Tariq M; Krogan, Nevan J; Sun, Ren; Wu, Ting-Ting

2016-11-09

Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication. Published by Elsevier Inc.

Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA.

PubMed

Pereira, Carina F; Read, Eliot K C; Wise, Helen M; Amorim, Maria J; Digard, Paul

2017-08-01

Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export. IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as previously identified roles in antagonizing the innate immune defenses of the cell and directly upregulating translation of viral mRNAs, it also promotes the nuclear export of the viral late gene mRNAs by acting as an adaptor between the viral mRNAs and the cellular mRNA nuclear export machinery. Copyright © 2017 Pereira et al.

Fasting increases the phosphorylation of AMPK and expression of sirtuin1 in muscle of adult male northern elephant seals (Mirounga angustirostris).

PubMed

Lee, Debby; Martinez, Bridget; Crocker, Daniel E; Ortiz, Rudy M

2017-02-01

Fasting typically suppresses thyroid hormone (TH)-mediated cellular events and increases sirtuin 1 (SIRT1) activity. THs may regulate metabolism through nongenomic pathways and directly through activation of adenosine monophosphate-activated protein kinase (AMPK). Adult male elephant seals ( Mirounga angustirostris ) are active, hypermetabolic, and normothermic during their annual breeding fast, which is characterized by stable TH levels. However, the contribution of TH to maintenance of their fasting metabolism is unknown. To investigate the fasting effects on cellular TH-mediated events and its potential association with SIRT1 and AMPK, we quantified plasma TH levels, mRNA expressions of muscle SIRT1 and TH-associated genes as well as the phosphorylation of AMPK in adult, male northern elephant seals ( n  = 10/fasting period) over 8 weeks of fasting (early vs. late). Deiodinase type I (DI1) expression increased twofold with fasting duration suggesting that the potential for TH-mediated cellular signaling is increased. AMPK phosphorylation increased 61 ± 21% with fasting suggesting that cellular metabolism is increased. The mRNA expression of the TH transporter, monocarboxylate transporter 10 (MCT10), increased 2.4-fold and the TH receptor (THr β -1) decreased 30-fold suggesting that cellular uptake of T 4 is increased, but its subsequent cellular effects such as activation of AMPK are likely nongenomic. The up-regulation of SIRT1 mRNA expression (2.6-fold) likely contributes to the nongenomic activation of AMPK by TH, which may be necessary to maintain the expression of PGC-1 α These coordinated changes likely contribute to the up-regulation of mitochondrial metabolism to support the energetic demands associated with prolonged fasting in adult seals. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Identification of a Mg2+-sensitive ORF in the 5′-leader of TRPM7 magnesium channel mRNA

PubMed Central

Nikonorova, Inna A.; Kornakov, Nikolay V.; Dmitriev, Sergey E.; Vassilenko, Konstantin S.; Ryazanov, Alexey G.

2014-01-01

TRPM7 is an essential and ubiquitous channel-kinase regulating cellular influx of Mg2+. Although TRPM7 mRNA is highly abundant, very small amount of the protein is detected in cells, suggesting post-transcriptional regulation of trpm7 gene expression. We found that TRPM7 mRNA 5′-leader contains two evolutionarily conserved upstream open reading frames that act together to drastically inhibit translation of the TRPM7 reading frame at high magnesium levels and ensure its optimal translation at low magnesium levels, when the activity of the channel-kinase is most required. The study provides the first example of magnesium channel synthesis being controlled by Mg2+ in higher eukaryotes. PMID:25326319

Differential Expression of NADPH Oxidases Depends on Skeletal Muscle Fiber Type in Rats.

PubMed

Loureiro, Adriano César Carneiro; do Rêgo-Monteiro, Igor Coutinho; Louzada, Ruy A; Ortenzi, Victor Hugo; de Aguiar, Angélica Ponte; de Abreu, Ewerton Sousa; Cavalcanti-de-Albuquerque, João Paulo Albuquerque; Hecht, Fabio; de Oliveira, Ariclécio Cunha; Ceccatto, Vânia Marilande; Fortunato, Rodrigo S; Carvalho, Denise P

2016-01-01

NADPH oxidases (NOX) are important sources of reactive oxygen species (ROS) in skeletal muscle, being involved in excitation-contraction coupling. Thus, we aimed to investigate if NOX activity and expression in skeletal muscle are fiber type specific and the possible contribution of this difference to cellular oxidative stress. Oxygen consumption rate, NOX activity and mRNA levels, and the activity of catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase (SOD), as well as the reactive protein thiol levels, were measured in the soleus (SOL), red gastrocnemius (RG), and white gastrocnemius (WG) muscles of rats. RG showed higher oxygen consumption flow than SOL and WG, while SOL had higher oxygen consumption than WG. SOL showed higher NOX activity, as well as NOX2 and NOX4 mRNA levels, antioxidant enzymatic activities, and reactive protein thiol contents when compared to WG and RG. NOX activity and NOX4 mRNA levels as well as antioxidant enzymatic activities were higher in RG than in WG. Physical exercise increased NOX activity in SOL and RG, specifically NOX2 mRNA levels in RG and NOX4 mRNA levels in SOL. In conclusion, we demonstrated that NOX activity and expression differ according to the skeletal muscle fiber type, as well as antioxidant defense.

[Effects of Jianpi Qinghua Decoctions on immune inflammatory injury in adriamycin-induced nephropathic rats MA].

PubMed

Ma, Xiao-Hong; He, Li-Qun

2014-01-01

To investigate the effects of Jianpi Qinghua Decoctions on the inflammation injury mediated by the cellular immunity in the focal segmental glomurular Sclerosis (FSGS) nephropathy rats. The FSGS nephropathy rat model was established by the method of intravenous injection of Adriamycin after the removal of one kidney. After the treatment of Jianpi Qinghua Decoctions, the blood, spleen and kidney samples of each rat were collected for the detection of splenocytes CD4+/CD8+ ratio, renal tubulointerstitial fibronectin (FN) mRNA, Col III mRNA, and the expression levels of TNF-alpha and IL6. The treatment of Jianpi Qinghua Decoctions decreased the levels of CD4+/CD8+, tubulointerstitial FN mRNA, Col III mRNA, TNF-alpha and IL6 significantly in FSGS nephropathy rats. Jianpi Qinghua Decoctions could improve renal FSGS damage in adriamycin-induced nephropathy rats.

Tissue-specific induction of Hsp90 mRNA and plasma cortisol response in chinook salmon following heat shock, seawater challenge, and handling challenge

USGS Publications Warehouse

Palmisano, Aldo N.; Winton, J.R.; Dickhoff, Walton W.

2000-01-01

In studying the whole-body response of chinook salmon (Oncorhynchus tshawytscha) to various stressors, we found that 5-hour exposure to elevated temperature (mean 21.6??C; + 10.6??C over ambient) induced a marked increase in Hsp90 messenger RNA accumulation in heart, brain, gill, muscle, liver, kidney, and tail fin tissues. The most vital tissues (heart, brain, gill, and muscle) showed the greatest Hsp90-mRNA response, with heart tissue increasing approximately 35-fold, Heat shock induced no increase in plasma cortisol. In contrast, a standard handling challenge induced high plasma cortisol levels, but no elevation in Hsp90 mRNA in any tissue, clearly separating the physiological and cellular stress responses. We saw no increase either in tissue Hsp90 mRNA levels or in plasma cortisol concentrations after exposing the fish to seawater overnight.

Behavior of Tumor Necrosis Factor-α and Tumor Necrosis Factor Receptor 1/Tumor Necrosis Factor Receptor 2 System in Mononuclear Cells Recovered From Peritoneal Fluid of Women With Endometriosis at Different Stages

PubMed Central

Salmeri, Francesca M.; Sofo, Vincenza; Triolo, Onofrio; Sturlese, Emanuele; Retto, Giovanni; Pizzo, Alfonsa; D'Ascola, Angela; Campo, Salvatore

2015-01-01

During endometriosis, a breakdown occurs in endometrial and peritoneal homeostasis caused by cytokine-induced cell proliferation and dysregulation of apoptosis. We studied tumor necrosis factor (TNF)-α, TNF receptor (TNFR) 1, and TNFR2 gene expression at both messenger RNA (mRNA) and protein levels in peritoneal fluid (PF) mononuclear cells (PFMCs), the percentages of these cells bearing the same markers, and soluble TNF-α (sTNF-α) values in PF of 80 women with endometriosis. We found that TNFR1 mRNA and protein levels, the percentages of TNFR1-bearing PFMCs, and sTNF-α values decreased from minimal to severe stages of the disease. Instead, TNF-α and TNFR2 mRNA and protein levels, the percentages of membrane TNF-α (mTNF-α)- and TNFR2-bearing PFMCs increased as the disease worsened. These data allow us to hypothesize that, in early stages, the high percentages of TNFR1-bearing PFMCs and the high levels of sTNF-α could address signal toward complex I pathway, favoring the inflammatory response. With the worsening of the disease, the low percentages of TNFR1-bearing PFMCs are probably due to decreased TNFR1 mRNA transcription and protein translation rate. In early stages (minimal and mild), the percentages of both TNFR2- and mTNF-α–bearing PFMCs are so low, due to decreased mRNA transcription and protein translation rate, that subsequent cellular events may depend minimally by this interaction. The high levels of sTNF-α may be rerouted to bind TNFR1. In contrast, in the moderate and severe stages, the high percentages of TNFR2-bearing PFMCs may be saturated by high percentages of mTNF-α–bearing PFMCs, triggering death process. So, in endometriosis, each component of the TNF-α/TNFRs system may trigger opposite cellular fate. PMID:24844917

Low-level lasers alter mRNA levels from traditional reference genes used in breast cancer cells

NASA Astrophysics Data System (ADS)

Teixeira, A. F.; Canuto, K. S.; Rodrigues, J. A.; Fonseca, A. S.; Mencalha, A. L.

2017-07-01

Cancer is among the leading causes of mortality worldwide, increasing the importance of treatment development. Low-level lasers are used in several diseases, but some concerns remains on cancers. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a technique used to understand cellular behavior through quantification of mRNA levels. Output data from target genes are commonly relative to a reference that cannot vary according to treatment. This study evaluated reference genes levels from MDA-MB-231 cells exposed to red or infrared lasers at different fluences. Cultures were exposed to red and infrared lasers, incubated (4 h, 37 °C), total RNA was extracted and cDNA synthesis was performed to evaluate mRNA levels from ACTB, GUSB and TRFC genes by RT-qPCR. Specific amplification was verified by melting curves and agarose gel electrophoresis. RefFinder enabled data analysis by geNorm, NormFinder and BestKeeper. Specific amplifications were obtained and, although mRNA levels from ACTB, GUSB or TRFC genes presented no significant variation through traditional statistical analysis, Excel-based tools revealed that the use of these reference genes are dependent of laser characteristics. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from ACTB, GUSB and TRFC in MDA-MB-231 cells.

Prolonged food deprivation increases mRNA expression of deiodinase 1 and 2, and thyroid hormone receptor β-1 in a fasting-adapted mammal

PubMed Central

Martinez, Bridget; Soñanez-Organis, José G.; Vázquez-Medina, José Pablo; Viscarra, Jose A.; MacKenzie, Duncan S.; Crocker, Daniel E.; Ortiz, Rudy M.

2013-01-01

SUMMARY Food deprivation in mammals is typically associated with reduced thyroid hormone (TH) concentrations and deiodinase content and activity to suppress metabolism. However, in prolonged-fasted, metabolically active elephant seal pups, TH levels are maintained, if not elevated. The functional relevance of this apparent paradox is unknown and demonstrates variability in the regulation of TH levels, metabolism and function in food-deprived mammals. To address our hypothesis that cellular TH-mediated activity is upregulated with fasting duration, we quantified the mRNA expression and protein content of adipose and muscle deiodinase type I (DI1) and type II (DI2), and TH receptor beta-1 (THrβ-1) after 1, 3 and 7 weeks of fasting in northern elephant seal pups (N=5–7 per week). Fasting did not decrease the concentrations of plasma thyroid stimulating hormone, total triiodothyronine (tT3), free T3, total thyroxine (tT4) or free T4, suggesting that the hypothalamic–pituitary–thyroid axis is not suppressed, but rather maintained during fasting. Mean mRNA expression of adipose DI1 and DI2 increased threefold and fourfold, respectively, and 20- and 30-fold, respectively, in muscle. With the exception of adipose DI1, protein expression of adipose DI2 and muscle DI1 and DI2 increased twofold to fourfold. Fasting also increased adipose (fivefold) and muscle (fourfold) THrβ-1 mRNA expression, suggesting that the mechanisms mediating cellular TH activity are upregulated with prolonged fasting. The data demonstrate a unique, atypical mechanism of TH activity and regulation in mammals adapted to prolonged food deprivation in which the potential responsiveness of peripheral tissues and cellular TH activity are increased, which may contribute to their lipid-based metabolism. PMID:24307712

Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability.

PubMed

Dudek, Sabine Eva; Nitzsche, Katja; Ludwig, Stephan; Ehrhardt, Christina

2016-06-06

Infection with influenza A viruses (IAV) provokes activation of cellular defence mechanisms contributing to the innate immune and inflammatory response. In this process the cyclooxygenase-2 (COX-2) plays an important role in the induction of prostaglandin-dependent inflammation. While it has been reported that COX-2 is induced upon IAV infection, in the present study we observed a down-regulation at later stages of infection suggesting a tight regulation of COX-2 by IAV. Our data indicate the pattern-recognition receptor RIG-I as mediator of the initial IAV-induced COX-2 synthesis. Nonetheless, during on-going IAV replication substantial suppression of COX-2 mRNA and protein synthesis could be detected, accompanied by a decrease in mRNA half-life. Interestingly, COX-2 mRNA stability was not only imbalanced by IAV replication but also by stimulation of cells with viral RNA. Our results reveal tristetraprolin (TTP), which is known to bind COX-2 mRNA and promote its rapid degradation, as regulator of COX-2 expression in IAV infection. During IAV replication and viral RNA accumulation TTP mRNA synthesis was induced, resulting in reduced COX-2 levels. Accordingly, the down-regulation of TTP resulted in increased COX-2 protein expression after IAV infection. These findings indicate a novel IAV-regulated cellular mechanism, contributing to the repression of host defence and therefore facilitating viral replication.

The altered expression of perineuronal net elements during neural differentiation.

PubMed

Eskici, Nazli F; Erdem-Ozdamar, Sevim; Dayangac-Erden, Didem

2018-01-01

Perineuronal nets (PNNs), which are localized around neurons during development, are specialized forms of neural extracellular matrix with neuroprotective and plasticity-regulating roles. Hyaluronan and proteoglycan link protein 1 (HAPLN1), tenascin-R (TNR) and aggrecan (ACAN) are key elements of PNNs. In diseases characterized by neuritogenesis defects, the expression of these proteins is known to be downregulated, suggesting that PNNs may have a role in neural differentiation. In this study, the mRNA and protein levels of HAPLN1, TNR and ACAN were determined and compared at specific time points of neural differentiation. We used PC12 cells as the in vitro model because they reflect this developmental process. On day 7, the HAPLN1 mRNA level showed a 2.9-fold increase compared to the non-differentiated state. However, the cellular HAPLN1 protein level showed a decrease, indicating that the protein may have roles in neural differentiation, and may be secreted during the early period of differentiation. By contrast, TNR mRNA and protein levels remained unchanged, and the amount of cellular ACAN protein showed a 3.7-fold increase at day 7. These results suggest that ACAN may be secreted after day 7, possibly due to its large amount of post-translational modifications. Our results provide preliminary data on the expression of PNN elements during neural differentiation. Further investigations will be performed on the role of these elements in neurological disease models.

Arsenic Induces Polyadenylation of Canonical Histone mRNA by Down-regulating Stem-Loop-binding Protein Gene Expression*

PubMed Central

Brocato, Jason; Fang, Lei; Chervona, Yana; Chen, Danqi; Kiok, Kathrin; Sun, Hong; Tseng, Hsiang-Chi; Xu, Dazhong; Shamy, Magdy; Jin, Chunyuan; Costa, Max

2014-01-01

The replication-dependent histone genes are the only metazoan genes whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3′-end. Instead, the histone mRNAs display a stem-loop structure at their 3′-end. Stem-loop-binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. Here we report that exposure to arsenic, a carcinogenic metal, decreased cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Notably, arsenic exposure dramatically increased polyadenylation of canonical histone H3.1 mRNA possibly through down-regulation of SLBP expression. The polyadenylated H3.1 mRNA induced by arsenic was not susceptible to normal degradation that occurs at the end of S phase, resulting in continued presence into mitosis, increased total H3.1 mRNA, and increased H3 protein levels. Excess expression of canonical histones have been shown to increase sensitivity to DNA damage as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic exposure may contribute to arsenic-induced carcinogenesis. PMID:25266719

Spiny Nanorod and Upconversion Nanoparticle Satellite Assemblies for Ultrasensitive Detection of Messenger RNA in Living Cells.

PubMed

Gao, Rui; Hao, Changlong; Xu, Liguang; Xu, Chuanlai; Kuang, Hua

2018-04-17

Quantitation and in situ monitoring of target mRNA (mRNA) in living cells remains a significant challenge for the chemical and biomedical communities. To quantitatively detect mRNA expression levels in living cells, we have developed DNA-driven gold nanorod coated platinum-upconversion nanoparticle satellite assemblies (termed Au NR@Pt-UCNP satellites) for intracellular thymidine kinase 1 (TK1) mRNA analysis. The nanostructures were capable of recognizing target mRNA in a sequence-specific manner as luminescence of UCNPs was effectively quenched by Au NR@Pt within the assemblies. Following recognition, UCNPs detached from Au NR@Pt, resulting in luminescence restoration to achieve effective in situ imaging and quantifiable detection of target mRNA. The upconversional luminescence intensity of confocal images showed a good linear relationship with intracellular TK1 mRNA ranging from 1.17 to 65.21 fmol/10 μg RNA and a limit of detection (LOD) of 0.67 fmol/10 μg RNA. We believe that our present assay can be broadly applied for detection of endogenous biomolecules at the cellular and tissue levels and restoration of tissue homeostasis in vivo.

Role of nitric oxide in cellular iron metabolism.

PubMed

Kim, Sangwon; Ponka, Prem

2003-03-01

Iron regulatory proteins (IRP1 and IRP2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements (IREs) which are located in the 3' untranslated region (UTR) and the 5' UTR of their respective mRNAs. Cellular iron levels affect binding of IRPs to IREs and consequently expression of TfR and ferritin. Moreover, NO*, a redox species of nitric oxide that interacts primarily with iron, can activate IRP1 RNA-binding activity resulting in an increase in TfR mRNA levels. We have shown that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO+ (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA-binding of IRP2, followed by IRP2 degradation, and these changes were associated with a decrease in TfR mRNA levels. Moreover, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) increased IRP1 binding activity, whereas RNA-binding of IRP2 decreased and was followed by a degradation of this protein. Furthermore, the decrease of IRP2 binding/protein levels was associated with a decrease in TfR mRNA levels in LPS/IFN-gamma-treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. These results suggest that NO+-mediated degradation of IRP2 plays a major role in iron metabolism during inflammation.

Principles of mRNA transport in yeast.

PubMed

Heym, Roland Gerhard; Niessing, Dierk

2012-06-01

mRNA localization and localized translation is a common mechanism by which cellular asymmetry is achieved. In higher eukaryotes the mRNA transport machinery is required for such diverse processes as stem cell division and neuronal plasticity. Because mRNA localization in metazoans is highly complex, studies at the molecular level have proven to be cumbersome. However, active mRNA transport has also been reported in fungi including Saccharomyces cerevisiae, Ustilago maydis and Candida albicans, in which these events are less difficult to study. Amongst them, budding yeast S. cerevisiae has yielded mechanistic insights that exceed our understanding of other mRNA localization events to date. In contrast to most reviews, we refrain here from summarizing mRNA localization events from different organisms. Instead we give an in-depth account of ASH1 mRNA localization in budding yeast. This approach is particularly suited to providing a more holistic view of the interconnection between the individual steps of mRNA localization, from transcriptional events to cytoplasmic mRNA transport and localized translation. Because of our advanced mechanistic understanding of mRNA localization in yeast, the present review may also be informative for scientists working, for example, on mRNA localization in embryogenesis or in neurons.

hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

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

Wang, Yimeng; Zhou, Jianhong; Du, Yuchun, E-mail: ydu@uark.edu

The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to themore » cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation. - Highlights: • Cellular protein hnRNP A2/B1 interacts with influenza viral protein NS1. • hnRNP A2/B1 suppresses the levels of NS1 protein, vRNA and mRNA in infected cells. • hnRNP A2/B1 protein is associated with NS1 and NS2 mRNAs. • hnRNP A2/B1 inhibits the nuclear export of NS1 mRNAs. • hnRNP A2/B1 inhibits influenza virus replication.« less

Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

PubMed

Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

2015-04-01

Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm. © 2014 John Wiley & Sons Ltd.

Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

PubMed Central

Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

2013-01-01

Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

Multihormonal induction of hepatic α2u-globulin mRNA as measured by hybridization to complementary DNA

PubMed Central

Kurtz, David T.; Feigelson, Philip

1977-01-01

A procedure is presented for the preparation of a 3H-labeled complementary DNA (cDNA) specific for the mRNA coding for α2u-globulin, a male rat liver protein under multihormonal control that represents approximately 1% of hepatic protein synthesis. Rat liver polysomes are incubated with monospecific rabbit antiserum to α2u-globulin, which binds to the nascent α2u-globulin chains on the polysomes. These antibody-polysome complexes are then adsorbed to goat antiserum to rabbit IgG that is covalently linked to p-aminobenzylcellulose. mRNA preparations are thus obtained that contain 30-40% α2u-globulin mRNA. A labeled cDNA is made to this α2u-globulin-enriched mRNA preparation by using RNA-dependent DNA polymerase (reverse transcriptase). To remove the non-α2u-globulin sequences, this cDNA preparation is hybridized to an RNA concentration × incubation time (R0t) of 1000 mol of ribonucleotide per liter × sec with female rat liver mRNA, which, though it shares the vast majority of mRNA sequences with male liver, contains no α2u-globulin mRNA sequences. The cDNA remaining single-stranded is isolated by hydroxylapatite chromatography and is shown to be specific for α2u-globulin mRNA by several criteria. Good correlation was found in all endocrine states studied between the hepatic level of α2u-globulin, the level of functional α2u-globulin mRNA as assayed in a wheat germ cell-free translational system, and the level of α2u-globulin mRNA sequences as measured by hybridization to the α2u-globulin cDNA. Thus, the hormonal control of hepatic α2u-globulin synthesis by sex steroids and thyroid hormone occurs through modulation of the cellular level of α2u-globulin mRNA sequences, presumably by hormonal control of transcriptive synthesis. PMID:73184

Combination of Fluorescent in situ Hybridization (FISH) and Immunofluorescence Imaging for Detection of Cytokine Expression in Microglia/Macrophage Cells

PubMed Central

Fe Lanfranco, Maria; Loane, David J.; Mocchetti, Italo; Burns, Mark P.; Villapol, Sonia

2017-01-01

Microglia and macrophage cells are the primary producers of cytokines in response to neuroinflammatory processes. But these cytokines are also produced by other glial cells, endothelial cells, and neurons. It is essential to identify the cells that produce these cytokines to target their different levels of activation. We used dual RNAscope® fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) techniques to visualize the mRNA expression pattern of pro- and anti-inflammatory cytokines in microglia/macrophages cells. Using these methods, we can associate one mRNA to specific cell types when combining with different cellular markers by immunofluorescence. Results from RNAscope® probes IL-1β, TNFα, TGFβ, IL-10 or Arg1, showed colocalization with antibodies for microglia/macrophage cells. These target probes showed adequate sensitivity and specificity to detect mRNA expression. New FISH detection techniques combined with immunohistochemical techniques will help to jointly determine the protein and mRNA localization, as well as provide reliable quantification of the mRNA expression levels. PMID:29238736

Using RNA interference to knock down the adhesion protein TES.

PubMed

Griffith, Elen

2007-01-01

RNA interference (RNAi) is a specific and efficient method to knock down protein levels using small interfering RNAs (siRNAs), which target mRNA degradation. RNAi can be used in mammalian cell culture systems to target any protein of interest, and several studies have used this method to knock down adhesion proteins. We used siRNAs to knock down the levels of TES, a focal adhesion protein, in HeLa cells. We demonstrated knockdown of both TES mRNA and TES protein. Although total knockdown of TES was not achieved, the observed reduction in TES protein was sufficient to result in a cellular phenotype of reduced actin stress fibers.

Exploring Uncoupling Proteins and Antioxidant Mechanisms under Acute Cold Exposure in Brains of Fish

PubMed Central

Lucassen, Magnus; Schmidt, Maike M.; Dringen, Ralf; Abele, Doris; Hwang, Pung-Pung

2011-01-01

Exposure to fluctuating temperatures accelerates the mitochondrial respiration and increases the formation of mitochondrial reactive oxygen species (ROS) in ectothermic vertebrates including fish. To date, little is known on potential oxidative damage and on protective antioxidative defense mechanisms in the brain of fish under cold shock. In this study, the concentration of cellular protein carbonyls in brain was significantly increased by 38% within 1 h after cold exposure (from 28°C to 18°C) of zebrafish (Danio rerio). In addition, the specific activity of superoxide dismutase (SOD) and the mRNA level of catalase (CAT) were increased after cold exposure by about 60% (6 h) and by 60%–90% (1 and 24 h), respectively, while the specific glutathione content as well as the ratio of glutathione disulfide to glutathione remained constant and at a very low level. In addition, cold exposure increased the protein level of hypoxia-inducible factor (HIF) by about 50% and the mRNA level of the glucose transporter zglut3 in brain by 50%–100%. To test for an involvement of uncoupling proteins (UCPs) in the cold adaptation of zebrafish, five UCP members were annotated and identified (zucp1-5). With the exception of zucp1, the mRNA levels of the other four zucps were significantly increased after cold exposure. In addition, the mRNA levels of four of the fish homologs (zppar) of the peroxisome proliferator-activated receptor (PPAR) were increased after cold exposure. These data suggest that PPARs and UCPs are involved in the alterations observed in zebrafish brain after exposure to 18°C. The observed stimulation of the PPAR-UCP axis may help to prevent oxidative damage and to maintain metabolic balance and cellular homeostasis in the brains of ectothermic zebrafish upon cold exposure. PMID:21464954

Accumulation and toxicity of monophenyl arsenicals in rat endothelial cells.

PubMed

Hirano, Seishiro; Kobayashi, Yayoi; Hayakawa, Toru; Cui, Xing; Yamamoto, Megumi; Kanno, Sanae; Shraim, Amjad

2005-01-01

Clark 1 (diphenylarsine chloride) and Clark 2 (diphenylarsine cyanide) were used as chemical weapon agents (CWA), and the soil contamination by these CWA and their degraded products, diphenyl and phenyl arsenicals, has been one of the most serious environmental issues. In a series of comparisons in toxicity between trivalent and pentavalent arsenicals we investigated differences in the accumulation and toxicity of phenylarsine oxide (PAO3+) and phenylarsonic acid (PAA5+) in rat heart microvascular endothelial cells. Both the cellular association and toxicity of PAO3+ were much higher than those of PAA5+, and LC50 values of PAO3+ and PAA5+ were calculated to be 0.295 microM and 1.93 mM, respectively. Buthionine sulfoximine, a glutathione depleter, enhanced the cytotoxicity of both PAO3+ and PAA5+. N-Acetyl-L-cysteine (NAC) reduced the cytotoxicity and induction of heme oxygenase-1 (HO-1) mRNA in PAO3+-exposed cells, while NAC affected neither the cytotoxicity nor the HO-1 mRNA level in PAA5+-exposed cells. The effect of NAC may be due to a strong affinity of PAO3+ to thiol groups because both NAC and GSH inhibited the cellular accumulation of PAO3+, but PAA3+ increased tyrosine phosphorylation levels of cellular proteins. These results indicate that the inhibition of protein phosphatases as well as the high affinity to cellular components may confer PAO3+ the high toxicity.

Mechanisms of ultraviolet disinfection and chlorination of Escherichia coli: Culturability, membrane permeability, metabolism, and genetic damage.

PubMed

Xu, Limei; Zhang, Chongmiao; Xu, Pengcheng; Wang, Xiaochang C

2018-03-01

Traditional culture methods may underestimate the tolerance of microorganisms to disinfectants because of the existence of viable but nonculturable or sublethally injured cells after disinfection. The selection of a strict method is crucial for the evaluation of disinfection performance. The actions of 2 typical disinfectants - ultraviolet (UV) and chlorine - on the fecal indicator Escherichia coli were investigated by the detection of culturability, membrane permeability, metabolic activity, deoxyribonucleic acid (DNA), and messenger ribonucleic acid (mRNA). During UV disinfection, the irreversible damages in the cell membrane and cellular adenosine triphosphate (ATP) were negligible at low UV doses (<80mJ/cm 2 ). However, membrane permeability was damaged at low doses of chlorine (<5mg/L), leading to leakage of cellular ATP. Our study showed that a slight lesion in DNA was detected even at high doses of UV (400mJ/cm 2 ) and chlorine (>5mg/L) treatments. The decay of mRNA was more rapid than that of DNA. The degradation level of mRNA depended on the choice of target genes. After exposure to 50mJ/cm 2 UV dose or 5mg/L chlorine for 30min, the DNA damage repair function (RecA mRNA) was inhibited. The mRNA involved in the DNA damage repair function can be a potential indicator of bacterial viability. Copyright © 2017. Published by Elsevier B.V.

Lipoprotein lipase regulation by insulin and glucocorticoid in subcutaneous and omental adipose tissues of obese women and men.

PubMed Central

Fried, S K; Russell, C D; Grauso, N L; Brolin, R E

1993-01-01

There are marked variations in the activity of lipoprotein lipase (LPL) among adipose depots, particularly in women. Consistent with data on LPL activity, the level of expression of LPL mRNA was lower in omental (OM) than subcutaneous (SQ) adipose tissue of women. To investigate the cellular basis of these differences, OM and SQ adipose tissues obtained at surgery from obese men and women were placed in organ culture for 7 d with varying concentrations of insulin and dexamethasone. Insulin increased levels of LPL mRNA and LPL activity in abdominal SQ but not OM adipose tissue. Dexamethasone also increased LPL mRNA and LPL activity, and these effects were more marked in the OM adipose tissue, particularly in men. When insulin and dexamethasone were added together, synergistic increases in LPL activity were seen in both depots, and this was in part explained at the level of LPL mRNA. The SQ depot was more sensitive to the effects of submaximal doses of dexamethasone in the presence of insulin. The maximum activity of LPL induced by insulin or insulin plus dexamethasone was higher in the SQ than in the OM depot of women, and this was associated with higher levels of LPL mRNA. Rates of LPL synthesis paralleled LPL mRNA levels. These data show that insulin and glucocorticoids influence human adipose tissue LPL activity at the level of LPL gene expression, as well as posttranslationally, and that responsiveness to these hormonal effects is dependent on adipose depot and gender. Images PMID:8227334

Cellular Antioxidant and Anti-Inflammatory Effects of Coffee Extracts with Different Roasting Levels.

PubMed

Jung, Soohan; Kim, Min Hyung; Park, Jae Hee; Jeong, Yoonhwa; Ko, Kwang Suk

2017-06-01

During roasting, major changes occur in the composition and physiological effects of coffee beans. In this study, in vitro antioxidant effects and anti-inflammatory effects of Coffea arabica green coffee extracts were investigated at different roasting levels corresponding to Light, Medium, City, and French roast. Total caffeine did not show huge difference according to roasting level, but total chlorogenic acid contents were higher in light roasted coffee extract than other roasted groups. In addition, light roasted coffee extract had the highest antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. To determine the in vitro antioxidant property, coffee extracts were used to treat AML-12 cells. Intracellular glutathione (GSH) concentration and mRNA expression levels of genes related to GSH synthesis were negatively related to roasting levels. The anti-inflammatory effects of coffee extracts were investigated in lipopolysaccharide-treated RAW 264.7 macrophage cells. The cellular antioxidant activity of coffee extracts exhibited similar patterns as the AML-12 cells. The expression of mRNA for tumor necrosis factor-alpha and interleukin-6 was decreased in cells treated with the coffee extracts and the expression decreased with increasing roasting levels. These data suggest that coffee has physiological antioxidant and anti-inflammatory activities and these effects are negatively correlated with roasting levels in the cell models.

Increasing cellular level of phosphatidic acid enhances FGF-1 production in long term-cultured rat astrocytes.

PubMed

Nagayasu, Yuko; Morita, Shin-Ya; Hayashi, Hideki; Miura, Yutaka; Yokoyama, Kazuki; Michikawa, Makoto; Ito, Jin-Ichi

2014-05-14

We found in a previous study that both mRNA expression and release of fibroblast growth factor 1 (FGF-1) are greater in rat astrocytes that are long term-cultured for one month (W/M cells) than in the cells cultured for one week (W/W cells). However, FGF-1 does not enhance phosphorylation of Akt, MEK, and ERK in W/M cells, while it does in W/W cells. In this work we studied the mechanism to cause these differences between W/W and W/M cells in culture. As it is known that long term culture generates oxidative stress, we characterized the stresses which W/M cells undergo in comparison with W/W cells. The levels of superoxide dismutase 1 (SOD1) and mitochondrial Bax were higher in W/M cells than in W/W cells. W/M cells recovered their ability to respond to FGF-1 to enhance phosphorylation of Akt, MEK, and ERK in the presence of antioxidants. Oxidative stress induced by hydrogen peroxide (H2O2) had no effect on mRNA expression of FGF-1 in W/W cells, although H2O2 enhances release of FGF-1 from W/W cells without inducing apoptosis. The influence of cell density was studied on mRNA expression of FGF-1 and cellular response to FGF-1, as an increasing cell density is observed in W/M cells. The increasing cell density enhanced mRNA expression of FGF-1 in W/W cells without suppression of responses to FGF-1. The decrease in cell density lowered the FGF-1 mRNA expression in W/M cells without recovery of the response to FGF-1 to enhance phosphorylation of Akt, MEK, and ERK. These findings suggest that oxidative stress attenuate sensitivity to FGF-1 and higher cell density may enhance FGF-1 expression in W/M cells. In addition, we found that the cellular level of phosphatidic acid (PA) increased in H2O2-treated W/W and W/M cells and decreased by the treatment with antioxidants, and that PA enhances the mRNA expression of FGF-1 in the W/W cells. These findings suggest that the increasing PA production may enhance FGF-1 expression to protect astrocytes against oxidative stress induced by long-term culture. Copyright © 2014 Elsevier B.V. All rights reserved.

Lower expression of glutamic acid decarboxylase 67 in the prefrontal cortex in schizophrenia: contribution of altered regulation by Zif268.

PubMed

Kimoto, Sohei; Bazmi, H Holly; Lewis, David A

2014-09-01

Cognitive deficits of schizophrenia may be due at least in part to lower expression of the 67-kDa isoform of glutamic acid decarboxylase (GAD67), a key enzyme for GABA synthesis, in the dorsolateral prefrontal cortex of individuals with schizophrenia. However, little is known about the molecular regulation of lower cortical GAD67 levels in schizophrenia. The GAD67 promoter region contains a conserved Zif268 binding site, and Zif268 activation is accompanied by increased GAD67 expression. Thus, altered expression of the immediate early gene Zif268 may contribute to lower levels of GAD67 mRNA in the dorsolateral prefrontal cortex in schizophrenia. The authors used polymerase chain reaction to quantify GAD67 and Zif268 mRNA levels in dorsolateral prefrontal cortex area 9 from 62 matched pairs of schizophrenia and healthy comparison subjects, and in situ hybridization to assess Zif268 expression at laminar and cellular levels of resolution. The effects of potentially confounding variables were assessed in human subjects, and the effects of antipsychotic treatments were tested in antipsychotic-exposed monkeys. The specificity of the Zif268 findings was assessed by quantifying mRNA levels for other immediate early genes. GAD67 and Zif268 mRNA levels were significantly lower and were positively correlated in the schizophrenia subjects. Both Zif268 mRNA-positive neuron density and Zif268 mRNA levels per neuron were significantly lower in the schizophrenia subjects. These findings were robust to the effects of the confounding variables examined and differed from other immediate early genes. Deficient Zif268 mRNA expression may contribute to lower cortical GAD67 levels in schizophrenia, suggesting a potential mechanistic basis for altered cortical GABA synthesis and impaired cognition in schizophrenia.

NONOates regulate KCl cotransporter-1 and -3 mRNA expression in vascular smooth muscle cells.

PubMed

Di Fulvio, Mauricio; Lauf, Peter K; Shah, Shalin; Adragna, Norma C

2003-05-01

Nitric oxide (NO) donors regulate KCl cotransport (KCC) activity and cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in sheep erythrocytes and in primary cultures of rat vascular smooth muscle cells (VSMCs), respectively. In this study, we used NONOates as rapid and slow NO releasers to provide direct evidence implicating NO as a regulator of KCC3 gene expression at the mRNA level. In addition, we used the expression of KCC3 mRNA to further investigate the mechanism of action of these NO donors at the cellular level. Treatment of VSMCs with rapid NO releasers, like NOC-5 and NOC-9, as well as with the direct NO-independent soluble guanylyl cyclase (sGC) stimulator YC-1, acutely increased KCC3 mRNA expression in a concentration- and time-dependent manner. The slow NO releaser NOC-18 had no effect on KCC3 gene expression. A specific NO scavenger completely prevented the NONOate-induced KCC3 mRNA expression. Inhibition of sGC with LY-83583 blocked the NONOate- and YC-1-induced KCC3 mRNA expression. This study shows that in primary cultures of rat VSMCs, the fast NO releasers NOC-9 and NOC-5, but not the slow NO releaser NOC-18, acutely upregulate KCC3 mRNA expression in a NO/sGC-dependent manner.

CELFish ways to modulate mRNA decay

PubMed Central

St. Louis, Irina Vlasova; Dickson, Alexa M.; Bohjanen, Paul R.; Wilusz, Carol J.

2013-01-01

The CELF family of RNA-binding proteins regulates many steps of mRNA metabolism. Although their best characterized function is in pre-mRNA splice site choice, CELF family members are also powerful modulators of mRNA decay. In this review we focus on the different modes of regulation that CELF proteins employ to mediate mRNA decay by binding to GU-rich elements. After starting with an overview of the importance of CELF proteins during development and disease pathogenesis, we then review the mRNA networks and cellular pathways these proteins regulate and the mechanisms by which they influence mRNA decay. Finally, we discuss how CELF protein activity is modulated during development and in response to cellular signals. We conclude by highlighting the priorities for new experiments in this field. PMID:23328451

Advanced cell-based modeling of the royal disease: characterization of the mutated F9 mRNA.

PubMed

Martorell, L; Luce, E; Vazquez, J L; Richaud-Patin, Y; Jimenez-Delgado, S; Corrales, I; Borras, N; Casacuberta-Serra, S; Weber, A; Parra, R; Altisent, C; Follenzi, A; Dubart-Kupperschmitt, A; Raya, A; Vidal, F; Barquinero, J

2017-11-01

Essentials The Royal disease (RD) is a form of hemophilia B predicted to be caused by a splicing mutation. We generated an iPSC-based model of the disease allowing mechanistic studies at the RNA level. F9 mRNA analysis in iPSC-derived hepatocyte-like cells showed the predicted abnormal splicing. Mutated F9 mRNA level was very low but we also found traces of wild type transcripts. Background The royal disease is a form of hemophilia B (HB) that affected many descendants of Queen Victoria in the 19th and 20th centuries. It was found to be caused by the mutation F9 c.278-3A>G. Objective To generate a physiological cell model of the disease and to study F9 expression at the RNA level. Methods Using fibroblasts from skin biopsies of a previously identified hemophilic patient bearing the F9 c.278-3A>G mutation and his mother, we generated induced pluripotent stem cells (iPSCs). Both the patient's and mother's iPSCs were differentiated into hepatocyte-like cells (HLCs) and their F9 mRNA was analyzed using next-generation sequencing (NGS). Results and Conclusion We demonstrated the previously predicted aberrant splicing of the F9 transcript as a result of an intronic nucleotide substitution leading to a frameshift and the generation of a premature termination codon (PTC). The F9 mRNA level in the patient's HLCs was significantly reduced compared with that of his mother, suggesting that mutated transcripts undergo nonsense-mediated decay (NMD), a cellular mechanism that degrades PTC-containing mRNAs. We also detected small proportions of correctly spliced transcripts in the patient's HLCs, which, combined with genetic variability in splicing and NMD machineries, could partially explain some clinical variability among affected members of the European royal families who had lifespans above the average. This work allowed the demonstration of the pathologic consequences of an intronic mutation in the F9 gene and represents the first bona fide cellular model of HB allowing the study of rare mutations at the RNA level. © 2017 International Society on Thrombosis and Haemostasis.

The effect of redox-related species of nitrogen monoxide on transferrin and iron uptake and cellular proliferation of erythroleukemia (K562) cells.

PubMed

Richardson, D R; Neumannova, V; Nagy, E; Ponka, P

1995-10-15

The iron-responsive element-binding protein (IRE-BP) modulates both ferritin mRNA translation and transferrin receptor (TfR) mRNA stability by binding to specific mRNA sequences called iron-responsive elements (IREs). The regulation of IRE-BP in situ could possibly occur either through its Fe-S cluster and/or via free cysteine sulphydryl groups such as cysteine 437 (Philpott et al, J Biol Chem 268:17655, 1993; and Hirling et al, EMBO J 13:453, 1994). Recently, nitrogen monoxide (NO) has been shown to have markedly different biologic effects depending on its redox state (Lipton et al, Nature 364:626, 1993). Considering this fact, it is conceivable that the NO group, as either the nitrosonium ion (NO+) or nitric oxide (NO+), may regulate IRE-BP activity by S-nitrosylation of key sulphydryl groups or via ligation of NO. to the Fe-S cluster, respectively. This hypothesis has been examined using the NO+ generator, sodium nitroprusside (SNP); the NO. generator, S-nitroso-N-acetylpenicillamine (SNAP); and the NO./peroxynitrite (ONOO-) generator, 3-morpholinosydnonimine hydrochloride (SIN-1). Treatment of K562 cells for 18 hours with SNP (1 mmol/L) resulted in a pronounced decrease in both the RNA-binding activity of IRE-BP and the level of TfR mRNA. In addition, Scatchard analysis showed a marked decrease in the number of specific Tf-binding sites, from 590,000/cell (control) to 170,000/cell (test), and there was also a distinct decrease in Fe uptake. Furthermore, SNP did not decrease cellular viability or proliferation. In contrast, the NO. generator, SNAP (1 mmol/L), increased RNA-binding activity of IRE-BP, the level of TfR mRNA, and the number of TfRs in K562 cells. Moreover, both SNAP (1 mmol/L) and SIN-1 (0.5 mmol/L) reduced cellular proliferation. The results are discussed in context of the possible physiologic role of redox-related species of NO in regulating iron metabolism.

Nitric oxide-mediated modulation of iron regulatory proteins: implication for cellular iron homeostasis.

PubMed

Kim, Sangwon; Ponka, Prem

2002-01-01

Iron regulatory proteins (IRP1 and IRP2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements (IREs) that are located in the 3' untranslated region (UTR) and the 5' UTR of their respective mRNAs. Cellular iron levels affect binding of IRPs to IREs and consequently expression of TfR and ferritin. Moreover, NO(.), a redox species of nitric oxide that interacts primarily with iron, can activate IRP1 RNA-binding activity resulting in an increase in TfR mRNA levels and a decrease in ferritin synthesis. We have shown that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO(+) (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA-binding of IRP2, followed by IRP2 degradation, and these changes were associated with a decrease in TfR mRNA levels and a dramatic increase in ferritin synthesis. Moreover, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) increased IRP1 binding activity, whereas RNA-binding of IRP2 decreased and was followed by a degradation of this protein. Furthermore, the decrease of IRP2 binding/protein levels was associated with a decrease in TfR mRNA levels and an increase in ferritin synthesis in LPS/IFN-gamma-treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. These results suggest that NO(+)-mediated degradation of IRP2 plays a major role in iron metabolism during inflammation.

HSP90 gene expression induced by aspirin is associated with damage remission in a chicken myocardial cell culture exposed to heat stress.

PubMed

Zhang, X; Qian, Z; Zhu, H; Tang, S; Wu, D; Zhang, M; Kemper, N; Hartung, J; Bao, E

2016-08-01

To understand the potential protection of heat shock protein 90 (HSP90) induced by aspirin against heat stress damage in chicken myocardial cells, enzyme activities related to stress damage, cytopathological changes, the expression and distribution of HSP90, and HSP90 mRNA levels in the myocardial cells exposed to heat stress (42°C) for different durations with or without aspirin administration (1 mg/ml, 2 h prior) in vitro were investigated. Significant increase of enzyme levels in the supernatant of heat-stressed myocardial cells and cellular lesions characterised by acute degeneration, karyopyknosis and karyorrhexis were observed, compared to non-treated cells. However, the lesions of cells treated with aspirin were milder, characterised by earlier recovery of enzyme levels to the control levels and no obvious heat stress-related cellular necrosis. Stronger positive signals in the cytoplasm and longer retention of HSP90 signal in nuclei were observed in aspirin-treated myocardial cells than those of only heat-stressed cells. HSP90 level in the aspirin-treated myocardial cells was 11.1-fold higher than that in non-treated cells, and remained at a high level at the early stage of heat stress, whereas it was just 4.1-fold higher in only heat-stressed cells and returned rapidly to a low level. Overexpression of HSP90 mRNA in aspirin-treated cells was observed throughout the experiment, whereas HSP90 mRNA decreased significantly only in heat-stressed cells. The early higher HSP90 expression induced by aspirin during heat stress was accompanied by decreased heat stress damage, suggesting that aspirin might play an important role in preventing myocardial cells from heat stress damage in vitro.

Influence of DNA-methylation on zinc homeostasis in myeloid cells: Regulation of zinc transporters and zinc binding proteins.

PubMed

Kessels, Jana Elena; Wessels, Inga; Haase, Hajo; Rink, Lothar; Uciechowski, Peter

2016-09-01

The distribution of intracellular zinc, predominantly regulated through zinc transporters and zinc binding proteins, is required to support an efficient immune response. Epigenetic mechanisms such as DNA methylation are involved in the expression of these genes. In demethylation experiments using 5-Aza-2'-deoxycytidine (AZA) increased intracellular (after 24 and 48h) and total cellular zinc levels (after 48h) were observed in the myeloid cell line HL-60. To uncover the mechanisms that cause the disturbed zinc homeostasis after DNA demethylation, the expression of human zinc transporters and zinc binding proteins were investigated. Real time PCR analyses of 14 ZIP (solute-linked carrier (SLC) SLC39A; Zrt/IRT-like protein), and 9 ZnT (SLC30A) zinc transporters revealed significantly enhanced mRNA expression of the zinc importer ZIP1 after AZA treatment. Because ZIP1 protein was also enhanced after AZA treatment, ZIP1 up-regulation might be the mediator of enhanced intracellular zinc levels. The mRNA expression of ZIP14 was decreased, whereas zinc exporter ZnT3 mRNA was also significantly increased; which might be a cellular reaction to compensate elevated zinc levels. An enhanced but not significant chromatin accessibility of ZIP1 promoter region I was detected by chromatin accessibility by real-time PCR (CHART) assays after demethylation. Additionally, DNA demethylation resulted in increased mRNA accumulation of zinc binding proteins metallothionein (MT) and S100A8/S100A9 after 48h. MT mRNA was significantly enhanced after 24h of AZA treatment also suggesting a reaction of the cell to restore zinc homeostasis. These data indicate that DNA methylation is an important epigenetic mechanism affecting zinc binding proteins and transporters, and, therefore, regulating zinc homeostasis in myeloid cells. Copyright © 2016 Elsevier GmbH. All rights reserved.

miR-128 inhibits telomerase activity by targeting TERT mRNA

PubMed Central

Guzman, Herlinda; Sanders, Katie; Idica, Adam; Bochnakian, Aurore; Jury, Douglas; Daugaard, Iben; Zisoulis, Dimitrios G; Pedersen, Irene Munk

2018-01-01

Telomerase is a unique cellular reverse transcriptase (RT) essential for maintaining telomere stability and required for the unlimited proliferation of cancer cells. The limiting determinant of telomerase activity is the catalytic component TERT, and TERT expression is closely correlated with telomerase activity and cancer initiation and disease progression. For this reason the regulation of TERT levels in the cell is of great importance. microRNAs (miRs) function as an additional regulatory level in cells, crucial for defining expression boundaries, proper cell fate decisions, cell cycle control, genome integrity, cell death and metastasis. We performed an anti-miR library screen to identity novel miRs, which participate in the control of telomerase. We identified the tumor suppressor miR (miR-128) as a novel endogenous telomerase inhibitor and determined that miR-128 significantly reduces the mRNA and protein levels of Tert in a panel of cancer cell lines. We further evaluated the mechanism by which miR-128 regulates TERT and demonstrated that miR-128 interacts directly with the coding sequence of TERT mRNA in both HeLa cells and teratoma cells. Interestingly, the functional miR-128 binding site in TERT mRNA, is conserved between TERT and the other cellular reverse transcriptase encoded by Long Interspersed Elements-1 (LINE-1 or L1), which can also contribute to the oncogenic phenotype of cancer. This finding supports the novel idea that miRs may function in parallel pathways to inhibit tumorigenesis, by regulating a group of enzymes (such as RT) by targeting conserved binding sites in the coding region of both enzymes. PMID:29568354

Mitomycin C induces apoptosis in cultured corneal fibroblasts derived from type II granular corneal dystrophy corneas.

PubMed

Kim, Tae-im; Choi, Seung-il; Lee, Hyung Keun; Cho, Young Jae; Kim, Eung Kweon

2008-06-30

The present study investigated the effect of mitomycin C (MMC) on cell viability, apoptosis, and transforming growth factor beta-induced protein (TGFBIp) expression in cultured normal corneal fibroblasts and heterozygote or homozygote granular corneal dystrophy type II (GCD II) corneal fibroblasts. Keratocytes were obtained from normal cornea or from heterozygote or homozygote GCD II patients after lamellar or penetrating keratoplasty. To measure cell viability, corneal fibroblasts were incubated with 0.02% MMC for 3 h, 6 h, and 24 h or with 0%, 0.01%, 0.02%, and 0.04% MMC for 24 h and then tested using lactate dehydrogenase (LDH) and 3-[4,5-demethylthiazol-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assays. To measure apoptosis, cells were analyzed by FACS analysis and annexin V staining. Bcl-xL, Bax, and TGFBI mRNA expression was measured using reverse transcription polymerase chain reaction (RT-PCR) assays. Cellular and media levels of TGFBIp protein were measured by immunoblotting. MTT and LDH assays showed that MMC reduced cell viability in all three cell types in a dose-dependent and time-dependent manner (p<0.05). FACS analysis and annexin V staining showed that MMC caused apoptosis with GCD II homozygote cells being most affected. RT-PCR analysis showed that MMC decreased Bcl-xL mRNA expression and increased Bax mRNA expression in all cell types. RT-PCR and immunoblotting analysis showed that MMC reduced TGFBI mRNA levels and cellular and media TGFBIp protein levels in all cell types. MMC induced apoptosis, and the effects of MMC were greatest in GCD II homozygote cells. MMC also reduced the production of TGFBIp in all three types of corneal fibroblasts. These findings may explain the additional therapeutic effect of MMC in GCD II patients.

Plant RNA Regulatory Network and RNA Granules in Virus Infection.

PubMed

Mäkinen, Kristiina; Lõhmus, Andres; Pollari, Maija

2017-01-01

Regulation of post-transcriptional gene expression on mRNA level in eukaryotic cells includes translocation, translation, translational repression, storage, mRNA decay, RNA silencing, and nonsense-mediated decay. These processes are associated with various RNA-binding proteins and cytoplasmic ribonucleoprotein complexes many of which are conserved across eukaryotes. Microscopically visible aggregations formed by ribonucleoprotein complexes are termed RNA granules. Stress granules where the translationally inactive mRNAs are stored and processing bodies where mRNA decay may occur present the most studied RNA granule types. Diverse RNP-granules are increasingly being assigned important roles in viral infections. Although the majority of the molecular level studies on the role of RNA granules in viral translation and replication have been conducted in mammalian systems, some studies link also plant virus infection to RNA granules. An increasing body of evidence indicates that plant viruses require components of stress granules and processing bodies for their replication and translation, but how extensively the cellular mRNA regulatory network is utilized by plant viruses has remained largely enigmatic. Antiviral RNA silencing, which is an important regulator of viral RNA stability and expression in plants, is commonly counteracted by viral suppressors of RNA silencing. Some of the RNA silencing suppressors localize to cellular RNA granules and have been proposed to carry out their suppression functions there. Moreover, plant nucleotide-binding leucine-rich repeat protein-mediated virus resistance has been linked to enhanced processing body formation and translational repression of viral RNA. Many interesting questions relate to how the pathways of antiviral RNA silencing leading to viral RNA degradation and/or repression of translation, suppression of RNA silencing and viral RNA translation converge in plants and how different RNA granules and their individual components contribute to these processes. In this review we discuss the roles of cellular RNA regulatory mechanisms and RNA granules in plant virus infection in the light of current knowledge and compare the findings to those made in animal virus studies.

APOBEC3G levels predict rates of progression to AIDS.

PubMed

Jin, Xia; Wu, Hulin; Smith, Harold

2007-03-20

APOBEC3G (hA3G) is a newly discovered cellular factor of innate immunity that inhibits HIV replication in vitro. Whether hA3G confers protection against HIV in vivo is not known. To investigate the possible anti-HIV activity of hA3G in vivo, we examined hA3G mRNA abundance in primary human cells isolated from either HIV-infected or HIV-uninfected individuals, and found that hA3G mRNA levels follow a hierarchical order of long-term nonprogressors>HIV-uninfected>Progressors; and, hA3G mRNA abundance is correlated with surrogates of HIV disease progression: viral load and CD4 count. Another group later confirmed that HIV-infected subjects have lower hA3G mRNA levels than HIV-uninfected controls, but did not find correlations between hA3G mRNA levels and viral load or CD4 count. These conflicting results indicate that a more comprehensive, conclusive investigation of hA3G expression levels in various patient cohorts is urgently needed. For exploring whether hA3G abundance might influence HIV disease progression, we have formulated a hypothesis that includes two parts: a) in vivo, the basal hA3G mRNA expression level per PBMC is a constant--with minor physiologic fluctuations--determined by host genetic and epigenetic elements in a healthy individual; and that the basal hA3G mRNA expression levels in a population follow a Normal (or Gaussian) distribution; b) that although HIV infects randomly, it results in more rapid disease progression in those with lower hA3G mRNA levels, and slower disease progression in those with higher hA3G mRNA levels. This hypothesis could be tested by a straight forward set of experiments to compare the distribution of hA3G mRNA levels in HIV-uninfected healthy individuals and that in HIV-infected, antiretroviral therapy-naïve subjects who are at early and late stages of infection. Testing this hypothesis will have significant implications for biomedical research. a) It will link hA3G to the mechanisms underlying slower disease progression in long-term nonprogressors. And, b) It may help to establish a new prognostic marker, the hA3G abundance measurement, for HIV-infected patients.

A Cellular High-Throughput Screening Approach for Therapeutic trans-Cleaving Ribozymes and RNAi against Arbitrary mRNA Disease Targets

PubMed Central

Yau, Edwin H.; Butler, Mark C.; Sullivan, Jack M.

2016-01-01

Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme (hhRz) or RNA interference (RNAi) constructs, using a model retinal degeneration target, human rod opsin (RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets. To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N=G,C,A,U; H=C,A,U) within the target mRNA of secreted alkaline phosphatase (SEAP), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening (HTS). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to evaluate a large set of potential VAI-hhRz expression plasmids against diverse NUH↓ cleavage sites uses cultured human HEK293S cells stably expressing a dicistronic Target-IRES-SEAP target fusion mRNA. Broad utility of this rational RNA drug discovery approach is feasible for any ophthalmological disease-relevant mRNA targets and any disease mRNA targets in general. The approach will permit rank ordering of PTGS agents based on potency to identify a lead therapeutic compound for further optimization. PMID:27233447

Arc/Arg3.1 mRNA expression reveals a sub-cellular trace of prior sound exposure in adult primary auditory cortex

PubMed Central

Ivanova, Tamara; Matthews, Andrew; Gross, Christina; Mappus, Rudolph C.; Gollnick, Clare; Swanson, Andrew; Bassell, Gary J.; Liu, Robert C.

2011-01-01

Acquiring the behavioral significance of a sound has repeatedly been shown to correlate with long term changes in response properties of neurons in the adult primary auditory cortex. However, the molecular and cellular basis for such changes is still poorly understood. To address this, we have begun examining the auditory cortical expression of an activity-dependent effector immediate early gene (IEG) with documented roles in synaptic plasticity and memory consolidation in the hippocampus: Arc/Arg3.1. For initial characterization, we applied a repeated 10 minute (24 hour separation) sound exposure paradigm to determine the strength and consistency of sound-evoked Arc/Arg3.1 mRNA expression in the absence of explicit behavioral contingencies for the sound. We used 3D surface reconstruction methods in conjunction with fluorescent in-situ hybridization (FISH) to assess the layer-specific sub-cellular compartmental expression of Arc/Arg3.1 mRNA. We unexpectedly found that both the intranuclear and cytoplasmic patterns of expression depended on the prior history of sound stimulation. Specifically, the percentage of neurons with expression only in the cytoplasm increased for repeated versus singular sound exposure, while intranuclear expression decreased. In contrast, the total cellular expression did not differ, consistent with prior IEG studies of primary auditory cortex. Our results were specific for cortical layers 3–6, as there was virtually no sound driven Arc/Arg3.1 mRNA in layers 1–2 immediately after stimulation. Our results are consistent with the kinetics and/or detectability of cortical sub-cellular Arc/Arg3.1 mRNA expression being altered by the initial exposure to the sound, suggesting exposure-induced modifications in the cytoplasmic Arc/Arg3.1 mRNA pool. PMID:21334422

Controlling nuclear RNA levels.

PubMed

Schmid, Manfred; Jensen, Torben Heick

2018-05-10

RNA turnover is an integral part of cellular RNA homeostasis and gene expression regulation. Whereas the cytoplasmic control of protein-coding mRNA is often the focus of study, we discuss here the less appreciated role of nuclear RNA decay systems in controlling RNA polymerase II (RNAPII)-derived transcripts. Historically, nuclear RNA degradation was found to be essential for the functionalization of transcripts through their proper maturation. Later, it was discovered to also be an important caretaker of nuclear hygiene by removing aberrant and unwanted transcripts. Recent years have now seen a set of new protein complexes handling a variety of new substrates, revealing functions beyond RNA processing and the decay of non-functional transcripts. This includes an active contribution of nuclear RNA metabolism to the overall cellular control of RNA levels, with mechanistic implications during cellular transitions.

Effects of glyceryl glucoside on AQP3 expression, barrier function and hydration of human skin.

PubMed

Schrader, A; Siefken, W; Kueper, T; Breitenbach, U; Gatermann, C; Sperling, G; Biernoth, T; Scherner, C; Stäb, F; Wenck, H; Wittern, K-P; Blatt, T

2012-01-01

Aquaporins (AQPs) present in the epidermis are essential hydration-regulating elements controlling cellular water and glycerol transport. In this study, the potential of glyceryl glucoside [GG; alpha-D-glucopyranosyl-alpha-(1->2)-glycerol], an enhanced glycerol derivative, to increase the expression of AQP3 in vitro and ex vivo was evaluated. In vitro studies with real-time RT-PCR and FACS measurements were performed to test the induction by GG (3% w/v) of AQP3 mRNA and protein in cultured human keratinocytes. GG-containing formulations were applied topically to volunteer subjects and suction blister biopsies were analyzed to assess whether GG (5%) could penetrate the epidermis of intact skin, and subsequently upregulate AQP3 mRNA expression and improve barrier function. AQP3 mRNA and protein levels were significantly increased in cultured human keratinocytes. In the studies on volunteer subjects, GG significantly increased AQP3 mRNA levels in the skin and reduced transepidermal water loss compared with vehicle-controlled areas. GG promotes AQP3 mRNA and protein upregulation and improves skin barrier function, and may thus offer an effective treatment option for dehydrated skin. Copyright © 2012 S. Karger AG, Basel.

Is central dogma a global property of cellular information flow?

PubMed Central

Piras, Vincent; Tomita, Masaru; Selvarajoo, Kumar

2012-01-01

The central dogma of molecular biology has come under scrutiny in recent years. Here, we reviewed high-throughput mRNA and protein expression data of Escherichia coli, Saccharomyces cerevisiae, and several mammalian cells. At both single cell and population scales, the statistical comparisons between the entire transcriptomes and proteomes show clear correlation structures. In contrast, the pair-wise correlations of single transcripts to proteins show nullity. These data suggest that the organizing structure guiding cellular processes is observed at omics-wide scale, and not at single molecule level. The central dogma, thus, globally emerges as an average integrated flow of cellular information. PMID:23189060

Is central dogma a global property of cellular information flow?

PubMed

Piras, Vincent; Tomita, Masaru; Selvarajoo, Kumar

2012-01-01

The central dogma of molecular biology has come under scrutiny in recent years. Here, we reviewed high-throughput mRNA and protein expression data of Escherichia coli, Saccharomyces cerevisiae, and several mammalian cells. At both single cell and population scales, the statistical comparisons between the entire transcriptomes and proteomes show clear correlation structures. In contrast, the pair-wise correlations of single transcripts to proteins show nullity. These data suggest that the organizing structure guiding cellular processes is observed at omics-wide scale, and not at single molecule level. The central dogma, thus, globally emerges as an average integrated flow of cellular information.

Cellular viability and genetic expression of human gingival fibroblasts to zirconia with enamel matrix derivative (Emdogain®)

PubMed Central

Kwon, Yong-Dae; Choi, Hyun-jung; Lee, Heesu; Lee, Jung-Woo; Weber, Hans-Peter

2014-01-01

PURPOSE The objective of this study was to investigate the biologic effects of enamel matrix derivative (EMD) with different concentrations on cell viability and the genetic expression of human gingival fibroblasts (HGF) to zirconia surfaces. MATERIALS AND METHODS Immortalized human gingival fibroblasts (HGF) were cultured (1) without EMD, (2) with EMD 25 µg/mL, and (3) with EMD 100 µg/mL on zirconia discs. MTT assay was performed to evaluate the cell proliferation activity and SEM was carried out to examine the cellular morphology and attachment. The mRNA expression of collagen type I, osteopontin, fibronectin, and TGF-β1 was evaluated with the real-time polymerase chain reaction (RT-PCR). RESULTS From MTT assay, HGF showed more proliferation in EMD 25 µg/mL group than control and EMD 100 µg/mL group (P<.05). HGFs showed more flattened cellular morphology on the experimental groups than on the control group after 4h culture and more cellular attachments were observed on EMD 25 µg/mL group and EMD 100 µg/mL group after 24h culture. After 48h of culture, cellular attachment was similar in all groups. The mRNA expression of type I collagen increased in a concentration dependent manner. The genetic expression of osteopontin, fibronectin, and TGF-β1 was increased at EMD 100 µg/mL. However, the mRNA expression of proteins associated with cellular attachment was decreased at EMD 25 µg/mL. CONCLUSION Through this short term culture of HGF on zirconium discs, we conclude that EMD affects the proliferation, attachment, and cell morphology of HGF cells. Also, EMD stimulates production of extracellular matrix collagen, osteopontin, and TGF-β1 in high concentration levels. CLINICAL RELEVANCE With the use of EMD, protective barrier between attached gingiva and transmucosal zirconia abutment may be enhanced leading to final esthetic results with implants. PMID:25352963

Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export

PubMed Central

Zhang, Liang; Das, Priyabrata; Schmolke, Mirco; Manicassamy, Balaji; Wang, Yaming; Deng, Xiaoyi; Cai, Ling; Tu, Benjamin P.; Forst, Christian V.; Roth, Michael G.; Levy, David E.; García-Sastre, Adolfo; de Brabander, Jef; Phillips, Margaret A.

2012-01-01

The NS1 protein of influenza virus is a major virulence factor essential for virus replication, as it redirects the host cell to promote viral protein expression. NS1 inhibits cellular messenger ribonucleic acid (mRNA) processing and export, down-regulating host gene expression and enhancing viral gene expression. We report in this paper the identification of a nontoxic quinoline carboxylic acid that reverts the inhibition of mRNA nuclear export by NS1, in the absence or presence of the virus. This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels. This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1. The release of NS1-mediated mRNA export block by DHODH inhibition also occurred in the presence of vesicular stomatitis virus M (matrix) protein, another viral inhibitor of mRNA export. This reversal of mRNA export block allowed expression of antiviral factors. Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors. PMID:22312003

Radiation-induced alternative transcripts as detected in total and polysome-bound mRNA.

PubMed

Wahba, Amy; Ryan, Michael C; Shankavaram, Uma T; Camphausen, Kevin; Tofilon, Philip J

2018-01-02

Alternative splicing is a critical event in the posttranscriptional regulation of gene expression. To investigate whether this process influences radiation-induced gene expression we defined the effects of ionizing radiation on the generation of alternative transcripts in total cellular mRNA (the transcriptome) and polysome-bound mRNA (the translatome) of the human glioblastoma stem-like cell line NSC11. For these studies, RNA-Seq profiles from control and irradiated cells were compared using the program SpliceSeq to identify transcripts and splice variations induced by radiation. As compared to the transcriptome (total RNA) of untreated cells, the radiation-induced transcriptome contained 92 splice events suggesting that radiation induced alternative splicing. As compared to the translatome (polysome-bound RNA) of untreated cells, the radiation-induced translatome contained 280 splice events of which only 24 were overlapping with the radiation-induced transcriptome. These results suggest that radiation not only modifies alternative splicing of precursor mRNA, but also results in the selective association of existing mRNA isoforms with polysomes. Comparison of radiation-induced alternative transcripts to radiation-induced gene expression in total RNA revealed little overlap (about 3%). In contrast, in the radiation-induced translatome, about 38% of the induced alternative transcripts corresponded to genes whose expression level was affected in the translatome. This study suggests that whereas radiation induces alternate splicing, the alternative transcripts present at the time of irradiation may play a role in the radiation-induced translational control of gene expression and thus cellular radioresponse.

An exploratory analysis of the effects of a weight loss plus exercise program on cellular quality control mechanisms in older overweight women.

PubMed

Wohlgemuth, Stephanie E; Lees, Hazel A; Marzetti, Emanuele; Manini, Todd M; Aranda, Juan M; Daniels, Michael J; Pahor, Marco; Perri, Michael G; Leeuwenburgh, Christian; Anton, Stephen D

2011-06-01

Obese older adults are particularly susceptible to sarcopenia and have a higher prevalence of disability than their peers of normal weight. Interventions to improve body composition in late life are crucial to maintaining independence. The main mechanisms underlying sarcopenia have not been determined conclusively, but chronic inflammation, apoptosis, and impaired mitochondrial function are believed to play important roles. It has yet to be determined whether impaired cellular quality control mechanisms contribute to this process. The objective of this study was to assess the effects of a 6-month weight loss program combined with moderate-intensity exercise on the cellular quality control mechanisms autophagy and ubiquitin-proteasome, as well as on inflammation, apoptosis, and mitochondrial function, in the skeletal muscle of older obese women. The intervention resulted in significant weight loss (8.0 ± 3.9 % vs. 0.4 ± 3.1% of baseline weight, p = 0.002) and improvements in walking speed (reduced time to walk 400 meters, - 20.4 ± 16% vs. - 2.5 ± 12%, p = 0.03). In the intervention group, we observed a three-fold increase in messenger RNA (mRNA) levels of the autophagy regulators LC3B, Atg7, and lysosome-associated membrane protein-2 (LAMP-2) compared to controls. Changes in mRNA levels of FoxO3A and its targets MuRF1, MAFBx, and BNIP3 were on average seven-fold higher in the intervention group compared to controls, but these differences were not statistically significant. Tumor necrosis factor-α (TNF-α) mRNA levels were elevated after the intervention, but we did not detect significant changes in the downstream apoptosis markers caspase 8 and 3. Mitochondrial biogenesis markers (PGC1α and TFAm) were increased by the intervention, but this was not accompanied by significant changes in mitochondrial complex content and activity. In conclusion, although exploratory in nature, this study is among the first to report the stimulation of cellular quality control mechanisms elicited by a weight loss and exercise program in older obese women.

Messenger RNA Delivery for Tissue Engineering and Regenerative Medicine Applications.

PubMed

Patel, Siddharth; Athirasala, Avathamsa; Menezes, Paula P; Ashwanikumar, N; Zou, Ting; Sahay, Gaurav; Bertassoni, Luiz E

2018-06-07

The ability to control cellular processes and precisely direct cellular reprogramming has revolutionized regenerative medicine. Recent advances in in vitro transcribed (IVT) mRNA technology with chemical modifications have led to development of methods that control spatiotemporal gene expression. Additionally, there is a current thrust toward the development of safe, integration-free approaches to gene therapy for translational purposes. In this review, we describe strategies of synthetic IVT mRNA modifications and nonviral technologies for intracellular delivery. We provide insights into the current tissue engineering approaches that use a hydrogel scaffold with genetic material. Furthermore, we discuss the transformative potential of novel mRNA formulations that when embedded in hydrogels can trigger controlled genetic manipulation to regenerate tissues and organs in vitro and in vivo. The role of mRNA delivery in vascularization, cytoprotection, and Cas9-mediated xenotransplantation is additionally highlighted. Harmonizing mRNA delivery vehicle interactions with polymeric scaffolds can be used to present genetic cues that lead to precise command over cellular reprogramming, differentiation, and secretome activity of stem cells-an ultimate goal for tissue engineering.

Induction of human spermine oxidase SMO(PAOh1) is regulated at the levels of new mRNA synthesis, mRNA stabilization and newly synthesized protein

PubMed Central

2004-01-01

The oxidation of polyamines induced by antitumour polyamine analogues has been associated with tumour response to specific agents. The human spermine oxidase, SMO(PAOh1), is one enzyme that may play a direct role in the cellular response to the antitumour polyamine analogues. In the present study, the induction of SMO(PAOh1) enzyme activity by CPENSpm [N1-ethyl-N11-(cyclopropyl)methyl-4,8,diazaundecane] is demonstrated to be a result of newly synthesized mRNA and protein. Inhibition of new RNA synthesis by actinomycin D inhibits both the appearance of SMO(PAOh1) mRNA and enzyme activity. Similarly, inhibition of newly synthesized protein with cycloheximide prevents analogue-induced enzyme activity. Half-life determinations indicate that stabilization of SMO(PAOh1) protein does not play a significant role in analogue-induced activity. However, half-life experiments using actinomycin D indicate that CPENSpm treatment not only increases mRNA expression, but also leads to a significant increase in mRNA half-life (17.1 and 8.8 h for CPENSpm-treated cells and control respectively). Using reporter constructs encompassing the SMO(PAOh1) promoter region, a 30–90% increase in transcription is observed after exposure to CPENSpm. The present results are consistent with the hypothesis that analogue-induced expression of SMO(PAOh1) is a result of increased transcription and stabilization of SMO(PAOh1) mRNA, leading to increased protein production and enzyme activity. These data indicate that the major level of control of SMO(PAOh1) expression in response to polyamine analogues exposure is at the level of mRNA. PMID:15496143

Identification and characterization of a DnaJ gene from red alga Pyropia yezoensis (Bangiales, Rhodophyta)

NASA Astrophysics Data System (ADS)

Liu, Jiao; Li, Xianchao; Tang, Xuexi; Zhou, Bin

2016-03-01

Members of the DnaJ family are proteins that play a pivotal role in various cellular processes, such as protein folding, protein transport and cellular responses to stress. In the present study, we identified and characterized the full-length DnaJ cDNA sequence from expressed sequence tags of Pyropia yezoensis ( PyDnaJ) via rapid identification of cDNA ends. This cDNA encoded a protein of 429 amino acids, which shared high sequence similarity with other identified DnaJ proteins, such as a heat shock protein 40/DnaJ from Pyropia haitanensis. The relative mRNA expression level of PyDnaJ was investigated using real-time PCR to determine its specific expression during the algal life cycle and during desiccation. The relative mRNA expression level in sporophytes was higher than that in gametophytes and significantly increased during the whole desiccation process. These results indicate that PyDnaJ is an authentic member of the DnaJ family in plants and red algae and might play a pivotal role in mitigating damage to P. yezoensis during desiccation.

Integrated MicroRNA and mRNA Signatures Associated with Survival in Triple Negative Breast Cancer

PubMed Central

Lovat, Francesca; Carasi, Stefania; Pulvirenti, Alfredo; Ferro, Alfredo; Alder, Hansjuerg; He, Gang; Vecchione, Andrea; Croce, Carlo M.; Shapiro, Charles L.; Huebner, Kay

2013-01-01

Triple negative breast cancer (TNBC) is a heterogeneous disease at the molecular, pathologic and clinical levels. To stratify TNBCs, we determined microRNA (miRNA) expression profiles, as well as expression profiles of a cancer-focused mRNA panel, in tumor, adjacent non-tumor (normal) and lymph node metastatic lesion (mets) tissues, from 173 women with TNBCs; we linked specific miRNA signatures to patient survival and used miRNA/mRNA anti-correlations to identify clinically and genetically different TNBC subclasses. We also assessed miRNA signatures as potential regulators of TNBC subclass-specific gene expression networks defined by expression of canonical signal pathways. Tissue specific miRNAs and mRNAs were identified for normal vs tumor vs mets comparisons. miRNA signatures correlated with prognosis were identified and predicted anti-correlated targets within the mRNA profile were defined. Two miRNA signatures (miR-16, 155, 125b, 374a and miR-16, 125b, 374a, 374b, 421, 655, 497) predictive of overall survival (P = 0.05) and distant-disease free survival (P = 0.009), respectively, were identified for patients 50 yrs of age or younger. By multivariate analysis the risk signatures were independent predictors for overall survival and distant-disease free survival. mRNA expression profiling, using the cancer-focused mRNA panel, resulted in clustering of TNBCs into 4 molecular subclasses with different expression signatures anti-correlated with the prognostic miRNAs. Our findings suggest that miRNAs play a key role in triple negative breast cancer through their ability to regulate fundamental pathways such as: cellular growth and proliferation, cellular movement and migration, Extra Cellular Matrix degradation. The results define miRNA expression signatures that characterize and contribute to the phenotypic diversity of TNBC and its metastasis. PMID:23405235

Integrated microRNA and mRNA signatures associated with survival in triple negative breast cancer.

PubMed

Cascione, Luciano; Gasparini, Pierluigi; Lovat, Francesca; Carasi, Stefania; Pulvirenti, Alfredo; Ferro, Alfredo; Alder, Hansjuerg; He, Gang; Vecchione, Andrea; Croce, Carlo M; Shapiro, Charles L; Huebner, Kay

2013-01-01

Triple negative breast cancer (TNBC) is a heterogeneous disease at the molecular, pathologic and clinical levels. To stratify TNBCs, we determined microRNA (miRNA) expression profiles, as well as expression profiles of a cancer-focused mRNA panel, in tumor, adjacent non-tumor (normal) and lymph node metastatic lesion (mets) tissues, from 173 women with TNBCs; we linked specific miRNA signatures to patient survival and used miRNA/mRNA anti-correlations to identify clinically and genetically different TNBC subclasses. We also assessed miRNA signatures as potential regulators of TNBC subclass-specific gene expression networks defined by expression of canonical signal pathways.Tissue specific miRNAs and mRNAs were identified for normal vs tumor vs mets comparisons. miRNA signatures correlated with prognosis were identified and predicted anti-correlated targets within the mRNA profile were defined. Two miRNA signatures (miR-16, 155, 125b, 374a and miR-16, 125b, 374a, 374b, 421, 655, 497) predictive of overall survival (P = 0.05) and distant-disease free survival (P = 0.009), respectively, were identified for patients 50 yrs of age or younger. By multivariate analysis the risk signatures were independent predictors for overall survival and distant-disease free survival. mRNA expression profiling, using the cancer-focused mRNA panel, resulted in clustering of TNBCs into 4 molecular subclasses with different expression signatures anti-correlated with the prognostic miRNAs. Our findings suggest that miRNAs play a key role in triple negative breast cancer through their ability to regulate fundamental pathways such as: cellular growth and proliferation, cellular movement and migration, Extra Cellular Matrix degradation. The results define miRNA expression signatures that characterize and contribute to the phenotypic diversity of TNBC and its metastasis.

Inhibition of Gαs/cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4(+) T Helper Cells.

PubMed

Hynes, Thomas R; Yost, Evan A; Yost, Stacy M; Hartle, Cassandra M; Ott, Braden J; Berlot, Catherine H

2015-07-06

The role of cAMP in regulating T cell activation and function has been controversial. cAMP is generally known as an immunosuppressant, but it is also required for generating optimal immune responses. As the effect of cAMP is likely to depend on its cellular context, the current study investigated whether the mechanism of activation of Gαs and adenylyl cyclase influences their effect on T cell receptor (TCR)-stimulated interleukin-2 (IL-2) mRNA levels. The effect of blocking Gs-coupled receptor (GsPCR)-mediated Gs activation on TCR-stimulated IL-2 mRNA levels in CD4(+) T cells was compared with that of knocking down Gαs expression or inhibiting adenylyl cyclase activity. The effect of knocking down Gαs expression on TCR-stimulated cAMP accumulation was compared with that of blocking GsPCR signaling. ZM-241385, an antagonist to the Gs-coupled A2A adenosine receptor (A2AR), enhanced TCR-stimulated IL-2 mRNA levels in primary human CD4(+) T helper cells and in Jurkat T cells. A dominant negative Gαs construct, GαsDN3, also enhanced TCR-stimulated IL-2 mRNA levels. Similar to GsPCR antagonists, GαsDN3 blocked GsPCR-dependent activation of both Gαs and Gβγ. In contrast, Gαs siRNA and 2',5'-dideoxyadenosine (ddA), an adenylyl cyclase inhibitor, decreased TCR-stimulated IL-2 mRNA levels. Gαs siRNA, but not GαsDN3, decreased TCR-stimulated cAMP synthesis. Potentiation of IL-2 mRNA levels by ZM-241385 required at least two days of TCR stimulation, and addition of ddA after three days of TCR stimulation enhanced IL-2 mRNA levels. GsPCRs play an inhibitory role in the regulation of TCR-stimulated IL-2 mRNA levels whereas Gαs and cAMP can play a stimulatory one. Additionally, TCR-dependent activation of Gαs does not appear to involve GsPCRs. These results suggest that the context of Gαs/cAMP activation and the stage of T cell activation and differentiation determine the effect on TCR-stimulated IL-2 mRNA levels.

Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA*

PubMed Central

Woo, Ho-Hyung; Baker, Terri; Laszlo, Csaba; Chambers, Setsuko K.

2013-01-01

CSF-1 mRNA 3′UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3′UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3′UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of nucleolin's actions on CSF-1 mRNA and describe the dependence of miR-130a- and miR-301a-directed CSF-1 mRNA decay and inhibition of ovarian cancer cell motility on nucleolin. PMID:23471483

Non-coding functions of alternative pre-mRNA splicing in development

PubMed Central

Mockenhaupt, Stefan; Makeyev, Eugene V.

2015-01-01

A majority of messenger RNA precursors (pre-mRNAs) in the higher eukaryotes undergo alternative splicing to generate more than one mature product. By targeting the open reading frame region this process increases diversity of protein isoforms beyond the nominal coding capacity of the genome. However, alternative splicing also frequently controls output levels and spatiotemporal features of cellular and organismal gene expression programs. Here we discuss how these non-coding functions of alternative splicing contribute to development through regulation of mRNA stability, translational efficiency and cellular localization. PMID:26493705

Involvement of lysosomal dysfunction in silver nanoparticle-induced cellular damage in A549 human lung alveolar epithelial cells.

PubMed

Miyayama, Takamitsu; Matsuoka, Masato

2016-01-01

While silver nanoparticles (AgNPs) are widely used in consumer and medical products, the mechanism by which AgNPs cause pulmonary cytotoxicity is not clear. AgNP agglomerates are found in endo-lysosomal structures within the cytoplasm of treated cells. In this study, the functional role of lysosomes in AgNP-induced cellular damage was examined in A549 human lung alveolar epithelial cells. We evaluated the intracellular distribution of AgNPs, lysosomal pH, cellular viability, Ag dissolution, and metallothionein (MT) mRNA levels in AgNP-exposed A549 cells that were treated with bafilomycin A1, the lysosomal acidification inhibitor. Exposure of A549 cells to citrate-coated AgNPs (20 nm diameter) for 24 h induced cellular damage and cell death at 100 and 200 μg Ag/ml, respectively. Confocal laser microscopic examination of LysoTracker-stained cells showed that AgNPs colocalized with lysosomes and their agglomeration increased in a dose-dependent manner (50-200 μg Ag/ml). In addition, the fluorescence signals of LysoTracker were reduced following exposure to AgNPs, suggesting the elevation of lysosomal pH. Treatment of A549 cells with 200 nM bafilomycin A1 and AgNPs (50 μg Ag/ml) further reduced the fluorescence signals of LysoTracker. AgNP-induced cell death was also increased by bafilomycin A1 treatment. Finally, treatment with bafilomycin A1 suppressed the dissolution of Ag and decreased the mRNA expression levels of MT-I and MT-II following exposure to AgNPs. The perturbation of lysosomal pH by AgNP exposure may play a role in AgNP agglomeration and subsequent cellular damage in A549 cells.

Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction.

PubMed

Ray, Swagat; Anderson, Emma C

2016-03-03

The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eukaryotic translation initiation factor 4G (eIF4G), we investigated whether Unr has a general role in translational control. We found that Unr strongly stimulates translation in vitro, and mutation of cold shock domains 2 or 4 inhibited its translation activity. The ability of Unr and its mutants to stimulate translation correlated with its ability to bind RNA, and to interact with PABP1. We found that Unr stimulated the binding of PABP1 to mRNA, and that Unr was required for the stable interaction of PABP1 and eIF4G in cells. siRNA-mediated knockdown of Unr reduced the overall level of cellular translation in cells, as well as that of cap-dependent and IRES-dependent reporters. These data describe a novel role for Unr in regulating cellular gene expression.

Matrin 3 binds and stabilizes mRNA.

PubMed

Salton, Maayan; Elkon, Ran; Borodina, Tatiana; Davydov, Aleksey; Yaspo, Marie-Laure; Halperin, Eran; Shiloh, Yosef

2011-01-01

Matrin 3 (MATR3) is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM), whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq) identified several small noncoding RNA species. Using microarray analysis to explore MATR3's role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts.

Matrin 3 Binds and Stabilizes mRNA

PubMed Central

Salton, Maayan; Elkon, Ran; Borodina, Tatiana; Davydov, Aleksey; Yaspo, Marie-Laure; Halperin, Eran; Shiloh, Yosef

2011-01-01

Matrin 3 (MATR3) is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM), whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq) identified several small noncoding RNA species. Using microarray analysis to explore MATR3′s role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts. PMID:21858232

Intracellular Kinases Mediate Increased Translation and Secretion of Netrin-1 from Renal Tubular Epithelial Cells

PubMed Central

Jayakumar, Calpurnia; Mohamed, Riyaz; Ranganathan, Punithavathi Vilapakkam; Ramesh, Ganesan

2011-01-01

Background Netrin-1 is a laminin-related secreted protein, is highly induced after tissue injury, and may serve as a marker of injury. However, the regulation of netrin-1 production is not unknown. Current study was carried out in mouse and mouse kidney cell line (TKPTS) to determine the signaling pathways that regulate netrin-1 production in response to injury. Methods and Principal Findings Ischemia reperfusion injury of the kidney was induced in mice by clamping renal pedicle for 30 minutes. Cellular stress was induced in mouse proximal tubular epithelial cell line by treating with pervanadate, cisplatin, lipopolysaccharide, glucose or hypoxia followed by reoxygenation. Netrin-1 expression was quantified by real time RT-PCR and protein production was quantified using an ELISA kit. Cellular stress induced a large increase in netrin-1 production without increase in transcription of netrin-1 gene. Mitogen activated protein kinase, ERK mediates the drug induced netrin-1 mRNA translation increase without altering mRNA stability. Conclusion Our results suggest that netrin-1 expression is suppressed at the translational level and MAPK activation leads to rapid translation of netrin-1 mRNA in the kidney tubular epithelial cells. PMID:22046354

Dual inhibition of γ-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase A-dependent mechanism.

PubMed

Jun, Hee-jin; Lee, Ji Hae; Cho, Bo-Ram; Seo, Woo-Duck; Kang, Hang-Won; Kim, Dong-Woo; Cho, Kang-Jin; Lee, Sung-Joon

2012-10-26

The in vitro effects on melanogenesis of γ-oryzanol (1), a rice bran-derived phytosterol, were investigated. The melanin content in B16F1 cells was significantly and dose-dependently reduced (-13% and -28% at 3 and 30 μM, respectively). Tyrosinase enzyme activity was inhibited by 1 both in a cell-free assay and when analyzed based on the measurement of cellular tyrosinase activity. Transcriptome analysis was performed to investigate the biological pathways altered by 1, and it was found that gene expression involving protein kinase A (PKA) signaling was markedly altered. Subsequent analyses revealed that 1 stimulation in B16 cells reduced cytosolic cAMP concentrations, PKA activity (-13% for cAMP levels and -40% for PKA activity), and phosphorylation of the cAMP-response element binding protein (-57%), which, in turn, downregulated the expression of microphthalmia-associated transcription factor (MITF; -59% for mRNA and -64% for protein), a key melanogenic gene transcription factor. Accordingly, tyrosinase-related protein 1 (TRP-1; -69% for mRNA and -82% for protein) and dopachrome tautomerase (-51% for mRNA and -92% for protein) in 1-stimulated B16F1 cells were also downregulated. These results suggest that 1 has dual inhibitory activities for cellular melanogenesis by inhibiting tyrosinase enzyme activity and reducing MITF and target genes in the PKA-dependent pathway.

Exon Microarray Analysis of Human Dorsolateral Prefrontal Cortex in Alcoholism

PubMed Central

Manzardo, Ann M.; Gunewardena, Sumedha; Wang, Kun; Butler, Merlin G.

2014-01-01

Background Alcohol abuse is associated with cellular and biochemical disturbances that impact upon protein and nucleic acid synthesis, brain development, function and behavioral responses. To further characterize the genetic influences in alcoholism and the effects of alcohol consumption on gene expression, we used a highly sensitive exon microarray to examine mRNA expression in human frontal cortex of alcoholics and control males. Methods Messenger RNA was isolated from the dorsolateral prefrontal cortex (dlPFC, Brodmann area 9) of 7 adult Alcoholic (6 males, 1 female, mean age 48 years) and 7 matched controls. Affymetrix Human Exon 1.0 ST Array was performed according to standard procedures and the results analyzed at the gene level. Microarray findings were validated using qRT-PCR, and the ontology of disturbed genes characterized using Ingenuity Pathway Analysis (IPA). Results Decreased mRNA expression was observed for genes involved in cellular adhesion (e.g., CTNNA3, ITGA2), transport (e.g., TF, ABCA8), nervous system development (e.g., LRP2, UGT8, GLDN) and signaling (e.g., RASGRP, LGR5) with influence over lipid and myelin synthesis (e.g., ASPA, ENPP2, KLK6). IPA identified disturbances in network functions associated with neurological disease, and development including cellular assembly and organization impacting on psychological disorders. Conclusions Our data in alcoholism support a reduction in expression of dlPFC mRNA for genes involved with neuronal growth, differentiation and signaling that targets white matter of the brain. PMID:24890784

Exon microarray analysis of human dorsolateral prefrontal cortex in alcoholism.

PubMed

Manzardo, Ann M; Gunewardena, Sumedha; Wang, Kun; Butler, Merlin G

2014-06-01

Alcohol abuse is associated with cellular and biochemical disturbances that impact upon protein and nucleic acid synthesis, brain development, function, and behavioral responses. To further characterize the genetic influences in alcoholism and the effects of alcohol consumption on gene expression, we used a highly sensitive exon microarray to examine mRNA expression in human frontal cortex of alcoholics and control males. Messenger RNA was isolated from the dorsolateral prefrontal cortex (dlPFC; Brodmann area 9) of 7 adult alcoholic (6 males, 1 female, mean age 49 years) and 7 matched controls. Affymetrix Human Exon 1.0 ST array was performed according to standard procedures and the results analyzed at the gene level. Microarray findings were validated using quantitative reverse transcription polymerase chain reaction, and the ontology of disturbed genes characterized using Ingenuity Pathway Analysis (IPA). Decreased mRNA expression was observed for genes involved in cellular adhesion (e.g., CTNNA3, ITGA2), transport (e.g., TF, ABCA8), nervous system development (e.g., LRP2, UGT8, GLDN), and signaling (e.g., RASGRP3, LGR5) with influence over lipid and myelin synthesis (e.g., ASPA, ENPP2, KLK6). IPA identified disturbances in network functions associated with neurological disease and development including cellular assembly and organization impacting on psychological disorders. Our data in alcoholism support a reduction in expression of dlPFC mRNA for genes involved with neuronal growth, differentiation, and signaling that targets white matter of the brain. Copyright © 2014 by the Research Society on Alcoholism.

Lysophosphatidylcholine acyltransferase1 overexpression promotes oral squamous cell carcinoma progression via enhanced biosynthesis of platelet-activating factor.

PubMed

Shida-Sakazume, Tomomi; Endo-Sakamoto, Yosuke; Unozawa, Motoharu; Fukumoto, Chonji; Shimada, Ken; Kasamatsu, Atsushi; Ogawara, Katsunori; Yokoe, Hidetaka; Shiiba, Masashi; Tanzawa, Hideki; Uzawa, Katsuhiro

2015-01-01

The relevance of lysophosphatidylcholine acyltransferase1 (LPCAT1), a cytosolic enzyme in the remodeling pathway of phosphatidylcholine metabolism, in oral squamous cell carcinoma (OSCC) is unknown. We investigated LPCAT1 expression and its functional mechanism in OSCCs. We analyzed LPCAT1 mRNA and protein expression levels in OSCC-derived cell lines. Immunohistochemistry was performed to identify correlations between LPCAT1 expression levels and primary OSCCs clinicopathological status. We established LPCAT1 knockdown models of the OSCC-derived cell lines (SAS, Ca9-22) for functional analysis and examined the association between LPCAT1 expression and the platelet-activating factor (PAF) concentration and PAF-receptor (PAFR) expression. LPCAT1 mRNA and protein were up-regulated significantly (p<0.05) in OSCC-derived cell lines compared with human normal oral keratinocytes. Immunohistochemistry showed significantly (p<0.05) elevated LPCAT1 expression in primary OSCCs compared with normal counterparts and a strong correlation between LPCAT1-positive OSCCs and tumoral size and regional lymph node metastasis. In LPCAT1 knockdown cells, cellular proliferation and invasiveness decreased significantly (p<0.05); cellular migration was inhibited compared with control cells. Down-regulation of LPCAT1 resulted in a decreased intercellular PAF concentration and PAFR expression. LPCAT1 was overexpressed in OSCCs and correlated with cellular invasiveness and migration. LPCAT1 may contribute to tumoral growth and metastasis in oral cancer.

Nogo-receptor gene activity: cellular localization and developmental regulation of mRNA in mice and humans.

PubMed

Josephson, Anna; Trifunovski, Alexandra; Widmer, Hans Ruedi; Widenfalk, Johan; Olson, Lars; Spenger, Christian

2002-11-18

Nogo (reticulon-4) is a myelin-associated protein that is expressed in three different splice variants, Nogo-A, Nogo-B, and Nogo-C. Nogo-A inhibits neurite regeneration in the central nervous system. Messenger RNA encoding Nogo is expressed in oligodendrocytes and central and peripheral neurons, but not in astrocytes or Schwann cells. Nogo is a transmembraneous protein; the extracellular domain is termed Nogo-66, and a Nogo-66-receptor (Nogo-R) has been identified. We performed in situ hybridization in human and mouse nervous tissues to map the cellular distribution of Nogo-R gene activity patterns in fetal and adult human spinal cord and sensory ganglia, adult human brain, and the nervous systems of developing and adult mice. In the human fetus Nogo-R was transcribed in the ventral horn of the spinal cord and in dorsal root ganglia. In adult human tissues Nogo-R gene activity was found in neocortex, hippocampus, amygdala, and a subset of large and medium-sized neurons of the dorsal root ganglia. Nogo-R mRNA was not expressed in the adult human spinal cord at detectable levels. In the fetal mouse, Nogo-R was diffusely expressed in brain, brainstem, trigeminal ganglion, spinal cord, and dorsal root ganglia at all stages. In the adult mouse strong Nogo-R mRNA expression was found in neurons in neocortex, hippocampus, amygdala, habenula, thalamic nuclei, brainstem, the granular cell layer of cerebellum, and the mitral cell layer of the olfactory bulb. Neurons in the adult mouse striatum, the medial septal nucleus, and spinal cord did not express Nogo-R mRNA at detectable levels. In summary, Nogo-66-R mRNA expression in humans and mice was observed in neurons of the developing nervous system Expression was downregulated in the adult spinal cord of both species, and specific expression patterns were seen in the adult brain. Copyright 2002 Wiley-Liss, Inc.

Comparison between chondroprotective effects of glucosamine, curcumin, and diacerein in IL-1beta-stimulated C-28/I2 chondrocytes.

PubMed

Toegel, S; Wu, S Q; Piana, C; Unger, F M; Wirth, M; Goldring, M B; Gabor, F; Viernstein, H

2008-10-01

To compare the effects of glucosamine (GlcN), curcumin, and diacerein in immortalized human C-28/I2 chondrocytes at the cellular and the gene expression level. This study aimed to provide insights into the proposed beneficial effects of these agents and to assess the applicability of the C-28/I2 cell line as a model for the evaluation of chondroprotective action. Interleukin-1beta (IL-1beta)-stimulated C-28/I2 cells were cultured in the presence of GlcN, curcumin, and diacerein prior to the evaluation of parameters such as viability, morphology and proliferation. The impact of GlcN, curcumin, and diacerein on gene expression was determined using quantitative real-time RT-PCR (qPCR). At the transcriptional level, 5 mM GlcN and 50 microM diacerein increased the expression of cartilage-specific genes such as aggrecan (AGC) and collagen type II (COL2), while reducing collagen type I (COL1) mRNA levels. Moreover, the IL-1beta-mediated shift in gene expression pattern was antagonized by GlcN and diacerein. These effects were associated with a significant reduction in cellular proliferation and the development of chondrocyte-specific cell morphology. In contrast, curcumin was not effective at lower concentrations but even damaged the cells at higher amounts. Both GlcN and diacerein promoted a differentiated chondrocytic phenotype of immortalized human C-28/I2 chondrocytes by altering proliferation, morphology, and COL2/COL1 mRNA ratios. Moreover, both agents antagonized inhibitory effects of IL-1beta by enhancing AGC and COL2 as well as by reducing COL1 mRNA levels.

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

Almeida, Luciana O.; Goto, Renata N.; Neto, Marinaldo P.C.

We hypothesized that SET, a protein accumulated in some cancer types and Alzheimer disease, is involved in cell death through mitochondrial mechanisms. We addressed the mRNA and protein levels of the mitochondrial uncoupling proteins UCP1, UCP2 and UCP3 (S and L isoforms) by quantitative real-time PCR and immunofluorescence as well as other mitochondrial involvements, in HEK293 cells overexpressing the SET protein (HEK293/SET), either in the presence or absence of oxidative stress induced by the pro-oxidant t-butyl hydroperoxide (t-BHP). SET overexpression in HEK293 cells decreased UCP1 and increased UCP2 and UCP3 (S/L) mRNA and protein levels, whilst also preventing lipid peroxidationmore » and decreasing the content of cellular ATP. SET overexpression also (i) decreased the area of mitochondria and increased the number of organelles and lysosomes, (ii) increased mitochondrial fission, as demonstrated by increased FIS1 mRNA and FIS-1 protein levels, an apparent accumulation of DRP-1 protein, and an increase in the VDAC protein level, and (iii) reduced autophagic flux, as demonstrated by a decrease in LC3B lipidation (LC3B-II) in the presence of chloroquine. Therefore, SET overexpression in HEK293 cells promotes mitochondrial fission and reduces autophagic flux in apparent association with up-regulation of UCP2 and UCP3; this implies a potential involvement in cellular processes that are deregulated such as in Alzheimer's disease and cancer. - Highlights: • SET, UCPs and autophagy prevention are correlated. • SET action has mitochondrial involvement. • UCP2/3 may reduce ROS and prevent autophagy. • SET protects cell from ROS via UCP2/3.« less

Regulation of Proinflammatory Cytokines in Human Lung Epithelial Cells Infected with Mycoplasma pneumoniae

PubMed Central

Yang, Jun; Hooper, W. Craig; Phillips, Donald J.; Talkington, Deborah F.

2002-01-01

Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans. It has also been associated with chronic conditions, such as arthritis, and extrapulmonary complications, such as encephalitis. Although the interaction of mycoplasmas with respiratory epithelial cells is a critical early phase of pathogenesis, little is known about the cascade of events initiated by infection of respiratory epithelial cells by mycoplasmas. Previous studies have shown that M. pneumoniae can induce proinflammatory cytokines in several different study systems including cultured murine and human monocytes. In this study, we demonstrate that M. pneumoniae infection also induces proinflammatory cytokine expression in A549 human lung carcinoma cells. Infection of A549 cells resulted in increased levels of interleukin-8 (IL-8) and tumor necrosis factor alpha mRNA, and both proteins were secreted into culture medium. IL-1β mRNA also increased after infection and IL-1β protein was synthesized, but it remained intracellular. In contrast, levels of IL-6 and gamma interferon mRNA and protein remained unchanged or undetectable. Using protease digestion and antibody blocking methods, we found that M. pneumoniae cytadherence is important for the induction of cytokines. On the other hand, while M. pneumoniae protein synthesis and DNA synthesis do not appear to be prerequisites for the induction of cytokine gene expression, A549 cellular de novo protein synthesis is responsible for the increased cytokine protein levels. These results suggest a novel role for lung epithelial cells in the pathogenesis of M. pneumoniae infection and provide a better understanding of M. pneumoniae pathology at the cellular level. PMID:12065506

A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18

PubMed Central

Chi, Binkai; Wang, Ke; Du, Yanhua; Gui, Bin; Chang, Xingya; Wang, Lantian; Fan, Jing; Chen, She; Wu, Xudong; Li, Guohui; Cheng, Hong

2014-01-01

Viral RNA elements that facilitate mRNA export are useful tools for identifying cellular RNA export factors. Here we show that hepatitis B virus post-transcriptional element (PRE) is one such element, and using PRE several new cellular mRNA export factors were identified. We found that PRE drastically enhances the cytoplasmic accumulation of cDNA transcripts independent of any viral protein. Systematic deletion analysis revealed the existence of a 116 nt functional Sub-Element of PRE (SEP1). The RNP that forms on the SEP1 RNA was affinity purified, in which TREX components as well as several other proteins were identified. TREX components and the SEP1-associating protein ZC3H18 are required for SEP1-mediated mRNA export. Significantly, ZC3H18 directly binds to the SEP1 RNA, interacts with TREX and is required for stable association of TREX with the SEP1-containing mRNA. Requirements for SEP1-mediated mRNA export are similar to those for splicing-dependent mRNA export. Consistent with these similarities, several SEP1-interacting proteins, including ZC3H18, ARS2, Acinus and Brr2, are required for efficient nuclear export of polyA RNAs. Together, our data indicate that SEP1 enhances mRNA export by recruiting TREX via ZC3H18. The new mRNA export factors that we identified might be involved in cap- and splicing-dependent TREX recruitment to cellular mRNAs. PMID:24782531

[Abnormal expression of genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer].

PubMed

Kuznetsova, E S; Zinovieva, O L; Oparina, N Yu; Prokofjeva, M M; Spirin, P V; Favorskaya, I A; Zborovskaya, I B; Lisitsyn, N A; Prassolov, V S; Mashkova, T D

2016-01-01

Retinoids are signaling molecules that control a wide variety of cellular processes and possess antitumor activity. This work presents a comprehensive description of changes in the expression of 23 genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer tumors compared to adjacent normal tissues obtained using RT-PCR. Even at early stages of malignant transformation, a significant decrease in ADH1B, ADH3, RDHL, and RALDH1 mRNA levels was observed in 82, 79, 73, and 64% of tumor specimens, respectively, and a considerable increase in AKR1B10 mRNA content was observed in 80% of tumors. Dramatic changes in the levels of these mRNAs can impair the synthesis of all-trans retinoic acid, a key natural regulatory retinoid. Apart from that, it was found that mRNA levels of nuclear retinoid receptor genes RXRγ, RARα, RXRα, and gene RDH11 were significantly decreased in 80, 67, 57, and 66% of tumor specimens, respectively. Thus, neoplastic transformation of lung tissue cells is accompanied with deregulated expression of key genes of retinoid metabolism and function.

Sequence features and phylogenetic analysis of the stress protein Hsp90α in chinook salmon Oncorhynchus tshawytscha, a poikilothermic vertebrate

USGS Publications Warehouse

Palmisano, Aldo N.; Winton, James R.; Dickhoff, Walton W.

1999-01-01

We cloned and sequenced a chinook salmon Hsp90 cDNA; sequence analysis shows it to be Hsp90??. Phylogenetic analysis supports the hypothesis that ?? and ?? paralogs of Hsp90 arose as a result of a gene duplication event and that they diverged early in the evolution of vertebrates, before tetrapods separated from the teleost lineage. Among several differences distinguishing poikilothermic Hsp90?? sequences from their bird and mammal orthologs, the teleost versions specifically lack a characteristic QTQDQP phosphorylation site near the N-terminus. We used the cDNA to develop an RNA (Northern) blot to quantify cellular Hsp90 mRNA levels. Chinook salmon embryonic (CHSE-214) cells responded to heat shock with a rapid rise in Hsp90 mRNA through 4 h, followed by a gradual decline over the next 20 h. Hsp90 mRNA level may be useful as a stress indicator, especially in a laboratory setting or in response to acute heat stress.

A biochemical approach to identifying microRNA targets

PubMed Central

Karginov, Fedor V.; Conaco, Cecilia; Xuan, Zhenyu; Schmidt, Bryan H.; Parker, Joel S.; Mandel, Gail; Hannon, Gregory J.

2007-01-01

Identifying the downstream targets of microRNAs (miRNAs) is essential to understanding cellular regulatory networks. We devised a direct biochemical method for miRNA target discovery that combined RNA-induced silencing complex (RISC) purification with microarray analysis of bound mRNAs. Because targets of miR-124a have been analyzed, we chose it as our model. We honed our approach both by examining the determinants of stable binding between RISC and synthetic target RNAs in vitro and by determining the dependency of both repression and RISC coimmunoprecipitation on miR-124a seed sites in two of its well characterized targets in vivo. Examining the complete spectrum of miR-124 targets in 293 cells yielded both a set that were down-regulated at the mRNA level, as previously observed, and a set whose mRNA levels were unaffected by miR-124a. Reporter assays validated both classes, extending the spectrum of mRNA targets that can be experimentally linked to the miRNA pathway. PMID:18042700

Myeloid-related proteins S100A8/S100A9 regulate joint inflammation and cartilage destruction during antigen-induced arthritis.

PubMed

van Lent, P L E M; Grevers, L; Blom, A B; Sloetjes, A; Mort, J S; Vogl, T; Nacken, W; van den Berg, W B; Roth, J

2008-12-01

To study the active involvement of Myeloid-related proteins S100A8 and S100A9 in joint inflammation and cartilage destruction during antigen-induced arthritis (AIA). Joint inflammation and cartilage destruction was measured with 99mTc uptake and histology. The role of S100A8/A9 was investigated by inducing AIA in S100A9-/- mice that also lack S100A8 at protein level, or after intra-articular injection of rS100A8 in mouse knee joints. Cartilage destruction was measured using immunolocalisation of the neoepitope VDIPEN or NITEGE. mRNA levels of matrix metalloproteinases (MMPs) and cytokines were measured using reverse transcriptase (RT)-PCR. Immunisation of S100A9-/- mice with the antigen mBSA induced normal cellular and humoral responses, not different from wild type (WT) controls. However, joint swelling measured at day 3 and 7 after AIA induction was significantly lower (36 and 70%, respectively). Histologically, at day 7 AIA, cellular mass was much lower (63-80%) and proteoglycan depletion from cartilage layers was significantly reduced (between 50-95%). Cartilage destruction mediated by MMPs was absent in S100A9-/- mice but clearly present in controls. MMP3, 9 and 13 mRNA levels were significantly lowered in arthritic synovia of S100A9-/-. In vitro stimulation of macrophages by the heterodimer S100A8/A9 or S100A8 elevated mRNA levels of MMP3, 9 and in particular MMP13. Intra-articular injection of S100A8 caused prominent joint inflammation and depletion of proteoglycans at day 1. Significant upregulation of mRNA levels of S100A8/A9, cytokines (interleukin 1 (IL1)), MMPs (MMP3, MMP13 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4) was found in the synovium and correlated with strong upregulation of NITEGE neoepitopes within the cartilage layers. S100A8/A9 regulate joint inflammation and cartilage destruction during antigen-induced arthritis.

A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1.

PubMed

Lin, Min-Hsuan; Sivakumaran, Haran; Jones, Alun; Li, Dongsheng; Harper, Callista; Wei, Ting; Jin, Hongping; Rustanti, Lina; Meunier, Frederic A; Spann, Kirsten; Harrich, David

2014-12-14

Previously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex. To seek a possible mechanism that could explain how Nullbasic inhibits Rev activity, we used a proteomics approach to identify host cellular proteins that interact with Nullbasic. Forty-six Nullbasic-binding proteins were identified by mass spectrometry including the DEAD-box RNA helicase, DDX1. To determine the effect of DDX1 on Nullbasic-mediated Rev activity, we performed cell-based immunoprecipitation assays, Rev reporter assays and bio-layer interferometry (BLI) assays. Interaction between DDX1 and Nullbasic was observed by co-immunoprecipitation of Nullbasic with endogenous DDX1 from cell lysates. BLI assays showed a direct interaction between Nullbasic and DDX1. Nullbasic affected DDX1 subcellular distribution in a Rev-independent manner. Interestingly overexpression of DDX1 in cells not only restored Rev-dependent mRNA export and gene expression in a Rev reporter assay but also partly reversed Nullbasic-induced Rev subcellular mislocalization. Moreover, HIV-1 wild type Tat co-immunoprecipitated with DDX1 and overexpression of Tat could rescue the unspliced viral mRNA levels inhibited by Nullbasic in HIV-1 expressing cells. Nullbasic was used to further define the complex mechanisms involved in the Rev-dependent nuclear export of the 9 kb and 4 kb viral RNAs. All together, these data indicate that DDX1 can be sequestered by Nullbasic leading to destabilization of the Rev nucleocytoplasmic transport complex and decreased levels of Rev-dependent viral transcripts. The outcomes support a role for DDX1 in maintenance of a Rev nuclear complex that transports viral RRE-containing mRNA to the cytoplasm. To our knowledge Nullbasic is the first anti-HIV protein that specifically targets the cellular protein DDX1 to block Rev's activity. Furthermore, our research raises the possibility that wild type Tat may play a previously unrecognized but very important role in Rev function.

Zearalenone and 17 β-estradiol induced damages in male rats reproduction potential; evidence for ERα and ERβ receptors expression and steroidogenesis.

PubMed

Adibnia, Elmira; Razi, Mazdak; Malekinejad, Hassan

2016-09-15

The estrogen receptors (ERs)-dependent effects of Zearalenone (ZEA) on structure and function of the testis as well as sperm parameters were compared with 17-β estradiol as endogenous substance. For this purpose, 30 mature male rats were assigned into five groups as; control (appropriate volume of normal saline, i. p.), ZEA-received (1, 2 and 4 mg/kg, b. w., i. p.) and 17 β-estradiol (E2)-received (appropriate dose of 0.1 mg/kg, i. p.). Following 28 days, the mRNA levels of estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) in the testis and sperms and the expression of them at protein levels in testicles were estimated. Mitochondrial content of germinal epithelium, Leydig cells steroid foci, sperm quality parameters and serum level of testosterone were assessed. Fluorescent techniques were used for analyzing apoptosis and mRNA damage in necrotic cells. ZEA reduced the mRNA and protein levels of ERα in testicles while up-regulated the ERβ expression. The mRNA level of ERα decreased in sperms of ZEA and E2-received animals. No remarkable changes were found for ERβ expression in sperms from ZEA and E2-received animals. ZEA reduced the Leydig cells steroidogenesis, mitochondrial content of germinal cells and elevated cellular apoptosis and necrosis dose-dependently. E2 reduced the testosterone concentration, enhanced the apoptosis and reduced sperm quality. Our data suggest that ZEA-induced detrimental effects in the structure and function of testis, may attribute to changing the ERs expression at mRNA and translational level. Copyright © 2016 Elsevier Ltd. All rights reserved.

Soya phytoestrogens, genistein and daidzein, decrease apolipoprotein B secretion from HepG2 cells through multiple mechanisms.

PubMed Central

Borradaile, Nica M; de Dreu, Linda E; Wilcox, Lisa J; Edwards, Jane Y; Huff, Murray W

2002-01-01

Diets containing the soya-derived phytoestrogens, genistein and daidzein, decrease plasma cholesterol in humans and experimental animals. The mechanisms responsible for the hypocholesterolaemic effects of these isoflavones are unknown. The present study was conducted to determine if genistein and daidzein regulate hepatocyte cholesterol metabolism and apolipoprotein (apo) B secretion in cultured human hepatoma (HepG2) cells. ApoB secretion was decreased dose-dependently by up to 63% and 71% by genistein and daidzein (100 microM; P<0.0001) respectively. In contrast, no effect on apoAI secretion was observed. Cellular cholesterol synthesis was inhibited 41% by genistein (100 microM; P<0.005) and 18% by daidzein (100 microM; P<0.05), which was associated with significant increases in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA. Cellular cholesterol esterification was decreased 56% by genistein (100 microM; P<0.04) and 29% by daidzein (100 microM; P<0.04); however, mRNA levels for acyl-CoA:cholesterol acyltransferase (ACAT) 1 and ACAT2 were unaffected. At 100 microM, both isoflavones equally inhibited the activities of both forms of ACAT in cells transfected with either ACAT1 or ACAT2. Genistein (100 microM) and daidzein (100 microM) significantly decreased the activity of microsomal triacylglycerol transfer protein (MTP) by 30% and 24% respectively, and significantly decreased MTP mRNA levels by 35% and 55%. Both isoflavones increased low-density lipoprotein (LDL)-receptor mRNA levels by 3- to 6-fold (100 microM; P<0.03) and significantly increased the binding, uptake and degradation of (125)I-labelled LDL, suggesting that enhanced reuptake of newly secreted apoB-containing lipoproteins contributed to the net decrease in apoB secretion. These results indicate that genistein and daidzein inhibit hepatocyte apoB secretion through several mechanisms, including inhibition of cholesterol synthesis and esterification, inhibition of MTP activity and expression and increased expression of the LDL-receptor. PMID:12030847

Mitomycin C induces apoptosis in cultured corneal fibroblasts derived from type II granular corneal dystrophy corneas

PubMed Central

Choi, Seung-il; Lee, Hyung Keun; Cho, Young Jae

2008-01-01

Purpose The present study investigated the effect of mitomycin C (MMC) on cell viability, apoptosis, and transforming growth factor beta-induced protein (TGFBIp) expression in cultured normal corneal fibroblasts and heterozygote or homozygote granular corneal dystrophy type II (GCD II) corneal fibroblasts. Methods Keratocytes were obtained from normal cornea or from heterozygote or homozygote GCD II patients after lamellar or penetrating keratoplasty. To measure cell viability, corneal fibroblasts were incubated with 0.02% MMC for 3 h, 6 h, and 24 h or with 0%, 0.01%, 0.02%, and 0.04% MMC for 24 h and then tested using lactate dehydrogenase (LDH) and 3-[4,5-demethylthiazol-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assays. To measure apoptosis, cells were analyzed by FACS analysis and annexin V staining. Bcl-xL, Bax, and TGFBI mRNA expression was measured using reverse transcription polymerase chain reaction (RT–PCR) assays. Cellular and media levels of TGFBIp protein were measured by immunoblotting. Results MTT and LDH assays showed that MMC reduced cell viability in all three cell types in a dose-dependent and time-dependent manner (p<0.05). FACS analysis and annexin V staining showed that MMC caused apoptosis with GCD II homozygote cells being most affected. RT–PCR analysis showed that MMC decreased Bcl-xL mRNA expression and increased Bax mRNA expression in all cell types. RT–PCR and immunoblotting analysis showed that MMC reduced TGFBI mRNA levels and cellular and media TGFBIp protein levels in all cell types. Conclusions MMC induced apoptosis, and the effects of MMC were greatest in GCD II homozygote cells. MMC also reduced the production of TGFBIp in all three types of corneal fibroblasts. These findings may explain the additional therapeutic effect of MMC in GCD II patients. PMID:18615204

An Interaction between KSHV ORF57 and UIF Provides mRNA-Adaptor Redundancy in Herpesvirus Intronless mRNA Export

PubMed Central

Jackson, Brian R.; Boyne, James R.; Noerenberg, Marko; Taylor, Adam; Hautbergue, Guillaume M.; Walsh, Matthew J.; Wheat, Rachel; Blackbourn, David J.; Wilson, Stuart A.; Whitehouse, Adrian

2011-01-01

The hTREX complex mediates cellular bulk mRNA nuclear export by recruiting the nuclear export factor, TAP, via a direct interaction with the export adaptor, Aly. Intriguingly however, depletion of Aly only leads to a modest reduction in cellular mRNA nuclear export, suggesting the existence of additional mRNA nuclear export adaptor proteins. In order to efficiently export Kaposi's sarcoma-associated herpesvirus (KSHV) intronless mRNAs from the nucleus, the KSHV ORF57 protein recruits hTREX onto viral intronless mRNAs allowing access to the TAP-mediated export pathway. Similarly however, depletion of Aly only leads to a modest reduction in the nuclear export of KSHV intronless mRNAs. Herein, we identify a novel interaction between ORF57 and the cellular protein, UIF. We provide the first evidence that the ORF57-UIF interaction enables the recruitment of hTREX and TAP to KSHV intronless mRNAs in Aly-depleted cells. Strikingly, depletion of both Aly and UIF inhibits the formation of an ORF57-mediated nuclear export competent ribonucleoprotein particle and consequently prevents ORF57-mediated mRNA nuclear export and KSHV protein production. Importantly, these findings highlight that redundancy exists in the eukaryotic system for certain hTREX components involved in the mRNA nuclear export of intronless KSHV mRNAs. PMID:21814512

Role of glutamic acid decarboxylase 67 in regulating cortical parvalbumin and GABA membrane transporter 1 expression: implications for schizophrenia.

PubMed

Curley, Allison A; Eggan, Stephen M; Lazarus, Matt S; Huang, Z Josh; Volk, David W; Lewis, David A

2013-02-01

Markers of GABA neurotransmission are altered in multiple regions of the neocortex in individuals with schizophrenia. Lower levels of glutamic acid decarboxylase 67 (GAD67) mRNA and protein, which is responsible for most cortical GABA synthesis, are accompanied by lower levels of GABA membrane transporter 1 (GAT1) mRNA. These alterations are thought to be most prominent in the parvalbumin (PV)-containing subclass of interneurons, which also contain lower levels of PV mRNA. Since GAT1 and PV each reduce the availability of GABA at postsynaptic receptors, lower levels of GAT1 and PV mRNAs have been hypothesized to represent compensatory responses to an upstream reduction in cortical GABA synthesis in schizophrenia. However, such cause-and-effect hypotheses cannot be directly tested in a human illness. Consequently, we used two mouse models with reduced GAD67 expression specifically in PV neurons (PV(GAD67+/-)) or in all interneurons (GABA(GAD67+/-)) and quantified GAD67, GAT1 and PV mRNA levels using methods identical to those employed in studies of schizophrenia. Cortical levels of PV or GAT1 mRNAs were not altered in PV(GAD67+/-) mice during postnatal development or in adulthood. Furthermore, cellular analyses confirmed the predicted reduction in GAD67 mRNA, but failed to show a deficit in PV mRNA in these animals. Levels of PV and GAT1 mRNAs were also unaltered in GABA(GAD67+/-) mice. Thus, mouse lines with cortical reductions in GAD67 mRNA that match or exceed those present in schizophrenia, and that differ in the developmental timing and cell type-specificity of the GAD67 deficit, failed to provide proof-of-concept evidence that lower PV and GAT1 expression in schizophrenia are a consequence of lower GAD67 expression. Together, these findings suggest that the correlated decrements in cortical GAD67, PV and GAT1 mRNAs in schizophrenia may be a common consequence of some other upstream factor. Copyright © 2012 Elsevier Inc. All rights reserved.

Temporal patterns of cardiac performance and genes encoding heat shock proteins and metabolic sensors of an intertidal limpet Cellana toreuma during sublethal heat stress.

PubMed

Zhang, Shu; Han, Guo-dong; Dong, Yun-wei

2014-04-01

Intertidal invertebrates develop effective physiological adaptations to cope with the rapidly changing thermal environment in the intertidal zone. In the present study, the temporal patterns of heart rate, protein carbonyl groups, and genes encoding heat shock proteins (hsp70 and hsp90) and metabolic sensors (ampkα, ampkβ and sirt1) were measured to study the effect of sublethal heat stress on the cardiac function, oxidative stress, heat shock response and cellular metabolism of an intertidal limpet Cellana toreuma. All the physiological parameters are sensitive to temperature and duration of heat stress. Spearman correlation analysis revealed that the correlations between heart rate and levels of heat shock proteins mRNA and metabolic sensors mRNA were statistically significant. These results further suggest that cardiac function plays crucial roles in cellular energy metabolism and heat shock responses. The significant increase of protein carbonyl groups at 34°C after 4h exposure indicated that the failure of cardiac function and the increase of anaerobic metabolism partly leads to the increase of protein carbonyl groups. Generally, the physiological responses to heat stress are sensitive to temperature and are energy-consumptive, as indicated by the upregulation of metabolic sensors mRNA. However, the upregulation of heat shock proteins and metabolic sensors at the post-transcriptional level and related functions need to be confirmed in further experiments. Copyright © 2014 Elsevier Ltd. All rights reserved.

Course of c-myc mRNA expression in the regenerating mouse testis determined by competitive reverse transcriptase polymerase chain reaction.

PubMed

Amendola, R

1994-11-01

The c-myc proto-oncogene is a reliable marker of the "G0-early G1" transition, and its down-regulation is believed to be necessary to obtain cellular differentiation. In murine spermatogenesis, the level of c-myc transcripts does not correlate with the rate of cellular division. Proliferation of supposed staminal spermatogonia to reproduce themselves is induced with a local 5 Gy X-ray dose in 90-day-old C57Bl/6 mice. c-myc quantification by a newly developed competitive reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to follow the expression course of this proto-oncogene. Damage and restoration of spermatogenesis were analyzed at days 3, 6, 9, 10, 13, 30, and 60 after injury by relative testes/body weight determination and histological examination. Proliferative status was determined by histone H3 Northern blot analysis. c-myc mRNA level was 10 times higher after 3 days in the irradiated animals compared to the controls. An increasing number of copies were noted up to 10 days, but promptly decreased to the base level found for irradiated mice from 13 to 60 days. Interestingly, the expression of histone H3 detected S phase only in testes at 60 days from damage.

Expression of Flk-1 and Cyclin D2 mRNA in the Myocardium of Rats with Doxorubicin-Induced Cardiomyopathy and after Treatment with Betulonic Acid Amide.

PubMed

Mzhelskaya, M M; Klinnikova, M G; Koldysheva, E V; Lushnikova, E L

2017-10-01

The expression of VEGFR2 (Flk-1, according to immunohistochemistry) and of cyclin D2 mRNA (according to real-time PCR) in the myocardium of rats is studied in doxorubicin-induced cardiomyopathy and in response to betulonic acid amide. Doxorubicin alone and in combination with betulonic acid amide causes after 3 days a manifest reduction of cyclin D2 mRNA expression (by 38 and 63%, respectively), while injection of betulonic acid amide alone causes a 23-fold increase of cyclin D2 mRNA expression. An increase of cyclin D2 mRNA expression has been detected in all experimental groups after 14 days of experiment, the most pronounced in response to betulonic acid amide (63 times). The expression of Flk-1 in cardiomyocytes increases significantly in response to both chemical agents starting from day 3 of experiment. These results indicate that doxorubicin and betulonic acid amide induce cytoprotective reactions in the myocardium, first at the intracellular, then at the cellular levels.

Gravity and Skeletal Growth

NASA Technical Reports Server (NTRS)

Morey-Holton, Emily; Turner, Russell T.

1999-01-01

Two simultaneous experiments were performed using 5-week-old male Sprague Dawley rats; in one study, the rats were flown in low earth orbit; in the other study, the hindlimbs of the growing rats were elevated to prevent weight bearing. Following 9 d of unloading, weight bearing was restored for 4, 28, and 76 hrs. Afterwards, additional hindlimb unloading experiments were performed to evaluate the skeletal response to 0, 2, 4, 6, 8, 10, 12, 16, and 24 hrs of restored weight bearing following 7 d of unloading. Cancellous and cortical bone histomorphometry were evaluated in the left tibia at the proximal metaphysis and in the left femur at mid-diaphysis, respectively. Steady-state mRNA levels for bone matrix proteins and skeletal signaling peptides were determined in total cellular RNA extracted from trabeculae from the right proximal tibiametaphysis and periosteum from the right femur. Spaceflight and hindlimb unloading each resulted in cancellous osteopenia, as well as a tendency towards decreased periosteal bone formation. Both models for skeletal unloading resulted in site specific reductions in mRNA levels for transforming growth factor-beta (sub 1) (TGF-beta) osteocalcin (OC), and prepro-alpha (I) subunit of type 1 collagen (collagen) and little or no changes in mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAP) and insulin-like growth factor I (IGF-I). Restoration of normal weight bearing resulted in transient increases in mRNA levels for the bone matrix proteins and TGF-beta in the proximal metaphysis and periosteum and no changes in either GAP or IGF-I mRNA levels. The timecourse for the response differed between the two skeletal compartments; the tibial metaphysis responded much more quickly to reloading. These results suggest that the skeletal adaptation to acute physiological changes in mechanical usage are mediated, in part, by changes in mRNA levels for bone matrix proteins and TGF-beta.

Fetal asphyctic preconditioning modulates the acute cytokine response thereby protecting against perinatal asphyxia in neonatal rats.

PubMed

Vlassaks, Evi; Strackx, Eveline; Vles, Johan Sh; Nikiforou, Maria; Martinez-Martinez, Pilar; Kramer, Boris W; Gavilanes, Antonio Wd

2013-01-26

Perinatal asphyxia (PA) is a major cause of brain damage and neurodevelopmental impairment in infants. Recent investigations have shown that experimental sublethal fetal asphyxia (FA preconditioning) protects against a subsequent more severe asphyctic insult at birth. The molecular mechanisms of this protection have, however, not been elucidated. Evidence implicates that inflammatory cytokines play a protective role in the induction of ischemic tolerance in the adult brain. Accordingly, we hypothesize that FA preconditioning leads to changes in the fetal cytokine response, thereby protecting the newborn against a subsequent asphyctic insult. In rats, FA preconditioning was induced at embryonic day 17 by clamping the uterine vasculature for 30 min. At term birth, global PA was induced by placing the uterine horns, containing the pups, in a saline bath for 19 min. We assessed, at different time points after FA and PA, mRNA and protein expression of several cytokines and related receptor mRNA levels in total hemispheres of fetal and neonatal brains. Additionally, we measured pSTAT3/STAT3 levels to investigate cellular responses to these cytokines. Prenatally, FA induced acute downregulation in IL-1β, TNF-α and IL-10 mRNA levels. At 96 h post FA, IL-6 mRNA and IL-10 protein expression were increased in FA brains compared with controls. Two hours after birth, all proinflammatory cytokines and pSTAT3/STAT3 levels decreased in pups that experienced FA and/or PA. Interestingly, IL-10 and IL-6 mRNA levels increased after PA. When pups were FA preconditioned, however, IL-10 and IL-6 mRNA levels were comparable to those in controls. FA leads to prenatal changes in the neuroinflammatory response. This modulation of the cytokine response probably results in the protective inflammatory phenotype seen when combining FA and PA and may have significant implications for preventing post-asphyctic perinatal encephalopathy.

Fetal asphyctic preconditioning modulates the acute cytokine response thereby protecting against perinatal asphyxia in neonatal rats

PubMed Central

2013-01-01

Background Perinatal asphyxia (PA) is a major cause of brain damage and neurodevelopmental impairment in infants. Recent investigations have shown that experimental sublethal fetal asphyxia (FA preconditioning) protects against a subsequent more severe asphyctic insult at birth. The molecular mechanisms of this protection have, however, not been elucidated. Evidence implicates that inflammatory cytokines play a protective role in the induction of ischemic tolerance in the adult brain. Accordingly, we hypothesize that FA preconditioning leads to changes in the fetal cytokine response, thereby protecting the newborn against a subsequent asphyctic insult. Methods In rats, FA preconditioning was induced at embryonic day 17 by clamping the uterine vasculature for 30 min. At term birth, global PA was induced by placing the uterine horns, containing the pups, in a saline bath for 19 min. We assessed, at different time points after FA and PA, mRNA and protein expression of several cytokines and related receptor mRNA levels in total hemispheres of fetal and neonatal brains. Additionally, we measured pSTAT3/STAT3 levels to investigate cellular responses to these cytokines. Results Prenatally, FA induced acute downregulation in IL-1β, TNF-α and IL-10 mRNA levels. At 96 h post FA, IL-6 mRNA and IL-10 protein expression were increased in FA brains compared with controls. Two hours after birth, all proinflammatory cytokines and pSTAT3/STAT3 levels decreased in pups that experienced FA and/or PA. Interestingly, IL-10 and IL-6 mRNA levels increased after PA. When pups were FA preconditioned, however, IL-10 and IL-6 mRNA levels were comparable to those in controls. Conclusions FA leads to prenatal changes in the neuroinflammatory response. This modulation of the cytokine response probably results in the protective inflammatory phenotype seen when combining FA and PA and may have significant implications for preventing post-asphyctic perinatal encephalopathy. PMID:23351591

Bridging the gap between high-throughput genetic and transcriptional data reveals cellular pathways responding to alpha-synuclein toxicity

PubMed Central

Yeger-Lotem, Esti; Riva, Laura; Su, Linhui Julie; Gitler, Aaron D.; Cashikar, Anil; King, Oliver D.; Auluck, Pavan K.; Geddie, Melissa L.; Valastyan, Julie S.; Karger, David R.; Lindquist, Susan; Fraenkel, Ernest

2009-01-01

Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens, yet the functional roles of the genes identified by these assays often remain enigmatic. By comparing the results of these two assays across various cellular responses, we found that they are consistently distinct. Moreover, genetic screens tend to identify response regulators, while mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We harnessed this approach to reveal cellular pathways related to alpha-synuclein, a small lipid-binding protein implicated in several neurodegenerative disorders including Parkinson disease. For this we screened an established yeast model for alpha-synuclein toxicity to identify genes that when overexpressed alter cellular survival. Application of our algorithm to these data and data from mRNA profiling provided functional explanations for many of these genes and revealed novel relations between alpha-synuclein toxicity and basic cellular pathways. PMID:19234470

Viral and Cellular mRNA Translation in Coronavirus-Infected Cells

PubMed Central

Nakagawa, K.; Lokugamage, K.G.; Makino, S.

2017-01-01

Coronaviruses have large positive-strand RNA genomes that are 5′ capped and 3′ polyadenylated. The 5′-terminal two-thirds of the genome contain two open reading frames (ORFs), 1a and 1b, that together make up the viral replicase gene and encode two large polyproteins that are processed by viral proteases into 15–16 nonstructural proteins, most of them being involved in viral RNA synthesis. ORFs located in the 3′-terminal one-third of the genome encode structural and accessory proteins and are expressed from a set of 5′ leader-containing subgenomic mRNAs that are synthesized by a process called discontinuous transcription. Coronavirus protein synthesis not only involves cap-dependent translation mechanisms but also employs regulatory mechanisms, such as ribosomal frameshifting. Coronavirus replication is known to affect cellular translation, involving activation of stress-induced signaling pathways, and employing viral proteins that affect cellular mRNA translation and RNA stability. This chapter describes our current understanding of the mechanisms involved in coronavirus mRNA translation and changes in host mRNA translation observed in coronavirus-infected cells. PMID:27712623

Non-coding functions of alternative pre-mRNA splicing in development.

PubMed

Mockenhaupt, Stefan; Makeyev, Eugene V

2015-12-01

A majority of messenger RNA precursors (pre-mRNAs) in the higher eukaryotes undergo alternative splicing to generate more than one mature product. By targeting the open reading frame region this process increases diversity of protein isoforms beyond the nominal coding capacity of the genome. However, alternative splicing also frequently controls output levels and spatiotemporal features of cellular and organismal gene expression programs. Here we discuss how these non-coding functions of alternative splicing contribute to development through regulation of mRNA stability, translational efficiency and cellular localization. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Differential restriction patterns of mRNA decay factor AUF1 during picornavirus infections.

PubMed

Cathcart, Andrea L; Semler, Bert L

2014-07-01

During infection by picornaviruses, the cellular environment is modified to favour virus replication. This includes the modification of specific host proteins, including the recently discovered viral proteinase cleavage of mRNA decay factor AU-rich binding factor 1 (AUF1). This cellular RNA-binding protein was shown previously to act as a restriction factor during poliovirus, rhinovirus and coxsackievirus infection. During infection by these viruses, AUF1 relocalizes to the cytoplasm and is cleaved by the viral 3C/3CD proteinase. In this study, we demonstrated that replication of encephalomyocarditis virus (EMCV), a picornavirus belonging to the genus Cardiovirus, is AUF1 independent. During EMCV infection, AUF1 relocalized to the cytoplasm; however, unlike what is seen during enterovirus infections, AUF1 was not cleaved to detectable levels, even at late times after infection. This suggests that AUF1 does not act broadly as an inhibitor of picornavirus infections but may instead act as a selective restriction factor targeting members of the genus Enterovirus. © 2014 The Authors.

The multiple roles of TDP-43 in pre-mRNA processing and gene expression regulation.

PubMed

Buratti, Emanuele; Baralle, Francisco Ernesto

2010-01-01

Heterogeneous ribonucleoproteins (hnRNPs) are multifunctional RNA-binding proteins (RBPs) involved in many cellular processes. They participate in most gene expression pathways, from DNA replication and repair to mRNA translation. Among this class of proteins, TDP-43 (and more recently FUS/TLS) have received considerable attention due to their involvement in several neurodegenerative diseases. This finding has prompted many research groups to focus on the gene expression pathways that are regulated by these proteins. The results have uncovered a considerable complexity of TDP-43 and FUS/TLS functions due to the many independent mechanisms by which they may act to influence various cellular processes (such as DNA transcription, pre-mRNA splicing, mRNA export/import). The aim of this chapter will be to review especially some of the novel functions that have been uncovered, such as role in miRNA synthesis, regulation of transcript levels, and potential autoregulatory mechanisms in order to provide the basis for further investigations.

Inhibition and Avoidance of mRNA Degradation by RNA Viruses

PubMed Central

Moon, Stephanie L.; Barnhart, Michael D.; Wilusz, Jeffrey

2012-01-01

The cellular mRNA decay machinery plays a major role in regulating the quality and quantity of gene expression in cells. This machinery involves multiple enzymes and pathways that converge to promote the exonucleolytic decay of mRNAs. The transcripts made by RNA viruses are susceptible to degradation by this machinery and, in fact, can be actively targeted. Thus, to maintain gene expression and replication, RNA viruses have evolved a number of strategies to avoid and/or inactivate aspects of the cellular mRNA decay machinery. Recent work uncovering the mechanisms used by RNA viruses to maintain the stability of their transcripts is described below. PMID:22626865

Inhibition of sirtuins 1 and 2 impairs cell survival and migration and modulates the expression of P-glycoprotein and MRP3 in hepatocellular carcinoma cell lines.

PubMed

Ceballos, María Paula; Decándido, Giulia; Quiroga, Ariel Darío; Comanzo, Carla Gabriela; Livore, Verónica Inés; Lorenzetti, Florencia; Lambertucci, Flavia; Chazarreta-Cifre, Lorena; Banchio, Claudia; Alvarez, María de Luján; Mottino, Aldo Domingo; Carrillo, María Cristina

2018-06-01

Sirtuins (SIRTs) 1 and 2 deacetylases are overexpressed in hepatocellular carcinoma (HCC) and are associated with tumoral progression and multidrug resistance (MDR). In this study we analyzed whether SIRTs 1 and 2 activities blockage was able to affect cellular survival and migration and to modulate p53 and FoxO1 acetylation in HepG2 and Huh7 cells. Moreover, we analyzed ABC transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 3 (MRP3) expression. We used cambinol and EX-527 as SIRTs inhibitors. Both drugs reduced cellular viability, number of colonies and cellular migration and augmented apoptosis. In 3D cultures, SIRTs inhibitors diminished spheroid growth and viability. 3D culture was less sensitive to drugs than 2D culture. The levels of acetylated p53 and FoxO1 increased after treatments. Drugs induced a decrease in ABC transporters mRNA and protein levels in HepG2 cells; however, only EX-527 was able to reduce MRP3 mRNA and protein levels in Huh7 cells. This is the first work demonstrating the regulation of MRP3 by SIRTs. In conclusion, both drugs decreased HCC cells survival and migration, suggesting SIRTs 1 and 2 activities blockage could be beneficial during HCC therapy. Downregulation of the expression of P-gp and MRP3 supports the potential application of SIRTs 1 and 2 inhibitions in combination with conventional chemotherapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Differential effects of histone deacetylase inhibitors on cellular drug transporters and their implications for using epigenetic modifiers in combination chemotherapy.

PubMed

Valdez, Benigno C; Li, Yang; Murray, David; Brammer, Jonathan E; Liu, Yan; Hosing, Chitra; Nieto, Yago; Champlin, Richard E; Andersson, Borje S

2016-09-27

HDAC inhibitors, DNA alkylators and nucleoside analogs are effective components of combination chemotherapy. To determine a possible mechanism of their synergism, we analyzed the effects of HDAC inhibitors on the expression of drug transporters which export DNA alkylators. Exposure of PEER lymphoma T-cells to 15 nM romidepsin (Rom) resulted in 40%-50% reduction in mRNA for the drug transporter MRP1 and up to ~500-fold increase in the MDR1 mRNA within 32-48 hrs. MRP1 protein levels concomitantly decreased while MDR1 increased. Other HDAC inhibitors - panobinostat, belinostat and suberoylanilide hydroxamic acid (SAHA) - had similar effects on these transporters. The protein level of MRP1 correlated with cellular resistance to busulfan and chlorambucil, and Rom exposure sensitized cells to these DNA alkylators. The decrease in MRP1 correlated with decreased cellular drug export activity, and increased level of MDR1 correlated with increased export of daunorubicin. A similar decrease in the level of MRP1 protein, and increase in MDR1, were observed when mononuclear cells derived from patients with T-cell malignancies were exposed to Rom. Decreased MRP1 and increased MDR1 expressions were also observed in blood mononuclear cells from lymphoma patients who received SAHA-containing chemotherapy in a clinical trial. This inhibitory effect of HDAC inhibitors on the expression of MRP1 suggests that their synergism with DNA alkylating agents is partly due to decreased efflux of these alkylators. Our results further imply the possibility of antagonistic effects when HDAC inhibitors are combined with anthracyclines and other MDR1 drug ligands in chemotherapy.

Extreme heterogeneity of influenza virus infection in single cells

PubMed Central

Russell, Alistair B; Trapnell, Cole

2018-01-01

Viral infection can dramatically alter a cell’s transcriptome. However, these changes have mostly been studied by bulk measurements on many cells. Here we use single-cell mRNA sequencing to examine the transcriptional consequences of influenza virus infection. We find extremely wide cell-to-cell variation in the productivity of viral transcription – viral transcripts comprise less than a percent of total mRNA in many infected cells, but a few cells derive over half their mRNA from virus. Some infected cells fail to express at least one viral gene, but this gene absence only partially explains variation in viral transcriptional load. Despite variation in viral load, the relative abundances of viral mRNAs are fairly consistent across infected cells. Activation of innate immune pathways is rare, but some cellular genes co-vary in abundance with the amount of viral mRNA. Overall, our results highlight the complexity of viral infection at the level of single cells. PMID:29451492

The herpes simplex virus 1 virion host shutoff protein enhances translation of viral late mRNAs by preventing mRNA overload.

PubMed

Dauber, Bianca; Saffran, Holly A; Smiley, James R

2014-09-01

We recently demonstrated that the virion host shutoff (vhs) protein, an mRNA-specific endonuclease, is required for efficient herpes simplex virus 1 (HSV-1) replication and translation of viral true-late mRNAs, but not other viral and cellular mRNAs, in many cell types (B. Dauber, J. Pelletier, and J. R. Smiley, J. Virol. 85:5363-5373, 2011, http://dx.doi.org/10.1128/JVI.00115-11). Here, we evaluated whether the structure of true-late mRNAs or the timing of their transcription is responsible for the poor translation efficiency in the absence of vhs. To test whether the highly structured 5' untranslated region (5'UTR) of the true-late gC mRNA is the primary obstacle for translation initiation, we replaced it with the less structured 5'UTR of the γ-actin mRNA. However, this mutation did not restore translation in the context of a vhs-deficient virus. We then examined whether the timing of transcription affects translation efficiency at late times. To this end, we engineered a vhs-deficient virus mutant that transcribes the true-late gene US11 with immediate-early kinetics (IEUS11-ΔSma). Interestingly, IEUS11-ΔSma showed increased translational activity on the US11 transcript at late times postinfection, and US11 protein levels were restored to wild-type levels. These results suggest that mRNAs can maintain translational activity throughout the late stage of infection if they are present before translation factors and/or ribosomes become limiting. Taken together, these results provide evidence that in the absence of the mRNA-destabilizing function of vhs, accumulation of viral mRNAs overwhelms the capacity of the host translational machinery, leading to functional exclusion of the last mRNAs that are made during infection. The process of mRNA translation accounts for a significant portion of a cell's energy consumption. To ensure efficient use of cellular resources, transcription, translation, and mRNA decay are tightly linked and highly regulated. However, during virus infection, the overall amount of mRNA may increase drastically, possibly overloading the capacity of the translation apparatus. Our results suggest that the HSV-1 vhs protein, an mRNA-specific endoribonuclease, prevents mRNA overload during infection, thereby allowing translation of late viral mRNAs. The requirement for vhs varies between cell types. Further studies of the basis for this difference likely will offer insights into how cells regulate overall mRNA levels and access to the translational apparatus. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis

PubMed Central

Qi, Lei; Yue, Lei; Feng, Deqin; Qi, Fengxia

2017-01-01

Abstract Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an ‘all-or-none’ pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry. PMID:28520982

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

Kim, Kyoung Mi; Cho, Hana; Kim, Yoon Ki, E-mail: yk-kim@korea.ac.kr

Highlights: Black-Right-Pointing-Pointer CDKN1A mRNA is a bona fide NMD substrate. Black-Right-Pointing-Pointer The uORF of CDKN1A mRNA is efficiently translated. Black-Right-Pointing-Pointer Translation of downstream main ORF is negatively regulated by translation of uORF in CDKN1A mRNA. -- Abstract: The first round of translation occurs on mRNAs bound by nuclear cap-binding complex (CBC), which is composed of nuclear cap-binding protein 80 and 20 (CBP80/20). During this round of translation, aberrant mRNAs are recognized and downregulated in abundance by nonsense-mediated mRNA decay (NMD), which is one of the mRNA quality control mechanisms. Here, our microarray analysis reveals that the level of cyclin-dependent kinasemore » inhibitor 1A (CDKN1A; also known as Waf1/p21) mRNAs increases in cells depleted of cellular NMD factors. Intriguingly, CDKN1A mRNA contains an upstream open reading frame (uORF), which is a NMD-inducing feature. Using chimeric reporter constructs, we find that the uORF of CDKN1A mRNA negatively modulates translation of the main downstream ORF. These findings provide biological insights into the possible role of NMD in diverse biological pathways mediated by CDKN1A.« less

Cellular distribution and regulation of ghrelin messenger ribonucleic acid in the rat pituitary gland.

PubMed

Caminos, J E; Nogueiras, R; Blanco, M; Seoane, L M; Bravo, S; Alvarez, C V; García-Caballero, T; Casanueva, F F; Diéguez, C

2003-11-01

Ghrelin, a 28-amino-acid acylated peptide, strongly stimulates GH release and food intake. In the present study, we found that ghrelin is expressed in somatotrophs, lactotrophs, and thyrotrophs but not in corticotrophs or gonadotrophs of rat pituitary. Persistent expression of the ghrelin gene is found during postnatal development in male and female rats, although the levels significantly decrease in both cases from pituitaries of 20-d-old rats onward, but at 60 d old, the levels were higher in male than female rats. This sexually dimorphic pattern appears to be mediated by estrogens because ovariectomy, but not orchidectomy, increases pituitary ghrelin mRNA levels. Taking into account that somatotroph cell function is markedly influenced by thyroid hormones, glucocorticoids, GH, and metabolic status, we also assessed such influence. We found that ghrelin mRNA levels decrease in hypothyroid- and glucocorticoid-treated rats, increase in GH-deficient rats (dwarf rats), and remain unaffected by food deprivation. In conclusion, we have defined the specific cell types that express ghrelin in the rat anterior pituitary gland. These data provide direct morphological evidence that ghrelin may well be acting in a paracrine-like fashion in the regulation of anterior pituitary cell function. In addition, we clearly demonstrate that pituitary ghrelin mRNA levels are age and gender dependent. Finally, we show that pituitary ghrelin mRNA levels are influenced by alteration on thyroid hormone, glucocorticoids, and GH levels but not by fasting, which indicates that the regulation of ghrelin gene expression is tissue specific.

Emphysema induced by elastase alters the mRNA relative levels from DNA repair genes in acute lung injury in response to sepsis induced by lipopolysaccharide administration in Wistar rats.

PubMed

Sergio, Luiz Philippe S; Lucinda, Leda M F; Reboredo, Maycon M; de Paoli, Flavia; Fonseca, Lídia M C; Pinheiro, Bruno V; Mencalha, Andre L; Fonseca, Adenilson S

2018-03-01

Purpose/Aim of the study: Patients suffering from chronic obstructive pulmonary disease (COPD) in association with acute respiratory distress syndrome (ARDS) present oxidative stress in lung cells, with production of free radicals and DNA lesions in pulmonary and adjacent cells. Once the DNA molecule is damaged, a set of enzymatic mechanisms are trigged to preserve genetic code integrity and cellular homeostasis. These enzymatic mechanisms include the base and the nucleotide excision repair pathways, as well as telomere regulation. Thus, the aim of this work was to evaluate the mRNA levels from APEX1, ERCC2, TP53, and TRF2 genes in lung tissue from Wistar rats affected by acute lung injury in response to sepsis and emphysema. Adult male Wistar rats were randomized into 4 groups (n = 6, for each group): control, emphysema, sepsis, and emphysema with sepsis. Pulmonary emphysema was induced by intratracheal instillation of elastase (12 IU/animal) and sepsis induced by intraperitoneal Escherichia coli lipopolysaccharide (LPS) injection (10 mg/kg). Lungs were removed, and samples were withdrawn for histological analysis and total RNA extraction, cDNA synthesis, and mRNA level evaluation by real time quantitative polymerase chain reaction. Data show acute lung injury by LPS and emphysema by elastase and that APEX1, ERCC2, TP53, and TRF2 mRNA levels are increased significantly (p < 0.01) in emphysema with sepsis group. Our results suggest that alteration in mRNA levels from DNA repair and genomic stability could be part of cell response to acute lung injury in response to emphysema and sepsis.

Moderate alcohol consumption aggravates high-fat diet induced steatohepatitis in rats.

PubMed

Wang, Yan; Seitz, Helmut K; Wang, Xiang-Dong

2010-03-01

Nonalcoholic steatohepatitis (NASH) develops in the absence of chronic and excessive alcohol consumption. However, it remains unknown whether moderate alcohol consumption aggravates liver inflammation in pre-existing NASH condition. Sprague-Dawley rats were first fed ad libitum with Lieber-DeCarli high-fat diet (71% energy from fat) for 6 weeks to induce NASH, as demonstrated previously. Afterwards, these rats were continuously fed with high-fat diet (HFD, 55% total energy from fat) or high fat plus alcohol diet (HFA, 55% energy from fat and 16% energy from alcohol) for an additional 4 weeks. Pathological lesions including fat accumulation and inflammatory foci in liver were examined and graded. Lipid peroxidation and apoptotic hepatocytes in the liver were assessed. The mRNA expressions of tumor necrosis factor-alpha (TNFalpha) and TNF receptor 1 (TNF-R1), Fas death receptor (Fas) and Fas ligant (FasL), IL-1beta and IL-12 were determined by real-time PCR. Protein levels of total and cleaved caspase-3, CYP2E1, Bax, and Bcl-2 were measured by western blotting. The number of hepatic inflammatory foci and apoptotic hepatocytes were significantly increased in rats fed with HFA as compared with those in HFD-fed rats. The aggravated inflammatory response and cellular apoptosis mediated by HFA were associated with elevated mRNA expression of Fas/FasL and cleaved caspase-3 protein. Although no significant differences were observed between HFD and HFA groups, the levels of lipid peroxidation, Bax and Bcl-2 protein concentration, and mRNA levels of other inflammatory cytokines were significantly higher in these 2 groups than those in the control group. These data suggest that even moderate alcohol consumption can cause more hepatic inflammation and cellular apoptosis in a pre-existing NASH condition.

Genome wide assessment of mRNA in astrocyte protrusions by direct RNA sequencing reveals mRNA localization for the intermediate filament protein nestin.

PubMed

Thomsen, Rune; Pallesen, Jonatan; Daugaard, Tina F; Børglum, Anders D; Nielsen, Anders L

2013-11-01

Subcellular RNA localization plays an important role in development, cell differentiation, and cell migration. For a comprehensive description of the population of protrusion localized mRNAs in astrocytes we separated protrusions from cell bodies in a Boyden chamber and performed high-throughput direct RNA sequencing. The mRNAs with localization in astrocyte protrusions encode proteins belonging to a variety of functional groups indicating involvement of RNA localization for a palette of cellular functions. The mRNA encoding the intermediate filament protein Nestin was among the identified mRNAs. By RT-qPCR and RNA FISH analysis we confirmed Nestin mRNA localization in cell protrusions and also protrusion localization of Nestin protein. Nestin mRNA localization was dependent of Fragile X mental retardation syndrome proteins Fmrp and Fxr1, and the Nestin 3'-UTR was sufficient to mediate protrusion mRNA localization. The mRNAs for two other intermediate filament proteins in astrocytes, Gfap and Vimentin, have moderate and no protrusion localization, respectively, showing that individual intermediate filament components have different localization mechanisms. The correlated localization of Nestin mRNA with Nestin protein in cell protrusions indicates the presence of a regulatory mechanism at the mRNA localization level for the Nestin intermediate filament protein with potential importance for astrocyte functions during brain development and maintenance. Copyright © 2013 Wiley Periodicals, Inc.

Organizing Principles of Mammalian Nonsense-Mediated mRNA Decay

PubMed Central

Popp, Maximilian Wei-Lin; Maquat, Lynne E.

2014-01-01

Cells use messenger RNAs (mRNAs) to ensure the accurate dissemination of genetic information encoded by DNA. Given that mRNAs largely direct the synthesis of a critical effector of cellular phenotype, i.e., proteins, tight regulation of both the quality and quantity of mRNA is a prerequisite for effective cellular homeostasis. Here, we review nonsense-mediated mRNA decay (NMD), which is the best-characterized posttranscriptional quality control mechanism that cells have evolved in their cytoplasm to ensure transcriptome fidelity. We use protein quality control as a conceptual framework to organize what is known about NMD, highlighting overarching similarities between these two polymer quality control pathways, where the protein quality control and NMD pathways intersect, and how protein quality control can suggest new avenues for research into mRNA quality control. PMID:24274751

Stress- and Rho-activated ZO-1–associated nucleic acid binding protein binding to p21 mRNA mediates stabilization, translation, and cell survival

PubMed Central

Nie, Mei; Balda, Maria S.; Matter, Karl

2012-01-01

A central component of the cellular stress response is p21WAF1/CIP1, which regulates cell proliferation, survival, and differentiation. Inflammation and cell stress often up-regulate p21 posttranscriptionally by regulatory mechanisms that are poorly understood. ZO-1–associated nucleic acid binding protein (ZONAB)/DbpA is a Y-box transcription factor that is regulated by components of intercellular junctions that are affected by cytokines and tissue damage. We therefore asked whether ZONAB activation is part of the cellular stress response. Here, we demonstrate that ZONAB promotes cell survival in response to proinflammatory, hyperosmotic, and cytotoxic stress and that stress-induced ZONAB activation involves the Rho regulator GEF-H1. Unexpectedly, stress-induced ZONAB activation does not stimulate ZONAB’s activity as a transcription factor but leads to the posttranscriptional up-regulation of p21 protein and mRNA. Up-regulation is mediated by ZONAB binding to specific sites in the 3′-untranslated region of the p21 mRNA, resulting in mRNA stabilization and enhanced translation. Binding of ZONAB to mRNA is activated by GEF-H1 via Rho stimulation and also mediates Ras-induced p21 expression. We thus identify a unique type of stress and Rho signaling activated pathway that drives mRNA stabilization and translation and links the cellular stress response to p21 expression and cell survival. PMID:22711822

GSK-3β activation index is a potential indicator for recurrent inflammation of chronic rhinosinusitis without nasal polyps.

PubMed

Hong, Haiyu; Chen, Fenghong; Qiao, Yongkang; Yan, Yan; Zhang, Rongkai; Zhu, Zhe; Li, Huabin; Fan, Yunping; Xu, Geng

2017-12-01

Chronic rhinosinusitis without nasal polyps (CRSsNP) is one of the most common otorhinolaryngologic diseases worldwide. However, the underlying mechanism remains unclear. In this study, the expression of glycogen synthase kinase 3 (GSK-3) was quantitatively evaluated in patients with CRSsNP (n = 20) and healthy controls (n = 20). The mRNA levels of GSK-3α and GSK-3β were examined by qPCR, the immunoreactivities of GSK-3β and nuclear factor-κB (NF-κB) were examined by immunohistochemistry (IHC) staining, and the protein levels of GSK-3β, phospho-GSK-3β (p-GSK-3β, s9) and NF-κB were examined using Western blot analysis. We found that GSK-3 was highly expressed in both CRSsNP and control groups without significant difference in both GSK-3β mRNA and protein levels. However, when compared with healthy control group, the GSK-3β activation index, defined as the ratio of GSK-3β over p-GSK-3β, was significantly decreased, whereas the NF-κB protein abundance was significantly increased in CRSsNP group (P < 0.05). Strikingly, the GSK-3β activation index, was highly correlated with NF-κB protein level, as well as CT scores in CRSsNP group (P < 0.05). It was also highly correlated with the mRNA expressions of inflammation-related genes, including T-bet, IFN-γ and IL-4 in CRSsNP group (P < 0.05). Our findings suggest that GSK-3β activation index, reflecting the inhibitory levels of GSK-3β through phosphorylation, may be a potential indicator for recurrent inflammation of CRSsNP, and that the insufficient inhibitory phosphorylation of GSK-3β may play a pivotal role in the pathogenesis of CRSsNP. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Effects of Varying Degrees of Intermittent Hypoxia on Proinflammatory Cytokines and Adipokines in Rats and 3T3-L1 Adipocytes

PubMed Central

Zhou, Qin; Zhu, Hui; Niu, Wen-yan; Feng, Jing; Wang, Yan; Cao, Jie; Chen, Bao-yuan

2014-01-01

Objectives Intermittent hypoxia (IH), resulted from recurring episodes of upper airway obstruction, is the hallmark feature and the most important pathophysiologic pathway of obstructive sleep apnea (OSA). IH is believed to be the most important factor causing systemic inflammation. Studies suggest that insulin resistance (IR) is positively associated with OSA. In this study, we hypothesized that the recurrence of IH might result in cellular and systemic inflammation, which was manifested through the levels of proinflammatory cytokines and adipokines after IH exposure, and because IR is linked with inflammation tightly, this inflammatory situation may implicate an IR status. Methods We developed an IH 3T3-L1 adipocyte and rat model respectively, recapitulating the nocturnal oxygen profile in OSA. In IH cells, nuclear factor kappa B (NF-κB) DNA binding reactions, hypoxia-inducible factor-1α (HIF-1α), glucose transporter-1 (Glut-1), necrosis factor alpha (TNF-α), interleukin (IL) -6, leptin, adiponectin mRNA transcriptional activities and protein expressions were measured. In IH rats, blood glucose, insulin, TNF-α, IL-6, leptin and adiponectin levels were analyzed. Results The insulin and blood glucose levels in rats and NF-κB DNA binding activities in cells had significantly statistical results described as severe IH>moderate IH>mild IH>sustained hypoxia>control. The mRNA and protein levels of HIF-1α and Glut-1 in severe IH group were the highest. In cellular and animal models, both the mRNA and protein levels of TNF-α, IL-6 and leptin were the highest in severe IH group, when the lowest in severe IH group for adiponectin. Conclusions Oxidative stress and the release of pro-inflammatory cytokines/adipokines, which are the systemic inflammatory markers, are associated with IH closely and are proportional to the severity of IH. Because IR and glucose intolerance are linked with inflammation tightly, our results may implicate the clinical relationships between OSA and IR. PMID:24466027

Comprehensive Survey of miRNA-mRNA Interactions Reveals That Ccr7 and Cd247 (CD3 zeta) are Posttranscriptionally Controlled in Pancreas Infiltrating T Lymphocytes of Non-Obese Diabetic (NOD) Mice.

PubMed

Fornari, Thais A; Donate, Paula B; Assis, Amanda F; Macedo, Claudia; Sakamoto-Hojo, Elza T; Donadi, Eduardo A; Passos, Geraldo A

2015-01-01

In autoimmune type 1 diabetes mellitus (T1D), auto-reactive clones of CD4+ and CD8+ T lymphocytes in the periphery evolve into pancreas-infiltrating T lymphocytes (PILs), which destroy insulin-producing beta-cells through inflammatory insulitis. Previously, we demonstrated that, during the development of T1D in non-obese diabetic (NOD) mice, a set of immune/inflammatory reactivity genes were differentially expressed in T lymphocytes. However, the posttranscriptional control involving miRNA interactions that occur during the evolution of thymocytes into PILs remains unknown. In this study, we postulated that miRNAs are differentially expressed during this period and that these miRNAs can interact with mRNAs involved in auto-reactivity during the progression of insulitis. To test this hypothesis, we used NOD mice to perform, for the first time, a comprehensive survey of miRNA and mRNA expression as thymocytes mature into peripheral CD3+ T lymphocytes and, subsequently, into PILs. Reconstruction of miRNA-mRNA interaction networks for target prediction revealed the participation of a large set of miRNAs that regulate mRNA targets related to apoptosis, cell adhesion, cellular regulation, cellular component organization, cellular processes, development and the immune system, among others. The interactions between miR-202-3p and the Ccr7 chemokine receptor mRNA or Cd247 (Cd3 zeta chain) mRNA found in PILs are highlighted because these interactions can contribute to a better understanding of how the lack of immune homeostasis and the emergence of autoimmunity (e.g., T1D) can be associated with the decreased activity of Ccr7 or Cd247, as previously observed in NOD mice. We demonstrate that these mRNAs are controlled at the posttranscriptional level in PILs.

Comprehensive Survey of miRNA-mRNA Interactions Reveals That Ccr7 and Cd247 (CD3 zeta) are Posttranscriptionally Controlled in Pancreas Infiltrating T Lymphocytes of Non-Obese Diabetic (NOD) Mice

PubMed Central

Macedo, Claudia; Sakamoto-Hojo, Elza T.; Donadi, Eduardo A.; Passos, Geraldo A.

2015-01-01

In autoimmune type 1 diabetes mellitus (T1D), auto-reactive clones of CD4+ and CD8+ T lymphocytes in the periphery evolve into pancreas-infiltrating T lymphocytes (PILs), which destroy insulin-producing beta-cells through inflammatory insulitis. Previously, we demonstrated that, during the development of T1D in non-obese diabetic (NOD) mice, a set of immune/inflammatory reactivity genes were differentially expressed in T lymphocytes. However, the posttranscriptional control involving miRNA interactions that occur during the evolution of thymocytes into PILs remains unknown. In this study, we postulated that miRNAs are differentially expressed during this period and that these miRNAs can interact with mRNAs involved in auto-reactivity during the progression of insulitis. To test this hypothesis, we used NOD mice to perform, for the first time, a comprehensive survey of miRNA and mRNA expression as thymocytes mature into peripheral CD3+ T lymphocytes and, subsequently, into PILs. Reconstruction of miRNA-mRNA interaction networks for target prediction revealed the participation of a large set of miRNAs that regulate mRNA targets related to apoptosis, cell adhesion, cellular regulation, cellular component organization, cellular processes, development and the immune system, among others. The interactions between miR-202-3p and the Ccr7 chemokine receptor mRNA or Cd247 (Cd3 zeta chain) mRNA found in PILs are highlighted because these interactions can contribute to a better understanding of how the lack of immune homeostasis and the emergence of autoimmunity (e.g., T1D) can be associated with the decreased activity of Ccr7 or Cd247, as previously observed in NOD mice. We demonstrate that these mRNAs are controlled at the posttranscriptional level in PILs. PMID:26606254

RNA-Binding Protein FXR1 Regulates p21 and TERC RNA to Bypass p53-Mediated Cellular Senescence in OSCC

PubMed Central

Majumder, Mrinmoyee; House, Reniqua; Palanisamy, Nallasivam; Qie, Shuo; Day, Terrence A.; Neskey, David; Diehl, J. Alan

2016-01-01

RNA-binding proteins (RBP) regulate numerous aspects of co- and post-transcriptional gene expression in cancer cells. Here, we demonstrate that RBP, fragile X-related protein 1 (FXR1), plays an essential role in cellular senescence by utilizing mRNA turnover pathway. We report that overexpressed FXR1 in head and neck squamous cell carcinoma targets (G-quadruplex (G4) RNA structure within) both mRNA encoding p21 (Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A, Cip1) and the non-coding RNA Telomerase RNA Component (TERC), and regulates their turnover to avoid senescence. Silencing of FXR1 in cancer cells triggers the activation of Cyclin-Dependent Kinase Inhibitors, p53, increases DNA damage, and ultimately, cellular senescence. Overexpressed FXR1 binds and destabilizes p21 mRNA, subsequently reduces p21 protein expression in oral cancer cells. In addition, FXR1 also binds and stabilizes TERC RNA and suppresses the cellular senescence possibly through telomerase activity. Finally, we report that FXR1-regulated senescence is irreversible and FXR1-depleted cells fail to form colonies to re-enter cellular proliferation. Collectively, FXR1 displays a novel mechanism of controlling the expression of p21 through p53-dependent manner to bypass cellular senescence in oral cancer cells. PMID:27606879

Whole-genome analysis of mRNA decay in Plasmodium falciparum reveals a global lengthening of mRNA half-life during the intra-erythrocytic development cycle.

PubMed

Shock, Jennifer L; Fischer, Kael F; DeRisi, Joseph L

2007-01-01

The rate of mRNA decay is an essential element of post-transcriptional regulation in all organisms. Previously, studies in several organisms found that the specific half-life of each mRNA is precisely related to its physiologic role, and plays an important role in determining levels of gene expression. We used a genome-wide approach to characterize mRNA decay in Plasmodium falciparum. We found that, globally, rates of mRNA decay increase dramatically during the asexual intra-erythrocytic developmental cycle. During the ring stage of the cycle, the average mRNA half-life was 9.5 min, but this was extended to an average of 65 min during the late schizont stage of development. Thus, a major determinant of mRNA decay rate appears to be linked to the stage of intra-erythrocytic development. Furthermore, we found specific variations in decay patterns superimposed upon the dominant trend of progressive half-life lengthening. These variations in decay pattern were frequently enriched for genes with specific cellular functions or processes. Elucidation of Plasmodium mRNA decay rates provides a key element for deciphering mechanisms of genetic control in this parasite, by complementing and extending previous mRNA abundance studies. Our results indicate that progressive stage-dependent decreases in mRNA decay rate function are a major determinant of mRNA accumulation during the schizont stage of intra-erythrocytic development. This type of genome-wide change in mRNA decay rate has not been observed in any other organism to date, and indicates that post-transcriptional regulation may be the dominant mechanism of gene regulation in P. falciparum.

Alternative Polyadenylation in Human Diseases

PubMed Central

Chang, Jae-Woong; Yeh, Hsin-Sung

2017-01-01

Varying length of messenger RNA (mRNA) 3′-untranslated region is generated by alternating the usage of polyadenylation sites during pre-mRNA processing. It is prevalent through all eukaryotes and has emerged as a key mechanism for controlling gene expression. Alternative polyadenylation (APA) plays an important role for cell growth, proliferation, and differentiation. In this review, we discuss the functions of APA related with various physiological conditions including cellular metabolism, mRNA processing, and protein diversity in a variety of disease models. We also discuss the molecular mechanisms underlying APA regulation, such as variations in the concentration of mRNA processing factors and RNA-binding proteins, as well as global transcriptome changes under cellular signaling pathway. PMID:29271615

Expression of calmodulin mRNA in rat olfactory neuroepithelium.

PubMed

Biffo, S; Goren, T; Khew-Goodall, Y S; Miara, J; Margolis, F L

1991-04-01

A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.

Protection against dexamethasone-induced muscle atrophy is related to modulation by testosterone of FOXO1 and PGC-1{alpha}

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

Qin, Weiping, E-mail: weiping.qin@mssm.edu; Department of Medicine, Mount Sinai School of Medicine, NY; Pan, Jiangping

Research highlights: {yields} In rat gastrocnemius muscle, dexamethasone reduced PGC-1{alpha} cellular and nuclear levels without altering mRNA levels for this factor. {yields} Dexamethasone reduced phosphorylating of p38 MAPK, which stabilizes PGC-1{alpha} and promotes its nuclear entry. {yields} Co-administration of testosterone with dexamethasone increased cellular and nuclear levels of PGC-1{alpha} protein without changing its mRNA levels. {yields} Co-administration of testosterone restored p38 MAPK levels to those of controls. -- Abstract: Glucocorticoid-induced muscle atrophy results from muscle protein catabolism and reduced protein synthesis, associated with increased expression of two muscle-specific ubiquitin ligases (MAFbx and MuRF1), and of two inhibitors of protein synthesis,more » REDD1 and 4EBP1. MAFbx, MuRF1, REDD1 and 4EBP1 are up-regulated by the transcription factors FOXO1 and FOXO3A. The transcriptional co-activator PGC-1{alpha} has been shown to attenuate many forms of muscle atrophy and to repress FOXO3A-mediated transcription of atrophy-specific genes. Dexamethasone-induced muscle atrophy can be prevented by testosterone, which blocks up-regulation by dexamethasone of FOXO1. Here, an animal model of dexamethasone-induced muscle atrophy was used to further characterize effects of testosterone to abrogate adverse actions of dexamethasone on FOXO1 levels and nuclear localization, and to determine how these agents affect PGC-1{alpha}, and its upstream activators, p38 MAPK and AMPK. In rat gastrocnemius muscle, testosterone blunted the dexamethasone-mediated increase in levels of FOXO1 mRNA, and FOXO1 total and nuclear protein. Dexamethasone reduced total and nuclear PGC-1{alpha} protein levels in the gastrocnemius; co-administration of testosterone with dexamethasone increased total and nuclear PGC-1{alpha} levels above those present in untreated controls. Testosterone blocked dexamethasone-induced decreases in activity of p38 MAPK in the gastrocnemius muscle. Regulation of FOXO1, PGC-1{alpha} and p38 MAPK by testosterone may represent a novel mechanism by which this agent protects against dexamethasone-induced muscle atrophy.« less

Impact of DBP on histology and expression of HSP 70 in gill and liver tissue of Cyprinus carpio.

PubMed

Agus, Hizlan H; Erkmen, Belda; Sümer, Sibel; Sepici-Dinçel, Aylin; Erkoç, Figen

2015-09-01

Di-n-butyl phthalate (DBP) widely used plasticizer in the plastic industry, affects regulation of the endocrine system and causes toxicity in animals. In the present study, the aim was to study the toxic effects/damages of DBP exposure using Hsp70 levels and histopathological changes in Carp liver and gill. Hsp70 expression levels were assessed as specific biomarker of in vivo ecotoxicological stress. Carp (Cyprinus carpio) were exposed to sub-lethal concentration of DBP (di-n-butyl phthalate, 1 mg/L) for 4, 24 and 96 h. Gill and liver tissues were evaluated histopathologically and RNA quantifications for Hsp70 expression levels were carried out using a two-step real-time RT-PCR. In liver, a rapid but non-significant increase in mRNA levels in the first 4 h was observed. mRNA levels significantly increased up to 2-3 fold after 24 and 96 h (p < 0.05). However, irregular mRNA level changes were also recorded: Gill specific and time-dependent regulation of Hsp70 expression were 4-5 fold inhibition after 4 and 24 h (p < 0.05), then increased up to 4 fold after 96 h (p < 0.05). Histopathological findings support altered transcription results as: Epithelial lifting, hyperplasia, fusion of secondary lamellae, telangiectasis, passive hyperemia and hydropic degeneration. Significant alterations of Hsp70 levels were likely due to a tissue specific response against chemical stress, cellular damage and lesions due to DBP. Carp was found to be a suitable experimental model for toxicology, and Hsp70 mRNA levels are reliable, specific biomarkers.

Immune-stimulatory effects of a bacteria-based probiotic on peripheral leukocyte subpopulations and cytokine mRNA expression levels in scouring holstein calves.

PubMed

Qadis, Abdul Qadir; Goya, Satoru; Yatsu, Minoru; Kimura, Atsushi; Ichijo, Toshihiro; Sato, Shigeru

2014-05-01

Subpopulations of peripheral leukocytes and cytokine mRNA expression levels were evaluated in scouring and healthy Holstein calves (age 10 ± 5 days; n=42) treated with a probiotic consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum. The calves were assigned to the scouring or healthy group and then subdivided into pathogen-positive treated (n=8), pathogen-positive control (n=8), pathogen-negative treated (n=6), pathogen-negative control (n=6), healthy treated (n=6) and healthy control (n=8) groups. A single dose of the probiotic (3.0 g/100 kg body weight) was given to each calf in the treatment groups for 5 days. Blood samples were collected on the first day of scour occurrence (day 0) and on day 7. In the scouring calves, smaller peripheral leukocyte subpopulations and cytokine mRNA expression levels were noted on day 0. The numbers of CD3(+) T cells; CD4(+), CD8(+) and WC1(+) γδ T cell subsets; and CD14(+), CD21(+) and CD282(+) (TLR2) cells were significantly increased in the scouring and healthy treated calves on day 7. Furthermore, interleukin-6, tumor necrosis factor-alpha and interferon-gamma mRNA expression was elevated in the peripheral leukocytes of the scouring and healthy treated calves on day 7. The scouring calves given the probiotic recovered on day 7. A significantly smaller number of peripheral leukocytes and lower cytokine mRNA expression level might be induced by scouring in calves. Repeated probiotic administration might stimulate cellular immunity and encourage recovery from scouring in pre-weaning Holstein calves.

Mechanoregulation of human articular chondrocyte aggrecan and type II collagen expression by intermittent hydrostatic pressure in vitro.

PubMed

Ikenoue, Takashi; Trindade, Michael C D; Lee, Mel S; Lin, Eric Y; Schurman, David J; Goodman, Stuart B; Smith, R Lane

2003-01-01

This study addressed the hypothesis that duration and magnitude of applied intermittent hydrostatic pressure (IHP) are critical parameters in regulation of normal human articular chondrocyte aggrecan and type II collagen expression. Articular chondrocytes were isolated from knee cartilage and maintained as primary, high-density monolayer cultures. IHP was applied at magnitudes of 1, 5 and 10 MPa at 1 Hz for durations of either 4 h per day for one day (4 x 1) or 4 h per day for four days (4 x 4). Total cellular RNA was isolated and analyzed for aggrecan and type II collagen mRNA signal levels using specific primers and reverse transcription polymerase chain reaction (RT-PCR) nested with beta-actin primers as internal controls. With a 4x1 loading regimen, aggrecan mRNA signal levels increased 1.3- and 1.5-fold at 5 and 10 MPa, respectively, relative to beta-actin mRNA when compared to unloaded cultures. Changing the duration of loading to a 4x4 regimen increased aggrecan mRNA signal levels by 1.4-, 1.8- and 1.9-fold at loads of 1, 5 and 10 MPa, respectively. In contrast to the effects of IHP on aggrecan, type II collagen mRNA signal levels were only upregulated at loads of 5 and 10 MPa with the 4x4 loading regimen. Analysis of cell-associated protein by western blotting confirmed that IHP increased aggrecan and type II collagen in chondrocyte extracts. These data demonstrate that duration and magnitude of applied IHP differentially alter chondrocyte matrix protein expression. The results show that IHP provides an important stimulus for increasing cartilage matrix anabolism and may contribute to repair and regeneration of damaged or diseased cartilage.

Immune-Stimulatory Effects of a Bacteria-Based Probiotic on Peripheral Leukocyte Subpopulations and Cytokine mRNA Expression Levels in Scouring Holstein Calves

PubMed Central

QADIS, Abdul Qadir; GOYA, Satoru; YATSU, Minoru; KIMURA, Atsushi; ICHIJO, Toshihiro; SATO, Shigeru

2014-01-01

ABSTRACT Subpopulations of peripheral leukocytes and cytokine mRNA expression levels were evaluated in scouring and healthy Holstein calves (age 10 ± 5 days; n=42) treated with a probiotic consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum. The calves were assigned to the scouring or healthy group and then subdivided into pathogen-positive treated (n=8), pathogen-positive control (n=8), pathogen-negative treated (n=6), pathogen-negative control (n=6), healthy treated (n=6) and healthy control (n=8) groups. A single dose of the probiotic (3.0 g/100 kg body weight) was given to each calf in the treatment groups for 5 days. Blood samples were collected on the first day of scour occurrence (day 0) and on day 7. In the scouring calves, smaller peripheral leukocyte subpopulations and cytokine mRNA expression levels were noted on day 0. The numbers of CD3+ T cells; CD4+, CD8+ and WC1+ γδ T cell subsets; and CD14+, CD21+ and CD282+ (TLR2) cells were significantly increased in the scouring and healthy treated calves on day 7. Furthermore, interleukin-6, tumor necrosis factor-alpha and interferon-gamma mRNA expression was elevated in the peripheral leukocytes of the scouring and healthy treated calves on day 7. The scouring calves given the probiotic recovered on day 7. A significantly smaller number of peripheral leukocytes and lower cytokine mRNA expression level might be induced by scouring in calves. Repeated probiotic administration might stimulate cellular immunity and encourage recovery from scouring in pre-weaning Holstein calves. PMID:24451928

Multi-level omics analysis in a murine model of dystrophin loss and therapeutic restoration.

PubMed

Roberts, Thomas C; Johansson, Henrik J; McClorey, Graham; Godfrey, Caroline; Blomberg, K Emelie M; Coursindel, Thibault; Gait, Michael J; Smith, C I Edvard; Lehtiö, Janne; El Andaloussi, Samir; Wood, Matthew J A

2015-12-01

Duchenne muscular dystrophy (DMD) is a classical monogenic disorder, a model disease for genomic studies and a priority candidate for regenerative medicine and gene therapy. Although the genetic cause of DMD is well known, the molecular pathogenesis of disease and the response to therapy are incompletely understood. Here, we describe analyses of protein, mRNA and microRNA expression in the tibialis anterior of the mdx mouse model of DMD. Notably, 3272 proteins were quantifiable and 525 identified as differentially expressed in mdx muscle (P < 0.01). Therapeutic restoration of dystrophin by exon skipping induced widespread shifts in protein and mRNA expression towards wild-type expression levels, whereas the miRNome was largely unaffected. Comparison analyses between datasets showed that protein and mRNA ratios were only weakly correlated (r = 0.405), and identified a multitude of differentially affected cellular pathways, upstream regulators and predicted miRNA-target interactions. This study provides fundamental new insights into gene expression and regulation in dystrophic muscle. © The Author 2015. Published by Oxford University Press.

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

Waltman, Peter H.; Guo, Jian; Reistetter, Emily Nahas

Micromonas is a unicellular green alga that belongs to the prasinophytes, a sister lineage to land plants. This picoeukaryotic (<2 μm diameter) alga is widespread in the marine environment but still not understood at the cellular level. Here, we examine the mRNA and protein level changes that take place over the course of the day-night cycle using mid-exponential nutrient replete cultures of Micromonas pusilla CCMP1545 grown and analyzed in biological triplicate. During the experiment, samples were collected at key transition points during the diel for evaluation using high-throughput LC-MS proteomics. We also sequenced matched mRNA samples from the same timemore » points, using pair-ended directional Illumina RNA-Seq to investigate the dynamics and relationship between the mRNA and protein expression programs of M. pusilla. Similar to a prior study of the marine cyanobacterium Prochlorococcus, we found significant divergence in the mRNA and proteomics expression dynamics in response to the light:dark cycle. Additionally, expressional responses of genes and the proteins they encoded could also be variable within the same metabolic pathway, such as the oxygenic photosynthesis pathway. A regression framework was used to predict protein levels using both mRNA expression and gene-specific sequence-based features. Several features in the genome sequence were found to influence protein abundance including the codon usage and the length of the 3’ UTR. Collectively, our studies provide insights into the regulation of the proteome over a diel as relationships between the transcriptional and translational programs in the widespread marine green alga Micromonas.« less

Molecular and Cellular Biology Animations: Development and Impact on Student Learning

PubMed Central

2005-01-01

Educators often struggle when teaching cellular and molecular processes because typically they have only two-dimensional tools to teach something that plays out in four dimensions. Learning research has demonstrated that visualizing processes in three dimensions aids learning, and animations are effective visualization tools for novice learners and aid with long-term memory retention. The World Wide Web Instructional Committee at North Dakota State University has used these research results as an inspiration to develop a suite of high-quality animations of molecular and cellular processes. Currently, these animations represent transcription, translation, bacterial gene expression, messenger RNA (mRNA) processing, mRNA splicing, protein transport into an organelle, the electron transport chain, and the use of a biological gradient to drive adenosine triphosphate synthesis. These animations are integrated with an educational module that consists of First Look and Advanced Look components that feature captioned stills from the animation representing the key steps in the processes at varying levels of complexity. These animation-based educational modules are available via the World Wide Web at http://vcell.ndsu.edu/animations. An in-class research experiment demonstrated that student retention of content material was significantly better when students received a lecture coupled with the animations and then used the animation as an individual study activity. PMID:15917875

Betaine Aldehyde Dehydrogenase expression during physiological cardiac hypertrophy induced by pregnancy.

PubMed

Rosas-Rodríguez, Jesús Alfredo; Soñanez-Organis, José Guadalupe; Godoy-Lugo, José Arquimides; Espinoza-Salazar, Juan Alberto; López-Jacobo, Cesar Jeravy; Stephens-Camacho, Norma Aurora; González-Ochoa, Guadalupe

2017-08-26

Betaine Aldehyde Dehydrogenase (betaine aldehyde: NAD(P) + oxidoreductase, (E.C. 1.2.1.8; BADH) catalyze the irreversible oxidation of betaine aldehyde (BA) to glycine betaine (GB) and is essential for polyamine catabolism, γ-aminobutyric acid synthesis, and carnitine biosynthesis. GB is an important osmolyte that regulates the homocysteine levels, contributing to a vascular risk factor reduction. In this sense, distinct investigations describe the physiological roles of GB, but there is a lack of information about the GB novo synthesis process and regulation during cardiac hypertrophy induced by pregnancy. In this work, the BADH mRNA expression, protein level, and activity were quantified in the left ventricle before, during, and after pregnancy. The mRNA expression, protein content and enzyme activity along with GB content of BADH increased 2.41, 1.95 and 1.65-fold respectively during late pregnancy compared to not pregnancy, and returned to basal levels at postpartum. Besides, the GB levels increased 1.53-fold during pregnancy and remain at postpartum. Our results demonstrate that physiological cardiac hypertrophy induced BADH mRNA expression and activity along with GB production, suggesting that BADH participates in the adaptation process of physiological cardiac hypertrophy during pregnancy, according to the described GB role in cellular osmoregulation, osmoprotection and reduction of vascular risk. Copyright © 2017 Elsevier Inc. All rights reserved.

Cell-cell contact regulates gene expression in CDK4-transformed mouse podocytes.

PubMed

Sakairi, Toru; Abe, Yoshifusa; Jat, Parmijit S; Kopp, Jeffrey B

2010-10-01

We transformed mouse podocytes by ectopic expression of cyclin-dependent kinase 4 (CDK4). Compared with podocytes transformed with a thermo-sensitive SV40 large T antigen mutant tsA58U19 (tsT podocytes), podocytes transformed with CDK4 (CDK4 podocytes) exhibited significantly higher expression of nephrin mRNA. Synaptopodin mRNA expression was significantly lower in CDK4 podocytes and in tsT podocytes under growth-permissive conditions (33°C) compared with tsT podocytes under growth-restricted conditions (37°C), which suggests a role for cell cycle arrest in synaptopodin mRNA expression. Confluent CDK4 podocytes showed significantly higher mRNA expression levels for nephrin, synaptopodin, Wilms tumor 1, podocalyxin, and P-cadherin compared with subconfluent cultures. We carried out experiments to clarify roles of various factors in the confluent podocyte cultures; our findings indicate that cell-cell contact promotes expression of five podocyte marker genes studied, that cellular quiescence increases synaptopodin and podocalyxin mRNA expression, and that soluble factors play a role in nephrin mRNA expression. Our findings suggest that CDK4 podocytes are useful tools to study podocyte biology. Furthermore, the role of cell-cell contact in podocyte gene expression may have relevance for podocyte function in vivo.

Polysome Fractionation to Analyze mRNA Distribution Profiles.

PubMed

Panda, Amaresh C; Martindale, Jennifer L; Gorospe, Myriam

2017-02-05

Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al. , 2014a and 2014b; Abdelmohsen et al. , 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ribosomes with a given mRNA. It provides valuable information about the translational status of that mRNA, depending on the number of ribosomes with which they are associated, and identifies mRNAs that are not translated (Panda et al. , 2016). mRNAs associated with many ribosomes form large polysomes that are predicted to be actively translated, while mRNAs associated with few or no ribosomes are expected to be translated poorly if at all. In sum, polysome fractionation analysis allows the direct determination of translation efficiencies at the level of the whole transcriptome as well as individual mRNAs.

Differential stability of host mRNAs in Friend erythroleukemia cells infected with herpes simplex virus type 1

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

Mayman, B.A.; Nishioka, Y.

1985-01-01

The consequences of herpes simplex virus type 1 infection on cellular macromolecules were investigated in Friend erythroleukemia cells. The patterns of protein synthesis, examined by polyacrylamide gel electrophoresis, demonstrated that by 4 h postinfection the synthesis of many host proteins, with the exception of histones, was inhibited. Examination of the steady-state level of histone H3 mRNA by molecular hybridization of total RNA to a cloned mouse histone H3 complementary DNA probe demonstrated that the ratio of histone H3 mRNA to total RNA remained unchanged for the first 4 h postinfection. In contrast, the steady-state levels of globin and actin mRNAsmore » decreased progressively at early intervals postinfection. Studies on RNA synthesis in isolated nuclei demonstrated that the transcription of the histone H3 gene was inhibited to approximately the same extent as that of actin gene. It was concluded that the stabilization of preexisting histone H3 mRNA was responsible for the persistence of H3 mRNA and histone protein synthesis in herpes simplex virus type 1-infected Friend erythroleukemia cells. The possible mechanisms influencing the differential stability of host mRNAs during the course of productive infection with herpes simplex virus type 1 are discussed.« less

Demonstration of variation in chondrocyte activity in different zones of articular cartilage: an assessment of the value of in-situ hybridization.

PubMed

Marles, P J; Hoyland, J A; Parkinson, R; Freemont, A J

1991-04-01

Several methods have been described for investigating chondrocyte metabolism in vitro. In this study, in-situ hybridization (ISH) using an oligonucleotide probe (i.e. a poly-d(T) probe) to detect total messenger RNA (mRNA) in cartilage explants has been compared with radiosulphate and radioleucine uptake studies in an attempt to assess the value of ISH in investigating chondrocyte metabolism. The relative results of the three parameters indicate qualitative similarities in cells in the intermediate, deep and calcified zones but differences in the superficial zone. The relative levels of mRNA and leucine and sulphate uptake in the midzone areas could be construed as indicating that the bulk of cellular activity was directed towards the synthesis of proteoglycans. A similar relation between the three parameters, but at a lower level, was seen in chondrocytes in the calcified zone demonstrating that these cells are viable and biosynthetic. Both quantitative and qualitative differences between the three methods were observed in the superficial chondrocytes regarding the amount of mRNA compared to sulphate and leucine uptake. The results suggest that ISH can detect differences in the amount of mRNA present in chondrocytes in differing zones of cartilage and, like the radioleucine and radiosulphate studies, particularly emphasizes their functional heterogeneity.

Knockdown of FABP5 mRNA decreases cellular cholesterol levels and results in decreased apoB100 secretion and triglyceride accumulation in ARPE-19 cells

PubMed Central

Wu, Tinghuai; Tian, Jane; Cutler, Roy G.; Telljohann, Richard S.; Bernlohr, David; Mattson, Mark P.; Handa, James T.

2010-01-01

To maintain normal retinal function, retinal pigment epithelial (RPE) cells engulf photoreceptor outer segments (ROS) enriched in free fatty acids (FFAs). We have previously demonstrated fatty acid-binding protein 5 (FABP5) down-regulation in the RPE/choroidal complex in a mouse model of aging and early age-related macular degeneration. FABPs are involved in intracellular transport of FFAs and their targeting to specific metabolic pathways. To elucidate the role of FABP5 in lipid metabolism, the production of the FABP5 protein in a human RPE cell line was inhibited using RNA interference technology. As a result, the levels of cholesterol and cholesterol ester were decreased by about 40%, whereas FFAs and triglycerides were increased by 18 and 67% after siRNA treatment, respectively. Some species of phospholipids were decreased in siRNA-treated cells. Cellular lipid droplets were evident and apoB secretion was decreased by 76% in these cells. Additionally, we discovered that ARPE-19 cells could synthesize and secrete Apolipoprotein B100 (apoB100), which may serve as a backbone structure for the formation of lipoprotein particles in these cells. Our results indicate that FABP5 mRNA knockdown results in the accumulation of cellular triglycerides, decreased cholesterol levels, and reduced secretion of apoB100 protein and lipoprotein-like particles. These observations indicated that FABP5 plays a critical role in lipid metabolism in RPE cells, suggesting that FABP5 down-regulation in the RPE/choroid complex in vivo might contribute to aging and early age-related macular degeneration. PMID:19434059

Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis.

PubMed

Qi, Lei; Yue, Lei; Feng, Deqin; Qi, Fengxia; Li, Jie; Dong, Xiuzhu

2017-07-07

Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an 'all-or-none' pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Expression of the cancer-testis antigen BORIS correlates with prostate cancer.

PubMed

Cheema, Zubair; Hari-Gupta, Yukti; Kita, Georgia-Xanthi; Farrar, Dawn; Seddon, Ian; Corr, John; Klenova, Elena

2014-02-01

BORIS, a paralogue of the transcription factor CTCF, is a member of the cancer-testis antigen (CT) family. BORIS is normally present at high levels in the testis; however it is aberrantly expressed in various tumors and cancer cell lines. The main objectives of this study were to investigate BORIS expression together with sub-cellular localization in both prostate cell lines and tumor tissues, and assess correlations between BORIS and clinical/pathological characteristics. We examined BORIS mRNA expression, protein levels and cellular localization in a panel of human prostate tissues, cancer and benign, together with a panel prostate cell lines. We also compared BORIS levels and localization with clinical/pathological characteristics in prostate tumors. BORIS was detected in all inspected prostate cancer cell lines and tumors, but was absent in benign prostatic hyperplasia. Increased levels of BORIS protein positively correlated with Gleason score, T-stage and androgen receptor (AR) protein levels in prostate tumors. The relationship between BORIS and AR was further highlighted in prostate cell lines by the ability of ectopically expressed BORIS to activate the endogenous AR mRNA and protein. BORIS localization in the nucleus plus cytoplasm was also associated with higher BORIS levels and Gleason score. Detection of BORIS in prostate tumors suggests potential applications of BORIS as a biomarker for prostate cancer diagnosis, as an immunotherapy target and, potentially, a prognostic marker of more aggressive prostate cancer. The ability of BORIS to activate the AR gene indicates BORIS involvement in the growth and development of prostate tumors. © 2013 Wiley Periodicals, Inc.

Effects of 1,25(OH)2 D3 and 25(OH)D3 on C2C12 Myoblast Proliferation, Differentiation, and Myotube Hypertrophy.

PubMed

van der Meijden, K; Bravenboer, N; Dirks, N F; Heijboer, A C; den Heijer, M; de Wit, G M J; Offringa, C; Lips, P; Jaspers, R T

2016-11-01

An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)2 D by 1α-hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)2 D3 and 25(OH)D3 affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D3 and 1,25(OH)2 D3 . We show that myoblasts not only responded to 1,25(OH)2 D3 , but also to the precursor 25(OH)D3 by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)2 D3 as well as 25(OH)D3 stimulated VDR mRNA expression and in myotubes 1,25(OH)2 D3 also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D3 metabolism. Although 1α-hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)2 D3 or 25(OH)D3 myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D3 to 24R,25(OH)2 D3 which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D3 metabolites, but is also able to metabolize 25(OH)D3 and 1,25(OH)2 D3 . J. Cell. Physiol. 231: 2517-2528, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

RNA Flow Cytometry Using the Branched DNA Technique.

PubMed

Soh, Kah Teong; Wallace, Paul K

2018-01-01

The systematic modulation of mRNA and proteins governs the complicated and intermingled biological functions of our cells. Traditionally, transcriptomic technologies such as DNA microarray and RNA-Seq have been used to identify, characterize, and profile gene expression data. These are, however, considered bulk methods as they are unable to measure gene expression at the single-cell level, unless the cells are pre-sorted. Branched DNA is a flow cytometry-based detection platform that has been developed recently to measure mRNA at the single-cell level. Originally adapted from microscopy, the current system has been modified to achieve compatibility with the detection of surface and intracellular antigens using monoclonal antibodies conjugated to fluorochromes, thus permitting simultaneous detection of mRNAs and proteins. The Branched DNA method offers a variety of advantages when compared to traditional or standard methods used for the quantification of mRNA, such as (a) the detection of specific mRNA on a per cell basis, (b) an alternate detection tool when the measurement of a protein is technically infeasible (i.e., no quality antibody exists) or the epitope is not assessable, and (c) correlate the analysis of mRNA with protein. Compared to earlier attempts at measuring nucleic acid by flow cytometry, the hybridization temperature applied in the Branched DNA assay is much lower, thus preserving the integrity of cellular structures for further characterization. It also has greatly increased specificity and sensitivity. Here, we provide detailed instruction for performing the Branched DNA method using it in a model system to correlate the expression of CD8 mRNA and CD8 protein by flow cytometry.

Small RNA Profiling of Influenza A Virus-Infected Cells Identifies miR-449b as a Regulator of Histone Deacetylase 1 and Interferon Beta

PubMed Central

Buggele, William A.; Krause, Katherine E.; Horvath, Curt M.

2013-01-01

The mammalian antiviral response relies on the alteration of cellular gene expression, to induce the production of antiviral effectors and regulate their activities. Recent research has indicated that virus infections can induce the accumulation of cellular microRNA (miRNA) species that influence the stability of host mRNAs and their protein products. To determine the potential for miRNA regulation of cellular responses to influenza A virus infection, small RNA profiling was carried out using next generation sequencing. Comparison of miRNA expression profiles in uninfected human A549 cells to cells infected with influenza A virus strains A/Udorn/72 and A/WSN/33, revealed virus-induced changes in miRNA abundance. Gene expression analysis identified mRNA targets for a cohort of highly inducible miRNAs linked to diverse cellular functions. Experiments demonstrate that the histone deacetylase, HDAC1, can be regulated by influenza-inducible miR-449b, resulting in altered mRNA and protein levels. Expression of miR-449b enhances virus and poly(I:C) activation of the IFNβ promoter, a process known to be negatively regulated by HDAC1. These findings demonstrate miRNA induction by influenza A virus infection and elucidate an example of miRNA control of antiviral gene expression in human cells, defining a role for miR-449b in regulation of HDAC1 and antiviral cytokine signaling. PMID:24086750

Bone morphogenetic protein 15 and growth differentiation factor 9 expression in the ovary of European sea bass (Dicentrarchus labrax): cellular localization, developmental profiles, and response to unilateral ovariectomy.

PubMed

García-López, Ángel; Sánchez-Amaya, María Isabel; Halm, Silke; Astola, Antonio; Prat, Francisco

2011-12-01

Vertebrate oocytes actively contribute to follicle development by secreting a variety of growth factors, among which bone morphogenetic protein 15 (BMP15/Bmp15) and growth differentiation factor 9 (GDF9/Gdf9) have been paid particular attention. In the present study, we describe the cellular localization, the developmental profiles, and the response to unilateral ovariectomy (a procedure implying the surgical removal of one of the ovaries) of protein and mRNA steady-state levels of Bmp15 and Gdf9 in the ovary of European sea bass, an important fish species for marine aquaculture industry. In situ hybridization and immunohistochemistry demonstrated that the oocyte is the main production site of Bmp15 and Gdf9 in European sea bass ovary. During oocyte development, Bmp15 protein expression started to be detected only from the lipid vesicle stage onwards but not in primary pre-vitellogenic (i.e. perinucleolar) oocytes as the bmp15 mRNA already did. Gdf9 protein and gdf9 mRNA expression were both detected in primary perinucleolar oocytes and followed similar decreasing patterns thereafter. Unilateral ovariectomy induced a full compensatory growth of the remaining ovary in the 2-month period following surgery (Á. García-López, M.I. Sánchez-Amaya, C.R. Tyler, F. Prat 2011). The compensatory growth elicited different changes in the expression levels of mRNA and protein of both factors, although the involvement of Bmp15 and Gdf9 in the regulatory network orchestrating such process remains unclear at present. Altogether, our results establish a solid base for further studies focused on elucidating the specific functions of Bmp15 and Gdf9 during primary and secondary oocyte growth in European sea bass. Copyright © 2011 Elsevier Inc. All rights reserved.

The RNA binding protein CsrA controls c-di-GMP metabolism by directly regulating the expression of GGDEF proteins

PubMed Central

Jonas, Kristina; Edwards, Adrianne N.; Simm, Roger; Romeo, Tony; Römling, Ute; Melefors, Öjar

2009-01-01

Summary The carbon storage regulator CsrA is an RNA binding protein that controls carbon metabolism, biofilm formation and motility in various eubacteria. Nevertheless, in Escherichia coli only five target mRNAs have been shown to be directly regulated by CsrA at the post-transcriptional level. Here we identified two new direct targets for CsrA, ycdT and ydeH, both of which encode proteins with GGDEF domains. A csrA mutation caused mRNA levels of ycdT and ydeH to increase more than 10-fold. RNA mobility shift assays confirmed the direct and specific binding of CsrA to the mRNA leaders of ydeH and ycdT. Overexpression of ycdT and ydeH resulted in a more than 20-fold increase in the cellular concentration of the second messenger c-di-GMP, implying that both proteins possess diguanylate cyclase activity. Phenotypic characterization revealed that both proteins are involved in the regulation of motility in a c-di-GMP dependent manner. CsrA was also found to regulate the expression of five additional GGDEF/EAL proteins and a csrA mutation led to modestly increased cellular levels of c-di-GMP. All together, these data demonstrate a global role for CsrA in the regulation of c-di-GMP metabolism by regulating the expression of GGDEF proteins at the post-transcriptional level. PMID:18713317

Proteomic Analysis of Zika Virus Infected Primary Human Fetal Neural Progenitors Suggests a Role for Doublecortin in the Pathological Consequences of Infection in the Cortex.

PubMed

Jiang, Xuan; Dong, Xiao; Li, Shi-Hua; Zhou, Yue-Peng; Rayner, Simon; Xia, Hui-Min; Gao, George F; Yuan, Hui; Tang, Ya-Ping; Luo, Min-Hua

2018-01-01

Zika virus (ZIKV) infection is associated with severe neurological defects in fetuses and newborns, such as microcephaly. However, the underlying mechanisms remain to be elucidated. In this study, proteomic analysis on ZIKV-infected primary human fetal neural progenitor cells (NPCs) revealed that virus infection altered levels of cellular proteins involved in NPC proliferation, differentiation and migration. The transcriptional levels of some of the altered targets were also confirmed by qRT-PCR. Among the altered proteins, doublecortin (DCX) plays an important role in NPC differentiation and migration. Results showed that ZIKV infection downregulated DCX, at both mRNA and protein levels, as early as 1 day post infection (1 dpi), and lasted throughout the virus replication cycle (4 days). The downregulation of DCX was also observed in a ZIKV-infected fetal mouse brain model, which displayed decreased body weight, brain size and weight, as well as defective cortex structure. By screening the ten viral proteins of ZIKV, we found that both the expression of NS4A and NS5 were correlated with the downregulation of both mRNA and protein levels of DCX in NPCs. These data suggest that DCX is modulated following infection of the brain by ZIKV. How these observed changes of DCX expression translate in the pathological consequences of ZIKV infection and if other cellular proteins are equally involved remains to be investigated.

Anatomy of ageing face.

PubMed

Ilankovan, V

2014-03-01

Ageing is a biological process that results from changes at a cellular level, particularly modification of mRNA. The face is affected by the same physiological process and results in skeletal, muscular, and cutaneous ageing; ligamentous attenuation, descent of fat, and ageing of the appendages. I describe these changes on a structural and clinical basis and summarise possible solutions for a rejuvenation surgeon. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Rig-I regulates NF-κB activity through binding to Nf-κb1 3′-UTR mRNA

PubMed Central

Zhang, Hong-Xin; Liu, Zi-Xing; Sun, Yue-Ping; Lu, Shun-Yuan; Liu, Xue-Song; Huang, Qiu-Hua; Xie, Yin-Yin; Dang, Su-Ying; Zheng, Guang-Yong; Li, Yi-Xue; Kuang, Ying; Fei, Jian; Chen, Zhu; Wang, Zhu-Gang

2013-01-01

Retinoic acid inducible gene I (RIG-I) senses viral RNAs and triggers innate antiviral responses through induction of type I IFNs and inflammatory cytokines. However, whether RIG-I interacts with host cellular RNA remains undetermined. Here we report that Rig-I interacts with multiple cellular mRNAs, especially Nf-κb1. Rig-I is required for NF-κB activity via regulating Nf-κb1 expression at posttranscriptional levels. It interacts with the multiple binding sites within 3′-UTR of Nf-κb1 mRNA. Further analyses reveal that three distinct tandem motifs enriched in the 3′-UTR fragments can be recognized by Rig-I. The 3′-UTR binding with Rig-I plays a critical role in normal translation of Nf-κb1 by recruiting the ribosomal proteins [ribosomal protein L13 (Rpl13) and Rpl8] and rRNAs (18S and 28S). Down-regulation of Rig-I or Rpl13 significantly reduces Nf-κb1 and 3′-UTR–mediated luciferase expression levels. These findings indicate that Rig-I functions as a positive regulator for NF-κB signaling and is involved in multiple biological processes in addition to host antivirus immunity. PMID:23553835

Bioinspired Nanocomplex for Spatiotemporal Imaging of Sequential mRNA Expression in Differentiating Neural Stem Cells

PubMed Central

2015-01-01

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions. PMID:25494492

Bioinspired nanocomplex for spatiotemporal imaging of sequential mRNA expression in differentiating neural stem cells.

PubMed

Wang, Zhe; Zhang, Ruili; Wang, Zhongliang; Wang, He-Fang; Wang, Yu; Zhao, Jun; Wang, Fu; Li, Weitao; Niu, Gang; Kiesewetter, Dale O; Chen, Xiaoyuan

2014-12-23

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions.

The importance of mRNA structure in determining the pathogenicity of synonymous and non-synonymous mutations in haemophilia

PubMed Central

Hamasaki-Katagiri, Nobuko; Lin, Brian C.; Simon, Jonathan; Hunt, Ryan C.; Schiller, Tal; Russek-Cohen, Estelle; Komar, Anton A.; Bar, Haim; Kimchi-Sarfaty, Chava

2016-01-01

Introduction Mutational analysis is commonly used to support the diagnosis and management of haemophilia. This has allowed for the generation of large mutation databases which provide unparalleled insight into genotype-phenotype relationships. Haemophilia is associated with inversions, deletions, insertions, nonsense and missense mutations. Both synonymous and non-synonymous mutations influence the base pairing of messenger RNA (mRNA), which can alter mRNA structure, cellular half-life and ribosome processivity/elongation. However, the role of mRNA structure in determining the pathogenicity of point mutations in haemophilia has not been evaluated. Aim To evaluate mRNA thermodynamic stability and associated RNA prediction software as a means to distinguish between neutral and disease-associated mutations in haemophilia. Methods Five mRNA structure prediction software programs were used to assess the thermodynamic stability of mRNA fragments carrying neutral vs. disease-associated and synonymous vs. non-synonymous point mutations in F8, F9 and a third X-linked gene, DMD (dystrophin). Results In F8 and DMD, disease-associated mutations tend to occur in more structurally stable mRNA regions, represented by lower MFE (minimum free energy) levels. In comparing multiple software packages for mRNA structure prediction, a 101–151 nucleotide fragment length appears to be a feasible range for structuring future studies. Conclusion mRNA thermodynamic stability is one predictive characteristic, which when combined with other RNA and protein features, may offer significant insight when screening sequencing data for novel disease-associated mutations. Our results also suggest potential utility in evaluating the mRNA thermodynamic stability profile of a gene when determining the viability of interchanging codons for biological and therapeutic applications. PMID:27933712

Heat-resistant variants of the Chinese hamster ovary cell: alteration of cellular structure and expression of vimentin.

PubMed

Lee, Y J; Hou, Z Z; Curetty, L; Armour, E P; al-Saadi, A; Bernstein, J; Corry, P M

1992-04-01

Three heat-resistant mutant cell lines (78-1, 78-2, 78-3) were previously selected from Chinese hamster ovary cells. In this study, we investigated whether the differences in intrinsic thermal sensitivity result from alteration of stress protein levels or cellular structural changes. Although there was no significant difference in the levels of stress proteins, i.e., constitutive HSP70 in wild type and three heat-resistant mutant strains, there were marked differences in the amounts of vimentin among the cell lines. Two-dimensional gel electrophoresis and Western blot showed a 2.3-2.9-fold increase in the level of vimentin in the mutant cells under normal growth conditions. Northern blot also revealed higher amounts of vimentin mRNA in the mutant cells. Electron microscopy and immunofluorescence suggest that increased amounts of the vimentin-containing intermediate filaments are correlated with the heat-resistant phenotypes.

Neuron-derived orphan receptor 1 transduces survival signals in neuronal cells in response to hypoxia-induced apoptotic insults.

PubMed

Chio, Chung-Ching; Wei, Li; Chen, Tyng Guey; Lin, Chien-Min; Shieh, Ja-Ping; Yeh, Poh-Shiow; Chen, Ruei-Ming

2016-06-01

OBJECT Hypoxia can induce cell death or trigger adaptive mechanisms to guarantee cell survival. Neuron-derived orphan receptor 1 (NOR-1) works as an early-response protein in response to a variety of environmental stresses. In this study, the authors evaluated the roles of NOR-1 in hypoxia-induced neuronal insults. METHODS Neuro-2a cells were exposed to oxygen/glucose deprivation (OGD). Cell viability, cell morphology, cas-pase-3 activity, DNA fragmentation, and cell apoptosis were assayed to determine the mechanisms of OGD-induced neuronal insults. RNA and protein analyses were carried out to evaluate the effects of OGD on expressions of NOR-1, cAMP response element-binding (CREB), and cellular inhibitor of apoptosis protein 2 (cIAP2) genes. Translations of these gene expressions were knocked down using RNA interference. Mice subjected to traumatic brain injury (TBI) and NOR-1 was immunodetected. RESULTS Exposure of neuro-2a cells to OGD decreased cell viability in a time-dependent manner. Additionally, OGD led to cell shrinkage, DNA fragmentation, and cell apoptosis. In parallel, treatment of neuro-2a cells with OGD time dependently increased cellular NOR-1 mRNA and protein expressions. Interestingly, administration of TBI also augmented NOR-1 levels in the impacted regions of mice. As to the mechanism, exposure to OGD increased nuclear levels of the transcription factor CREB protein. Downregulating CREB expression using RNA interference simultaneously inhibited OGD-induced NOR-1 mRNA expression. Also, levels of cIAP2 mRNA and protein in neuro-2a cells were augmented by OGD. After reducing cIAP2 translation, OGD-induced cell death was reduced. Sequentially, application of NOR-1 small interfering RNA to neuro-2a cells significantly inhibited OGD-induced cIAP2 mRNA expression and concurrently alleviated hypoxia-induced alterations in cell viability, caspase-3 activation, DNA damage, and cell apoptosis. CONCLUSIONS This study shows that NOR-1 can transduce survival signals in neuronal cells responsible for hypoxiainduced apoptotic insults through activation of a CREB/cIAP2-dependent mechanism.

5-HT2C Receptor Desensitization Moderates Anxiety in 5-HTT Deficient Mice: From Behavioral to Cellular Evidence

PubMed Central

Martin, Cédric BP; Martin, Vincent S.; Trigo, José M.; Chevarin, Caroline; Maldonado, Rafael; Fink, Latham H.; Cunningham, Kathryn A.; Hamon, Michel; Lanfumey, Laurence

2015-01-01

Background: Desensitization and blockade of 5-HT2C receptors (5-HT2CR) have long been thought to be central in the therapeutic action of antidepressant drugs. However, besides behavioral pharmacology studies, there is little in vivo data documenting antidepressant-induced 5-HT2CR desensitization in specific brain areas. Methods: Mice lacking the 5-HT reuptake carrier (5-HTT-/-) were used to model the consequences of chronic 5-HT reuptake inhibition with antidepressant drugs. The effect of this mutation on 5-HT2CR was evaluated at the behavioral (social interaction, novelty-suppressed feeding, and 5-HT2CR–induced hypolocomotion tests), the neurochemical, and the cellular (RT-qPCR, mRNA editing, and c-fos–induced expression) levels. Results: Although 5-HTT-/- mice had an anxiogenic profile in the novelty-suppressed feeding test, they displayed less 5-HT2CR–mediated anxiety in response to the agonist m-chlorophenylpiperazine in the social interaction test. In addition, 5-HT2CR–mediated inhibition of a stress-induced increase in 5-HT turnover, measured in various brain areas, was markedly reduced in 5-HTT-/- mutants. These indices of tolerance to 5-HT2CR stimulation were associated neither with altered levels of 5-HT2CR protein and mRNA nor with changes in pre-mRNA editing in the frontal cortex. However, basal c-fos mRNA production in cells expressing 5-HT2CR was higher in 5-HTT-/- mutants, suggesting an altered basal activity of these cells following sustained 5-HT reuptake carrier inactivation. Furthermore, the increased c-fos mRNA expression in 5-HT2CR–like immune-positive cortical cells observed in wild-type mice treated acutely with the 5-HT2CR agonist RO-60,0175 was absent in 5-HTT-/- mutants. Conclusions: Such blunted responsiveness of the 5-HT2CR system, observed at the cell signaling level, probably contributes to the moderation of the anxiety phenotype in 5-HTT-/- mice. PMID:25522398

Responses of skeletal muscle lipid metabolism in rat gastrocnemius to hypothyroidism and iodothyronine administration: a putative role for FAT/CD36.

PubMed

Lombardi, Assunta; De Matteis, Rita; Moreno, Maria; Napolitano, Laura; Busiello, Rosa Anna; Senese, Rosalba; de Lange, Pieter; Lanni, Antonia; Goglia, Fernando

2012-11-15

Iodothyronines such as triiodothyronine (T(3)) and 3,5-diiodothyronine (T(2)) influence energy expenditure and lipid metabolism. Skeletal muscle contributes significantly to energy homeostasis, and the above iodothyronines are known to act on this tissue. However, little is known about the cellular/molecular events underlying the effects of T(3) and T(2) on skeletal muscle lipid handling. Since FAT/CD36 is involved in the utilization of free fatty acids by skeletal muscle, specifically in their import into that tissue and presumably their oxidation at the mitochondrial level, we hypothesized that related changes in lipid handling and in FAT/CD36 expression and subcellular redistribution would occur due to hypothyroidism and to T(3) or T(2) administration to hypothyroid rats. In gastrocnemius muscles isolated from hypothyroid rats, FAT/CD36 was upregulated (mRNA levels and total tissue, sarcolemmal, and mitochondrial protein levels). Administration of either T(3) or T(2) to hypothyroid rats resulted in 1) little or no change in FAT/CD36 mRNA level, 2) a decreased total FAT/CD36 protein level, and 3) further increases in FAT/CD36 protein level in sarcolemma and mitochondria. Thus, the main effect of each iodothyronine seemed to be exerted at the level of FAT/CD36 cellular distribution. The effect of further increases in FAT/CD36 protein level in sarcolemma and mitochondria was already evident at 1 h after iodothyronine administration. Each iodothyronine increased the mitochondrial fatty acid oxidation rate. However, the mechanisms underlying their rapid effects seem to differ; T(2) and T(3) each induce FAT/CD36 translocation to mitochondria, but only T(2) induces increases in carnitine palmitoyl transferase system activity and in the mitochondrial substrate oxidation rate.

Common variants of OPA1 conferring genetic susceptibility to leprosy in Han Chinese from Southwest China.

PubMed

Xiang, Yang-Lin; Zhang, Deng-Feng; Wang, Dong; Li, Yu-Ye; Yao, Yong-Gang

2015-11-01

Leprosy is an ancient chronic infection caused by Mycobacterium leprae. Onset of leprosy was highly affected by host nutritional condition and energy production, (partially) due to genomic loss and parasitic life style of M. leprae. The optic atrophy 1 (OPA1) gene plays an essential role in mitochondria, which function in cellular energy supply and innate immunity. To investigate the potential involvement of OPA1 in leprosy. We analyzed 7 common genetic variants of OPA1 in 1110 Han Chinese subjects with and without leprosy, followed by mRNA expression profiling and protein-protein interaction (PPI) network analysis. We observed positive associations between OPA1 variants rs9838374 (Pgenotypic=0.003) and rs414237 (Pgenotypic=0.002) with lepromatous leprosy. expression quantitative trait loci (eQTL) analysis showed that the leprosy-related risk allele C of rs414237 is correlated with lower OPA1 mRNA expression level. Indeed, we identified a decrease of OPA1 mRNA expression in both with patients and cellular model of leprosy. In addition, the PPI analysis showed that OPA1 protein was actively involved in the interaction network of M. leprae induced differentially expressed genes. Our results indicated that OPA1 variants confer risk of leprosy and may affect OPA1 expression, mitochondrial function and antimicrobial pathways. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Selective regulation of YB-1 mRNA translation by the mTOR signaling pathway is not mediated by 4E-binding protein.

PubMed

Lyabin, D N; Ovchinnikov, L P

2016-03-02

The Y-box binding protein 1 (YB-1) is a key regulator of gene expression at the level of both translation and transcription. The mode of its action on cellular events depends on its subcellular distribution and the amount in the cell. So far, the regulatory mechanisms of YB-1 synthesis have not been adequately studied. Our previous finding was that selective inhibition of YB-1 mRNA translation was caused by suppression of activity of the mTOR signaling pathway. It was suggested that this event may be mediated by phosphorylation of the 4E-binding protein (4E-BP). Here, we report that 4E-BP alone can only slightly inhibit YB-1 synthesis both in the cell and in vitro, although it essentially decreases binding of the 4F-group translation initiation factors to mRNA. With inhibited mTOR kinase, the level of mRNA binding to the eIF4F-group factors was decreased, while that to 4E-BP1 was increased, as was observed for both mTOR kinase-sensitive mRNAs and those showing low sensitivity. This suggests that selective inhibition of translation of YB-1 mRNA, and probably some other mRNAs as well, by mTOR kinase inhibitors is not mediated by the action of the 4E-binding protein upon functions of the 4F-group translation initiation factors.

The actin-binding protein profilin 2 is a novel regulator of iron homeostasis.

PubMed

Luscieti, Sara; Galy, Bruno; Gutierrez, Lucia; Reinke, Michael; Couso, Jorge; Shvartsman, Maya; Di Pascale, Antonio; Witke, Walter; Hentze, Matthias W; Pilo Boyl, Pietro; Sanchez, Mayka

2017-10-26

Cellular iron homeostasis is controlled by the iron regulatory proteins (IRPs) 1 and 2 that bind cis -regulatory iron-responsive elements (IRE) on target messenger RNAs (mRNA). We identified profilin 2 ( Pfn2 ) mRNA, which encodes an actin-binding protein involved in endocytosis and neurotransmitter release, as a novel IRP-interacting transcript, and studied its role in iron metabolism. A combination of electrophoretic mobility shift assay experiments and bioinformatic analyses led to the identification of an atypical and conserved IRE in the 3' untranslated region of Pfn2 mRNA. Pfn2 mRNA levels were significantly reduced in duodenal samples from mice with intestinal IRP ablation, suggesting that IRPs exert a positive effect on Pfn2 mRNA expression in vivo. Overexpression of Pfn2 in HeLa and Hepa1-6 cells reduced their metabolically active iron pool. Importantly, Pfn2-deficient mice showed iron accumulation in discrete areas of the brain (olfactory bulb, hippocampus, and midbrain) and reduction of the hepatic iron store without anemia. Despite low liver iron levels, hepatic hepcidin expression remained high, likely because of compensatory activation of hepcidin by mild inflammation. Splenic ferroportin was increased probably to sustain hematopoiesis. Overall, our results indicate that Pfn2 expression is controlled by the IRPs in vivo and that Pfn2 contributes to maintaining iron homeostasis in cell lines and mice. © 2017 by The American Society of Hematology.

Update: Mechanisms underlying N6-methyladenosine modification of eukaryotic mRNA

PubMed Central

Wang, Yang; Zhao, Jing Crystal

2016-01-01

Summary Eukaryotic messenger RNA (mRNA) undergoes chemical modification both at the 5′cap [1, 2] and internally [3–14]. Among internal modifications, m6A, by far the most abundant, is present in all eukaryotes examined, including mammals [3–6], flies [15], plants [16, 17] and yeast [18, 19]. m6A modification plays an essential role in diverse biological processes. Over the past few years, our knowledge relevant to establishment and function of this modification has grown rapidly. This review focuses on technologies that have facilitated m6A detection in mRNAs, identification of m6A methylation enzymes and binding proteins, and potential functions of the modification at the molecular level. Regarding m6A function at cellular or organismal levels or in disease, please refer to other recent reviews [20–23]. PMID:27793360

Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA.

PubMed

Kim, Bo-Eun; Choi, Soon Won; Shin, Ji-Hee; Kim, Jae-Jun; Kang, Insung; Lee, Byung-Chul; Lee, Jin Young; Kook, Myoung Geun; Kang, Kyung-Sun

2018-01-01

Neural stem cells (NSCs) are a prominent cell source for understanding neural pathogenesis and for developing therapeutic applications to treat neurodegenerative disease because of their regenerative capacity and multipotency. Recently, a variety of cellular reprogramming technologies have been developed to facilitate in vitro generation of NSCs, called induced NSCs (iNSCs). However, the genetic safety aspects of established virus-based reprogramming methods have been considered, and non-integrating reprogramming methods have been developed. Reprogramming with in vitro transcribed (IVT) mRNA is one of the genetically safe reprogramming methods because exogenous mRNA temporally exists in the cell and is not integrated into the chromosome. Here, we successfully generated expandable iNSCs from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via transfection with IVT mRNA encoding SOX2 (SOX2 mRNA) with properly optimized conditions. We confirmed that generated human UCB-MSC-derived iNSCs (UM-iNSCs) possess characteristics of NSCs, including multipotency and self-renewal capacity. Additionally, we transfected human dermal fibroblasts (HDFs) with SOX2 mRNA. Compared with human embryonic stem cell-derived NSCs, HDFs transfected with SOX2 mRNA exhibited neural reprogramming with similar morphologies and NSC-enriched mRNA levels, but they showed limited proliferation ability. Our results demonstrated that human UCB-MSCs can be used for direct reprogramming into NSCs through transfection with IVT mRNA encoding a single factor, which provides an integration-free reprogramming tool for future therapeutic application.

A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells.

PubMed

Ly, Tony; Ahmad, Yasmeen; Shlien, Adam; Soroka, Dominique; Mills, Allie; Emanuele, Michael J; Stratton, Michael R; Lamond, Angus I

2014-01-01

Technological advances have enabled the analysis of cellular protein and RNA levels with unprecedented depth and sensitivity, allowing for an unbiased re-evaluation of gene regulation during fundamental biological processes. Here, we have chronicled the dynamics of protein and mRNA expression levels across a minimally perturbed cell cycle in human myeloid leukemia cells using centrifugal elutriation combined with mass spectrometry-based proteomics and RNA-Seq, avoiding artificial synchronization procedures. We identify myeloid-specific gene expression and variations in protein abundance, isoform expression and phosphorylation at different cell cycle stages. We dissect the relationship between protein and mRNA levels for both bulk gene expression and for over ∼6000 genes individually across the cell cycle, revealing complex, gene-specific patterns. This data set, one of the deepest surveys to date of gene expression in human cells, is presented in an online, searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/). DOI: http://dx.doi.org/10.7554/eLife.01630.001.

Pellino-1 Protects Periodontal Ligament Stem Cells Against H2O2-Induced Apoptosis via Activation of NF-κB Signaling.

PubMed

Tian, Jiangang; Gu, Liufang; Adams, Andrew; Wang, Xueliang; Huang, Ruizhe

2018-06-02

To determine the protective effects of Pellino-1 against H 2 O 2 -induced apoptosis in periodontal ligament stem cells (PDLSC). We demonstrated that H 2 O 2 decreases PDLSC viability by 40 and 50% with the concentrations of 400 and 500 μM, respectively, with an observed downregulation of Pellino-1 mRNA and protein; we further concluded that overexpression of Pellino-1 significantly lowers 8-hydroxy-2'-deoxyguanosine levels by 10% and upregulates superoxide dismutase 1, glutathione peroxidase levels, and catalase mRNA levels by 200, 40, and 250%, respectively. More importantly, we found that overexpression of Pellino-1 inhibited H 2 O 2 -induced cellular apoptosis through the activation of the NF-κB signaling pathway. Pellino-1 may be critically important for cell survival in the presence of oxidative elements; activation of the NF-κB signaling cascade was required for the overexpression of Pellino-1 to protect the cells from H 2 O 2 -induced apoptosis.

mRNA levels of enzymes and receptors implicated in arachidonic acid metabolism in gliomas.

PubMed

De Armas, Rafael; Durand, Karine; Guillaudeau, Angélique; Weinbreck, Nicolas; Robert, Sandrine; Moreau, Jean-Jacques; Caire, François; Acosta, Gisela; Pebet, Matias; Chaunavel, Alain; Marin, Benoît; Labrousse, François; Denizot, Yves

2010-07-01

Gliomas are tumors of the central nervous system derived from glial cells. They show cellular heterogeneity and lack specific diagnostic markers. Although a possible role for the eicosanoid cascade has been suggested in glioma tumorigenesis, the relationship between enzymes and receptors implicated in arachidonic acid metabolism, with histological tumor type has not yet been determined. Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure and compare transcript levels of enzymes and receptors implicated in both lipoxygenase and cyclooxygenase pathways between oligodendrogliomas, astrocytomas, glioblastomas and mixed oligoastrocytomas. Arachidonic acid metabolism-related enzymes and receptor transcripts (i) were underexpressed in classical oligodendrogliomas compared to astrocytomas and/or glioblastomas, (ii) differed between astrocytomas and glioblastomas and (iii) had an intermediate expression in mixed oligoastrocytomas. mRNA levels of enzymes and receptors implicated both in lipoxygenase and cyclooxygenase pathways differed significantly in gliomas according to the histological type. Copyright 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells

PubMed Central

Ly, Tony; Ahmad, Yasmeen; Shlien, Adam; Soroka, Dominique; Mills, Allie; Emanuele, Michael J; Stratton, Michael R; Lamond, Angus I

2014-01-01

Technological advances have enabled the analysis of cellular protein and RNA levels with unprecedented depth and sensitivity, allowing for an unbiased re-evaluation of gene regulation during fundamental biological processes. Here, we have chronicled the dynamics of protein and mRNA expression levels across a minimally perturbed cell cycle in human myeloid leukemia cells using centrifugal elutriation combined with mass spectrometry-based proteomics and RNA-Seq, avoiding artificial synchronization procedures. We identify myeloid-specific gene expression and variations in protein abundance, isoform expression and phosphorylation at different cell cycle stages. We dissect the relationship between protein and mRNA levels for both bulk gene expression and for over ∼6000 genes individually across the cell cycle, revealing complex, gene-specific patterns. This data set, one of the deepest surveys to date of gene expression in human cells, is presented in an online, searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/). DOI: http://dx.doi.org/10.7554/eLife.01630.001 PMID:24596151

Oxytocin Intranasal Administration Affects Neural Networks Upstream of GNRH Neurons.

PubMed

Salehi, Mohammad Saied; Khazali, Homayoun; Mahmoudi, Fariba; Janahmadi, Mahyar

2017-08-01

The last decade has witnessed a surge in studies on the clinical applications of intranasal oxytocin as a method of enhancing social interaction. However, the molecular and cellular mechanisms underlying its function are not completely understood. Since oxytocin is involved in the regulation of hypothalamic-pituitary-gonadal axis by affecting the gonadotropin-releasing hormone (GNRH) system, the present study addressed whether intranasal application of oxytocin has a role in affecting GNRH expression in the male rat hypothalamus. In addition, we assessed expression of two excitatory (kisspeptin and neurokinin B) and two inhibitory (dynorphin and RFamide-related peptide-3) neuropeptides upstream of GNRH neurons as a possible route to relay oxytocin information. Here, adult male rats received 20, 40, or 80 μg oxytocin intranasally once a day for 10 consecutive days, and then, the posterior (PH) and anterior hypothalamus (AH) dissected for evaluation of target genes. Using qRT-PCR, we found that oxytocin treatment increased Gnrh mRNA levels in both the PH and AH. In addition, oxytocin at its highest dose increased kisspeptin expression in the AH by around 400%, whereas treatments, dose dependently decreased kisspeptin mRNA in the PH. The expression of neurokinin B was increased from the basal levels following the intervention. Furthermore, although intranasal-applied oxytocin decreased hypothalamic RFamide-related peptide-3 mRNA level, the dynorphin mRNA was not affected. These observations are consistent with the hypothesis that applications of intranasal oxytocin can affect the GNRH system.

Avian leukosis virus subgroup J induces its receptor--chNHE1 up-regulation.

PubMed

Feng, Weiguo; Meng, Wei; Cai, Liming; Cui, Xiyao; Pan, Zhifang; Wang, Guihua; Cheng, Ziqiang

2016-04-02

Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus which causes immunosuppression and neoplasia in meat-type and egg-type chickens. ALV-J infects host cells via specific interaction between the viral Env and the cell surface receptor -chicken sodium hydrogen exchanger type 1 (chNHE1). NHE1 involved in altering the cellular pH and playing a critical role in tumorigenesis. However, little is known about the other relationship between ALV-J and chNHE1. In ALV-J infected DF-1 cells, the mRNA level of chNHE1 was up-regulated with time-dependent manner tested by real time PCR, and accordingly, intracellular pH was increased tested by spectrofluorometer. In vivo, the mRNA level of chNHE1 was determined by real time PCR in ALV-J infected experimental chickens and field cases. The result showed that the mRNA level of chNHE1 was up-regulated after virus shedding, especially in continuous viremic shedders (CS group). However, no significant difference was found between non-shedding group (NS group) and control group. In field cases, mRNA level of chNHE1 was positively correlated with increasing ALV-J load in tumor bearing and immune tolerance chickens. Furthermore, immunohistochemistry results showed that the protein expression of chNHE1 was up-regulated in different organs of both experimental chickens and tumor bearing chickens compared with the control. Taken together, we conclude that ALV-J induces chNHE1 up-regulation in viremia and neoplasia chickens.

TREM-1 SNP rs2234246 regulates TREM-1 protein and mRNA levels and is associated with plasma levels of L-selectin

PubMed Central

Aldasoro Arguinano, Alex-Ander; Dadé, Sébastien; Stathopoulou, Maria; Derive, Marc; Coumba Ndiaye, Ndeye; Xie, Ting; Masson, Christine; Gibot, Sébastien

2017-01-01

High levels of TREM-1 are associated with cardiovascular and inflammatory diseases risks and the most recent studies have showed that TREM-1 deletion or blockade is associated with up to 60% reduction of the development of atherosclerosis. So far, it is unknown whether the levels of TREM-1 protein are genetically regulated. Moreover, TREM family receptors have been suggested to regulate the cellular adhesion process. The goal of this study was to investigate whether polymorphisms within TREM-1 are regulating the variants of serum TREM-1 levels and the expression levels of their mRNA. Furthermore, we aimed to point out associations between polymorphisms on TREM-1 and blood levels of selectins. Among the 10 SNPs studied, the minor allele T of rs2234246, was associated with increased sTREM-1 in the discovery population (p-value = 0.003), explaining 33% of its variance, and with increased levels of mRNA (p-value = 0.007). The same allele was associated with increased soluble L-selectin levels (p-value = 0.011). The higher levels of sTREM-1 and L-selectin were confirmed in the replication population (p-value = 0.0007 and p-value = 0.018 respectively). We demonstrated for the first time one SNP on TREM-1, affecting its expression levels. These novel results, support the hypothesis that TREM-1 affects monocytes extravasation and accumulation processes leading to atherogenesis and atherosclerotic plaque progression, possibly through increased inflammation and subsequent higher expression of sL-selectin. PMID:28771614

Identification of Type VI Collagen Synthesizing Cells in Human Diabetic Glomerulosclerosis Using Renal Biopsy Sections

PubMed Central

Razzaque, Mohammed Shawkat; Koji, Takehiko; Harada, Takashi; Taguchi, Takashi

1997-01-01

Although the role of extracellular matrices in the development of glomerulosclerosis has been discussed widely, the cellular origin of type VI collagen in diabetic nephropathy (DN) has remained relatively unexplored. This study reports the distribution and cellular origin of type VI collagen in DN. Type VI collagen‐specific oligonucleotide probes and monoclonal antibody were used to assess the relative expression of mRNA for \\alpha1 (VI) chain and its translated protein in paraffin‐embedded renal biopsy sections of DN. By immunohistochemistry, compared to the control, increased deposition of type VI collagen was noted in the diffuse and nodular lesions of diabetic glomeruli. For cellular localization of type VI collagen mRNA, paraffin‐embedded renal sections of the control and DN were hybridized in situ with digoxigenin (Dig)‐labeled antisense oligo‐DNA probe complementary to a part of \\alpha1 (VI) mRNA. In comparison to the control kidney sections, increased numbers of intraglomerular cells (both mesangial and epithelial cells) were positive for α1 (VI) mRNA in renal biopsy sections of DN. From the results, we conclude that overexpression of type VI collagen by intraglomerular cells with its increased deposition might significantly contribute to the glomerulosclerosis found in DN. PMID:9497854

Protective Effect of Garlic on Cellular Senescence in UVB-Exposed HaCaT Human Keratinocytes.

PubMed

Kim, Hye Kyung

2016-07-29

Ultraviolet (UV) irradiation generates reactive oxygen species (ROS) in the cells, which induces the cellular senescence and photoaging. The present study investigated the protective effects of garlic on photo-damage and cellular senescence in UVB-exposed human keratinocytes, HaCaT cells. An in vitro cell free system was used to examine the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and nitric oxide (NO). The effect of garlic extract on ROS formation, MMP-1 protein and mRNA expressions, cytokines such as interleukin (IL)-1β and IL-6, senescence associated-β-galactosidase (SA-β-gal) activity, and silent information regulator T1 (SIRT1) activity were determined in UVB-irradiated HaCaT cells. Garlic exhibited strong DPPH radical and NO scavenging activity in cell free system exhibiting IC50 values of 2.50 mg/mL and 4.38 mg/mL, respectively. Garlic pretreatment attenuated the production of UVB-induced intracellular ROS. MMP-1 level, which has been known to be induced by ROS, was dramatically elevated by UVB irradiation, and UVB-induced MMP-1 mRNA and protein expressions were significantly reduced by garlic treatment (50 µg/mL) comparable to those of UV-unexposed control cells. UV-induced pro-inflammatory cytokine productions (IL-6 and IL-1β) were significantly inhibited by pretreatment with garlic in a dose-dependent manner. SA-β-gal activity, a classical biomarker of cellular senescence, and SIRT1 activity, which has attracted attention as an anti-aging factor in recent years, were ameliorated by garlic treatment in UV-irradiated HaCaT cells. The present study provides the first evidence of garlic inhibiting UVB-induced photoaging as a result of augmentation of cellular senescence in HaCaT human keratinocytes.

Protective Effect of Garlic on Cellular Senescence in UVB-Exposed HaCaT Human Keratinocytes

PubMed Central

Kim, Hye Kyung

2016-01-01

Ultraviolet (UV) irradiation generates reactive oxygen species (ROS) in the cells, which induces the cellular senescence and photoaging. The present study investigated the protective effects of garlic on photo-damage and cellular senescence in UVB-exposed human keratinocytes, HaCaT cells. An in vitro cell free system was used to examine the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and nitric oxide (NO). The effect of garlic extract on ROS formation, MMP-1 protein and mRNA expressions, cytokines such as interleukin (IL)-1β and IL-6, senescence associated-β-galactosidase (SA-β-gal) activity, and silent information regulator T1 (SIRT1) activity were determined in UVB-irradiated HaCaT cells. Garlic exhibited strong DPPH radical and NO scavenging activity in cell free system exhibiting IC50 values of 2.50 mg/mL and 4.38 mg/mL, respectively. Garlic pretreatment attenuated the production of UVB-induced intracellular ROS. MMP-1 level, which has been known to be induced by ROS, was dramatically elevated by UVB irradiation, and UVB-induced MMP-1 mRNA and protein expressions were significantly reduced by garlic treatment (50 µg/mL) comparable to those of UV-unexposed control cells. UV-induced pro-inflammatory cytokine productions (IL-6 and IL-1β) were significantly inhibited by pretreatment with garlic in a dose-dependent manner. SA-β-gal activity, a classical biomarker of cellular senescence, and SIRT1 activity, which has attracted attention as an anti-aging factor in recent years, were ameliorated by garlic treatment in UV-irradiated HaCaT cells. The present study provides the first evidence of garlic inhibiting UVB-induced photoaging as a result of augmentation of cellular senescence in HaCaT human keratinocytes. PMID:27483310

Early life stress stimulates hippocampal reelin gene expression in a sex-specific manner: evidence for corticosterone-mediated action.

PubMed

Gross, Claus M; Flubacher, Armin; Tinnes, Stefanie; Heyer, Andrea; Scheller, Marie; Herpfer, Inga; Berger, Mathias; Frotscher, Michael; Lieb, Klaus; Haas, Carola A

2012-03-01

Early life stress predisposes to the development of psychiatric disorders. In this context the hippocampal formation is of particular interest, because it is affected by stress on the structural and cognitive level. Since little is known how early life stress is translated on the molecular level, we mimicked early life stress in mouse models and analyzed the expression of the glycoprotein Reelin, a master molecule for development and differentiation of the hippocampus. From postnatal day 1 (P1) to P14, mouse pups were subjected to one of the following treatments: nonhandling (NH), handling (H), maternal separation (MS), and early deprivation (ED) followed by immediate (P15) or delayed (P70) real time RT-PCR analysis of reelin mRNA expression. We show that at P15, reelin mRNA levels were significantly increased in male H and ED groups when compared with the NH group. In contrast, no stress-induced alterations of reelin mRNA expression were found in female animals. This sex difference in stress-mediated stimulation of reelin expression was maintained into adulthood, since at P70 intergroup differences were still found in male, but not in female mice. On the cellular level, however, we did not find any significant differences in cell densities of Reelin-immunolabeled neurons between treatment groups or sexes, but an overall reduction of Reelin-expressing neurons in the adult hippocampus when compared to P15. To address the question whether corticosterone mediates the stress-induced up-regulation of reelin gene expression, we used age-matched hippocampal slice cultures derived from male and female mouse pups. Quantitative determination of mRNA levels revealed that corticosterone treatment significantly up-regulated reelin mRNA expression in male, but not in female hippocampi. Taken together, these results show a sex-specific regulation of reelin gene expression by early life experience, most likely mediated by corticosterone. Copyright © 2010 Wiley Periodicals, Inc.

Glutamatergic and Dopaminergic Neurons in the Mouse Ventral Tegmental Area

PubMed Central

Yamaguchi, Tsuyoshi; Qi, Jia; Wang, Hui-Ling; Zhang, Shiliang; Morales, Marisela

2014-01-01

The ventral tegmental area (VTA) comprises dopamine (DA), GABA and glutamate (Glu) neurons. Some rat VTA Glu neurons, expressing vesicular glutamate transporter 2 (VGluT2), co-express tyrosine hydroxylase (TH). While transgenic mice are now being used in attempts to determine the role of VGluT2/TH neurons in reward and neuronal signaling, such neurons have not been characterized in mouse tissue. By cellular detection of VGluT2-mRNA and TH-immunoreactivity (TH-IR), we determined the cellular expression of VGluT2-mRNA within VTA TH-IR neurons in the mouse. We found that some mouse VGluT2 neurons co-expressed TH-IR, but their frequency was lower than in the rat. To determine whether low expression of TH mRNA or TH-IR accounts for this low frequency, we evaluated VTA cellular co-expression of TH-transcripts and TH-protein. Within the medial aspects of the VTA, some neurons expressed TH mRNA but lacked TH-IR; among them a subset co-expressed VGluT2 mRNA. To determine if lack of VTA TH-IR was due to TH trafficking, we tagged VTA TH neurons by cre-inducible expression of mCherry in TH::Cre mice. By dual immunofluorescence, we detected axons containing mCherry, but lacking TH-IR, in the lateral habenula, indicating that mouse low frequency of VGluT2 mRNA (+)/TH-IR (+) neurons is due to lack of synthesis of TH protein, rather than TH-protein trafficking. In conclusion, VGluT2 neurons are present in the rat and mouse VTA, but they differ in the populations of VGluT2/TH and TH neurons. We reveal that under normal conditions, the translation of TH protein is suppressed in the mouse mesohabenular TH neurons. PMID:25572002

Extraribosomal L13a Is a Specific Innate Immune Factor for Antiviral Defense

PubMed Central

Poddar, Darshana; Basu, Abhijit; Kour, Ravinder; Verbovetskaya, Valentina

2014-01-01

ABSTRACT We report a novel extraribosomal innate immune function of mammalian ribosomal protein L13a, whereby it acts as an antiviral agent. We found that L13a is released from the 60S ribosomal subunit in response to infection by respiratory syncytial virus (RSV), an RNA virus of the Pneumovirus genus and a serious lung pathogen. Unexpectedly, the growth of RSV was highly enhanced in L13a-knocked-down cells of various lineages as well as in L13a knockout macrophages from mice. In all L13a-deficient cells tested, translation of RSV matrix (M) protein was specifically stimulated, as judged by a greater abundance of M protein and greater association of the M mRNA with polyribosomes, while general translation was unaffected. In silico RNA folding analysis and translational reporter assays revealed a putative hairpin in the 3′untranslated region (UTR) of M mRNA with significant structural similarity to the cellular GAIT (gamma-activated inhibitor of translation) RNA hairpin, previously shown to be responsible for assembling a large, L13a-containing ribonucleoprotein complex that promoted translational silencing in gamma interferon (IFN-γ)-activated myeloid cells. However, RNA-protein interaction studies revealed that this complex, which we named VAIT (respiratory syncytial virus-activated inhibitor of translation) is functionally different from the GAIT complex. VAIT is the first report of an extraribosomal L13a-mediated, IFN-γ-independent innate antiviral complex triggered in response to virus infection. We provide a model in which the VAIT complex strongly hinders RSV replication by inhibiting the translation of the rate-limiting viral M protein, which is a new paradigm in antiviral defense. IMPORTANCE The innate immune mechanisms of host cells are diverse in nature and act as a broad-spectrum cellular defense against viruses. Here, we report a novel innate immune mechanism functioning against respiratory syncytial virus (RSV), in which the cellular ribosomal protein L13a is released from the large ribosomal subunit soon after infection and inhibits the translation of a specific viral mRNA, namely, that of the matrix protein M. Regarding its mechanism, we show that the recognition of a specific secondary structure in the 3′ untranslated region of the M mRNA leads to translational arrest of the mRNA. We also show that the level of M protein in the infected cell is rate limiting for viral morphogenesis, providing a rationale for L13a to target the M mRNA for suppression of RSV growth. Translational silencing of a viral mRNA by a deployed ribosomal protein is a new paradigm in innate immunity. PMID:24899178

Extraribosomal l13a is a specific innate immune factor for antiviral defense.

PubMed

Mazumder, Barsanjit; Poddar, Darshana; Basu, Abhijit; Kour, Ravinder; Verbovetskaya, Valentina; Barik, Sailen

2014-08-01

We report a novel extraribosomal innate immune function of mammalian ribosomal protein L13a, whereby it acts as an antiviral agent. We found that L13a is released from the 60S ribosomal subunit in response to infection by respiratory syncytial virus (RSV), an RNA virus of the Pneumovirus genus and a serious lung pathogen. Unexpectedly, the growth of RSV was highly enhanced in L13a-knocked-down cells of various lineages as well as in L13a knockout macrophages from mice. In all L13a-deficient cells tested, translation of RSV matrix (M) protein was specifically stimulated, as judged by a greater abundance of M protein and greater association of the M mRNA with polyribosomes, while general translation was unaffected. In silico RNA folding analysis and translational reporter assays revealed a putative hairpin in the 3'untranslated region (UTR) of M mRNA with significant structural similarity to the cellular GAIT (gamma-activated inhibitor of translation) RNA hairpin, previously shown to be responsible for assembling a large, L13a-containing ribonucleoprotein complex that promoted translational silencing in gamma interferon (IFN-γ)-activated myeloid cells. However, RNA-protein interaction studies revealed that this complex, which we named VAIT (respiratory syncytial virus-activated inhibitor of translation) is functionally different from the GAIT complex. VAIT is the first report of an extraribosomal L13a-mediated, IFN-γ-independent innate antiviral complex triggered in response to virus infection. We provide a model in which the VAIT complex strongly hinders RSV replication by inhibiting the translation of the rate-limiting viral M protein, which is a new paradigm in antiviral defense. The innate immune mechanisms of host cells are diverse in nature and act as a broad-spectrum cellular defense against viruses. Here, we report a novel innate immune mechanism functioning against respiratory syncytial virus (RSV), in which the cellular ribosomal protein L13a is released from the large ribosomal subunit soon after infection and inhibits the translation of a specific viral mRNA, namely, that of the matrix protein M. Regarding its mechanism, we show that the recognition of a specific secondary structure in the 3' untranslated region of the M mRNA leads to translational arrest of the mRNA. We also show that the level of M protein in the infected cell is rate limiting for viral morphogenesis, providing a rationale for L13a to target the M mRNA for suppression of RSV growth. Translational silencing of a viral mRNA by a deployed ribosomal protein is a new paradigm in innate immunity. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Methylglyoxal, a reactive glucose metabolite, increases renin angiotensin aldosterone and blood pressure in male Sprague-Dawley rats.

PubMed

Dhar, Indu; Dhar, Arti; Wu, Lingyun; Desai, Kaushik M

2014-03-01

The majority of people with diabetes develop hypertension along with increased activity of the renin-angiotensin system. Methylglyoxal, a reactive glucose metabolite, is elevated in diabetic patients. We investigated the effects of methylglyoxal on the renin-angiotensin system and blood pressure. Male Sprague-Dawley rats were treated with a continuous infusion of methylglyoxal with a minipump for 4 weeks. Organs/tissues and cultured vascular smooth muscle cells (VSMCs) were used for molecular studies. High-performance liquid chromatography, Western blotting, and quantitative real-time polymerase chain reaction were used to measure methylglyoxal, proteins, and mRNA, respectively. Small interfering RNA for angiotensinogen and the receptor for advanced glycation endproducts (RAGE) were used to study mechanisms. Methylglyoxal-treated rats developed a significant increase in blood pressure and plasma levels of aldosterone, renin, angiotensin, and catecholamines. Methylglyoxal level and protein and mRNA for angiotensin, AT1 receptor, adrenergic α1D receptor, and renin were significantly increased in the aorta and/or kidney of methylglyoxal-treated rats, a novel finding. Alagebrium attenuated the above effects of methylgloyxal. Treatment of cultured VSMCs with methylglyoxal or high glucose (25 mM) significantly increased cellular methylglyoxal and protein and mRNA for nuclear factor kappa B (NF-κB), angiotensin, AT1 receptor, and α1D receptor, which were prevented by inhibition of NF-κB, and by alagebrium. Silencing of mRNA for RAGE prevented the increase in NF-kB induced by methylglyoxal. Silencing of mRNA for angiotensinogen prevented the increase in NF-κB, angiotensin, AT1 receptor, and α1D receptor. Methylglyoxal activates NF-κB through RAGE and thereby increases renin-angiotensin levels, a novel finding, and a probable mechanism of increase in blood pressure.

Quantitation of CYP1A1 and 1B1 mRNA in polycyclic aromatic hydrocarbon-treated human T-47D and HepG2 cells by a modified bDNA assay using fluorescence detection.

PubMed

Wu, Susan J; Spink, David C; Spink, Barbara C; Kaminsky, Laurence S

2003-01-15

The quantitation of mRNA, essential for assessing mechanisms of enzyme regulation, is normally carried out using reverse transcriptase-polymerase chain reaction (RT-PCR). An alternative method uses a signal-amplification nucleic acid probe assay, which measures RNA directly by the QuantiGene Expression Kit and incorporates branched DNA technology from Bayer and luminometer-based readings of a chemilumigenic alkaline phosphatase substrate. To broaden the utility of this assay, we investigated substitution of a fluorescent substrate, 2'-(2-benzothiazol)-6'-hydroxybenzothiazole phosphate and a fluorometer, and applied the method to quantitation of CYP1A1 and 1B1 mRNA in human T-47D and HepG2 cells following induction by benzo[a]pyrene (B[a]P) and dibenzo[a,h]anthracene (DB[a,h]A). The fluorescence response increased linearly for 200 min without photobleaching and increased linearly (r2=0.997) up to at least 0.2 microg total RNA. The data revealed that at 0.5 and 1.0 microM inducing agent, the induction of CYP1A1 mRNA in HepG2 cells by DB[a,h]A exceeded that by B[a]P by 18- and 6-fold, respectively. In T-47D cells B[a]P induced CYP1A1 mRNA by 23-fold and CYP1B1 mRNA by 3.9-fold. A B[a]P cocontaminant in the environment, arsenite, did not affect B[a]P-induced levels of CYP1A1 or 1B1 mRNA in these cells. The modified analytical system provides a rapid-throughput, reproducible, and less labor-intensive method than RT-PCR for quantifying cellular mRNA levels.

Differential expression of chicken dimerization cofactor of hepatocyte nuclear factor-1 (DcoH) and its novel counterpart, DcoHalpha.

PubMed Central

Kim, H; You, S; Foster, L K; Farris, J; Choi, Y J; Foster, D N

2001-01-01

We have used differential display PCR to study altered gene expression in immortalized chicken embryo fibroblasts (CEFs) that have been established in our laboratory. This technique resulted in the cloning of a novel counterpart of the previously cloned chicken dimerization cofactor of hepatocyte nuclear factor (HNF)-1 (cDcoH), which was identified as cDcoHalpha. The steady-state mRNA levels of cDcoHalpha were up-regulated in all immortal CEFs tested compared with primary CEF cells. cDcoH and cDcoHalpha showed opposite patterns of mRNA expression due to differential regulation of transcription rates, but not mRNA half-lives, in primary and immortal CEFs. Expression of cDcoHalpha increased in the late G1 and early S phases of the cell cycle, while cDcoH mRNA increased in the late S and G2/M phases. In contrast with consistent expression of both genes in primary quiescent cells, cDcoH mRNA, but not cDcoHalpha mRNA, was dramatically decreased in primary senescent cells. The highest levels of cDcoHalpha mRNA were found in the kidney, liver, heart and ovarian follicles, while the major tissues expressing cDcoH were hypothalamus, kidney and liver. cDcoH and cDcoHalpha probes did not cross-hybridize to human hepatocyte mRNA. When transfected into human HepG2 cells, both cDcoH and cDcoHalpha showed similar functional activity as measured by increased expression of a reporter gene, as well as alpha-fetoprotein and albumin genes that both contain HNF-1 binding elements in their promoters. Our results suggest that the novel chicken DcoHalpha might function as a transcriptional cofactor for HNF-1 in specific cellular-environmental states. PMID:11237869

Effect of patchouli alcohol on the regulation of heat shock-induced oxidative stress in IEC-6 cells.

PubMed

Liu, Xiaoxi; Jiang, Linshu; Liu, Fenghua; Chen, Yuping; Xu, Lei; Li, Deyin; Ma, Yunfei; Li, Huanrong; Xu, Jianqin

2016-08-01

Purpose Patchouli alcohol (PA) is used to treat gastrointestinal dysfunction. The purpose of this study was to ascertain the function of PA in the regulated process of oxidative stress in rat intestinal epithelial cells (IEC-6). Materials and methods Oxidative stress was stimulated by exposing IEC-6 cells to heat shock (42 °C for 3 h). IEC-6 cells in treatment groups were pretreated with various concentrations of PA (10, 40, and 80 ng/mL) for 3 h before heat shock. Results Heat shock caused damage to the morphology of IEC-6 cells, and increased reactive oxygen species (ROS) level and malondialdehyde (MDA) content. Moreover, mRNA and protein expression by target genes related to oxidative stress in heat shock were also altered. Specifically, the mRNA expression by HSP70, HSP90, GSH-px, NRF2 nd HO-1were all increased, and Nrf2 and Keap1 protein expression were increased after heat shock. However, pretreatment with PA weakened the level of damage to the cellular morphology, and decreased the MDA content caused by heat shock, indicating PA had cytoprotective activities. Pretreatment with PA at high dose significantly increased generation of intracellular ROS. Compared with the heat shock group alone, PA pretreatment significantly decreased the mRNA expression by HSP70, HSP90, SOD, CAT, GSH-px, KEAP1 and HO-1. Furthermore, the high dose of PA significantly increased Nrf2 protein expression, while both the intermediate and high dose of PA significantly increased HO-1 protein expression. Conclusion Heat-shock-induced oxidative stress in IEC-6 cells, and PA could alleviate the Nrf2-Keap1 cellular oxidative stress responses.

Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A challenging correlation.

PubMed

Krawczenko, Agnieszka; Bielawska-Pohl, Aleksandra; Wojtowicz, Karolina; Jura, Roksana; Paprocka, Maria; Wojdat, Elżbieta; Kozłowska, Urszula; Klimczak, Aleksandra; Grillon, Catherine; Kieda, Claudine; Duś, Danuta

2017-01-01

Active cellular transporters of harmful agents-multidrug resistance (mdr) proteins-are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins-MDR1, BCRP, MRP1, MRP4 and MRP5-in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific.

S-Enantiomer of 19-Hydroxyeicosatetraenoic Acid Preferentially Protects Against Angiotensin II-Induced Cardiac Hypertrophy.

PubMed

Shoieb, Sherif M; El-Kadi, Ayman O S

2018-06-07

We have recently demonstrated that the racemic mixture of 19-hydroxyeicosatetraenoic acid (19-HETE) protects against angiotensin II (Ang II) induced cardiac hypertrophy. Therefore, the purpose of this study was to investigate whether R- or S-enantiomer of 19-HETE confers cardioprotection against Ang II-induced cellular hypertrophy in RL-14 and H9c2 cells. Both cell lines were treated with vehicle or 10 μM Ang II in the absence and presence of 20 &#[mu]M 19(R)-HETE or 19(S)-HETE for 24 h. Thereafter, the level of mid-chain HETEs was determined using liquid chromatography - mass spectrometry (LC/MS). Gene and protein expression levels were measured using real-time PCR and Western blot analysis, respectively. The results showed that both 19(R)-HETE and 19(S)-HETE significantly decreased the metabolite formation rate of midchain HETEs namely 8-, 9-, 12- and 15-HETE compared to control group while the level of 5-HETE was selectively decreased by S-enantiomer. Moreover, both 19(R)-HETE and 19(S)-HETE significantly inhibited the catalytic activity of CYP1B1 and decreased the protein expression of 5- and 12-lipoxxygenase (LOX) as well as cyclooxygenase-2 (COX-2). Notably, the decrease in 15-LOX protein expression was only mediated by 19(S)-HETE. Moreover, both enantiomers protected against Ang II-induced cellular hypertrophy as evidenced by a significant decrease in mRNA expression of β/α-myosin heavy chain ratio, ANP, IL-6 and IL-8. Our data demonstrated that S-enantiomer of 19-HETE preferentially protected against Ang II-induced cellular hypertrophy via decreasing the level of mid-chain HETEs, inhibiting catalytic activity of CYP1B1, decreasing protein expression of LOX and COX-2 enzymes and decreasing mRNA expression of IL-6 and IL-8. The American Society for Pharmacology and Experimental Therapeutics.

One-Week Exposure to a Free-Choice High-Fat High-Sugar Diet Does Not Interfere With the Lipopolysaccharide-Induced Acute Phase Response in the Hypothalamus of Male Rats.

PubMed

Belegri, Evita; Eggels, Leslie; la Fleur, Susanne E; Boelen, Anita

2018-01-01

Obesity has been associated with increased susceptibility to infection in humans and rodents. Obesity is also associated with low-grade hypothalamic inflammation that depends not only on body weight but also on diet. In the present study, we investigated if the bacterial endotoxin [lipopolysaccharide (LPS)]-induced acute phase response is aggravated in rats on a 1-week free-choice high-fat high-sugar (fcHFHS) diet and explained by diet-induced hypothalamic inflammation. Male Wistar rats were on an fcHFHS diet or chow for 1 week and afterwards intraperitoneally injected with LPS or saline. Hypothalamic inflammatory intermediates and plasma cytokines were measured after LPS. Both LPS and the fcHFHS diet altered hypothalamic Nfkbia mRNA and nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha (NFKBIA) protein levels, whereas Il1 β, Il6 , and Tnf α mRNA expression was solely induced upon LPS. We observed an interaction in hypothalamic Nfkbia and suppressor of cytokine signaling (SOCS) 3 mRNA upon LPS; both were higher in rats on a fcHFHS diet compared with chow animals. Despite this, plasma cytokine levels between fcHFHS diet-fed and chow-fed rats were similar after LPS administration. Consuming a fcHFHS diet but not LPS injections increased hypothalamic Atf4 (a cellular stress marker) mRNA expression, whereas Tlr4 mRNA was decreased only upon LPS. Our study does not support a role for diet-induced mild hypothalamic inflammation in the increased susceptibility to infection despite altered Nfkbia and Socs3 mRNA expression after the diet. Additional factors, related to increased fat mass, might be involved.

The regulation of mitochondrial transcription factor A (Tfam) expression during skeletal muscle cell differentiation.

PubMed

Collu-Marchese, Melania; Shuen, Michael; Pauly, Marion; Saleem, Ayesha; Hood, David A

2015-05-19

The ATP demand required for muscle development is accommodated by elevations in mitochondrial biogenesis, through the co-ordinated activities of the nuclear and mitochondrial genomes. The most important transcriptional activator of the mitochondrial genome is mitochondrial transcription factor A (Tfam); however, the regulation of Tfam expression during muscle differentiation is not known. Thus, we measured Tfam mRNA levels, mRNA stability, protein expression and localization and Tfam transcription during the progression of muscle differentiation. Parallel 2-fold increases in Tfam protein and mRNA were observed, corresponding with 2-3-fold increases in mitochondrial content. Transcriptional activity of a 2051 bp promoter increased during this differentiation period and this was accompanied by a 3-fold greater Tfam mRNA stabilization. Interestingly, truncations of the promoter at 1706 bp, 978 bp and 393 bp promoter all exhibited 2-3-fold higher transcriptional activity than the 2051 bp construct, indicating the presence of negative regulatory elements within the distal 350 bp of the promoter. Activation of AMP kinase augmented Tfam transcription within the proximal promoter, suggesting the presence of binding sites for transcription factors that are responsive to cellular energy state. During differentiation, the accumulating Tfam protein was progressively distributed to the mitochondrial matrix where it augmented the expression of mtDNA and COX (cytochrome c oxidase) subunit I, an mtDNA gene product. Our data suggest that, during muscle differentiation, Tfam protein levels are regulated by the availability of Tfam mRNA, which is controlled by both transcription and mRNA stability. Changes in energy state and Tfam localization also affect Tfam expression and action in differentiating myotubes. © 2015 Authors.

CORSEN, a new software dedicated to microscope-based 3D distance measurements: mRNA-mitochondria distance, from single-cell to population analyses.

PubMed

Jourdren, Laurent; Delaveau, Thierry; Marquenet, Emelie; Jacq, Claude; Garcia, Mathilde

2010-07-01

Recent improvements in microscopy technology allow detection of single molecules of RNA, but tools for large-scale automatic analyses of particle distributions are lacking. An increasing number of imaging studies emphasize the importance of mRNA localization in the definition of cell territory or the biogenesis of cell compartments. CORSEN is a new tool dedicated to three-dimensional (3D) distance measurements from imaging experiments especially developed to access the minimal distance between RNA molecules and cellular compartment markers. CORSEN includes a 3D segmentation algorithm allowing the extraction and the characterization of the cellular objects to be processed--surface determination, aggregate decomposition--for minimal distance calculations. CORSEN's main contribution lies in exploratory statistical analysis, cell population characterization, and high-throughput assays that are made possible by the implementation of a batch process analysis. We highlighted CORSEN's utility for the study of relative positions of mRNA molecules and mitochondria: CORSEN clearly discriminates mRNA localized to the vicinity of mitochondria from those that are translated on free cytoplasmic polysomes. Moreover, it quantifies the cell-to-cell variations of mRNA localization and emphasizes the necessity for statistical approaches. This method can be extended to assess the evolution of the distance between specific mRNAs and other cellular structures in different cellular contexts. CORSEN was designed for the biologist community with the concern to provide an easy-to-use and highly flexible tool that can be applied for diverse distance quantification issues.

Sodium 4-phenylbutyrate downregulates HSC70 expression by facilitating mRNA degradation.

PubMed

Rubenstein, R C; Lyons, B M

2001-07-01

Intracellular trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) is repaired by sodium 4-phenylbutyrate (4PBA) by an undetermined mechanism. 4PBA downregulates protein and mRNA expression of the heat shock cognate protein HSC70 (the constitutively expressed member of the 70-kDa heat shock protein family) by approximately 40-50% and decreases formation of a HSC70-DeltaF508 CFTR complex that may be important in the intracellular degradation of DeltaF508 CFTR. We examined the potential mechanisms by which 4PBA decreases HSC70 mRNA and protein expression. In IB3-1 cells, 1 mM 4PBA did not alter the activity of the Chinese hamster ovary HSC70 promoter or of a human HSC70 promoter fragment in luciferase reporter assays nor did it alter HSC70 mRNA synthesis in nuclear runoff assays. In contrast, preincubation with 4PBA increased the rate of HSC70 mRNA degradation by approximately 40%. The initial rate of 35S-HSC70 protein synthesis in 4PBA-treated IB3-1 cells was reduced by approximately 40%, consistent with the steady-state mRNA level, whereas its rate of degradation was unaltered by 4PBA. 4PBA also reduced the steady-state accumulation of (35)S-HSC70 by approximately 40%. These data suggest that 4PBA decreases the expression of HSC70 mRNA and protein by inducing cellular adaptations that result in the decreased stability of HSC70 mRNA.

Multiple-Targeted Graphene-based Nanocarrier for Intracellular Imaging of mRNAs

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

Wang, Ying; Li, Zhaohui; Liu, Misha

Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) mRNA and 2.45 nM for β-actin mRNA. Additionally, this nanosensing platform composed of fluorescent labeledmore » single strand DNA probes and GO nanocarrier can identify Mn-SOD mRNA and endogenous mRNA of β-actin in living cancer cells, showing rapid response, high specificity, nuclease stability, and good biocompatibility during the cell imaging. Thirdly, changes of the expression levels of mRNA in living cells before or after the drug treatment can be monitored successfully. By using multiple ssDNA as probes and GO nanocarrier as the cellular delivery cargo, the proposed simultaneous multiple-targeted sensing platform will be of great potential as a powerful tool for intracellular trafficking process from basic research to clinical diagnosis.« less

Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

PubMed Central

Martins, Rute; Proença, Daniela; Silva, Bruno; Barbosa, Cristina; Silva, Ana Luísa; Faustino, Paula; Romão, Luísa

2012-01-01

Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 3′ untranslated region (UTR), along with exon seven. Therefore, this 3′ UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 3′-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements. PMID:22530027

MicroRNAs and cancer.

PubMed

Cowland, Jack B; Hother, Christoffer; Grønbaek, Kirsten

2007-10-01

MicroRNAs (miRNAs) are a recently discovered group of small RNA molecules involved in the regulation of gene expression. Analogously to mRNAs, the non-protein-encoding pri-miRNAs are synthesized by RNA polymerase II and post-transcriptionally modified by addition of a 5'-cap and a 3'-poly (A) tail. Subsequently, the pri-miRNA undergoes a number of processing steps in the nucleus and cytoplasm, and ends up as a mature approximately 22 nt miRNA, which can exert its function by binding to the 3'-untranslated region of a subset of mRNAs. Binding of the miRNA to the mRNA results in a reduced translation rate and/or increased degradation of the mRNA. In this way a large number of cellular pathways, such as cellular proliferation, differentiation, and apoptosis, are regulated by mi-RNAs. As corruption of these pathways is the hallmark of many cancers, dysregulation of miRNA biogenesis or expression levels may lead to tumorigenesis. The mechanisms that alter the expression of miRNAs are similar to those that change the expression levels of mRNAs of tumor suppressor- and oncogenes, i.e. gross genomic aberrations, epigenetic changes, and minor mutations affecting the expression level, processing, or target-interaction potential of the miRNA. Furthermore, expression profiling of miRNAs has been found to be useful for classification of different tumor types. Taken together, miRNAs can be classified as onco-miRs or tumor suppressor-miRs, and may turn out to be potential targets for cancer therapy.

Asymmetric cell division requires specific mechanisms for adjusting global transcription.

PubMed

Mena, Adriana; Medina, Daniel A; García-Martínez, José; Begley, Victoria; Singh, Abhyudai; Chávez, Sebastián; Muñoz-Centeno, Mari C; Pérez-Ortín, José E

2017-12-01

Most cells divide symmetrically into two approximately identical cells. There are many examples, however, of asymmetric cell division that can generate sibling cell size differences. Whereas physical asymmetric division mechanisms and cell fate consequences have been investigated, the specific problem caused by asymmetric division at the transcription level has not yet been addressed. In symmetrically dividing cells the nascent transcription rate increases in parallel to cell volume to compensate it by keeping the actual mRNA synthesis rate constant. This cannot apply to the yeast Saccharomyces cerevisiae, where this mechanism would provoke a never-ending increasing mRNA synthesis rate in smaller daughter cells. We show here that, contrarily to other eukaryotes with symmetric division, budding yeast keeps the nascent transcription rates of its RNA polymerases constant and increases mRNA stability. This control on RNA pol II-dependent transcription rate is obtained by controlling the cellular concentration of this enzyme. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Altered Markers of Cortical γ-Aminobutyric Acid Neuronal Activity in Schizophrenia

PubMed Central

Kimoto, Sohei; Zaki, Mark M.; Bazmi, H. Holly; Lewis, David A.

2016-01-01

IMPORTANCE In schizophrenia, working memory deficits appear to reflect abnormalities in the generation of gamma oscillations in the dorsolateral prefrontal cortex. The generation of gamma oscillations requires the phasic excitation of inhibitory parvalbumin-containing interneurons. Thus, gamma oscillations depend, in part, on the number of synaptic glutamate receptors on parvalbumin interneurons. However, little is known about the molecular factors that regulate glutamate receptor–mediated excitation of parvalbumin interneurons in schizophrenia. OBJECTIVE To quantify in individuals with schizophrenia the expression of immediate early genes (NARP, ARC, and SGK1) regulating glutamate synaptic neurotransmission. DESIGN, SETTING, AND PARTICIPANTS Postmortem brain specimens (n = 206) were obtained from individuals with schizophrenia, bipolar disorder, or major depressive disorder and from well-matched healthy persons (controls). For a study of brain tissue, quantitative polymerase chain reaction, in situ hybridization, or microarray analyses were used to measure transcript levels in the dorsolateral prefrontal cortex at gray matter, laminar, and cellular levels of resolutions. This study was conducted between January 1, 2013, and November 30, 2014. MAIN OUTCOMES AND MEASURES Expression levels for NARP, ARC, and SGK1 messenger RNA (mRNA) were compared between specimens from individuals with schizophrenia and controls. Diagnostic specificity was assessed by quantifying NARP mRNA levels in specimens from individuals with mood disorders. RESULTS By quantitative polymerase chain reaction, levels of NARP mRNA were significantly lower by 25.6%in specimens from individuals with schizophrenia compared with the controls (mean [SD], 0.036 [0.018] vs 0.049 [0.015]; F1,114 = 21.0; P < .001). Levels of ARC (F1,112 = 0.93; P = .34) and SGK1 (F1,110 = 2.52; P = .12) were not significant. These findings were supported by in situ hybridization (NARP; individuals with schizophrenia vs controls: 40.1% lower [P = .003]) and microarray analyses (NARP; individuals with schizophrenia vs controls: 12.2%lower in layer 3 [P = .11] and 14.6%lower in layer 5 pyramidal cells [P = .001]). In schizophrenia specimens, NARP mRNA levels were positively correlated with GAD67 mRNA (r = 0.55; P < .001); the expression of GAD67 mRNA in parvalbumin interneurons is activity dependent. The NARP mRNA levels were also lower than healthy controls in bipolar disorder (−18.2%; F1,60 = 11.39; P = .001) and major depressive disorder (−21.7%; F1,30 = 5.36; P = .03) specimens, especially those from individuals with psychosis. In all 3 diagnostic groups, NARP mRNA levels were positively correlated (all r ≥ 0.53; all P ≤ .02) with somatostatin mRNA, the expression of which is activity dependent. CONCLUSIONS AND RELEVANCE Given the role of NARP in the formation of excitatory inputs to parvalbumin (and perhaps somatostatin) interneurons, our findings suggest that lower NARP mRNA expression contributes to lower excitatory drive onto parvalbumin interneurons in schizophrenia. This reduced excitatory drive may lead to lower synthesis of γ-aminobutyric acid in these interneurons, contributing to a reduced capacity to generate the gamma oscillations required for working memory. PMID:26038830

Altered Markers of Cortical γ-Aminobutyric Acid Neuronal Activity in Schizophrenia: Role of the NARP Gene.

PubMed

Kimoto, Sohei; Zaki, Mark M; Bazmi, H Holly; Lewis, David A

2015-08-01

In schizophrenia, working memory deficits appear to reflect abnormalities in the generation of gamma oscillations in the dorsolateral prefrontal cortex. The generation of gamma oscillations requires the phasic excitation of inhibitory parvalbumin-containing interneurons. Thus, gamma oscillations depend, in part, on the number of synaptic glutamate receptors on parvalbumin interneurons. However, little is known about the molecular factors that regulate glutamate receptor-mediated excitation of parvalbumin interneurons in schizophrenia. To quantify in individuals with schizophrenia the expression of immediate early genes (NARP, ARC, and SGK1) regulating glutamate synaptic neurotransmission. Postmortem brain specimens (n = 206) were obtained from individuals with schizophrenia, bipolar disorder, or major depressive disorder and from well-matched healthy persons (controls). For a study of brain tissue, quantitative polymerase chain reaction, in situ hybridization, or microarray analyses were used to measure transcript levels in the dorsolateral prefrontal cortex at gray matter, laminar, and cellular levels of resolutions. This study was conducted between January 1, 2013, and November 30, 2014. Expression levels for NARP, ARC, and SGK1 messenger RNA (mRNA) were compared between specimens from individuals with schizophrenia and controls. Diagnostic specificity was assessed by quantifying NARP mRNA levels in specimens from individuals with mood disorders. By quantitative polymerase chain reaction, levels of NARP mRNA were significantly lower by 25.6% in specimens from individuals with schizophrenia compared with the controls (mean [SD], 0.036 [0.018] vs 0.049 [0.015]; F1,114 = 21.0; P < .001). Levels of ARC (F1,112 = 0.93; P = .34) and SGK1 (F1,110 = 2.52; P = .12) were not significant. These findings were supported by in situ hybridization (NARP; individuals with schizophrenia vs controls: 40.1% lower [P = .003]) and microarray analyses (NARP; individuals with schizophrenia vs controls: 12.2% lower in layer 3 [P = .11] and 14.6% lower in layer 5 pyramidal cells [P = .001]). In schizophrenia specimens, NARP mRNA levels were positively correlated with GAD67 mRNA (r = 0.55; P < .001); the expression of GAD67 mRNA in parvalbumin interneurons is activity dependent. The NARP mRNA levels were also lower than healthy controls in bipolar disorder (-18.2%; F1,60 = 11.39; P = .001) and major depressive disorder (-21.7%; F1,30 = 5.36; P = .03) specimens, especially those from individuals with psychosis. In all 3 diagnostic groups, NARP mRNA levels were positively correlated (all r ≥ 0.53; all P ≤ .02) with somatostatin mRNA, the expression of which is activity dependent. Given the role of NARP in the formation of excitatory inputs to parvalbumin (and perhaps somatostatin) interneurons, our findings suggest that lower NARP mRNA expression contributes to lower excitatory drive onto parvalbumin interneurons in schizophrenia. This reduced excitatory drive may lead to lower synthesis of γ-aminobutyric acid in these interneurons, contributing to a reduced capacity to generate the gamma oscillations required for working memory.

Cellular antioxidant effects of atorvastatin in vitro and in vivo.

PubMed

Wassmann, Sven; Laufs, Ulrich; Müller, Kirsten; Konkol, Christian; Ahlbory, Katja; Bäumer, Anselm T; Linz, Wolfgang; Böhm, Michael; Nickenig, Georg

2002-02-01

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may exert direct effects on vascular cells and beneficially influence endothelial dysfunction. Because reactive oxygen species (ROS) may lead to vascular damage and dysfunction, we investigated the effect of atorvastatin on ROS production and the underlying mechanisms in vitro and in vivo. Cultured rat aortic vascular smooth muscle cells were incubated with 10 micromol/L atorvastatin. Angiotensin II-induced and epidermal growth factor-induced ROS production were significantly reduced by atorvastatin (dichlorofluorescein fluorescence laser microscopy). Atorvastatin downregulated mRNA expression of the NAD(P)H oxidase subunit nox1, whereas p22phox mRNA expression was not significantly altered (reverse transcription-polymerase chain reaction, Northern analysis). Membrane translocation of rac1 GTPase, which is required for the activation of NAD(P)H oxidase, was inhibited by atorvastatin (Western blot). mRNA expression of superoxide dismutase isoforms and glutathione peroxidase was not modified by atorvastatin, whereas catalase expression was upregulated at mRNA and protein levels, resulting in an increased enzymatic activity. Effects of atorvastatin on ROS production and nox1, rac1, and catalase expression were inhibited by L-mevalonate but not by 25-hydroxycholesterol. In addition, spontaneously hypertensive rats were treated with atorvastatin for 30 days. ROS production in aortic segments was significantly reduced in statin-treated rats (lucigenin chemiluminescence). Treatment with atorvastatin reduced vascular mRNA expression of p22phox and nox1 and increased aortic catalase expression. mRNA expression of superoxide dismutases, glutathione peroxidase, and NAD(P)H oxidase subunits gp91phox, p40phox, p47phox, and p67phox remained unchanged. Translocation of rac1 from the cytosol to the cell membrane was also reduced in vivo. Thus, atorvastatin exerts cellular antioxidant effects in cultured rat vascular smooth muscle cells and in the vasculature of spontaneously hypertensive rats mediated by decreased expression of essential NAD(P)H oxidase subunits and by upregulation of catalase expression. These effects of atorvastatin may contribute to the vasoprotective effects of statins.

Effect of Negative Pressure Wound Therapy on Cellular Fibronectin and Transforming Growth Factor-β1 Expression in Diabetic Foot Wounds.

PubMed

Yang, Shao Ling; Zhu, Lv Yun; Han, Rui; Sun, Lei Lei; Dou, Jing Tao

2017-08-01

Chronic diabetic foot wounds are a leading cause of amputation, morbidity, and hospitalization for patients with diabetes. Negative-pressure wound therapy (NPWT) can putatively facilitate wound healing, but the underlying mechanisms remain unclear. Cellular fibronectin (cFN) and transforming growth factor-β1 (TGF-β1) play an important role in wound healing. This prospective randomized controlled trial evaluated the effects of NPWT on the production of cFN and the expression of TGF-β1 in diabetic foot wounds of patients. From January 2012 to January 2015, 40 patients with diabetic foot wounds were randomly and equally apportioned to receive either NPWT or advanced moist wound therapy (control) for 7 days. Granulation tissue was harvested before and after treatment. Immunohistochemistry and Western blot were performed to evaluate protein levels of cFN and TGF-β1, and real-time polymerase chain reaction (PCR) to measure corresponding mRNA expressions. NPWT facilitated the expression of cFN and TGF-β1 in diabetic foot wounds. Immunohistochemical analysis revealed higher levels of cFN and TGF-β1 in the NPWT group than in the control group. Western blot and real-time PCR analysis further showed that protein and mRNA levels of cFN or TGF-β1 were higher in the NPWT group than that in the control group ( P < .01, both). Our results showed that NPWT facilitated the production of cFN and the expression of TGF-β1 in granulation tissue in diabetic foot ulcers. Level I, randomized controlled study.

Imbalance in leptin-adiponectin levels and leptin receptor expression as chief contributors to triple negative breast cancer progression in Northeast India.

PubMed

Sultana, Rizwana; Kataki, Amal Ch; Borthakur, Bibhuti Bhusan; Basumatary, Tarun K; Bose, Sujoy

2017-07-20

Triple-Negative breast cancer (TNBC), accounts for a large percentage of breast cancer cases in India including Northeast India. TNBC has an unclear molecular aetiology and hence limited targeted therapies. Human breast is comprised of glandular, ductal, connective, and adipose tissues. Adipose tissue is composed of adipocytes. The adipocytes apart from being energy storage depots, are also active sources of adipocytokines and/or adipokines. The role of adipokines in breast cancer including TNBC has been sporadically documented. Two adipokines in particular, leptin and adiponectin, have come to be recognized for their influence on breast cancer risk and tumour biology. Therefore, the aim of this study was to understand the association of differential expression of critical adipokines and associated cellular mechanism in the susceptibility and severity of TNBC in northeast Indian population. We collected 68 TNBC and 63 controls cases and examined for serum leptin and adiponectin levels using enzyme linked immunosorbent assay (ELISA). Leptin Receptor (Ob-R) mRNA expression was determined by real-time polymerase chain reaction (RT-PCR) assay. Differential Ob-R mRNA expression and correlation with cancer stem cell (CSC) markers was evaluated, and correlated with severity. The serum leptin levels were significantly associated with TNBC severity, while the adiponectin levels were comparative. The serum leptin levels correlated inversely with the adiponetin levels. Serum leptin levels were unaffected with difference in parity. The difference in leptin levels in pre and post menopausal cases were found to be statistically non-significant. Higher leptin levels were also found to be associated obesity, mortality and recurrence. Obesity was found to be a factor for TNBC pathogenesis and severity. Increased Ob-R mRNA expression was associated with TNBC, significantly with TNBC severity, and was significantly higher in obese patients with higher grade TNBC cases. The Ob-R gene mRNA expression was significantly higher in the obese TNBC cases showing recurrence or mortality. The higher Ob-R gene mRNA expression correlated significantly with higher serum leptin levels and lower serum adiponectin levels in TNBC cases. The Ob-R mRNA expression with associated with modulation of CSC oct4 and nanog. In conclusion, the present study is first of its kind on TNBC from northeast India, indicates that adipocytokines does play a role in TNBC pathogenesis. Thus, the understanding of molecular mechanisms of both leptin and adiponectin and their interplay in TNBC offer the prospects for new therapeutic approaches targeting similar signalling pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

The roles of MMP-9/TIMP-1 in cerebral edema following experimental acute cerebral infarction in rats.

PubMed

Li, Dan-Dong; Song, Jin-Ning; Huang, Huan; Guo, Xiao-Ye; An, Ji-Yang; Zhang, Ming; Li, Yu; Sun, Peng; Pang, Hong-Gang; Zhao, Yong-Lin; Wang, Jun-Feng

2013-08-29

Matrix metalloproteinases 9 (MMP-9) and its endogenous inhibitor, tissue inhibitor of metalloproteinases 1 (TIMP-1), regulate homeostasis and turnover of the extra cellular matrix (ECM). They play important roles in acute cerebral infarction (ACI). The contributions of MMP-9 and TIMP-1 to the early stages of ACI are not completely understood. This study investigates the time course of MMP-9 and TIMP-1 and their relations to edema after ACI in rats. Serum concentrations of MMP-9 and TIMP-1 protein were measured using ELISA and mRNA level were measured using real-time PCR. Brain samples were harvested and the brain water content (BWC) was measured. Results revealed that MMP-9 concentration increased fast during the first 12 h after ACI, while after 12 h the increase was much slower. The MMP-9 protein concentration was elevated earlier than the mRNA level. BWC increased starting at 6 h after ACI to reach a peak at 12 h and decreased back to normal levels at 72 h. Both the MMP-9 protein and its mRNA were positively correlated with BWC, however no correlation was found between TIMP-1 levels and BWC. The MMP-9/TIMP-1 protein ratio was more closely correlated with BWC than the MMP-9 concentration. These results indicate that brain edema induced by ACI is associated with increased MMP-9 levels and MMP-9/TIMP-1 ratio in serum. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Distinct expression patterns of the E3 ligase SIAH-1 and its partner Kid/KIF22 in normal tissues and in the breast tumoral processes.

PubMed

Bruzzoni-Giovanelli, Heriberto; Fernandez, Plinio; Veiga, Lucía; Podgorniak, Marie-Pierre; Powell, Darren J; Candeias, Marco M; Mourah, Samia; Calvo, Fabien; Marín, Mónica

2010-02-09

SIAH proteins are the human members of an highly conserved family of E3 ubiquitin ligases. Several data suggest that SIAH proteins may have a role in tumor suppression and apoptosis. Previously, we reported that SIAH-1 induces the degradation of Kid (KIF22), a chromokinesin protein implicated in the normal progression of mitosis and meiosis, by the ubiquitin proteasome pathway. In human breast cancer cells stably transfected with SIAH-1, Kid/KIF22 protein level was markedly reduced whereas, the Kid/KIF22 mRNA level was increased. This interaction has been further elucidated through analyzing SIAH and Kid/KIF22 expression in both paired normal and tumor tissues and cell lines. It was observed that SIAH-1 protein is widely expressed in different normal tissues, and in cells lines but showing some differences in western blotting profiles. Immunofluorescence microscopy shows that the intracellular distribution of SIAH-1 and Kid/KIF22 appears to be modified in human tumor tissues compared to normal controls. When mRNA expression of SIAH-1 and Kid/KIF22 was analyzed by real-time PCR in normal and cancer breast tissues from the same patient, a large variation in the number of mRNA copies was detected between the different samples. In most cases, SIAH-1 mRNA is decreased in tumor tissues compared to their normal counterparts. Interestingly, in all breast tumor tissues analyzed, variations in the Kid/KIF22 mRNA levels mirrored those seen with SIAH-1 mRNAs. This concerted variation of SIAH-1 and Kid/KIF22 messengers suggests the existence of an additional level of control than the previously described protein-protein interaction and protein stability regulation. Our observations also underline the need to re-evaluate the results of gene expression obtained by qRT-PCR and relate it to the protein expression and cellular localization when matched normal and tumoral tissues are analyzed.

Selenium Pretreatment Alleviated LPS-Induced Immunological Stress Via Upregulation of Several Selenoprotein Encoding Genes in Murine RAW264.7 Cells.

PubMed

Wang, Longqiong; Jing, Jinzhong; Yan, Hui; Tang, Jiayong; Jia, Gang; Liu, Guangmang; Chen, Xiaoling; Tian, Gang; Cai, Jingyi; Shang, Haiying; Zhao, Hua

2018-04-18

This study was conducted to profile selenoprotein encoding genes in mouse RAW264.7 cells upon lipopolysaccharide (LPS) challenge and integrate their roles into immunological regulation in response to selenium (Se) pretreatment. LPS was used to develop immunological stress in macrophages. Cells were pretreated with different levels of Se (0, 0.5, 1.0, 1.5, 2.0 μmol Se/L) for 2 h, followed by LPS (100 ng/mL) stimulation for another 3 h. The mRNA expression of 24 selenoprotein encoding genes and 9 inflammation-related genes were investigated. The results showed that LPS (100 ng/mL) effectively induced immunological stress in RAW264.7 cells with induced inflammation cytokines, IL-6 and TNF-α, mRNA expression, and cellular secretion. LPS increased (P < 0.05) mRNA profiles of 9 inflammation-related genes in cells, while short-time Se pretreatment modestly reversed (P < 0.05) the LPS-induced upregulation of 7 genes (COX-2, ICAM-1, IL-1β, IL-6, IL-10, iNOS, and MCP-1) and further increased (P < 0.05) expression of IFN-β and TNF-α in stressed cells. Meanwhile, LPS decreased (P < 0.05) mRNA levels of 18 selenoprotein encoding genes and upregulated mRNA levels of TXNRD1 and TXNRD3 in cells. Se pretreatment recovered (P < 0.05) expression of 3 selenoprotein encoding genes (GPX1, SELENOH, and SELENOW) in a dose-dependent manner and increased (P < 0.05) expression of another 5 selenoprotein encoding genes (SELENOK, SELENOM, SELENOS, SELENOT, and TXNRD2) only at a high level (2.0 μmol Se/L). Taken together, LPS-induced immunological stress in RAW264.7 cells accompanied with the global downregulation of selenoprotein encoding genes and Se pretreatment alleviated immunological stress via upregulation of a subset of selenoprotein encoding genes.

Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

PubMed

Fu, Glenn K; Wilhelmy, Julie; Stern, David; Fan, H Christina; Fodor, Stephen P A

2014-03-18

We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification.

The Anabolic Androgenic Steroid Nandrolone Decanoate Disrupts Redox Homeostasis in Liver, Heart and Kidney of Male Wistar Rats

PubMed Central

Frankenfeld, Stephan P.; Oliveira, Leonardo P.; Ortenzi, Victor H.; Rego-Monteiro, Igor CC.; Chaves, Elen A.; Ferreira, Andrea C.; Leitão, Alvaro C.; Carvalho, Denise P.; Fortunato, Rodrigo S.

2014-01-01

The abuse of anabolic androgenic steroids (AAS) may cause side effects in several tissues. Oxidative stress is linked to the pathophysiology of most of these alterations, being involved in fibrosis, cellular proliferation, tumorigenesis, amongst others. Thus, the aim of this study was to determine the impact of supraphysiological doses of nandrolone decanoate (DECA) on the redox balance of liver, heart and kidney. Wistar male rats were treated with intramuscular injections of vehicle or DECA (1 mg.100 g−1 body weight) once a week for 8 weeks. The activity and mRNA levels of NADPH Oxidase (NOX), and the activity of catalase, glutathione peroxidase (GPx) and total superoxide dismutase (SOD), as well as the reduced thiol and carbonyl residue proteins, were measured in liver, heart and kidney. DECA treatment increased NOX activity in heart and liver, but NOX2 mRNA levels were only increased in heart. Liver catalase and SOD activities were decreased in the DECA-treated group, but only catalase activity was decreased in the kidney. No differences were detected in GPx activity. Thiol residues were decreased in the liver and kidney of treated animals in comparison to the control group, while carbonyl residues were increased in the kidney after the treatment. Taken together, our results show that chronically administered DECA is able to disrupt the cellular redox balance, leading to an oxidative stress state. PMID:25225984

The anabolic androgenic steroid nandrolone decanoate disrupts redox homeostasis in liver, heart and kidney of male Wistar rats.

PubMed

Frankenfeld, Stephan P; Oliveira, Leonardo P; Ortenzi, Victor H; Rego-Monteiro, Igor C C; Chaves, Elen A; Ferreira, Andrea C; Leitão, Alvaro C; Carvalho, Denise P; Fortunato, Rodrigo S

2014-01-01

The abuse of anabolic androgenic steroids (AAS) may cause side effects in several tissues. Oxidative stress is linked to the pathophysiology of most of these alterations, being involved in fibrosis, cellular proliferation, tumorigenesis, amongst others. Thus, the aim of this study was to determine the impact of supraphysiological doses of nandrolone decanoate (DECA) on the redox balance of liver, heart and kidney. Wistar male rats were treated with intramuscular injections of vehicle or DECA (1 mg.100 g(-1) body weight) once a week for 8 weeks. The activity and mRNA levels of NADPH Oxidase (NOX), and the activity of catalase, glutathione peroxidase (GPx) and total superoxide dismutase (SOD), as well as the reduced thiol and carbonyl residue proteins, were measured in liver, heart and kidney. DECA treatment increased NOX activity in heart and liver, but NOX2 mRNA levels were only increased in heart. Liver catalase and SOD activities were decreased in the DECA-treated group, but only catalase activity was decreased in the kidney. No differences were detected in GPx activity. Thiol residues were decreased in the liver and kidney of treated animals in comparison to the control group, while carbonyl residues were increased in the kidney after the treatment. Taken together, our results show that chronically administered DECA is able to disrupt the cellular redox balance, leading to an oxidative stress state.

Cellular mechanisms of renal adaptation of sodium dependent sulfate cotransport to altered dietary sulfate in rats.

PubMed

Sagawa, K; DuBois, D C; Almon, R R; Murer, H; Morris, M E

1998-12-01

The renal transport and fractional reabsorption of inorganic sulfate is altered under conditions of sulfate deficiency or excess. The objective of this study was to examine the cellular mechanisms of adaptation of renal sodium/sulfate cotransport after varying dietary intakes of a sulfur containing amino acid, methionine. Female Lewis rats were divided into four groups and fed diets containing various concentrations of methionine (0, 0.3, 0.82 and 2.46%) for 8 days. Urinary excretion rates and renal clearance of sulfate were significantly decreased in the animals fed a 0% methionine diet or a 0.3% methionine diet, and significantly increased in the animals fed a 2.46% methionine diet when evaluated on days 4 and 7. Serum sulfate concentrations were unchanged by diet treatment in all animals. The fractional reabsorption of sulfate was significantly increased in the animals fed the 0% methionine diet and the 0.3% methionine diets, and decreased in the animals fed the 2.46% methionine diet. Increased mRNA and protein levels for the sodium/sulfate transporter (NaSi-1) were found in the kidney cortex following treatment with the 0 and 0.3% methionine diet groups. Sulfate homeostasis by renal reabsorption is maintained by an up-regulation of steady state levels of NaSi-1 mRNA and protein when the diet is low in methionine.

Double activity imaging reveals distinct cellular targets of haloperidol, clozapine and dopamine D(3) receptor selective RGH-1756.

PubMed

Kovács, K J; Csejtei, M; Laszlovszky, I

2001-03-01

Acute administration of typical (haloperidol) and atypical (clozapine) antipsychotics results in distinct and overlapping regions of immediate-early gene expression in the rat brain. RGH-1756 is a recently developed atypical antipsychotic with high affinity to dopamine D(3) receptors that results in a unique pattern of c-Fos induction. A single injection of either antipsychotic results in c-fos mRNA expression that peaks around 30 min after drug administration, while the maximum of c-Fos protein induction is seen 2 h after challenge. The transient and distinct temporal inducibility of c-fos mRNA and c-Fos protein was exploited to reveal and compare cellular targets of different antipsychotic drugs by concomitant localization of c-fos mRNA and c-Fos immunoreactivity in brain sections of rats that were timely challenged with two different antipsychotics. Double activity imaging revealed that haloperidol, clozapine and RGH-1756 share cellular targets in the nucleus accumbens, where 40% of all labeled neurons displayed both c-fos mRNA and c-Fos protein. Haloperidol activates cells in the caudate putamen, while clozapine-responsive, single labeled neurons were dominant in the prefrontal cortex and major island of Calleja. RGH-1756 targets haloperidol-sensitive cells in the caudate putamen, but cells that are activated by clozapine and RGH-1756 in the major island of Calleja are different.

Derepression of microRNA-mediated protein translation inhibition by apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) and its family members.

PubMed

Huang, Jialing; Liang, Zhihui; Yang, Bin; Tian, Heng; Ma, Jin; Zhang, Hui

2007-11-16

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. A3G interacts with a vast spectrum of RNA-binding proteins and is located in processing bodies and stress granules. However, its cellular function remains to be further clarified. Using a luciferase reporter gene and green fluorescent protein reporter gene, we demonstrate that A3G and other APOBEC family members can counteract the inhibition of protein synthesis by various microRNAs (miRNAs) such as mir-10b, mir-16, mir-25, and let-7a. A3G could also enhance the expression level of miRNA-targeted mRNA. Further, A3G facilitated the association of microRNA-targeted mRNA with polysomes rather than with processing bodies. Intriguingly, experiments with a C288A/C291A A3G mutant indicated that this function of A3G is separable from its cytidine deaminase activity. Our findings suggest that the major cellular function of A3G, in addition to inhibiting the mobility of retrotransposons and replication of endogenous retroviruses, is most likely to prevent the decay of miRNA-targeted mRNA in processing bodies.

Titanium dioxide nanoparticles increase inflammatory responses in vascular endothelial cells

PubMed Central

Han, Sung Gu; Newsome, Bradley; Hennig, Bernhard

2013-01-01

Atherosclerosis is a chronic inflammatory disease that remains the leading cause of death in the United States. Numerous risk factors for endothelial cell inflammation and the development of atherosclerosis have been identified, including inhalation of ultrafine particles. Recently, engineered nanoparticles (NPs) such as titanium (TiO2) NPs have attracted much attention due to their wide range of applications. However, there are also great concerns surrounding potential adverse health effects in vascular systems. Although TiO2 NPs are known to induce oxidative stress and inflammation, the associated signaling pathways have not been well studied. The focus of this work, therefore, deals with examination of the cellular signaling pathways responsible for TiO2 NP-induced endothelial oxidative stress and inflammation. In this study, primary vascular endothelial cells were treated with TiO2 NPs for 2–16 h at concentrations of 0–50 µg/mL. TiO2 NP exposure increased cellular oxidative stress and DNA binding of NF-κB. Further, phosphorylation of Akt, ERK, JNK and p38 was increased in cells exposed to TiO2 NPs. TiO2 NPs also significantly increased induction of mRNA and protein levels of vascular cell adhesion molecule-1 (VCAM-1) and mRNA levels of monocyte chemoattractant protein-1 (MCP-1). Pretreatment with inhibitors for NF-κB (pyrrolidine dithiocarbamate), oxidative stress (epigallocatechin gallate and apocynin), Akt (LY294002), ERK (PD98059), JNK (SP600125) and p38 (SB203580) significantly attenuated TiO2 NP-induced MCP-1 and VCAM-1 gene expression, as well as activation of NF-κB. These data indicate that TiO2 NPs can induce endothelial inflammatory responses via redox-sensitive cellular signaling pathways. PMID:23380242

Molecular cloning of heat shock protein 60 (PtHSP60) from Portunus trituberculatus and its expression response to salinity stress.

PubMed

Xu, Qianghua; Qin, Ye

2012-09-01

Heat shock protein 60 (HSP60) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Portunus trituberculatus is an important marine fishery and aquaculture species, and water salinity condition influenced its artificial propagations significantly. In order to investigate the function of P. trituberculatus HSP60 against osmotic stress, P. trituberculatus HSP60 gene was firstly cloned. The full-length cDNA of PtHSP60 contains 1,743 nucleotides encoding 577 amino acids with a calculated molecular weight of 61.25 kDa. Multiple alignments indicated that the deduced amino acid sequences of PtHSP60 shared a high level of identity with invertebrate and vertebrate HSP60 sequence including shrimp, fruit fly, zebrafish, and human. The expression profiles of PtHSP60 at mRNA and protein levels under salinity treatment were investigated by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. It was found that the mRNA transcripts of PtHSP60 gene varied among different tissues under normal salinity conditions, and the antennal gland showed the highest expression level among the tissues tested. As for low salinity challenge, the mRNA expression of PtHSP60 gene was higher in the gill and appendicular muscle compared with other tissues, and gill and hypodermis represented the higher gene expressions during the hyperosmotic stress, which indicated that those tissues were salinity-sensitive tissues. In addition, salinity challenges significantly altered the expression of PtHSP60 at mRNA and protein level in a salinity- and time-dependent manner in P. trituberculatus gill tissue. The results indicate that PtHSP60 played important roles in mediating the salinity stress in P. trituberculatus.

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in early Xenopus embryos induces cell dissociation and inhibits transition from the blastula to gastrula stage.

PubMed

Shibata, M; Shinga, J; Yasuhiko, Y; Kai, M; Miura, K; Shimogori, T; Kashiwagi, K; Igarashi, K; Shiokawa, K

1998-07-01

Xenopus early embryos contain relatively low levels of S-adenosyl-methionine decarboxylase (SAMDC) and its mRNA. When SAMDC mRNA was injected into Xenopus embryos, it was preserved until the blastula stage and induced a large increase in SAMDC activity. The SAMDC-overexpressed embryos developed normally until the blastula stage but at the early gastrula stage cells which received the mRNA, dissociated autonomously and stopped synthesizing protein. In a hypotonic medium, the dissociated cells, and hence whole embryos, autolyzed. However, in isotonic media dissociated cells did not autolyze, although they did not divide and their DNA and RNA synthesis activity was greatly inhibited. The effects of SAMDC overexpression were abolished by coinjection of ethylglyoxal-bis(guanylhydrazone) (EGBG), a specific inhibitor of SAMDC. In SAMDC-overexpressed embryos the level of putrescine decreased and that of spermidine increased, though to limited extents, resulting in a considerable decrease in the putrescine/spermidine ratio. However, direct injection of spermidine did not mimic the effect of SAMDC overexpression, and putrescine coinjected with SAMDC mRNA to maintain the normal putrescine/spermidine ratio did not rescue the embryos. Conversely, the level of S-adenosylmethionine (SAM) greatly decreased and coinjection of SAM, which restored the level of SAM, rescued the embryos. We concluded that in SAMDC-overexpressed embryos a SAM-deficient state was induced and this caused cell dissociation and inhibition of transition from the blastula to gastrula stage. We suggest that the SAM-deficient embryos obtained in the present study provide a unique system for studying the cellular control mechanism underlying the blastula-gastrula transition.

A Nascent Peptide Signal Responsive to Endogenous Levels of Polyamines Acts to Stimulate Regulatory Frameshifting on Antizyme mRNA.

PubMed

Yordanova, Martina M; Wu, Cheng; Andreev, Dmitry E; Sachs, Matthew S; Atkins, John F

2015-07-17

The protein antizyme is a negative regulator of cellular polyamine concentrations from yeast to mammals. Synthesis of functional antizyme requires programmed +1 ribosomal frameshifting at the 3' end of the first of two partially overlapping ORFs. The frameshift is the sensor and effector in an autoregulatory circuit. Except for Saccharomyces cerevisiae antizyme mRNA, the frameshift site alone only supports low levels of frameshifting. The high levels usually observed depend on the presence of cis-acting stimulatory elements located 5' and 3' of the frameshift site. Antizyme genes from different evolutionary branches have evolved different stimulatory elements. Prior and new multiple alignments of fungal antizyme mRNA sequences from the Agaricomycetes class of Basidiomycota show a distinct pattern of conservation 5' of the frameshift site consistent with a function at the amino acid level. As shown here when tested in Schizosaccharomyces pombe and mammalian HEK293T cells, the 5' part of this conserved sequence acts at the nascent peptide level to stimulate the frameshifting, without involving stalling detectable by toe-printing. However, the peptide is only part of the signal. The 3' part of the stimulator functions largely independently and acts at least mostly at the nucleotide level. When polyamine levels were varied, the stimulatory effect was seen to be especially responsive in the endogenous polyamine concentration range, and this effect may be more general. A conserved RNA secondary structure 3' of the frameshift site has weaker stimulatory and polyamine sensitizing effects on frameshifting. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Low salinity affects cellularity, DNA methylation, and mRNA expression of igf1 in the liver of half smooth tongue sole (Cynoglossus semilaevis).

PubMed

Li, Siping; He, Feng; Wen, Haishen; Li, Jifang; Si, Yufeng; Liu, Mingyuan; Huang, Yajuan; Meng, Lingcai

2017-12-01

Animal growth depends on feedback regulation of hormone levels and environmental conditions. Insulin-like growth factor-1 (Igf1) promotes cell growth and differentiation and represses apoptosis and is highly regulated by the environment. Moreover, animals modify physiological homeostasis under stressful conditions through epigenetics and genetic regulatory mechanisms. Therefore, a comprehensive understanding of the effects of salt on fish growth is needed. In this study, half smooth tongue sole (Cynoglossus semilaevis) were subjected to 15‰ salinity for 0, 7, and 60 days (D) to assess the effects of low salinity on liver cellularity and growth. The results show that low salinity changed liver morphology, suggesting an increase in energy expenditure to recover from the osmotic disruption. igf1 was upregulated in female fish under 15‰ salinity after 7D and may participate in molecular repair. igf1 was downregulated after 60D of salt stress, resulting in retarded growth. Methylation levels were opposite to those of gene expression, suggesting inhibited regulation. Furthermore, three exons in the igf1 gene had significantly different methylation levels in fish under salt stress. Notably, more putative transcription factor binding sites were located in CpG sites at higher methylation levels. igf1 is not a sex-related gene, as no difference in methylation level was detected between males and females in the control group. These results clarify liver damage and changes in DNA methylation and mRNA expression of igf1, providing insight into the adverse effects of low salt on growth of C. semilaevis and the epigenetics and regulatory mechanisms involved in stressful conditions.

Antihypertensive methyldopa, labetalol, hydralazine, and clonidine reversed tumour necrosis factor-α inhibited endothelial nitric oxide synthase expression in endothelial-trophoblast cellular networks.

PubMed

Xu, Bei; Bobek, Gabriele; Makris, Angela; Hennessy, Annemarie

2017-03-01

Medications used to control hypertension in pregnancy also improve trophoblast and endothelial cellular interaction in vitro. Tumour necrosis factor-α (TNF-α) inhibits trophoblast and endothelial cellular interactions and simultaneously decreases endothelial nitric oxide synthase (eNOS) expression. This study investigated whether antihypertensive medications improved these cellular interactions by modulating eNOS and inducible nitric oxide synthase (iNOS) expression. Human uterine myometrial microvascular endothelial cells (UtMVECs) were pre-incubated with (or without) low dose TNF-α (0.5 ng/mL) or TNF-α plus soluble fms-like tyrosine kinase-1 (sFlt-1) (100 ng/mL). The endothelial cells were cultured on Matrigel. After endothelial cellular networks appeared, trophoblast derived HTR-8/SVneo cells were co-cultured in the presence of clinically relevant doses of methyldopa, labetalol, hydralazine or clonidine for 24 hours. Cells were retrieved from the Matrigel to extract mRNA and eNOS and iNOS expression were examined by quantitative PCR. Methyldopa, labetalol, hydralazine and clonidine reversed the inhibitory effect of TNF-α on eNOS mRNA expression. After pre-incubating endothelial cells with TNF-α and sFlt-1, all the medications except methyldopa lost their effect on eNOS mRNA expression. In the absence of TNF-α, antihypertensive medications did not change eNOS expression. The mRNA expression of iNOS was not affected by TNF-α or any medications. This study shows that selected antihypertensive medications used in the treatment of hypertension in pregnancy increase eNOS expression in vitro when induced by the inflammatory TNF-α. The anti-angiogenic molecule sFlt-1 may antagonise the potential benefit of these medications by interfering with the NOS pathway. © 2016 John Wiley & Sons Australia, Ltd.

Fluoride exposure changed the structure and the expressions of HSP related genes in testes of pubertal rats.

PubMed

Zhao, Yangfei; Zhao, Jun; Wang, Jinming; Wang, Jundong

2017-10-01

Previous studies have indicated that fluoride exposure damaged the male reproductive function; however, the cellular mechanism of fluoride-induced testicular toxicity is still unclear. In this study, twenty-two female pregnant Wistar rats were allotted randomly to two groups: control (deionized water) and sodium fluoride (NaF, contain F - : 67.86 mg/L) groups. After delivery, the dosage was continued for 15 weeks for puppies. Twelve rats in each group were tested at 6 and 9 (pubertal); 12 and 15 (mature) weeks of age. Our results suggested that organ coefficient of epididymis was significantly decreased in the mature (12 and 15 week-old) rats. Epididymal sperm abnormality and femur fluoride concentration were increased with the concomitant decrease in sperm motility and concentration in these experimental periods. Compared to the control, in the NaF group, the seminiferous tubules of each age were reduced in terms of diameter and thickness. The sperm cells were lost and shedding and finally disappeared after 9 weeks. mRNA and protein levels of HSP27 and 90 were decreased with a concomitant increase in HSP70 and HSF mRNA and protein levels in NaF exposed rats. The mRNA and protein levels of HSP27 and HSF (only mRNA) were significantly increased in NaF treated rats at 9 and 15 weeks of age, respectively. In summary, these results emphasize that NaF induces testicular and sperm abnormalities through the involvement of HSPs especially during the pubertal period. Copyright © 2017 Elsevier Ltd. All rights reserved.

Differential regulation of duplicate light-dependent protochlorophyllide oxidoreductases in the diatom Phaeodactylum tricornutum

DOE PAGES

Hunsperger, Heather M.; Ford, Christopher J.; Miller, James S.; ...

2016-07-01

Diatoms (Bacilliariophyceae) encode two light-dependent protochlorophyllide oxidoreductases (POR1 and POR2) that catalyze the penultimate step of chlorophyll biosynthesis in the light. Algae live in dynamic environments whose changing light levels induce photoacclimative metabolic shifts, including altered cellular chlorophyll levels. We hypothesized that the two POR proteins may be differentially adaptive under varying light conditions. Using the diatom Phaeodactylum tricornutum as a test system, differences in POR protein abundance and por gene expression were examined when this organism was grown on an alternating light:dark cycles at different irradiances; exposed to continuous light; and challenged by a significant decrease in light availability.more » As a result, for cultures maintained on a 12h light: 12h dark photoperiod at 200μEm –2 s –1 ( 200L/D), both por genes were up-regulated during the light and down-regulated in the dark, though por1 transcript abundance rose and fell earlier than that of por2. Little concordance occurred between por1 mRNA and POR1 protein abundance. In contrast, por2 mRNA and POR2 protein abundances followed similar diurnal patterns. When 200L/D P. tricornutum cultures were transferred to continuous light ( 200L/L), the diurnal regulatory pattern of por1 mRNA abundance but not of por2 was disrupted, and POR1 but not POR2 protein abundance dropped steeply. Under 1200μEm –2 s –1 ( 1200L/D), both por1 mRNA and POR1 protein abundance displayed diurnal oscillations. A compromised diel por2 mRNA response under 1200L/D did not impact the oscillation in POR2 abundance. When cells grown at 1200L/D were then shifted to 50μEm –2 s –1 (50L/D), por1 and por2 mRNA levels decreased swiftly but briefly upon light reduction. Thereafter, POR1 but not POR2 protein levels rose significantly in response to this light stepdown.« less

Urinary bladder organ hypertrophy is partially regulated by Akt1-mediated protein synthesis pathway.

PubMed

Qiao, Li-Ya; Xia, Chunmei; Shen, Shanwei; Lee, Seong Ho; Ratz, Paul H; Fraser, Matthew O; Miner, Amy; Speich, John E; Lysiak, Jeffrey J; Steers, William D

2018-05-15

The present study aims to investigate the role of Akt in the regulation of urinary bladder organ hypertrophy caused by partial bladder outlet obstruction (pBOO). Male rats were surgically induced for pBOO. Real-time PCR and western blot were used to examine the levels of mRNA and protein. A phosphoinositide 3-kinase (PI3K) inhibitor LY294002 was used to inhibit the activity of endogenous Akt. The urinary bladder developed hypertrophy at 2 weeks of pBOO. The protein but not mRNA levels of type I collagen and α-smooth muscle actin (αSMA) were increased in pBOO bladder when compared to sham control. The phosphorylation (activation) levels of Akt1 (p-Ser 473 ), mammalian target of rapamycin (mTOR), p70S6 kinase (p70S6K), and 4E-BP1 were also increased in pBOO bladder. LY294002 treatment reduced the phosphorylation levels of Akt1 and 4E-BP1, and the protein levels of type I collagen and αSMA in pBOO bladder. The mRNA and protein levels of proliferating cell nuclear antigen (PCNA) were increased in pBOO bladder, and PCNA up-regulation occurred in urothelial not muscular layer. LY294002 treatment had no effect on the mRNA and protein levels of PCNA in pBOO bladder. LY294002 treatment partially reduced the bladder weight caused by pBOO. pBOO-induced urinary bladder hypertrophy is attributable to fibrosis, smooth muscle cellular hypertrophy, and urothelium cell hyper-proliferation. Akt1-mediated protein synthesis in pBOO bladder contributes to type I collagen and αSMA but not PCNA up-regulation. Target of Akt1 is necessary but not sufficient in treatment of urinary bladder hypertrophy following pBOO. Copyright © 2018 Elsevier Inc. All rights reserved.

Expression of BMI-1 and Mel-18 in breast tissue - a diagnostic marker in patients with breast cancer

PubMed Central

2010-01-01

Background Polycomb Group (PcG) proteins are epigenetic silencers involved in maintaining cellular identity, and their deregulation can result in cancer. Expression of Mel-18 and Bmi-1 has been studied in tumor tissue, but not in adjacent non-cancerous breast epithelium. Our study compares the expression of the two genes in normal breast epithelium of cancer patients and relates it to the level of expression in the corresponding tumors as well as in breast epithelium of healthy women. Methods A total of 79 tumors, of which 71 malignant tumors of the breast, 6 fibroadenomas, and 2 DCIS were studied and compared to the reduction mammoplastic specimens of 11 healthy women. In addition there was available adjacent cancer free tissue for 23 of the malignant tumors. The tissue samples were stored in RNAlater, RNA was isolated to create expression microarray profile. These two genes were then studied more closely first on mRNA transcription level by microarrays (Agilent 44 K) and quantitative RT-PCR (TaqMan) and then on protein expression level using immunohistochemistry. Results Bmi-1 mRNA is significantly up-regulated in adjacent normal breast tissue in breast cancer patients compared to normal breast tissue from noncancerous patients. Conversely, mRNA transcription level of Mel-18 is lower in normal breast from patients operated for breast cancer compared to breast tissue from mammoplasty. When protein expression of these two genes was evaluated, we observed that most of the epithelial cells were positive for Bmi-1 in both groups of tissue samples, although the expression intensity was stronger in normal tissue from cancer patients compared to mammoplasty tissue samples. Protein expression of Mel-18 showed inversely stronger intensity in tissue samples from mammoplasty compared to normal breast tissue from patients operated for breast cancer. Conclusion Bmi-1 mRNA level is consistently increased and Mel-18 mRNA level is consistently decreased in adjacent normal breast tissue of cancer patients as compared to normal breast tissue in women having had reduction mammoplasties. Bmi-1/Mel-18 ratio can be potentially used as a tool for stratifying women at risk of developing malignancy. PMID:21162745

Expression of fructose-1,6-bisphosphatase mRNA isoforms in normal and basal forebrain cholinergic lesioned rat brain.

PubMed

Löffler, T; Al-Robaiy, S; Bigl, M; Eschrich, K; Schliebs, R

2001-06-01

Fructose-1,6-bisphosphatase is one of the key enzymes in the gluconeogenic pathway predominantly occurring in liver, kidney and muscle. In the brain, fructose-1,6-bisphosphatase has been suggested to be an astrocyte-specific enzyme but the functional importance of glyconeogenesis in the brain is still unclear. To further elucidate the cellular source of fructose-1,6-bisphosphatase in the brain, non-radioactive in situ hybridizations were performed using digoxigenin-labeled RNA probes based on the sequence of recently cloned rat liver and muscle fructose-1,6-bisphosphatase cDNAs. In situ hybridization using a riboprobe for the liver isoform revealed a location of the hybridization signal mainly in neurons, while rat muscle fructose-1,6-bisphosphatase mRNA was detected in both neurons and astrocytes in the hippocampal formation and in layer I of the cerebral cortex.RT-PCR using RNA preparations of rat astrocytes, neurons, and adult whole brain demonstrated a localization of liver fructose-1,6-bisphosphatase mRNA isoform in neurons but not in astrocytes. The muscle fructose-1,6-bisphosphatase mRNA isoform could be detected by RT-PCR in total rat brain, astrocytic, and neuronal mRNA preparations. The isoforms of fructose-1,6-bisphosphatase mRNA seemingly demonstrate a distinct cellular expression pattern in rat brain suggesting a role of glyconeogenesis in both neurons and glial cells.

Modulation by geraniol of gene expression involved in lipid metabolism leading to a reduction of serum-cholesterol and triglyceride levels.

PubMed

Galle, Marianela; Kladniew, Boris Rodenak; Castro, María Agustina; Villegas, Sandra Montero; Lacunza, Ezequiel; Polo, Mónica; de Bravo, Margarita García; Crespo, Rosana

2015-07-15

Geraniol (G) is a natural isoprenoid present in the essential oils of several aromatic plants, with various biochemical and pharmacologic properties. Nevertheless, the mechanisms of action of G on cellular metabolism are largely unknown. We propose that G could be a potential agent for the treatment of hyperlipidemia that could contribute to the prevention of cardiovascular disease. The aim of the present study was to advance our understanding of its mechanism of action on cholesterol and TG metabolism. NIH mice received supplemented diets containing 25, 50, and 75 mmol G/kg chow. After a 3-week treatment, serum total-cholesterol and triglyceride levels were measured by commercial kits and lipid biosynthesis determined by the [(14)C] acetate incorporated into fatty acids plus nonsaponifiable and total hepatic lipids of the mice. The activity of the mRNA encoding HMGCR-the rate-limiting step in cholesterol biosynthesis-along with the enzyme levels and catalysis were assessed by real-time RT-PCR, Western blotting, and HMG-CoA-conversion assays, respectively. In-silico analysis of several genes involved in lipid metabolism and regulated by G in cultured cells was also performed. Finally, the mRNA levels encoded by the genes for the low-density-lipoprotein receptor (LDLR), the sterol-regulatory-element-binding transcription factor (SREBF2), the very-low-density-lipoprotein receptor (VLDLR), and the acetyl-CoA carboxylase (ACACA) were determined by real-time RT-PCR. Plasma total-cholesterol and triglyceride levels plus hepatic fatty-acid, total-lipid, and nonsaponifiable-lipid biosynthesis were significantly reduced by feeding with G. Even though an up-regulation of the mRNA encoding HMGCR occurred in the G treated mouse livers, the protein levels and specific activity of the enzyme were both inhibited. G also enhanced the mRNAs encoding the LDL and VLDL receptors and reduced ACACA mRNA, without altering the transcription of the mRNA encoding the SREBF2. The following mechanisms may have mediated the decrease in plasma lipids levels in mice: a down-regulation of hepatocyte-cholesterol synthesis occurred as a result of decreased HMGCR protein levels and catalytic activity; the levels of LDLR mRNA became elevated, thus suggesting an increase in the uptake of serum LDL, especially by the liver; and TG synthesis became reduced very likely because of a decrease in fatty-acid synthesis. Copyright © 2015 Elsevier GmbH. All rights reserved.

The MicroRNA and MessengerRNA Profile of the RNA-Induced Silencing Complex in Human Primary Astrocyte and Astrocytoma Cells

PubMed Central

Moser, Joanna J.; Fritzler, Marvin J.

2010-01-01

Background GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. Methodology/Principal Findings RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. Conclusions/Significance The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies. PMID:20976148

The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

PubMed

Moser, Joanna J; Fritzler, Marvin J

2010-10-18

GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

Arsenic augments the uptake of oxidized LDL by upregulating the expression of lectin-like oxidized LDL receptor in mouse aortic endothelial cells

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

Hossain, Ekhtear; Ota, Akinobu, E-mail: aota@aichi-med-u.ac.jp; Karnan, Sivasundaram

Although chronic arsenic exposure is a well-known risk factor for cardiovascular diseases, including atherosclerosis, the molecular mechanism underlying arsenic-induced atherosclerosis remains obscure. Therefore, this study aimed to elucidate this molecular mechanism. We examined changes in the mRNA level of the lectin-like oxidized LDL (oxLDL) receptor (LOX-1) in a mouse aortic endothelial cell line, END-D, after sodium arsenite (SA) treatment. SA treatment significantly upregulated LOX-1 mRNA expression; this finding was also verified at the protein expression level. Flow cytometry and fluorescence microscopy analyses showed that the cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with SA treatment. In addition, anmore » anti-LOX-1 antibody completely abrogated the augmented uptake of Dil-oxLDL. We observed that SA increased the levels of the phosphorylated forms of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB)/p65. SA-induced upregulation of LOX-1 protein expression was clearly prevented by treatment with an antioxidant, N-acetylcysteine (NAC), or an NF-κB inhibitor, caffeic acid phenethylester (CAPE). Furthermore, SA-augmented uptake of Dil-oxLDL was also prevented by treatment with NAC or CAPE. Taken together, our results indicate that arsenic upregulates LOX-1 expression through the reactive oxygen species-mediated NF-κB signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of the aberrant LOX-1 signaling pathway in the pathogenesis of arsenic-induced atherosclerosis. - Highlights: • Sodium arsenite (SA) increases LOX-1 expression in mouse aortic endothelial cells. • SA enhances cellular uptake of oxidized LDL in dose-dependent manner. • SA-induced ROS generation enhances phosphorylation of NF-κB. • SA upregulates LOX-1 expression through ROS-activated NF-κB signaling pathway.« less

TNF-alpha, but not IFN-gamma, regulates CCN2 (CTGF), collagen type I, and proliferation in mesangial cells: possible roles in the progression of renal fibrosis.

PubMed

Cooker, Laurinda A; Peterson, Darryl; Rambow, Joann; Riser, Melisa L; Riser, Rebecca E; Najmabadi, Feridoon; Brigstock, David; Riser, Bruce L

2007-07-01

Connective tissue growth factor (CCN2) is a profibrotic factor acting downstream and independently of TGF-beta to mediate renal fibrosis. Although inflammation is often involved in the initiation and/or progression of fibrosis, the role of inflammatory cytokines in regulation of glomerular CCN2 expression, cellular proliferation, and extracellular matrix accumulation is unknown. We studied two such cytokines, TNF-alpha and IFN-gamma, for their effects on cultured mesangial cells in the presence or absence of TGF-beta, as a model for progressive renal fibrosis. Short-term treatment with TNF-alpha, like TGF-beta, significantly increased secreted CCN2 per cell, but unlike TGF-beta inhibited cellular replication. TNF-alpha combined with TGF-beta further increased CCN2 secretion and mRNA levels and reduced proliferation. Surprisingly, however, TNF-alpha treatment decreased baseline collagen type I protein and mRNA levels and largely blocked their stimulation by TGF-beta. Long-term treatment with TGF-beta or TNF-alpha alone no longer increased CCN2 protein levels. However, the combination synergistically increased CCN2. IFN-gamma had no effect on either CCN2 or collagen activity and produced a mild inhibition of TGF-beta-induced collagen only at a high concentration (500 U/ml). In summary, we report a strong positive regulatory role for TNF-alpha, but not IFN-gamma, in CCN2 production and secretion, including that driven by TGF-beta. The stimulation of CCN2 release by TNF-alpha, unlike TGF-beta, is independent of cellular proliferation and not linked to increased collagen type I accumulation. This suggests that the paradigm of TGF-beta-driven CCN2 with subsequent collagen production may be overridden by an as yet undefined inhibitory mechanism acting either directly or indirectly on matrix metabolism.

Hsp27 binding to the 3′UTR of bim mRNA prevents neuronal death during oxidative stress–induced injury: a novel cytoprotective mechanism

PubMed Central

Dávila, David; Jiménez-Mateos, Eva M.; Mooney, Claire M.; Velasco, Guillermo; Henshall, David C.; Prehn, Jochen H. M.

2014-01-01

Neurons face a changeable microenvironment and therefore need mechanisms that allow rapid switch on/off of their cytoprotective and apoptosis-inducing signaling pathways. Cellular mechanisms that control apoptosis activation include the regulation of pro/antiapoptotic mRNAs through their 3′-untranslated region (UTR). This region holds binding elements for RNA-binding proteins, which can control mRNA translation. Here we demonstrate that heat shock protein 27 (Hsp27) prevents oxidative stress–induced cell death in cerebellar granule neurons by specific regulation of the mRNA for the proapoptotic BH3-only protein, Bim. Hsp27 depletion induced by oxidative stress using hydrogen peroxide (H2O2) correlated with bim gene activation and subsequent neuronal death, whereas enhanced Hsp27 expression prevented these. This effect could not be explained by proteasomal degradation of Bim or bim promoter inhibition; however, it was associated with a specific increase in the levels of bim mRNA and with its binding to Hsp27. Finally, we determined that enhanced Hsp27 expression in neurons exposed to H2O2 or glutamate prevented the translation of a reporter plasmid where bim-3′UTR mRNA sequence was cloned downstream of a luciferase gene. These results suggest that repression of bim mRNA translation through binding to the 3′UTR constitutes a novel cytoprotective mechanism of Hsp27 during stress in neurons. PMID:25187648

RT-PCR amplification of RNA extracted from formalin-fixed, paraffin-embedded oral cancer sections: analysis of p53 pathway.

PubMed

Tachibana, Masatsugu; Shinagawa, Yasuhiro; Kawamata, Hitoshi; Omotehara, Fumie; Horiuchi, Hideki; Ohkura, Yasuo; Kubota, Keiichi; Imai, Yutaka; Fujibayashi, Takashi; Fujimori, Takahiro

2003-01-01

We present a new approach towards the detection of the mRNAs in formalin-fixed, paraffin-embedded samples using a reverse transcriptase (RT)-polymerase chain reaction (PCR). The total RNAs were extracted from 10-micron-thick sections and were reverse-transcribed, then the RT-products were subjected to PCR amplification of GAPDH mRNA for screening the mRNA degradation. Next, nested PCR was performed for examining the expression of p53-related genes, p21WAF1, MDM2, p33ING1 and p14ARF. GAPDH mRNA expression was detectable in 12 out of 21 oral squamous cell carcinoma (SCC) samples. p21WAF1 mRNA expression was detectable in 5 out of 12 SCC samples, MDM2 mRNA expression was detectable in 5 our of 12 SCC samples and p33ING1 mRNA expression was detectable in 6 out of 12 SCC samples. However, the expression of p14ARF mRNA was not detectable in any of the samples. Seven out of 12 oral SCC samples showed abnormal nuclear accumulation of p53 protein by immunohistochemical staining, whereas 5 out of 12 oral SCCs showed negative staining for p53 protein. Of of p33ING1 mRNA. One of these was a verrucous carcinoma in which the p53 gene products might be inactivated by the oncoprotein E6 of human papilloma virus. Thus, the p53 tumor suppressor pathway was disrupted in most oral SCCs at the cellular levels, due to either an abnormality in p53 itself or loss of expression of p53 regulatory factors. This method would assist in making diagnosis, determining therapeutic strategy and predicting the prognosis of various cancers including oral SCCs.

Poly(A)-binding proteins and mRNA localization: who rules the roost?

PubMed

Gray, Nicola K; Hrabálková, Lenka; Scanlon, Jessica P; Smith, Richard W P

2015-12-01

RNA-binding proteins are often multifunctional, interact with a variety of protein partners and display complex localizations within cells. Mammalian cytoplasmic poly(A)-binding proteins (PABPs) are multifunctional RNA-binding proteins that regulate multiple aspects of mRNA translation and stability. Although predominantly diffusely cytoplasmic at steady state, they shuttle through the nucleus and can be localized to a variety of cytoplasmic foci, including those associated with mRNA storage and localized translation. Intriguingly, PABP sub-cellular distribution can alter dramatically in response to cellular stress or viral infection, becoming predominantly nuclear and/or being enriched in induced cytoplasmic foci. However, relatively little is known about the mechanisms that govern this distribution/relocalization and in many cases PABP functions within specific sites remain unclear. Here we discuss the emerging evidence with respect to these questions in mammals. © 2015 Authors; published by Portland Press Limited.

Long-term culture of bovine nucleus pulposus explants in a native environment.

PubMed

van Dijk, Bart G M; Potier, Esther; Ito, Keita

2013-04-01

Chronic low back pain is a disease with tremendous financial and social implications, and it is often caused by intervertebral disc degeneration. Regenerative therapies for disc repair are promising treatments, but they need to be tested in physiological models. To develop a physiological in vitro explant model that incorporates the native environment of the intervertebral disc, for example, hypoxia, low glucose, and high tissue osmolarity. Bovine nucleus pulposus (NP) explants were cultured for 42 days in conditions mimicking the native physiological environment. Two different approaches were used to balance the swelling pressure of the NP: raised medium osmolarity or an artificial annulus. Bovine NP explants were either cultured in media with osmolarity balanced at isotonic and hypertonic levels compared with the native tissue or cultured inside a fiber jacket used as an artificial annulus. Oxygen and glucose levels were set at either standard (21% O2 and 4.5 g/L glucose) or physiological (5% O2 and 1 g/L glucose) levels. Samples were analyzed at Day 0, 3, and 42 for tissue composition (water, sulfated glycosaminoglycans, DNA, and hydroxyproline contents and fixed charge density), tissue histology, cell viability, and cellular behavior with messenger RNA (mRNA) expression. Both the hypertonic culture and the artificial annulus approach maintained the tissue matrix composition for 42 days. At Day 3, mRNA expressions of aggrecan, collagen Type I, and collagen Type II in both hypertonic and artificial annulus cultures were not different from Day 0; however, at Day 42, the artificial annulus preserved the mRNA expression closer to Day 0. Gene expressions of matrix metalloprotease 13, tissue inhibitor of matrix metalloprotease 1, and tissue inhibitor of matrix metalloprotease 2 were downregulated under physiological O2 and glucose levels, whereas the other parameters analyzed were not affected. Although the hypertonic culture and the artificial annulus approach are both promising models to test regenerative therapies, the artificial annulus was better able to maintain a cellular behavior closer to the native tissue in longer term cultures. Copyright © 2013 Elsevier Inc. All rights reserved.

The Polyadenosine RNA-binding Protein, Zinc Finger Cys3His Protein 14 (ZC3H14), Regulates the Pre-mRNA Processing of a Key ATP Synthase Subunit mRNA*

PubMed Central

Wigington, Callie P.; Morris, Kevin J.; Newman, Laura E.; Corbett, Anita H.

2016-01-01

Polyadenosine RNA-binding proteins (Pabs) regulate multiple steps in gene expression. This protein family includes the well studied Pabs, PABPN1 and PABPC1, as well as the newly characterized Pab, zinc finger CCCH-type containing protein 14 (ZC3H14). Mutations in ZC3H14 are linked to a form of intellectual disability. To probe the function of ZC3H14, we performed a transcriptome-wide analysis of cells depleted of either ZC3H14 or the control Pab, PABPN1. Depletion of PABPN1 affected ∼17% of expressed transcripts, whereas ZC3H14 affected only ∼1% of expressed transcripts. To assess the function of ZC3H14 in modulating target mRNAs, we selected the gene encoding the ATP synthase F0 subunit C (ATP5G1) transcript. Knockdown of ZC3H14 significantly reduced ATP5G1 steady-state mRNA levels. Consistent with results suggesting that ATP5G1 turnover increases upon depletion of ZC3H14, double knockdown of ZC3H14 and the nonsense-mediated decay factor, UPF1, rescues ATP5G1 transcript levels. Furthermore, fractionation reveals an increase in the amount of ATP5G1 pre-mRNA that reaches the cytoplasm when ZC3H14 is depleted and that ZC3H14 binds to ATP5G1 pre-mRNA in the nucleus. These data support a role for ZC3H14 in ensuring proper nuclear processing and retention of ATP5G1 pre-mRNA. Consistent with the observation that ATP5G1 is a rate-limiting component for ATP synthase activity, knockdown of ZC3H14 decreases cellular ATP levels and causes mitochondrial fragmentation. These data suggest that ZC3H14 modulates pre-mRNA processing of select mRNA transcripts and plays a critical role in regulating cellular energy levels, observations that have broad implications for proper neuronal function. PMID:27563065

In vitro fatty acid enrichment of macrophages alters inflammatory response and net cholesterol accumulation

PubMed Central

Wang, Shu; Wu, Dayong; Lamon-Fava, Stefania; Matthan, Nirupa R.; Honda, Kaori L.; Lichtenstein, Alice H.

2010-01-01

Dietary long-chain PUFA, both n-3 and n-6, have unique benefits with respect to CVD risk. The aim of the present study was to determine the mechanisms by which n-3 PUFA (EPA, DHA) and n-6 PUFA (linoleic acid (LA), arachidonic acid (AA)) relative to SFA (myristic acid (MA), palmitic acid (PA)) alter markers of inflammation and cholesterol accumulation in macrophages (MΦ). Cells treated with AA and EPA elicited significantly less inflammatory response than control cells or those treated with MA, PA and LA, with intermediate effects for DHA, as indicated by lower levels of mRNA and secretion of TNFα, IL-6 and monocyte chemoattractant protein-1. Differences in cholesterol accumulation after exposure to minimally modified LDL were modest. AA and EPA resulted in significantly lower MΦ scavenger receptor 1 mRNA levels relative to control or MA-, PA-, LA- and DHA-treated cells, and ATP-binding cassette A1 mRNA levels relative to control or MA-, PA- and LA-treated cells. These data suggest changes in the rate of bidirectional cellular cholesterol flux. In summary, individual long-chain PUFA have differential effects on inflammatory response and markers of cholesterol flux in MΦ which are not related to the n position of the first double bond, chain length or degree of saturation. PMID:19660150

Loss of ATRX, associated with DNA methylation pattern of chromosome end, impacted biological behaviors of astrocytic tumors

PubMed Central

Zhang, Wei; Yang, Pei; Zhang, Chuanbao; Li, Mingyang; Yao, Kun; Wang, Hongjun; Li, Qingbin; Jiang, Chuanlu; Jiang, Tao

2015-01-01

Loss of ATRX leads to epigenetic alterations, including abnormal levels of DNA methylation at repetitive elements such as telomeres in murine cells. We conducted an extensive DNA methylation and mRNA expression profile study on a cohort of 82 patients with astrocytic tumors to study whether ATRX expression was associated with DNA methylation level in astrocytic tumors and in which cellular functions it participated. We observed that astrocytic tumors with lower ATRX expression harbored higher DNA methylation level at chromatin end and astrocytic tumors with ATRX-low had distinct gene expression profile and DNA methylation profile compared with ATRX-high tumors. Then, we uncovered that several ATRX associated biological functions in the DNA methylation and mRNA expression profile (GEP), including apoptotic process, DNA-dependent positive regulation of transcription, chromatin modification, and observed that ATRX expression was companied by MGMT methylation and expression. We also found that loss of ATRX caused by siRNA induced apoptotic cells increasing, reduced tumor cell proliferation and repressed the cell migration in glioma cells. Our results showed ATRX-related regulatory functions of the combined profiles from DNA methylation and mRNA expression in astrocytic tumors, and delineated that loss of ATRX impacted biological behaviors of astrocytic tumor cells, providing important resources for future dissection of ATRX role in glioma. PMID:25971279

Loss of ATRX, associated with DNA methylation pattern of chromosome end, impacted biological behaviors of astrocytic tumors.

PubMed

Cai, Jinquan; Chen, Jing; Zhang, Wei; Yang, Pei; Zhang, Chuanbao; Li, Mingyang; Yao, Kun; Wang, Hongjun; Li, Qingbin; Jiang, Chuanlu; Jiang, Tao

2015-07-20

Loss of ATRX leads to epigenetic alterations, including abnormal levels of DNA methylation at repetitive elements such as telomeres in murine cells. We conducted an extensive DNA methylation and mRNA expression profile study on a cohort of 82 patients with astrocytic tumors to study whether ATRX expression was associated with DNA methylation level in astrocytic tumors and in which cellular functions it participated. We observed that astrocytic tumors with lower ATRX expression harbored higher DNA methylation level at chromatin end and astrocytic tumors with ATRX-low had distinct gene expression profile and DNA methylation profile compared with ATRX-high tumors. Then, we uncovered that several ATRX associated biological functions in the DNA methylation and mRNA expression profile (GEP), including apoptotic process, DNA-dependent positive regulation of transcription, chromatin modification, and observed that ATRX expression was companied by MGMT methylation and expression. We also found that loss of ATRX caused by siRNA induced apoptotic cells increasing, reduced tumor cell proliferation and repressed the cell migration in glioma cells. Our results showed ATRX-related regulatory functions of the combined profiles from DNA methylation and mRNA expression in astrocytic tumors, and delineated that loss of ATRX impacted biological behaviors of astrocytic tumor cells, providing important resources for future dissection of ATRX role in glioma.

17β-Estradiol Alters Oxidative Stress Response Protein Expression and Oxidative Damage in the Uterus

PubMed Central

Yuan, Lisi; Dietrich, Alicia K.; Nardulli, Ann M.

2014-01-01

The steroid hormone 17β-estradiol (E2) has profound effects on the uterus. However, with the E2-induced increase in uterine cell proliferation and metabolism comes increased production of reactive oxygen species (ROS). We examined the expression of an interactive network of oxidative stress response proteins including thioredoxin (Trx), Cu/Zn superoxide dismutase (SOD1), apurinic endonuclease (Ape1), and protein disulfide isomerase (PDI). We demonstrated that treatment of ovariectomized C57BL/6J female mice with E2 increased the mRNA and protein levels of Trx, but decreased SOD1 and Ape1 mRNA and protein expression. In contrast, E2 treatment increased PDI protein levels but had no effect on PDI transcript levels.Interestingly, E2 treatment also increased two markers of cellular damage, lipid peroxidation and protein carbonylation. Our studies suggest that the decreased expression of SOD1 and Ape1 caused by E2 treatment may in the long term result in disruption of ROS regulation and play a role in endometrial carcinogenesis. PMID:24103313

Taxol and LPS Modulation of c-kit and nm23 Expression in Macrophages and Normal vs. Malignant Breast Cancer Cell Lines.

DTIC Science & Technology

1999-07-01

medium only, LPS (100 ng/ml), or paclitaxel (35 ^iM), concentrations found to induce maximal levels of mRNA in murine macrophages. Total RNA was...not detected in RNA derived from the DA-3 cells over an 8 h timecourse, even after 40 cycles of PCR amplification, without or with treatment...indicated times after stimulation with LPS or paclitaxel. Isolation of total cellular RNA . For in vitro experiments, culture supematants were removed

Macrophage colony-stimulating factor accelerates wound healing and upregulates TGF-beta1 mRNA levels through tissue macrophages.

PubMed

Wu, L; Yu, Y L; Galiano, R D; Roth, S I; Mustoe, T A

1997-10-01

Macrophage colony-stimulating factor (M-CSF) is produced by many cell types involved in wound repair, yet it acts specifically on monocytes and macrophages. The monocyte-derived cell is thought to be important in wound healing, but the importance of the role of tissue macrophages in wound healing has not been well defined. Dermal ulcers were created in normal and ischemic ears of young rabbits. Either rhM-CSF (17 microg/wound) or buffer was applied to each wound. Wounds were bisected and analyzed histologically at Days 7 and 10 postwounding. The amounts of epithelial growth and granulation tissue deposition were measured in all wounds. The level of increase of TGF-beta1 mRNA level in M-CSF-treated wounds was examined using competitive RT-PCR. M-CSF increased new granulation tissue formation by 37% (N = 21, P < 0.01) and 50% (P < 0.01) after single and multiple treatments, respectively, in nonischemic wounds. TGF-beta1 mRNA levels in rhM-CSF-treated wounds increased 5.01-fold (N = 8) over vehicle-treated wounds under nonischemic conditions. In contrast, no effect could be detected in ischemic wounds treated with rhM-CSF, and these wounds only showed a 1.66-fold increase in TGF-beta1 mRNA levels when compared to ischemic wounds treated with vehicle alone. GAPDH, a housekeeping gene, showed no change. As mesenchymal cells lack receptors for M-CSF, the improved healing of wounds treated with topical rhM-CSF must reflect a generalized enhancement of activation and function of tissue macrophages, as demonstrated by upregulation of TGF-beta. The lack of effect under ischemic conditions suggests that either macrophage activity and/or response to M-CSF is adversely affected under those conditions; this may suggest the pathogenesis of impaired wound healing at the cellular level. Copyright 1997 Academic Press.

1950MHz Radio Frequency Electromagnetic Radiation Inhibits Testosterone Secretion of Mouse Leydig Cells

PubMed Central

Lin, Yan-Yun; Wu, Tao; Liu, Jun-Ye; Gao, Peng; Li, Kang-Chu; Guo, Qi-Yan; Yuan, Meng; Lang, Hai-Yang; Zeng, Li-Hua; Guo, Guo-Zhen

2017-01-01

More studies that are focused on the bioeffects of radio-frequency (RF) electromagnetic radiation that is generated from the communication devices, but there were few reports with confirmed results about the bioeffects of RF radiation on reproductive cells. To explore the effects of 1950 MHz RF electromagnetic radiation (EMR) on mouse Leydig (TM3) cells. TM3 cells were irradiated or sham-irradiated continuously for 24 h by the specific absorption rate (SAR) 3 W/kg radiation. At 0, 1, 2, 3, 4, and 5 days after irradiation, cell proliferation was detected by cell counting kit-8 (CCK-8) method, cell cycle distribution, percentage of apoptosis, and cellular reactive oxygen species (ROS) were examined by flow cytometry, Testosterone level was measured using enzyme-linked immunosorbent assay (ELISA) assay, messenger ribonucleic acid (mRNA) expression level of steroidogenic acute regulatory protein (StAR) and P450scc in TM3 cells was detected by real-time polymerase chain reaction (PCR). After being irradiated for 24 h, cell proliferation obviously decreased and cell cycle distribution, secretion capacity of Testosterone, and P450scc mRNA level were reduced. While cell apoptosis, ROS, and StAR mRNA level did not change significantly. The current results indicated that 24 h of exposure at 1950 MHz 3 W/kg radiation could cause some adverse effects on TM3 cells proliferation and Testosterone secretion, further studies about the biological effects in the reproductive system that are induced by RF radiation are also needed. PMID:29295490

1950MHz Radio Frequency Electromagnetic Radiation Inhibits Testosterone Secretion of Mouse Leydig Cells.

PubMed

Lin, Yan-Yun; Wu, Tao; Liu, Jun-Ye; Gao, Peng; Li, Kang-Chu; Guo, Qi-Yan; Yuan, Meng; Lang, Hai-Yang; Zeng, Li-Hua; Guo, Guo-Zhen

2017-12-23

More studies that are focused on the bioeffects of radio-frequency (RF) electromagnetic radiation that is generated from the communication devices, but there were few reports with confirmed results about the bioeffects of RF radiation on reproductive cells. To explore the effects of 1950 MHz RF electromagnetic radiation (EMR) on mouse Leydig (TM3) cells. TM3 cells were irradiated or sham-irradiated continuously for 24 h by the specific absorption rate (SAR) 3 W/kg radiation. At 0, 1, 2, 3, 4, and 5 days after irradiation, cell proliferation was detected by cell counting kit-8 (CCK-8) method, cell cycle distribution, percentage of apoptosis, and cellular reactive oxygen species (ROS) were examined by flow cytometry, Testosterone level was measured using enzyme-linked immunosorbent assay (ELISA) assay, messenger ribonucleic acid (mRNA) expression level of steroidogenic acute regulatory protein (StAR) and P450scc in TM3 cells was detected by real-time polymerase chain reaction (PCR). After being irradiated for 24 h, cell proliferation obviously decreased and cell cycle distribution, secretion capacity of Testosterone, and P450scc mRNA level were reduced. While cell apoptosis, ROS, and StAR mRNA level did not change significantly. The current results indicated that 24 h of exposure at 1950 MHz 3 W/kg radiation could cause some adverse effects on TM3 cells proliferation and Testosterone secretion, further studies about the biological effects in the reproductive system that are induced by RF radiation are also needed.

Multi-functional regulation of 4E-BP gene expression by the Ccr4-Not complex.

PubMed

Okada, Hirokazu; Schittenhelm, Ralf B; Straessle, Anna; Hafen, Ernst

2015-01-01

The mechanistic target of rapamycin (mTOR) signaling pathway is highly conserved from yeast to humans. It senses various environmental cues to regulate cellular growth and homeostasis. Deregulation of the pathway has been implicated in many pathological conditions including cancer. Phosphorylation cascades through the pathway have been extensively studied but not much is known about the regulation of gene expression of the pathway components. Here, we report that the mRNA level of eukaryotic translation initiation factor (eIF) subunit 4E-binding protein (4E-BP) gene, one of the key mTOR signaling components, is regulated by the highly conserved Ccr4-Not complex. RNAi knockdown of Not1, a putative scaffold protein of this protein complex, increases the mRNA level of 4E-BP in Drosophila Kc cells. Examination of the gene expression mechanism using reporter swap constructs reveals that Not1 depletion increases reporter mRNAs with the 3'UTR of 4E-BP gene, but decreases the ones with the 4E-BP promoter region, suggesting that Ccr4-Not complex regulates both degradation and transcription of 4E-BP mRNA. These results indicate that the Ccr4-Not complex controls expression of a single gene at multiple levels and adjusts the magnitude of the total effect. Thus, our study reveals a novel regulatory mechanism of a key component of the mTOR signaling pathway at the level of gene expression.

Putrescine-Dependent Re-Localization of TvCP39, a Cysteine Proteinase Involved in Trichomonas vaginalis Cytotoxicity

PubMed Central

Carvajal-Gamez, Bertha Isabel; Quintas-Granados, Laura Itzel; Arroyo, Rossana; Vázquez-Carrillo, Laura Isabel; Ramón-Luing, Lucero De los Angeles; Carrillo-Tapia, Eduardo; Alvarez-Sánchez, María Elizbeth

2014-01-01

Polyamines are involved in the regulation of some Trichomonas vaginalis virulence factors such as the transcript, proteolytic activity, and cytotoxicity of TvCP65, a cysteine proteinase (CP) involved in the trichomonal cytotoxicity. In this work, we reported the putrescine effect on TvCP39, other CP that also participate in the trichomonal cytotoxicity. Parasites treated with 1,4-diamino-2-butanone (DAB) (an inhibitor of putrescine biosynthesis), diminished the amount and proteolytic activity of TvCP39 as compared with untreated parasites. Inhibition of putrescine biosynthesis also reduced ∼80% the tvcp39 mRNA levels according to RT-PCR and qRT-PCR assays. Additionally, actinomycin D-treatment showed that the tvcp39 mRNA half-life decreased in the absence of putrescine. However, this reduction was restored by exogenous putrescine addition, suggesting that putrescine is necessary for tvcp39 mRNA stability. TvCP39 was localized in the cytoplasm but, in DAB treated parasites transferred into exogenous putrescine culture media, TvCP39 was re-localized to the nucleus and nuclear periphery of trichomonads. Interestingly, the amount and proteolytic activity of TvCP39 was recovered as well as the tvcp39 mRNA levels were restored when putrescine exogenous was added to the DAB-treated parasites. In conclusion, our data show that putrescine regulate the TvCP39 expression, protein amount, proteolytic activity, and cellular localization. PMID:25251406

LARP4 Is Regulated by Tumor Necrosis Factor Alpha in a Tristetraprolin-Dependent Manner

PubMed Central

Mattijssen, Sandy

2015-01-01

LARP4 is a protein with unknown function that independently binds to poly(A) RNA, RACK1, and the poly(A)-binding protein (PABPC1). Here, we report on its regulation. We found a conserved AU-rich element (ARE) in the human LARP4 mRNA 3′ untranslated region (UTR). This ARE, but not its antisense version or a point-mutated version, significantly decreased the stability of β-globin reporter mRNA. We found that overexpression of tristetraprolin (TTP), but not its RNA binding mutant or the other ARE-binding proteins tested, decreased cellular LARP4 levels. RNA coimmunoprecipitation showed that TTP specifically associated with LARP4 mRNA in vivo. Consistent with this, mouse LARP4 accumulated to higher levels in TTP gene knockout (KO) cells than in control cells. Stimulation of WT cells with tumor necrosis factor alpha (TNF-α), which rapidly induces TTP, robustly decreased LARP4 with a coincident time course but had no such effect on LARP4B or La protein or on LARP4 in the TTP KO cells. The TNF-α-induced TTP pulse was followed by a transient decrease in LARP4 mRNA that was quickly followed by a subsequent transient decrease in LARP4 protein. Involvement of LARP4 as a target of TNF-α–TTP regulation provides a clue as to how its functional activity may be used in a physiologic pathway. PMID:26644407

Characterizing the Effects of Inorganic Acid and Alkaline Shock on the Staphylococcus aureus Transcriptome and Messenger RNA Turnover

PubMed Central

Anderson, Kelsi L.; Roux, Christelle M.; Olson, Matthew W.; Luong, Thanh T.; Lee, Chia Y.; Olson, Robert; Dunman, Paul M.

2010-01-01

Staphylococcus aureus pathogenesis can be partially attributed to its ability to adapt to otherwise deleterious host-associated stresses. Here, Affymetrix GeneChips® were used to examine the S. aureus responses to inorganic acid and alkaline shock and to assess whether stress dependent changes in mRNA turnover are likely to facilitate the organism’s ability to tolerate pH challenge. Results indicate that S. aureus adapts to pH shock by eliciting responses expected of cells coping with pH alteration, including neutralizing cellular pH, DNA repair, amino acid biosynthesis and virulence factor expression. Further, the S. aureus response to alkaline conditions is strikingly similar to that of stringent response induced cells. Indeed, we show that alkaline shock stimulates accumulation of the stringent response activator (p)ppGpp. Results also revealed that pH shock significantly alters the mRNA properties of the cell. A comparison of the mRNA degradation properties of transcripts whose titers either increased or decreased in response to sudden pH change revealed that alterations in mRNA degradation may, in part, account for the changes in the mRNA levels of factors predicted to mediate pH tolerance. A set of small stable RNA molecules were induced in response to acid or alkaline shock conditions and may mediate adaptation to pH stress. PMID:21039920

Oleate ameliorates palmitate-induced reduction of NAMPT activity and NAD levels in primary human hepatocytes and hepatocarcinoma cells.

PubMed

Penke, Melanie; Schuster, Susanne; Gorski, Theresa; Gebhardt, Rolf; Kiess, Wieland; Garten, Antje

2017-10-03

Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide adenine dinucleotide (NAD) levels are crucial for liver function. The saturated fatty acid palmitate and the unsaturated fatty acid oleate are the main free fatty acids in adipose tissue and human diet. We asked how these fatty acids affect cell survival, NAMPT and NAD levels in HepG2 cells and primary human hepatocytes. HepG2 cells were stimulated with palmitate (0.5mM), oleate (1mM) or a combination of both (0.5mM/1mM) as well as nicotinamide mononucleotide (NMN) (0.5 mM) or the specific NAMPT inhibitor FK866 (10nM). Cell survival was measured by WST-1 assay and Annexin V/propidium iodide staining. NAD levels were determined by NAD/NADH Assay or HPLC. Protein and mRNA levels were analysed by Western blot analyses and qPCR, respectively. NAMPT enzyme activity was measured using radiolabelled 14 C-nicotinamide. Lipids were stained by Oil red O staining. Palmitate significantly reduced cell survival and induced apoptosis at physiological doses. NAMPT activity and NAD levels significantly declined after 48h of palmitate. In addition, NAMPT mRNA expression was enhanced which was associated with increased NAMPT release into the supernatant, while intracellular NAMPT protein levels remained stable. Oleate alone did not influence cell viability and NAMPT activity but ameliorated the negative impact of palmitate on cell survival, NAMPT activity and NAD levels, as well as the increased NAMPT mRNA expression and secretion. NMN was able to normalize intracellular NAD levels but did not ameliorate cell viability after co-stimulation with palmitate. FK866, a specific NAMPT inhibitor did not influence lipid accumulation after oleate-treatment. Palmitate targets NAMPT activity with a consequent cellular depletion of NAD. Oleate protects from palmitate-induced apoptosis and variation of NAMPT and NAD levels. Palmitate-induced cell stress leads to an increase of NAMPT mRNA and accumulation in the supernatant. However, the proapoptotic action of palmitate seems not to be mediated by decreased NAD levels.

Selenium Deficiency Induces Autophagy in Immune Organs of Chickens.

PubMed

Khoso, Pervez Ahmed; Pan, Tingru; Wan, Na; Yang, Zijiang; Liu, Ci; Li, Shu

2017-05-01

The aim of the present study was to investigate the effects of selenium (Se) deficiency on autophagy-related genes and on ultrastructural changes in the spleen, bursa of Fabricius, and thymus of chickens. The Se deficiency group was fed a basal diet containing Se at 0.033 mg/kg and the control group was fed the same basal diet containing Se at 0.15 mg/kg. The messenger RNA (mRNA) levels of the autophagy genes microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, Beclin 1, dynein, autophagy associated gene 5 (ATG5), and target of rapamycin complex 1 (TORC1) were assessed using real-time qPCR. The protein levels of LC3-II, Beclin 1, and dynein were investigated using western blot analysis. Furthermore, the ultrastructure was observed using an electron microscope. The results indicated that spleen mRNA levels of LC3-I, LC3-II, Beclin 1, dynein, ATG5, and TORC1 and the protein levels of LC3-II, Beclin 1, and dynein were increased in the Se deficiency group compared with the control group. In the bursa of Fabricius, the mRNA levels of LC3-I, LC3-II, Beclin 1, dynein, ATG5, and TORC1 and the protein levels of Beclin 1 and dynein were increased; furthermore, the protein level of LC3-II was decreased in the Se deficiency group compared to the control group. In the thymus, the mRNA levels of LC3-I, Beclin 1, and ATG5 increased; the levels of LC3-II, dynein, and TORC1 were decreased; the protein level of Beclin 1 increased; and the levels of LC3-II and dynein decreased in the Se deficiency group compared to those in the control group. Further cellular morphological changes, such as autophagy vacuoles, autolysosomes, and lysosomal degradation, were observed in the spleen, bursa of Fabricius, and thymus of the Se-deficiency group. In summary, Se deficiency caused changes in autophagy-related genes, which increased the autophagic process and also caused structural damages to the immune organs of chickens.

Aiding and Abetting Cancer: mRNA export and the nuclear pore

PubMed Central

Culjkovic-Kraljacic, Biljana; Borden, Katherine L.B

2013-01-01

mRNA export is a critical step in gene expression. Export of transcripts can be modulated in response to cellular signaling or stress. Consistently, mRNA export is dysregulated in primary human specimens derived from many different forms of cancer. Aberrant expression of export factors can alter export of specific transcripts encoding proteins involved in proliferation, survival and oncogenesis. These specific factors, which are not used for bulk mRNA export, are obvious therapeutic targets. Indeed, given the emerging role of mRNA export in cancer, it is not surprising that efforts to target different aspects of this pathway have reached the clinical trial stage. Thus, like transcription and translation, mRNA export may also play a critical role in cancer genesis and maintenance. PMID:23582887

Markedly Lower Glutamic Acid Decarboxylase 67 Protein Levels in a Subset of Boutons in Schizophrenia.

PubMed

Rocco, Brad R; Lewis, David A; Fish, Kenneth N

2016-06-15

Convergent findings indicate that cortical gamma-aminobutyric acid (GABA)ergic circuitry is altered in schizophrenia. Postmortem studies have consistently found lower levels of glutamic acid decarboxylase 67 (GAD67) messenger RNA (mRNA) in the prefrontal cortex (PFC) of subjects with schizophrenia. At the cellular level, the density of GABA neurons with detectable levels of GAD67 mRNA is ~30% lower across cortical layers. Knowing how this transcript deficit translates to GAD67 protein levels in axonal boutons is important for understanding the impact it might have on GABA synthesis. In addition, because reductions in GAD67 expression before, but not after, the maturation of GABAergic boutons results in a lower density of GABAergic boutons in mouse cortical cultures, knowing if GABAergic bouton density is altered in schizophrenia would provide insight into the timing of the GAD67 deficit. PFC tissue sections from 20 matched pairs of schizophrenia and comparison subjects were immunolabeled for the vesicular GABA transporter (vGAT) and GAD67. vGAT+ bouton density did not differ between subject groups, consistent with findings that vGAT mRNA levels are unaltered in the illness and confirming that the number of cortical GABAergic boutons is not lower in schizophrenia. In contrast, in schizophrenia subjects, the proportion of vGAT+ boutons with detectable GAD67 levels (vGAT+/GAD67+ boutons) was 16% lower and mean GAD67 levels were 14% lower in the remaining vGAT+/GAD67+ boutons. Our findings suggest that GABA production is markedly reduced in a subset of boutons in the PFC of schizophrenia subjects and that this reduction likely occurs after the maturation of GABAergic boutons. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Suppression of Iron-Regulatory Hepcidin by Vitamin D

PubMed Central

Bacchetta, Justine; Zaritsky, Joshua J.; Sea, Jessica L.; Chun, Rene F.; Lisse, Thomas S.; Zavala, Kathryn; Nayak, Anjali; Wesseling-Perry, Katherine; Westerman, Mark; Hollis, Bruce W.; Salusky, Isidro B.

2014-01-01

The antibacterial protein hepcidin regulates the absorption, tissue distribution, and extracellular concentration of iron by suppressing ferroportin-mediated export of cellular iron. In CKD, elevated hepcidin and vitamin D deficiency are associated with anemia. Therefore, we explored a possible role for vitamin D in iron homeostasis. Treatment of cultured hepatocytes or monocytes with prohormone 25-hydroxyvitamin D or active 1,25-dihydroxyvitamin D decreased expression of hepcidin mRNA by 0.5-fold, contrasting the stimulatory effect of 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D on related antibacterial proteins such as cathelicidin. Promoter-reporter and chromatin immunoprecipitation analyses indicated that direct transcriptional suppression of hepcidin gene (HAMP) expression mediated by 1,25-dihydroxyvitamin D binding to the vitamin D receptor caused the decrease in hepcidin mRNA levels. Suppression of HAMP expression was associated with a concomitant increase in expression of the cellular target for hepcidin, ferroportin protein, and decreased expression of the intracellular iron marker ferritin. In a pilot study with healthy volunteers, supplementation with a single oral dose of vitamin D (100,000 IU vitamin D2) increased serum levels of 25D-hydroxyvitamin D from 27±2 ng/ml before supplementation to 44±3 ng/ml after supplementation (P<0.001). This response was associated with a 34% decrease in circulating levels of hepcidin within 24 hours of vitamin D supplementation (P<0.05). These data show that vitamin D is a potent regulator of the hepcidin-ferroportin axis in humans and highlight a potential new strategy for the management of anemia in patients with low vitamin D and/or CKD. PMID:24204002

Modeling RNA interference in mammalian cells

PubMed Central

2011-01-01

Background RNA interference (RNAi) is a regulatory cellular process that controls post-transcriptional gene silencing. During RNAi double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous mRNA via the generation of smaller dsRNA oligomers of length between 21-23nt (siRNAs). siRNAs are then loaded onto the RNA-Induced Silencing multiprotein Complex (RISC), which uses the siRNA antisense strand to specifically recognize mRNA species which exhibit a complementary sequence. Once the siRNA loaded-RISC binds the target mRNA, the mRNA is cleaved and degraded, and the siRNA loaded-RISC can degrade additional mRNA molecules. Despite the widespread use of siRNAs for gene silencing, and the importance of dosage for its efficiency and to avoid off target effects, none of the numerous mathematical models proposed in literature was validated to quantitatively capture the effects of RNAi on the target mRNA degradation for different concentrations of siRNAs. Here, we address this pressing open problem performing in vitro experiments of RNAi in mammalian cells and testing and comparing different mathematical models fitting experimental data to in-silico generated data. We performed in vitro experiments in human and hamster cell lines constitutively expressing respectively EGFP protein or tTA protein, measuring both mRNA levels, by quantitative Real-Time PCR, and protein levels, by FACS analysis, for a large range of concentrations of siRNA oligomers. Results We tested and validated four different mathematical models of RNA interference by quantitatively fitting models' parameters to best capture the in vitro experimental data. We show that a simple Hill kinetic model is the most efficient way to model RNA interference. Our experimental and modeling findings clearly show that the RNAi-mediated degradation of mRNA is subject to saturation effects. Conclusions Our model has a simple mathematical form, amenable to analytical investigations and a small set of parameters with an intuitive physical meaning, that makes it a unique and reliable mathematical tool. The findings here presented will be a useful instrument for better understanding RNAi biology and as modelling tool in Systems and Synthetic Biology. PMID:21272352

Changes in gene expression and cellular localization of insulin-like growth factors 1 and 2 in the ovaries during ovary development of the yellowtail, Seriola quinqueradiata.

PubMed

Higuchi, Kentaro; Gen, Koichiro; Izumida, Daisuke; Kazeto, Yukinori; Hotta, Takuro; Takashi, Toshinori; Aono, Hideaki; Soyano, Kiyoshi

2016-06-01

A method of controlling the somatic growth and reproduction of yellowtail fish (Seriola quinqueradiata) is needed in order to establish methods for the efficient aquaculture production of the species. However, little information about the hormonal interactions between somatic growth and reproduction is available for marine teleosts. There is accumulating evidence that insulin-like growth factor (IGF), a major hormone related somatic growth, plays an important role in fish reproduction. As the first step toward understanding the physiological role of IGF in the development of yellowtail ovaries, we characterized the expression and cellular localization of IGF-1 and IGF-2 in the ovary during development. We histologically classified the maturity of two-year-old females with ovaries at various developmental stages into the perinucleolar (Pn), yolk vesicle (Yv), primary yolk (Py), secondary yolk and tertiary yolk (Ty) stages, according to the most advanced type of oocyte present. The IGF-1 gene expression showed constitutively high levels at the different developmental stages, although IGF-1 mRNA levels tended to increase from the Py to the Ty stage with vitellogenesis, reaching maximum levels during the Ty stage. The IGF-2 mRNA levels increased as ovarian development advanced. Using immunohistochemistry methods, immunoreactive IGF-1 was mainly detected in the theca cells of ovarian follicles during late secondary oocyte growth, and in part of the granulosa cells of Ty stage oocytes. IGF-2 immunoreactivity was observed in all granulosa cells in layer in Ty stage oocytes. These results indicate that follicular IGFs may be involved in yellowtail reproduction via autocrine/paracrine mechanisms. Copyright © 2016 Elsevier Inc. All rights reserved.

Zinc chelation decreases IFN-β-induced STAT1 upregulation and iNOS expression in RAW 264.7 macrophages.

PubMed

Reiber, Cathleen; Brieger, Anne; Engelhardt, Gabriela; Hebel, Silke; Rink, Lothar; Haase, Hajo

2017-12-01

One consequence of lipopolysaccharide (LPS)-induced stimulation of macrophages is the release of Interferon (IFN)-β, and subsequently the activation of the JAK-STAT1 pathway, resulting in the expression of inducible nitric oxide synthase (iNOS). Free intracellular zinc ions (Zn 2+ ) have a profound impact as a second messenger in LPS-dependent gene expression. Previous work had indicated a Zn 2+ -dependent upregulation of STAT1 mRNA in response to LPS and IFN-β, potentially affecting STAT1-dependent downstream signaling upon pre-incubation with these agents. The aim of the present study was to investigate the long-term influence of Zn 2+ chelation on cellular STAT1 levels and their effect on protein levels and activity of iNOS. The LPS- and IFN-β-mediated increase of STAT1 mRNA and protein levels was abrogated by chelation of Zn 2+ with the membrane permeable chelator N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) in RAW 264.7 macrophages. After 48h pre-incubation together with IFN-β, TPEN also led to reduced nitric monoxide formation in response to a second stimulation with LPS. Nonetheless, the latter was observed regardless of any pre-incubation with IFN-β, suggesting that the effect of treatment with TPEN negatively affects iNOS induction independently from cellular STAT1 levels. In conclusion, long term Zn 2+ chelation does affect STAT1 protein expression, but interferes with NO production by a different, yet unknown pathway not involving STAT1. However, as there are many additional STAT1-dependent genes, there might still be effects on targets other than iNOS. Copyright © 2017 Elsevier GmbH. All rights reserved.

Genomic analysis suggests that mRNA destabilization by the microprocessor is specialized for the auto-regulation of Dgcr8.

PubMed

Shenoy, Archana; Blelloch, Robert

2009-09-11

The Microprocessor, containing the RNA binding protein Dgcr8 and RNase III enzyme Drosha, is responsible for processing primary microRNAs to precursor microRNAs. The Microprocessor regulates its own levels by cleaving hairpins in the 5'UTR and coding region of the Dgcr8 mRNA, thereby destabilizing the mature transcript. To determine whether the Microprocessor has a broader role in directly regulating other coding mRNA levels, we integrated results from expression profiling and ultra high-throughput deep sequencing of small RNAs. Expression analysis of mRNAs in wild-type, Dgcr8 knockout, and Dicer knockout mouse embryonic stem (ES) cells uncovered mRNAs that were specifically upregulated in the Dgcr8 null background. A number of these transcripts had evolutionarily conserved predicted hairpin targets for the Microprocessor. However, analysis of deep sequencing data of 18 to 200nt small RNAs in mouse ES, HeLa, and HepG2 indicates that exonic sequence reads that map in a pattern consistent with Microprocessor activity are unique to Dgcr8. We conclude that the Microprocessor's role in directly destabilizing coding mRNAs is likely specifically targeted to Dgcr8 itself, suggesting a specialized cellular mechanism for gene auto-regulation.

sHLA-I Contamination, a novel mechanism to explain ex vivo/in vitro modulation of IL-10 synthesis and release in CD8(+) T lymphocytes and in neutrophils following intravenous immunoglobulin infusion.

PubMed

Ghio, Massimo; Contini, Paola; Setti, Maurizio; Ubezio, Gianluca; Mazzei, Clemente; Tripodi, Gino

2010-05-01

Numerous mechanisms have been proposed to explain the beneficial action of intravenous immune globulin (IVIG) in autoimmune and systemic inflammatory disorders; among others, they could decrease pro-inflammatory cytokine levels and also induce anti-inflammatory cytokines. Ex vivo analysis of cells from ten IVIG recipients showed significant increase of IL-10 mRNA and intra-cellular IL-10 molecules in both leukotypes. In vitro comparable results were obtained incubating CD8(+) T lymphocytes and neutrophils from healthy donors with IVIG. sHLA-I and/or sFasL immunodepletion abolished IL-10 modulation. Co-culture with contaminant-free IgM or MabThera did not exert any mRNA modulation. Finally, IgM or MabThera plus purified sHLA-I molecules enhanced IL-10-mRNA in both leukotypes to levels comparable to those obtained with IVIG incubation. As IVIG infusion involves administration of soluble contaminants, these data consent to speculate that IVIG might modulate IL-10 via the immunomodulatory activities of sHLA-I contaminant molecules inducing transcriptional and post-transcriptional modulation of IL-10 in CD8(+) T lymphocytes and neutrophils.

Effects of cyclic heat stress or vitamin C supplementation during cyclic heat stress on HSP70, inflammatory cytokines, and the antioxidant defense system in Sprague Dawley rats.

PubMed

Yun, Seo-Hyun; Moon, Yang-Soo; Sohn, Sea-Hwan; Jang, In-Surk

2012-01-01

A total of 21 male SD rats were divided into three groups to investigate the effects of consecutive cyclic heat stress or vitamin C under heat stress on heat shock protein (HSP) 70, inflammatory cytokines, and antioxidant systems. The heat stress (HS) and vitamin C supplementation during heat stress (HS+VC) groups were exposed to cyclic heat stress (23 to 38 to 23°C) for 2 h on each of seven consecutive days. The HS+VC group had free access to water containing 0.5% vitamin C throughout the experiment. Hepatic HSP70 mRNA in the HS group was significantly (P<0.05) higher than that in the control (CON) or HS+VC group. The mRNA levels of tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) in the HS group were greater (P<0.05) than those in the CON group. The HS+VC group showed significantly (P<0.05) lower mRNA levels of hepatic interleukin-6 and TNF-α than the HS group. However, thymic HSP70 and inflammatory cytokines were unaffected by treatments. In the hepatic antioxidant system, the mRNA and activity of glutathione peroxidase (GPX) were greater (P<0.05) in the HS than in the CON group, whereas the HS+VC group showed markedly (P<0.05) lower GPX mRNA and activity than the HS group. However, superoxide dismutase, glutathione S-transferase, and malondialdehyde were unaffected by treatments. In conclusion, cyclic heat stress activated hepatic HSP70, TNF-α, iNOS, and GPX genes, whereas vitamin C during heat stress ameliorated heat stress-induced cellular responses in rats.

The relative expression levels of insulin-like growth factor 1 and myostatin mRNA in the asynchronous development of skeletal muscle in ducks during early development.

PubMed

Hu, Yan; Liu, Hongxiang; Shan, Yanju; Ji, Gaige; Xu, Wenjuan; Shu, Jingting; Li, Huifang

2015-08-10

Genetic selection is a powerful tool for modifying poultry muscle yield. Insulin-like growth factor I (IGF-I) and myostatin (MSTN) are important regulators of muscle growth, especially in the myogenesis stage. This study compared the developmental pattern of the pectoralis major (PM) and lateral gastrocnemius (LM) muscles, mRNA expression characterization of IGF-I and MSTN-A and their correlation between 14 days in ovo and 1 week post-hatch in two Chinese local duck breeds. During early development, the growth of duck PM and LM followed an asynchronous pattern. Variations in PM growth rate observed with development followed the relative variations of MSTN and IGF-I expression; however, the same behavior was not observed in LM. Moreover, the profile of IGF-I expression in duck skeletal muscles indicated that genetic selection for high meat-yield poultry has altered the temporal expression of IGF-I and affected cellular characteristics and mass by hatch in a PM-specific manner. The MSTN-A expression profile showed synchronization with the growth of skeletal muscle and peaks of myofiber proliferation. The expression patterns of IGF-I and MSTN suggest that duck pectoralis fibers are prioritized for proliferation in embryogenesis. The IGF-1/MSTN-A mRNA ratios in PM and LM presented very similar trends in the changes of myofiber characteristics, and differences in the IGF-1/MSTN-A mRNA ratio in PM between the two breeds corresponded to the timing of differences in PM mass between the varieties. Our results support the hypothesis that relative levels of IGF-I and MSTN mRNA may participate in ordering muscle growth rates with selected development. Copyright © 2015 Elsevier B.V. All rights reserved.

Adenovirus-mediated interference of FABP4 regulates mRNA expression of ADIPOQ, LEP and LEPR in bovine adipocytes.

PubMed

Wei, S; Zan, L S; Wang, H B; Cheng, G; Du, M; Jiang, Z; Hausman, G J; McFarland, D C; Dodson, M V

2013-02-27

Fatty acid binding protein 4 (FABP4) is an important adipocyte gene, with roles in fatty acid transport and fat deposition in animals as well as human metabolic syndrome. However, little is known about the functional regulation of FABP4 at the cellular level in bovine. We designed and selected an effective shRNA (small hairpin RNA) against bovine FABP4, constructed a corresponding adenovirus (AD-FABP4), and then detected its influence on mRNA expression of four differentiation-related genes (PPAR(y), CEBPA, CEBPB, and SREBF1) and three lipid metabolism-related genes (ADIPOQ, LEP and LEPR) of adipocytes. The FABP4 mRNA content, derived from bovine adipocytes, decreased by 41% (P < 0.01) after 24 h and 66% (P < 0.01) after 72 h of AD-FABP4 infection. However, lower mRNA content of FABP4 did not significantly alter levels of differentiation-related gene expression at 24 h following AD-FABP4 treatment of bovine-derived preadipocytes (P = 0.54, 0.78, 0.89, and 0.94, respectively). Meanwhile, knocking down (partially silencing) FABP4 significantly decreased ADIPOQ (P < 0.05) and LEP (P < 0.01) gene expression after 24 h of AD-FABP4 treatment, decreased ADIPOQ (P < 0.01) and LEP (P < 0.01) gene expression, but increased LEPR mRNA expression (P < 0.01) after a 72-h treatment of bovine preadipocytes. We conclude that FABP4 plays a role in fat deposition and metabolic syndrome by regulating lipid metabolism-related genes (such as ADIPOQ, LEP and LEPR), without affecting the ability of preadipocytes to differentiate into adipocytes.

Self-amplifying mRNA vaccines.

PubMed

Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

2015-01-01

This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization and biological function analysis of the TRIM47 gene from common carp (Cyprinus carpio).

PubMed

Wang, Yeda; Kuang, Ming; Lu, Yuanan; Lin, Li; Liu, Xueqin

2017-09-05

The TRIM family protein was known to play an important role in many cellular processes, including potential antiviral activity, which has attracted lots of attention. In this study, a TRIM47 homolog from common carp (Cyprinus carpio) was cloned and the full length coding DNA sequence (CDS) of this gene was analyzed, results showed that there was a 97% similarity between common carp and zebrafish (Danio rerio), but only 18% similarity with that of human (Homo sapiens) and mouse (Mus musculus). The tissue distribution analysis showed TRIM47 had the highest mRNA level in the brain, a few immune related organs such as liver and kidney also had a relatively high level of TRIM47 expression. SVCV infection decreased TRIM47 mRNA level significantly both in vitro and in vivo, but its expression was not affected by the virus at the protein level. The recombinant plasmid pcDNA4-TRIM47-His was constructed, the subcellular localization in FHM cells showed that TRIM47 uniformly distributed in the cytoplasm at the form of tiny spots, and partially localized in the mitochondria. Overexpression TRIM47 in FHM cells significantly decreased the mRNA level of SVCV-G gene, and it was accompanied with the increasing of IFN1, a member of type I IFN, at the case of SVCV stimulation. In summary, our results had first demonstrated that TRIM47 of the common carp played an important role in viral resistance processes as well as the regulation of IFN signaling pathway. Copyright © 2017. Published by Elsevier B.V.

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism.

PubMed

Rodríguez, Andrea E; López-Crisosto, Camila; Peña-Oyarzún, Daniel; Salas, Daniela; Parra, Valentina; Quiroga, Clara; Morawe, Tobias; Chiong, Mario; Behl, Christian; Lavandero, Sergio

2016-01-01

Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells.

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism

PubMed Central

Rodríguez, Andrea E.; López-Crisosto, Camila; Peña-Oyarzún, Daniel; Salas, Daniela; Parra, Valentina; Quiroga, Clara; Morawe, Tobias; Chiong, Mario; Behl, Christian; Lavandero, Sergio

2016-01-01

ABSTRACT Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells. PMID:26654586

Peroxide reduction by a metal-dependent catalase in Nostoc punctiforme (cyanobacteria).

PubMed

Hudek, L; Torriero, A A J; Michalczyk, A A; Neilan, B A; Ackland, M L; Bräu, Lambert

2017-05-01

This study investigated the role of a novel metal-dependent catalase (Npun_R4582) that reduces hydrogen peroxide in the cyanobacterium Nostoc punctiforme. Quantitative real-time PCR showed that npun_R4582 relative mRNA levels were upregulated by over 16-fold in cells treated with either 2 μM added Co, 0.5 μM added Cu, 500 μM Mn, 1 μM Ni, or 18 μM Zn. For cells treated with 60 μM H 2 O 2 , no significant alteration in Npun_R4582 relative mRNA levels was detected, while in cells treated with Co, Cu, Mn, Ni, or Zn and 60 μM peroxide, relative mRNA levels were generally above control or peroxide only treated cells. Disruption or overexpression of npun_R4582 altered sensitivity to cells exposed to 60 μM H 2 O 2 and metals for treatments beyond the highest viable concentrations, or in a mixed metal solution for Npun_R4582 - cells. Moreover, overexpression of npun_R4582 increased cellular peroxidase activity in comparison with wild-type and Npun_R4582 - cells, and reduced peroxide levels by over 50%. The addition of cobalt, manganese, nickel, and zinc increased the capacity of Npun_R4582 to reduce the rate or total levels of peroxide produced by cells growing under photooxidative conditions. The work presented confirms the function of NpunR4582 as a catalase and provides insights as to how cells reduce potentially lethal peroxide levels produced by photosynthesis. The findings also show how trace elements play crucial roles as enzymatic cofactors and how the role of Npun_R4582 in hydrogen peroxide breakdown is dependent on the type of metal and the level available to cells.

Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.

PubMed

Azimi, Sayyed M; Sheridan, Steven D; Ghannad-Rezaie, Mostafa; Eimon, Peter M; Yanik, Mehmet Fatih

2018-05-01

Identification of optimal transcription-factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription-factor copy-numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH -expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition. © 2018, Azimi et al.

Viral Evasion and Manipulation of Host RNA Quality Control Pathways

PubMed Central

2016-01-01

Viruses have evolved diverse strategies to maximize the functional and coding capacities of their genetic material. Individual viral RNAs are often used as substrates for both replication and translation and can contain multiple, sometimes overlapping open reading frames. Further, viral RNAs engage in a wide variety of interactions with both host and viral proteins to modify the activities of important cellular factors and direct their own trafficking, packaging, localization, stability, and translation. However, adaptations increasing the information density of small viral genomes can have unintended consequences. In particular, viral RNAs have developed features that mark them as potential targets of host RNA quality control pathways. This minireview focuses on ways in which viral RNAs run afoul of the cellular mRNA quality control and decay machinery, as well as on strategies developed by viruses to circumvent or exploit cellular mRNA surveillance. PMID:27226372

Viral Evasion and Manipulation of Host RNA Quality Control Pathways.

PubMed

Hogg, J Robert

2016-08-15

Viruses have evolved diverse strategies to maximize the functional and coding capacities of their genetic material. Individual viral RNAs are often used as substrates for both replication and translation and can contain multiple, sometimes overlapping open reading frames. Further, viral RNAs engage in a wide variety of interactions with both host and viral proteins to modify the activities of important cellular factors and direct their own trafficking, packaging, localization, stability, and translation. However, adaptations increasing the information density of small viral genomes can have unintended consequences. In particular, viral RNAs have developed features that mark them as potential targets of host RNA quality control pathways. This minireview focuses on ways in which viral RNAs run afoul of the cellular mRNA quality control and decay machinery, as well as on strategies developed by viruses to circumvent or exploit cellular mRNA surveillance. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

PubMed Central

Halter, David; Collart, Martine A.; Panasenko, Olesya O.

2014-01-01

Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome. PMID:24465968

Low intracellular iron increases the stability of matriptase-2.

PubMed

Zhao, Ningning; Nizzi, Christopher P; Anderson, Sheila A; Wang, Jiaohong; Ueno, Akiko; Tsukamoto, Hidekazu; Eisenstein, Richard S; Enns, Caroline A; Zhang, An-Sheng

2015-02-13

Matriptase-2 (MT2) is a type II transmembrane serine protease that is predominantly expressed in hepatocytes. It suppresses the expression of hepatic hepcidin, an iron regulatory hormone, by cleaving membrane hemojuvelin into an inactive form. Hemojuvelin is a bone morphogenetic protein (BMP) co-receptor. Here, we report that MT2 is up-regulated under iron deprivation. In HepG2 cells stably expressing the coding sequence of the MT2 gene, TMPRSS6, incubation with apo-transferrin or the membrane-impermeable iron chelator, deferoxamine mesylate salt, was able to increase MT2 levels. This increase did not result from the inhibition of MT2 shedding from the cells. Rather, studies using a membrane-permeable iron chelator, salicylaldehyde isonicotinoyl hydrazone, revealed that depletion of cellular iron was able to decrease the degradation of MT2 independently of internalization. We found that lack of the putative endocytosis motif in its cytoplasmic domain largely abolished the sensitivity of MT2 to iron depletion. Neither acute nor chronic iron deficiency was able to alter the association of Tmprss6 mRNA with polyribosomes in the liver of rats indicating a lack of translational regulation by low iron levels. Studies in mice showed that Tmprss6 mRNA was not regulated by iron nor the BMP-mediated signaling with no evident correlation with either Bmp6 mRNA or Id1 mRNA, a target of BMP signaling. These results suggest that regulation of MT2 occurs at the level of protein degradation rather than by changes in the rate of internalization and translational or transcriptional mechanisms and that the cytoplasmic domain of MT2 is necessary for its regulation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Low Intracellular Iron Increases the Stability of Matriptase-2*

PubMed Central

Zhao, Ningning; Nizzi, Christopher P.; Anderson, Sheila A.; Wang, Jiaohong; Ueno, Akiko; Tsukamoto, Hidekazu; Eisenstein, Richard S.; Enns, Caroline A.; Zhang, An-Sheng

2015-01-01

Matriptase-2 (MT2) is a type II transmembrane serine protease that is predominantly expressed in hepatocytes. It suppresses the expression of hepatic hepcidin, an iron regulatory hormone, by cleaving membrane hemojuvelin into an inactive form. Hemojuvelin is a bone morphogenetic protein (BMP) co-receptor. Here, we report that MT2 is up-regulated under iron deprivation. In HepG2 cells stably expressing the coding sequence of the MT2 gene, TMPRSS6, incubation with apo-transferrin or the membrane-impermeable iron chelator, deferoxamine mesylate salt, was able to increase MT2 levels. This increase did not result from the inhibition of MT2 shedding from the cells. Rather, studies using a membrane-permeable iron chelator, salicylaldehyde isonicotinoyl hydrazone, revealed that depletion of cellular iron was able to decrease the degradation of MT2 independently of internalization. We found that lack of the putative endocytosis motif in its cytoplasmic domain largely abolished the sensitivity of MT2 to iron depletion. Neither acute nor chronic iron deficiency was able to alter the association of Tmprss6 mRNA with polyribosomes in the liver of rats indicating a lack of translational regulation by low iron levels. Studies in mice showed that Tmprss6 mRNA was not regulated by iron nor the BMP-mediated signaling with no evident correlation with either Bmp6 mRNA or Id1 mRNA, a target of BMP signaling. These results suggest that regulation of MT2 occurs at the level of protein degradation rather than by changes in the rate of internalization and translational or transcriptional mechanisms and that the cytoplasmic domain of MT2 is necessary for its regulation. PMID:25550162

Leptin downregulates heat shock protein-70 (HSP-70) gene expression in chicken liver and hypothalamus.

PubMed

Figueiredo, Denise; Gertler, Arieh; Cabello, Gérard; Decuypere, Eddy; Buyse, Johan; Dridi, Sami

2007-07-01

Heat shock protein (HSP)-70 is expressed in normal and stressed cells but is highly stress-inducible. Although leptin has long been suggested to be involved in the regulation of stress response, its interaction with the HSP-70 gene is still unknown, under both unstressed and stressed conditions. The present study has aimed to investigate the effect of leptin on HSP-70 gene expression in normal chicken liver, hypothalamus, and muscle. Continuous infusion of recombinant chicken leptin (8 mug/kg per hour) at a constant rate of 3 ml/h for 6 h in 3-week-old broiler chickens significantly (P < 0.05) decreased food intake and HSP-70 mRNA levels in liver and hypothalamus, but not in muscle. In an attempt to discriminate between the effect of leptin and of leptin-reduced food intake on HSP-70 gene expression, we also evaluated the effect of food deprivation on the same cellular responses in two broiler chicken lines genetically selected for low (LL) or high (FL) abdominal fat pad size. Food deprivation for 16 h did not affect HSP-70 gene expression in any of the studied tissues indicating that the effect of leptin was independent of the inhibition of food intake. Regardless of the nutritional status, HSP-70 mRNA levels were significantly (P < 0.05) higher in the hypothalamus of FL compared with LL chickens consistent with higher mRNA levels for hypothalamic corticotropin-releasing factor. To assess, whether the effects of leptin were direct or indirect, we carried out in vitro studies. Leptin treatments did not affect HSP-70 mRNA levels in a leghorn male hepatoma cell line or quail myoblast cell line suggesting that the effect of leptin on HSP-70 gene expression is mediated through the central nervous system. Furthermore, HSP-70 gene expression was gender-dependent with significantly (P < 0.05) higher levels in male than in female chickens.

Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib.

PubMed

Zhai, Yang; Zhang, Yanjun; Nan, Kejun; Liang, Xuan

2017-05-01

The clinical resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been linked to EGFR T790M resistance mutations or MET amplifications. Additional mechanisms underlying EGFR-TKI drug resistance remain unclear. The present study demonstrated that icotinib significantly inhibited the proliferation and increased the apoptosis rate of HCC827 cells; the cellular mRNA and protein expression levels of phosphatase and tensin homolog (PTEN) were also significantly downregulated. To investigate the effect of PTEN expression levels on the sensitivity of HCC827 cells to icotinib, PTEN expression was silenced using a PTEN-specific small interfering RNA. The current study identified that the downregulation of PTEN expression levels may promote cellular proliferation in addition to decreasing the apoptosis of HCC827 cells, and may reduce the sensitivity of HCC827 cells to icotinib. These results suggested that reduced PTEN expression levels were associated with the decreased sensitivity of HCC827 cells to icotinib. Furthermore, PTEN expression levels may be a useful marker for predicting icotinib resistance and elucidating the resistance mechanisms underlying EGFR-mutated NSCLC.

Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib

PubMed Central

Zhai, Yang; Zhang, Yanjun; Nan, Kejun; Liang, Xuan

2017-01-01

The clinical resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been linked to EGFR T790M resistance mutations or MET amplifications. Additional mechanisms underlying EGFR-TKI drug resistance remain unclear. The present study demonstrated that icotinib significantly inhibited the proliferation and increased the apoptosis rate of HCC827 cells; the cellular mRNA and protein expression levels of phosphatase and tensin homolog (PTEN) were also significantly downregulated. To investigate the effect of PTEN expression levels on the sensitivity of HCC827 cells to icotinib, PTEN expression was silenced using a PTEN-specific small interfering RNA. The current study identified that the downregulation of PTEN expression levels may promote cellular proliferation in addition to decreasing the apoptosis of HCC827 cells, and may reduce the sensitivity of HCC827 cells to icotinib. These results suggested that reduced PTEN expression levels were associated with the decreased sensitivity of HCC827 cells to icotinib. Furthermore, PTEN expression levels may be a useful marker for predicting icotinib resistance and elucidating the resistance mechanisms underlying EGFR-mutated NSCLC. PMID:28521430

Detection of ovarian matrix metalloproteinase mRNAs by in situ hybridization.

PubMed

Rosewell, Katherine L; Curry, Thomas E

2009-01-01

In situ hybridization represents a powerful technique to localize DNA or RNA of interest at the chromosomal or cellular level. In endocrine tissues composed of diverse and varied cell types, in situ hybridization has allowed the identification of specific cells responsible for the expression of genes controlling the function of the tissue. Our laboratory has routinely used this approach to understand the cellular expression of genes associated with the growth of the ovarian follicle, rupture of the follicle, and transformation of the ruptured follicle into the corpus luteum. The current study outlines the procedural details of in situ detection of mRNA in tissues and illustrates the utility of this approach in identifying the ovarian cells expressing the matrix metalloproteinases and their endogenous inhibitors, the TIMPs, in the human ovary.

Thin film bioreactors in space

NASA Technical Reports Server (NTRS)

Hughes-Fulford, M.; Scheld, H. W.

1989-01-01

Studies from the Skylab, SL-3 and D-1 missions have demonstrated that biological organisms grown in microgravity have changes in basic cellular functions such as DNA, mRNA and protein synthesis, cytoskeleton synthesis, glucose utilization, and cellular differentiation. Since microgravity could affect prokaryotic and eukaryotic cells at a subcellular and molecular level, space offers an opportunity to learn more about basic biological systems with one inmportant variable removed. The thin film bioreactor will facilitate the handling of fluids in microgravity, under constant temperature and will allow multiple samples of cells to be grown with variable conditions. Studies on cell cultures grown in microgravity would make it possible to identify and quantify changes in basic biological function in microgravity which are needed to develop new applications of orbital research and future biotechnology.

Caveolin-1 mediates tissue plasminogen activator-induced MMP-9 up-regulation in cultured brain microvascular endothelial cells.

PubMed

Jin, Xinchun; Sun, Yanyun; Xu, Ji; Liu, Wenlan

2015-03-01

Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates blood-brain barrier injury and increases the risk of symptomatic cerebral hemorrhage. The mechanism through which tPA enhances MMP-9 activity is not well understood. Here we report an important role of caveolin-1 in mediating tPA-induced MMP-9 synthesis. Brain microvascular endothelial cell line bEnd3 cells were incubated with 5 or 20 μg/ml tPA for 24 hrs before analyzing MMP-9 levels in the conditioned media and cellular extracts by gelatin zymography. tPA at a dose of 20 μg/mL tPA, but not 5 μg/mL, significantly increased MMP-9 level in cultured media while decreasing it in cellular extracts. Concurrently, tPA treatment induced a 2.3-fold increase of caveolin-1 protein levels in endothelial cells. Interestingly, knockdown of Cav-1 with siRNA inhibited tPA-induced MMP-9 mRNA up-regulation and MMP-9 increase in the conditioned media, but did not affect MMP-9 decrease in cellular extracts. These results suggest that caveolin-1 critically contributes to tPA-mediated MMP-9 up-regulation, but may not facilitate MMP-9 secretion in endothelial cells. Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates ischemic blood brain barrier (BBB) injury and increases the risk of symptomatic cerebral hemorrhage. Our results suggest a novel mechanism underlying this tPA-MMP 9 axis. In response to tPA treatment, caveolin-1 protein levels increased in endothelial cells, which mediate MMP-9 mRNA up-regulation and its secretion into extracellular space. Caveolin-1 may, however, not facilitate MMP-9 secretion in endothelial cells. Our data suggest caveolin-1 as a novel therapeutic target for protecting the BBB against ischemic damage. The schematic outlines tPA-induced MMP-9 upreguation. © 2015 International Society for Neurochemistry.

A Novel Role for SIRT3 in Regulating Mediators Involved in the Terminal Pathways of Human Labor and Delivery.

PubMed

Lim, Ratana; Barker, Gillian; Menon, Ramkumar; Lappas, Martha

2016-11-01

Preterm birth remains the major cause of neonatal mortality and morbidity, mediated largely by an inflammatory process. The sirtuin (SIRT) family of cellular regulators has been implicated as key inhibitors of inflammation. We have previously reported a role for SIRT1, SIRT2, and SIRT6 in regulating inflammation-induced prolabor mediators. In this study, we determined the effect of term labor and pro-inflammatory cytokines on SIRT3, SIRT4, SIRT5, and SIRT7 expression in human myometrium. Functional studies were also used to investigate the effect of small interfering RNA (siRNA) knockdown of SIRTs in regulating inflammation-induced prolabor mediators. Western blot analysis and qRT-PCR were used to determine SIRT3, SIRT4, SIRT5, and SIRT7 mRNA and protein expression in human myometrium. Small interfering RNA knockdown of SIRT3 in myometrial primary cells determined its role in response to inflammatory stimuli IL1B and TNF. SIRT3 mRNA and protein expression levels were significantly lower in term laboring myometrium compared with term nonlaboring myometrium. There was no effect of labor on SIRT4, SIRT5 or SIRT7 protein expression. The pro-inflammatory cytokines IL1B and TNF significantly decreased levels of SIRT3 mRNA and protein expression. SIRT3 knockdown by siRNA significantly augmented IL1B- and TNF-stimulated IL6, CXCL8, and CCL2 mRNA expression and release; PTGS2 mRNA expression and subsequent PGF 2alpha release; the mRNA expression and secretion of the adhesion molecule ICAM1 and the extracellular matrix remodeling enzyme MMP9; and nuclear factor kappa B1 (NFkappaB1) transcriptional activity. In human myometrium, SIRT3 expression decreases with term labor and regulates the mediators involved in the terminal effector pathways of human labor and delivery through the NFkappaB1 pathway. © 2016 by the Society for the Study of Reproduction, Inc.

Genetic and Epigenetic Changes in Chromosomally Stable and Unstable Progeny of Irradiated Cells

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

Baulch, Janet E.; Aypar, Umut; Waters, Katrina M.

2014-09-24

Radiation induced genomic instability is a well-studied phenomenon, the underlying mechanisms of which are poorly understood. Persistent oxidative stress, mitochondrial dysfunction, elevated cytokine levels and epigenetic changes are among the mechanisms invoked in the perpetuation of the phenotype. To determine whether epigenetic aberrations affect genomic instability we measured DNA methylation, mRNA and microRNA (miR) levels in well characterized chromosomally stable and unstable clonally expanded single cell survivors of irradiation. While no changes in DNA methylation were observed for the gene promoters evaluated, increased LINE-1 methylation was observed for two unstable clones (LS12, CS9) and decreased Alu element methylation was observedmore » for the other two unstable clones (115, Fe5.0-8). These relationships also manifested for mRNA and miR expression. mRNA identified for the LS12 and CS9 clones were most similar to each other (261 mRNA), while the 115 and Fe5.0-8 clones were more similar to each other, and surprisingly also similar to the two stable clones, 114 and 118 (286 mRNA among these four clones). Pathway analysis showed enrichment for pathways involved in mitochondrial function and cellular redox, themes routinely invoked in genomic instability. The commonalities between the two subgroups of clones were also observed for miR. The number of miR for which anti-correlated mRNA were identified suggests that these miR exert functional effects in each clone. The results of this study demonstrate significant genetic and epigenetic changes in unstable cells, but similar changes almost equally common in chromosomally stable cells. Possible conclusions might be that the chromosomally stable clones have some other form of instability, or that some of the observed changes represent a sort of radiation signature for and that other changes are related to genomic instability. Irrespective, these findings again suggest that a spectrum of changes both drive genomic instability and permit unstable cells to persist and proliferate.« less

Mitotic inheritance of mRNA facilitates translational activation of the osteogenic-lineage commitment factor Runx2 in progeny of osteoblastic cells

PubMed Central

Varela, Nelson; Aranguiz, Alejandra; Lizama, Carlos; Sepulveda, Hugo; Antonelli, Marcelo; Thaler, Roman; Moreno, Ricardo D.; Montecino, Martin; Stein, Gary S.; van Wijnen, Andre J.; Galindo, Mario

2017-01-01

Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype-committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post-mitotic re-activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that ‘bookmark’ the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone-related runt-related transcription factor Runx2. Here, we characterized a non-genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self-renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non-chromosomal tubulin-containing compartment. Subsequently, transcription-independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast-specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis. PMID:26381402

Cancer-targeting siRNA delivery from porous silicon nanoparticles.

PubMed

Wan, Yuan; Apostolou, Sinoula; Dronov, Roman; Kuss, Bryone; Voelcker, Nicolas H

2014-10-01

Porous silicon nanoparticles (pSiNPs) with tunable pore size are biocompatible and biodegradable, suggesting that they are suitable biomaterials as vehicles for drug delivery. Loading of small interfering RNA (siRNA) into the pores of pSiNPs can protect siRNA from degradation as well as improve the cellular uptake. We aimed to deliver MRP1 siRNA loaded into pSiNPs to glioblastoma cells, and to demonstrate downregulation of MRP1 at the mRNA and protein levels. 50-220 nm pSiNPs with an average pore size of 26 nm were prepared, followed by electrostatic adsorption of siRNA into pores. Oligonucleotide loading and release profiles were investigated; MRP1 mRNA and protein expression, cell viability and cell apoptosis were studied. Approximately 7.7 µg of siRNA was loaded per mg of pSiNPs. Cells readily took up nanoparticles after 30 min incubation. siRNA-loaded pSiNPs were able to effectively downregulate target mRNA (~40%) and protein expression (31%), and induced cell apoptosis and necrosis (33%). siRNA loaded pSiNPs downregulated mRNA and protein expression and induced cell death. This novel siRNA delivery system may pave the way towards developing more effective tumor therapies.

HFE mRNA expression is responsive to intracellular and extracellular iron loading: short communication.

PubMed

Mehta, Kosha J; Farnaud, Sebastien; Patel, Vinood B

2017-10-01

In liver hepatocytes, the HFE gene regulates cellular and systemic iron homeostasis by modulating cellular iron-uptake and producing the iron-hormone hepcidin in response to systemic iron elevation. However, the mechanism of iron-sensing in hepatocytes remain enigmatic. Therefore, to study the effect of iron on HFE and hepcidin (HAMP) expressions under distinct extracellular and intracellular iron-loading, we examined the effect of holotransferrin treatment (1, 2, 5 and 8 g/L for 6 h) on intracellular iron levels, and mRNA expressions of HFE and HAMP in wild-type HepG2 and previously characterized iron-loaded recombinant-TfR1 HepG2 cells. Gene expression was analyzed by real-time PCR and intracellular iron was measured by ferrozine assay. Data showed that in the wild-type cells, where intracellular iron content remained unchanged, HFE expression remained unaltered at low holotransferrin treatments but was upregulated upon 5 g/L (p < 0.04) and 8 g/L (p = 0.05) treatments. HAMP expression showed alternating elevations and increased upon 1 g/L (p < 0.05) and 5 g/L (p < 0.05). However, in the recombinant cells that showed higher intracellular iron levels than wild-type cells, HFE and HAMP expressions were elevated only at low 1 g/L treatment (p < 0.03) and were repressed at 2 g/L treatment (p < 0.03). Under holotransferrin-untreated conditions, the iron-loaded recombinant cells showed higher expressions of HFE (p < 0.03) and HAMP (p = 0.05) than wild-type cells. HFE mRNA was independently elevated by extracellular and intracellular iron-excess. Thus, it may be involved in sensing both, extracellular and intracellular iron. Repression of HAMP expression under simultaneous intracellular and extracellular iron-loading resembles non-hereditary iron-excess pathologies.

Molecular evidence and clinical importance of β-arrestins expression in patients with acromegaly.

PubMed

Coelho, Maria Caroline Alves; Vasquez, Marina Lipkin; Wildemberg, Luiz Eduardo; Vázquez-Borrego, Mari C; Bitana, Luciana; Camacho, Aline Helen da Silva; Silva, Débora; Ogino, Liana Lumi; Ventura, Nina; Chimelli, Leila; Luque, Raul M; Kasuki, Leandro; Gadelha, Mônica R

2018-04-01

β-arrestins seem to have a role in endocytosis and desensitization of somatostatin receptor subtype 2 (sst2) and could be associated with the responsiveness to somatostatin receptor ligands (SRL) in patients with acromegaly. To investigate the in vivo correlation between β-arrestins 1 and 2 with sst2, sst5 and dopamine receptor subtype 2 (D2) expressions, and the association of β-arrestins with response to first-generation SRL and invasiveness in somatotropinomas. β-arrestins 1 and 2, sst2, sst5 and D2 mRNA expressions were evaluated by quantitative real-time RT-PCR on tumoral tissue of 96 patients. Moreover, sst2 and sst5 protein expressions were also evaluated in 40 somatotropinomas by immunohistochemistry. Response to SRL, defined as GH <1 μg/l and normal IGF-I levels, was assessed in 40 patients. The Knosp-Steiner criteria were used to define invasiveness. Median β-arrestin 1, β-arrestin 2, sst2, sst5 and D2 mRNA copy numbers were 478; 9375; 731; 156; and 3989, respectively. There was a positive correlation between β-arrestins 1 and 2 (R = 0.444, P < 0.001). However, no correlation between β-arrestins and sst2, sst5 (mRNA and protein levels) or D2 was found. No association was found between β-arrestins expression and SRL responsiveness or tumour invasiveness. Although previous data suggest a putative correlation between β-arrestins and sst2, our data clearly indicated that no association existed between β-arrestins and sst2, sst5 or D2 expression, nor with response to SRL or tumour invasiveness. Therefore, further studies are required to clarify whether β-arrestins have a role in the response to treatment with SRL in acromegaly. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

The Splicing History of an mRNA Affects Its Level of Translation and Sensitivity to Cleavage by the Virion Host Shutoff Endonuclease during Herpes Simplex Virus Infections

PubMed Central

Sadek, Jouliana

2016-01-01

ABSTRACT During lytic herpes simplex virus (HSV) infections, the virion host shutoff (Vhs) (UL41) endoribonuclease degrades many cellular and viral mRNAs. In uninfected cells, spliced mRNAs emerge into the cytoplasm bound by exon junction complexes (EJCs) and are translated several times more efficiently than unspliced mRNAs that have the same sequence but lack EJCs. Notably, most cellular mRNAs are spliced, whereas most HSV mRNAs are not. To examine the effect of splicing on gene expression during HSV infection, cells were transfected with plasmids harboring an unspliced renilla luciferase (RLuc) reporter mRNA or RLuc constructs with introns near the 5′ or 3′ end of the gene. After splicing of intron-containing transcripts, all three RLuc mRNAs had the same primary sequence. Upon infection in the presence of actinomycin D, spliced mRNAs were much less sensitive to degradation by copies of Vhs from infecting virions than were unspliced mRNAs. During productive infections (in the absence of drugs), RLuc was expressed at substantially higher levels from spliced than from unspliced mRNAs. Interestingly, the stimulatory effect of splicing on RLuc expression was significantly greater in infected than in uninfected cells. The translational stimulatory effect of an intron during HSV-1 infections could be replicated by artificially tethering various EJC components to an unspliced RLuc transcript. Thus, the splicing history of an mRNA, and the consequent presence or absence of EJCs, affects its level of translation and sensitivity to Vhs cleavage during lytic HSV infections. IMPORTANCE Most mammalian mRNAs are spliced. In contrast, of the more than 80 mRNAs harbored by herpes simplex virus 1 (HSV-1), only 5 are spliced. In addition, synthesis of the immediate early protein ICP27 causes partial inhibition of pre-mRNA splicing, with the resultant accumulation of both spliced and unspliced versions of some mRNAs in the cytoplasm. A common perception is that HSV-1 infection necessarily inhibits the expression of spliced mRNAs. In contrast, this study demonstrates two instances in which pre-mRNA splicing actually enhances the synthesis of proteins from mRNAs during HSV-1 infections. Specifically, splicing stabilized an mRNA against degradation by copies of the Vhs endoribonuclease from infecting virions and greatly enhanced the amount of protein synthesized from spliced mRNAs at late times after infection. The data suggest that splicing, and the resultant presence of exon junction complexes on an mRNA, may play an important role in gene expression during HSV-1 infections. PMID:27681125

Gill structural integrity changes in fish deficient or excessive in dietary isoleucine: Towards the modulation of tight junction protein, inflammation, apoptosis and antioxidant defense via NF-κB, TOR and Nrf2 signaling pathways.

PubMed

Feng, Lin; Gan, Lu; Jiang, Wei-Dan; Wu, Pei; Liu, Yang; Jiang, Jun; Tang, Ling; Kuang, Sheng-Yao; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

2017-04-01

This study firstly aimed to test the impact of dietary isoleucine (Ile) on tight junction protein, inflammation, apoptosis, antioxidant defense and related signaling molecule gene expression in the gill of fish. Young grass carp (Ctenopharyngodon idella) (weighing 256.8 ± 3.5 g) were fed six diets containing graded levels of Ile, namely, 3.8, 6.6, 9.3, 12.5, 15.2 and 18.5 g/kg diet for 8 weeks. The results firstly revealed that Ile deficiency down-regulated the mRNA expressions of claudin-3, claudin-b, claudin-c, occludin and zonula occludens-1 (ZO-1) and up-regulated the mRNA expression of claudin-12, which led to the intercellular structure damage of fish gill. These effects were partially ascribed to the up-regulation of pro-inflammatory cytokines [interleukin 1β (IL-1β), interleukin 8 (IL-8) and tumor necrosis factor-α (TNF-α)] mRNA expressions that referring to up-regulated nuclear factor κB P65 (NF-κB P65) mRNA expression and down-regulated inhibitor factor κBα (IκBα) mRNA expression, and the down-regulation of anti-inflammatory cytokines [interleukin 10 (IL-10) and transforming growth factor β1 (TGF-β1)] mRNA expressions that referring to the down-regulated TOR and S6K1 mRNA expression. Interestingly, no change in claudin 15 mRNA level was observed among every treatment. At the same time, the results firstly indicated that Ile deficiency also resulted in the cellular structure damage of fish gill: (1) DNA fragmentation partially due to the up-regulation of caspase-3, caspase-8 and caspase-9 mRNA expression; (2) increase in protein carbonyl (PC), malondialdehyde (MDA) and ROS contents, which may be partially attributed to the impaired antioxidant defense [indicated by decreased glutathione (GSH) level and depressed anti-superoxide anion (ASA), anti-hydroxyl radical (a-HR), copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPx) activities] that referring to the down-regulation of corresponding antioxidant enzyme mRNA expressions and the related signaling molecules Nrf2 mRNA expression. Ile excess caused similar negative effects that observed in Ile-deficient group, whereas these negative effects were reversed with appropriate Ile supplementation. In conclusion, our results indicated that Ile deficiency or excess disrupted the structural integrity of fish gill, partially due to the trigger of apoptosis, the impairment of antioxidant defense, and the regulation of tight junction protein, inflammatory cytokines, apoptosis-related, antioxidant enzymes and related signaling molecules mRNA expressions in the fish gill. Copyright © 2017 Elsevier Ltd. All rights reserved.

Changes in Brain 14-3-3 Proteins in Response to Insulin Resistance Induced by a High Palatable Diet.

PubMed

Bock, Hugo; Zimmer, Aline Rigon; Zimmer, Eduardo Rigon; de Souza, Diogo Onofre Gomes; Portela, Luis Valmor Cruz; Saraiva-Pereira, Maria Luiza

2015-08-01

The 14-3-3 protein family takes part in a wide range of cellular processes and is expressed in all eukaryotic organisms. In mammals, seven isoforms (β, ε, η, γ, τ, ζ, and σ) have been identified. 14-3-3 proteins are suggested to modulate the insulin-signaling cascade in the brain. The aim of this study was to investigate whether insulin resistance state induced by high palatable diet modulates expression of the 14-3-3 proteins in brain. Wistar male rats (n = 8) were divided into two experimental groups: insulin resistant (IR), induced by high palatable diet, and control (CO) group. Biochemical parameters (glucose tolerance test and plasma lipid profile) were evaluated after 130 days. Brain structures (cortex and hippocampus) were dissected for evaluation of messenger RNA (mRNA) and protein levels of different 14-3-3 proteins. Statistical analyses included Student t test and Pearson correlation. Significant decrease was observed in Ywhah and in Ywahq mRNA levels in the cortex of IR group, while no changes were observed in the hippocampus. Significant increase of θ isoform was observed in hippocampus IR group by immunodetection, while no differences were detected in the remaining isoforms. Inverse correlation was observed between blood glucose levels in cortex IR group and both Ywhah and Ywhaq mRNA levels. Protein levels of Creb and phosphatidylinositide 3-kinases (PI3K) showed to be increased in the hippocampus. These alterations may be due to a compensatory effect of impaired insulin signaling. We demonstrated differential expression of 14-3-3 isoforms throughout brain regions of rats with IR. As a whole, our results indicate that brain 14-3-3 levels are influenced by different diets.

Nandrolone Normalizes Determinants of Muscle Mass and Fiber Type after Spinal Cord Injury

PubMed Central

Wu, Yong; Zhao, Jingbo; Zhao, Weidong; Pan, Jiangping; Bauman, William A.

2012-01-01

Abstract Spinal cord injury (SCI) results in atrophy of skeletal muscle and changes from slow oxidative to fast glycolytic fibers, which may reflect reduced levels of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), increased myostatin signaling, or both. In animals, testosterone reduces loss of muscle fiber cross-sectional area and activity of enzymes of energy metabolism. To identify the molecular mechanisms behind the benefits of androgens on paralyzed muscle, male rats were spinal cord transected and treated for 8 weeks with vehicle, testosterone at a physiological replacement dose, or testosterone plus nandrolone, an anabolic steroid. Treatments were initiated immediately after SCI and continued until the day animals were euthanized. In the SCI animals, gastrocnemius muscle mass was significantly increased by testosterone plus nandrolone, but not by testosterone alone. Both treatments significantly reduced nuclear content of Smad2/3 and mRNA levels of activin receptor IIB and follistatin-like 3. Testosterone alone or with nandrolone reversed SCI-induced declines in cellular and nuclear levels of PGC-1α protein and PGC-1α mRNA levels. For PGC-1α target genes, testosterone plus nandrolone partially reversed SCI-induced decreases in levels of proteins without corresponding increases in their mRNA levels. Thus, the findings demonstrate that following SCI, signaling through activin receptors and Smad2/3 is increased, and that androgens suppress activation of this signaling pathway. The findings also indicate that androgens upregulate PGC-1α in paralyzed muscle and promote its nuclear localization, but that these effects are insufficient to fully activate transcription of PGC-1α target genes. Furthermore, the transcription of these genes is not tightly coupled with their translation. PMID:22208735

Nandrolone normalizes determinants of muscle mass and fiber type after spinal cord injury.

PubMed

Wu, Yong; Zhao, Jingbo; Zhao, Weidong; Pan, Jiangping; Bauman, William A; Cardozo, Christopher P

2012-05-20

Spinal cord injury (SCI) results in atrophy of skeletal muscle and changes from slow oxidative to fast glycolytic fibers, which may reflect reduced levels of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), increased myostatin signaling, or both. In animals, testosterone reduces loss of muscle fiber cross-sectional area and activity of enzymes of energy metabolism. To identify the molecular mechanisms behind the benefits of androgens on paralyzed muscle, male rats were spinal cord transected and treated for 8 weeks with vehicle, testosterone at a physiological replacement dose, or testosterone plus nandrolone, an anabolic steroid. Treatments were initiated immediately after SCI and continued until the day animals were euthanized. In the SCI animals, gastrocnemius muscle mass was significantly increased by testosterone plus nandrolone, but not by testosterone alone. Both treatments significantly reduced nuclear content of Smad2/3 and mRNA levels of activin receptor IIB and follistatin-like 3. Testosterone alone or with nandrolone reversed SCI-induced declines in cellular and nuclear levels of PGC-1α protein and PGC-1α mRNA levels. For PGC-1α target genes, testosterone plus nandrolone partially reversed SCI-induced decreases in levels of proteins without corresponding increases in their mRNA levels. Thus, the findings demonstrate that following SCI, signaling through activin receptors and Smad2/3 is increased, and that androgens suppress activation of this signaling pathway. The findings also indicate that androgens upregulate PGC-1α in paralyzed muscle and promote its nuclear localization, but that these effects are insufficient to fully activate transcription of PGC-1α target genes. Furthermore, the transcription of these genes is not tightly coupled with their translation.

Paclitaxel-resistant HeLa cells have up-regulated levels of reactive oxygen species and increased expression of taxol resistance gene 1.

PubMed

Bi, Wenxiang; Wang, Yuxia; Sun, Gaoying; Zhang, Xiaojin; Wei, Yongqing; Li, Lu; Wang, Xiaoyuan

2014-07-01

This study is to establish a paclitaxel (PTX)-resistant human cervical carcinoma HeLa cell line (HeLa/PTX) and to investigate its redox characteristics and the expression of taxol resistance gene 1 (Txr1). HeLa cells were treated with PTX and effects of PTX on cell proliferation were detected through cell counting and the MTT assay. Levels of cellular reactive oxygen species (ROS), reduced glutathione (GSH), and oxidized glutathione (GSSG) as well as the ratio of GSH to GSSG were measured by the 2,7-difluorescein diacetate (DCFH-DA) method and the 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) method. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were determined by the nitrite formation method, the molybdate colorimetric method, and the DTNB colorimetric method, respectively. The level of Txr1 mRNA was determined by real-time PCR. Compared with the regular HeLa cells, HeLa/PTX cells were larger in size and had more cytoplasmic granules. The population doubling time for HeLa/PTX cells was 1.32 times of that of HeLa cells (P<0.01). HeLa/PTX cells showed stronger resistance to PTX than HeLa cells with a resistance index of 122.69. HeLa/PTX cells had higher levels of ROS (P<0.01) and Txr1 mRNA (P<0.01), lower level of GSH (P < 0.05), and lower activities of SOD (P<0.01) and GPx (P < 0.05) than HeLa cells. HeLa/PTX cells, with higher levels of ROS and Txr1 mRNA expression, are more resistant to PTX than HeLa cells.

Integrated regulation of PIKK-mediated stress responses by AAA+ proteins RUVBL1 and RUVBL2

PubMed Central

Izumi, Natsuko; Yamashita, Akio; Ohno, Shigeo

2012-01-01

Proteins of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family are activated by various cellular stresses, including DNA damage, premature termination codon and nutritional status, and induce appropriate cellular responses. The importance of PIKK functions in the maintenance of genome integrity, accurate gene expression and the proper control of cell growth/proliferation is established. Recently, ATPase associated diverse cellular activities (AAA+) proteins RUVBL1 and RUVBL2 (RUVBL1/2) have been shown to be common regulators of PIKKs. The RUVBL1/2 complex regulates PIKK-mediated stress responses through physical interactions with PIKKs and by controlling PIKK mRNA levels. In this review, the functions of PIKKs in stress responses are outlined and the physiological significance of the integrated regulation of PIKKs by the RUVBL1/2 complex is presented. We also discuss a putative “PIKK regulatory chaperone complex” including other PIKK regulators, Hsp90 and the Tel2 complex. PMID:22540023

Reverse engineering of gene regulatory networks.

PubMed

Cho, K H; Choo, S M; Jung, S H; Kim, J R; Choi, H S; Kim, J

2007-05-01

Systems biology is a multi-disciplinary approach to the study of the interactions of various cellular mechanisms and cellular components. Owing to the development of new technologies that simultaneously measure the expression of genetic information, systems biological studies involving gene interactions are increasingly prominent. In this regard, reconstructing gene regulatory networks (GRNs) forms the basis for the dynamical analysis of gene interactions and related effects on cellular control pathways. Various approaches of inferring GRNs from gene expression profiles and biological information, including machine learning approaches, have been reviewed, with a brief introduction of DNA microarray experiments as typical tools for measuring levels of messenger ribonucleic acid (mRNA) expression. In particular, the inference methods are classified according to the required input information, and the main idea of each method is elucidated by comparing its advantages and disadvantages with respect to the other methods. In addition, recent developments in this field are introduced and discussions on the challenges and opportunities for future research are provided.

Interplay of bistable kinetics of gene expression during cellular growth

NASA Astrophysics Data System (ADS)

Zhdanov, Vladimir P.

2009-02-01

In cells, the bistable kinetics of gene expression can be observed on the level of (i) one gene with positive feedback between protein and mRNA production, (ii) two genes with negative mutual feedback between protein and mRNA production, or (iii) in more complex cases. We analyse the interplay of two genes of type (ii) governed by a gene of type (i) during cellular growth. In particular, using kinetic Monte Carlo simulations, we show that in the case where gene 1, operating in the bistable regime, regulates mutually inhibiting genes 2 and 3, also operating in the bistable regime, the latter genes may eventually be trapped either to the state with high transcriptional activity of gene 2 and low activity of gene 3 or to the state with high transcriptional activity of gene 3 and low activity of gene 2. The probability to get to one of these states depends on the values of the model parameters. If genes 2 and 3 are kinetically equivalent, the probability is equal to 0.5. Thus, our model illustrates how different intracellular states can be chosen at random with predetermined probabilities. This type of kinetics of gene expression may be behind complex processes occurring in cells, e.g., behind the choice of the fate by stem cells.

β-Catenin recognizes a specific RNA motif in the cyclooxygenase-2 mRNA 3′-UTR and interacts with HuR in colon cancer cells

PubMed Central

Kim, Inae; Kwak, Hoyun; Lee, Hee Kyu; Hyun, Soonsil; Jeong, Sunjoo

2012-01-01

RNA-binding proteins regulate multiple steps of RNA metabolism through both dynamic and combined binding. In addition to its crucial roles in cell adhesion and Wnt-activated transcription in cancer cells, β-catenin regulates RNA alternative splicing and stability possibly by binding to target RNA in cells. An RNA aptamer was selected for specific binding to β-catenin to address RNA recognition by β-catenin more specifically. Here, we characterized the structural properties of the RNA aptamer as a model and identified a β-catenin RNA motif. Similar RNA motif was found in cellular RNA, Cyclooxygenase-2 (COX-2) mRNA 3′-untranslated region (3′-UTR). More significantly, the C-terminal domain of β-catenin interacted with HuR and the Armadillo repeat domain associated with RNA to form the RNA–β-catenin–HuR complex in vitro and in cells. Furthermore, the tertiary RNA–protein complex was predominantly found in the cytoplasm of colon cancer cells; thus, it might be related to COX-2 protein level and cancer progression. Taken together, the β-catenin RNA aptamer was valuable for deducing the cellular RNA aptamer and identifying novel and oncogenic RNA–protein networks in colon cancer cells. PMID:22544606

Lactuca capensis reverses memory deficits in Aβ1-42-induced an animal model of Alzheimer's disease.

PubMed

Postu, Paula Alexandra; Noumedem, Jaures A K; Cioanca, Oana; Hancianu, Monica; Mihasan, Marius; Ciorpac, Mitica; Gorgan, Dragos Lucian; Petre, Brindusa Alina; Hritcu, Lucian

2018-01-01

We investigated the neuropharmacological effects of the methanolic extract from Lactuca capensis Thunb. leaves (100 and 200 mg/kg) for 21 days on memory impairment in an Alzheimer's disease (AD) rat model produced by direct intraventricular delivery of amyloid-β1-42 (Aβ1-42). Behavioural assays such as Y-maze and radial arm maze test were used for assessing memory performance. Aβ1-42 decreased cognitive performance in the behavioural tests which were ameliorated by pre-treatment with the methanolic extract. Acetylcholinesterase activity and oxidant-antioxidant balance in the rat hippocampus were abnormally altered by Aβ1-42 treatment while these deficits were recovered by pre-treatment with the methanolic extract. In addition, rats were given Aβ1-42 exhibited in the hippocampus decreased brain-derived neurotrophic factor (BDNF) mRNA copy number and increased IL-1β mRNA copy number which was reversed by the methanolic extract administration. These findings suggest that the methanolic extract could be a potent neuropharmacological agent against dementia via modulating cholinergic activity, increasing of BDNF levels and promoting antioxidant action in the rat hippocampus. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Facing warm temperatures during migration: cardiac mRNA responses of two adult Oncorhynchus nerka populations to warming and swimming challenges.

PubMed

Anttila, K; Eliason, E J; Kaukinen, K H; Miller, K M; Farrell, A P

2014-05-01

The main findings of the current study were that exposing adult sockeye salmon Onchorhynchus nerka to a warm temperature that they regularly encounter during their river migration induced a heat shock response at an mRNA level, and this response was exacerbated with forced swimming. Similar to the heat shock response, increased immune defence-related responses were also observed after warm temperature treatment and with a swimming challenge in two different populations (Chilko and Nechako), but with some important differences. Microarray analyses revealed that 347 genes were differentially expressed between the cold (12-13° C) and warm (18-19° C) treated fish, with stress response (GO:0006950) and response to fungus (GO:0009620) elevated with warm treatment, while expression for genes involved in oxidative phosphorylation (GO:0006119) and electron transport chain (GO:0022900) elevated for cold-treated fish. Analysis of single genes with real-time quantitative PCR revealed that temperature had the most significant effect on mRNA expression levels, with swimming and population having secondary influences. Warm temperature treatment for the Chilko population induced expression of heat shock protein (hsp) 90α, hsp90β and hsp30 as well as interferon-inducible protein. The Nechako population, which is known to have a narrower thermal tolerance window than the Chilko population, showed even more pronounced stress responses to the warm treatment and there was significant interaction between population and temperature treatment for hsp90β expression. Moreover, significant interactions were noted between temperature treatment and swimming challenge for hsp90α and hsp30, and while swimming challenge alone increased expression of these hsps, the expression levels were significantly elevated in warm-treated fish swum to exhaustion. In conclusion, it seems that adult O. nerka currently encounter conditions that induce several cellular defence mechanisms during their once-in-the-lifetime migration. As river temperatures continue to increase, it remains to be seen whether or not these cellular defences provide sufficient protection for all O. nerka populations. © 2014 The Fisheries Society of the British Isles.

Differential Regulation of Duplicate Light-Dependent Protochlorophyllide Oxidoreductases in the Diatom Phaeodactylum tricornutum

PubMed Central

Hunsperger, Heather M.; Cattolico, Rose Ann

2016-01-01

Background Diatoms (Bacilliariophyceae) encode two light-dependent protochlorophyllide oxidoreductases (POR1 and POR2) that catalyze the penultimate step of chlorophyll biosynthesis in the light. Algae live in dynamic environments whose changing light levels induce photoacclimative metabolic shifts, including altered cellular chlorophyll levels. We hypothesized that the two POR proteins may be differentially adaptive under varying light conditions. Using the diatom Phaeodactylum tricornutum as a test system, differences in POR protein abundance and por gene expression were examined when this organism was grown on an alternating light:dark cycles at different irradiances; exposed to continuous light; and challenged by a significant decrease in light availability. Results For cultures maintained on a 12h light: 12h dark photoperiod at 200μE m−2 s−1 (200L/D), both por genes were up-regulated during the light and down-regulated in the dark, though por1 transcript abundance rose and fell earlier than that of por2. Little concordance occurred between por1 mRNA and POR1 protein abundance. In contrast, por2 mRNA and POR2 protein abundances followed similar diurnal patterns. When 200L/D P. tricornutum cultures were transferred to continuous light (200L/L), the diurnal regulatory pattern of por1 mRNA abundance but not of por2 was disrupted, and POR1 but not POR2 protein abundance dropped steeply. Under 1200μE m−2 s−1 (1200L/D), both por1 mRNA and POR1 protein abundance displayed diurnal oscillations. A compromised diel por2 mRNA response under 1200L/D did not impact the oscillation in POR2 abundance. When cells grown at 1200L/D were then shifted to 50μE m−2 s−1 (50L/D), por1 and por2 mRNA levels decreased swiftly but briefly upon light reduction. Thereafter, POR1 but not POR2 protein levels rose significantly in response to this light stepdown. Conclusion Given the sensitivity of diatom por1/POR1 to real-time light cues and adherence of por2/POR2 regulation to the diurnal cycle, we suggest that POR1 supports photoacclimation, whereas POR2 is the workhorse for daily chlorophyll synthesis. PMID:27367227

Nuclear imprisonment of host cellular mRNA by nsp1β protein of porcine reproductive and respiratory syndrome virus.

PubMed

Han, Mingyuan; Ke, Hanzhong; Zhang, Qingzhan; Yoo, Dongwan

2017-05-01

Positive-strand RNA genomes function as mRNA for viral protein synthesis which is fully reliant on host cell translation machinery. Competing with cellular protein translation apparatus needs to ensure the production of viral proteins, but this also stifles host innate defense. In the present study, we showed that porcine reproductive and respiratory syndrome virus (PRRSV), whose replication takes place in the cytoplasm, imprisoned host cell mRNA in the nucleus, which suggests a novel mechanism to enhance translation of PRRSV genome. PRRSV nonstructural protein (nsp) 1β was identified as the nuclear protein playing the role for host mRNA nuclear retention and subversion of host protein synthesis. A SAP (SAF-A/B, Acinus, and PIAS) motif was identified in nsp1β with the consensus sequence of 126 -LQxxLxxxGL- 135 . In situ hybridization unveiled that SAP mutants were unable to cause nuclear retention of host cell mRNAs and did not suppress host protein synthesis. In addition, these SAP mutants reverted PRRSV-nsp1β-mediated suppression of interferon (IFN) production, IFN signaling, and TNF-α production pathway. Using reverse genetics, a series of SAP mutant PRRS viruses, vK124A, vL126A, vG134A, and vL135A were generated. No mRNA nuclear retention was observed during vL126A and vL135A infections. Importantly, vL126A and vL135A did not suppress IFN production. For other arteriviruses, mRNA nuclear accumulation was also observed for LDV-nsp1β and SHFV-nsp1β. EAV-nsp1 was exceptional and did not block the host mRNA nuclear export. Copyright © 2017 Elsevier Inc. All rights reserved.

Distinct expression patterns of the E3 ligase SIAH-1 and its partner Kid/KIF22 in normal tissues and in the breast tumoral processes

PubMed Central

2010-01-01

SIAH proteins are the human members of an highly conserved family of E3 ubiquitin ligases. Several data suggest that SIAH proteins may have a role in tumor suppression and apoptosis. Previously, we reported that SIAH-1 induces the degradation of Kid (KIF22), a chromokinesin protein implicated in the normal progression of mitosis and meiosis, by the ubiquitin proteasome pathway. In human breast cancer cells stably transfected with SIAH-1, Kid/KIF22 protein level was markedly reduced whereas, the Kid/KIF22 mRNA level was increased. This interaction has been further elucidated through analyzing SIAH and Kid/KIF22 expression in both paired normal and tumor tissues and cell lines. It was observed that SIAH-1 protein is widely expressed in different normal tissues, and in cells lines but showing some differences in western blotting profiles. Immunofluorescence microscopy shows that the intracellular distribution of SIAH-1 and Kid/KIF22 appears to be modified in human tumor tissues compared to normal controls. When mRNA expression of SIAH-1 and Kid/KIF22 was analyzed by real-time PCR in normal and cancer breast tissues from the same patient, a large variation in the number of mRNA copies was detected between the different samples. In most cases, SIAH-1 mRNA is decreased in tumor tissues compared to their normal counterparts. Interestingly, in all breast tumor tissues analyzed, variations in the Kid/KIF22 mRNA levels mirrored those seen with SIAH-1 mRNAs. This concerted variation of SIAH-1 and Kid/KIF22 messengers suggests the existence of an additional level of control than the previously described protein-protein interaction and protein stability regulation. Our observations also underline the need to re-evaluate the results of gene expression obtained by qRT-PCR and relate it to the protein expression and cellular localization when matched normal and tumoral tissues are analyzed. PMID:20144232

Interplay between Selenium Levels, Selenoprotein Expression, and Replicative Senescence in WI-38 Human Fibroblasts*

PubMed Central

Legrain, Yona; Touat-Hamici, Zahia; Chavatte, Laurent

2014-01-01

Selenium is an essential trace element, which is incorporated as selenocysteine into at least 25 selenoproteins using a unique translational UGA-recoding mechanism. Selenoproteins are important enzymes involved in antioxidant defense, redox homeostasis, and redox signaling pathways. Selenium levels decline during aging, and its deficiency is associated with a marked increase in mortality for people over 60 years of age. Here, we investigate the relationship between selenium levels in the culture medium, selenoprotein expression, and replicative life span of human embryonic lung fibroblast WI-38 cells. Selenium levels regulate the entry into replicative senescence and modify the cellular markers characteristic for senescent cells. Whereas selenium supplementation extends the number of population doublings, its deficiency impairs the proliferative capacity of WI-38 cells. We observe that the expression of several selenoproteins involved in antioxidant defense is specifically affected in response to cellular senescence. Their expression is selectively controlled by the modulation of mRNA levels and translational recoding efficiencies. Our data provide novel mechanistic insights into how selenium impacts the replicative life span of mammalian cells by identifying several selenoproteins as new targets of senescence. PMID:24425862

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

PubMed

Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell W; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay A A

2014-10-01

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.

Gene specific epigenetic regulation of hepatic folate transport system is responsible for perturbed cellular folate status during aging and exogenous modulation.

PubMed

Ahmad Najar, Rauf; Rahat, Beenish; Hussain, Aashiq; Thakur, Shilpa; Kaur, Jaspreet; Kaur, Jyotdeep; Hamid, Abid

2016-06-01

The present study was designed to identify the molecular mechanism of folate modulation and aging on aberrant liver folate transporter system. An in vivo rat model was used, in which weanling, young and adult rats were given folate deficient diet for 3 and 5 months and after 3 months of folate deficiency, one group received physiological folate repletion (2 mg/kg diet) and another group received over supplemented folate diet (8 mg/kg diet) for another 2 months. In adult group, 3 and 5 months of folate deficiency decreased serum and tissue folate levels with decreased uptake of folate, further associated with decreased expression levels of reduced folate carrier (RFC) and increased expression levels of folate exporter (ABCG2) at both mRNA and protein levels, which in turn regulated by promoter hypermethylation of RFC and promoter hypomethylation of ABCG2 gene. Promoter hypermethylation of RFC and promoter hypomethylation of ABCG2 may be attributed to the down regulation of RFC and up regulation of ABCG2 at mRNA and protein levels in conditions of 3 and 5 months of folate deficiency in the adult group. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differences in brain gene expression between sleep and waking as revealed by mRNA differential display and cDNA microarray technology.

PubMed

Cirelli, C; Tononi, G

1999-06-01

The consequences of sleep and sleep deprivation at the molecular level are largely unexplored. Knowledge of such molecular events is essential to understand the restorative processes occurring during sleep as well as the cellular mechanisms of sleep regulation. Here we review the available data about changes in neural gene expression across different behavioural states using candidate gene approaches such as in situ hybridization and immunocytochemistry. We then describe new techniques for systematic screening of gene expression in the brain, such as subtractive hybridization, mRNA differential display, and cDNA microarray technology, outlining advantages and disadvantages of these methods. Finally, we summarize our initial results of a systematic screening of gene expression in the rat brain across behavioural states using mRNA differential display and cDNA microarray technology. The expression pattern of approximately 7000 genes was analysed in the cerebral cortex of rats after 3 h of spontaneous sleep, 3 h of spontaneous waking, or 3 h of sleep deprivation. While the majority of transcripts were expressed at the same level among these three conditions, 14 mRNAs were modulated by sleep and waking. Six transcripts, four more expressed in waking and two more expressed in sleep, corresponded to novel genes. The eight known transcripts were all expressed at higher levels in waking than in sleep and included transcription factors and mitochondrial genes. A possible role for these known transcripts in mediating neural plasticity during waking is discussed.

Venlafaxine treatment after endothelin-1-induced cortical stroke modulates growth factor expression and reduces tissue damage in rats.

PubMed

Zepeda, Rodrigo; Contreras, Valentina; Pissani, Claudia; Stack, Katherine; Vargas, Macarena; Owen, Gareth I; Lazo, Oscar M; Bronfman, Francisca C

2016-08-01

Neuromodulators, such as antidepressants, may contribute to neuroprotection by modulating growth factor expression to exert anti-inflammatory effects and to support neuronal plasticity after stroke. Our objective was to study whether early treatment with venlafaxine, a serotonin-norepinephrine reuptake inhibitor, modulates growth factor expression and positively contributes to reducing the volume of infarcted brain tissue resulting in increased functional recovery. We studied the expression of BDNF, FGF2 and TGF-β1 by examining their mRNA and protein levels and cellular distribution using quantitative confocal microscopy at 5 days after venlafaxine treatment in control and infarcted brains. Venlafaxine treatment did not change the expression of these growth factors in sham rats. In infarcted rats, BDNF mRNA and protein levels were reduced, while the mRNA and protein levels of FGF2 and TGF-β1 were increased. Venlafaxine treatment potentiated all of the changes that were induced by cortical stroke alone. In particular, increased levels of FGF2 and TGF-β1 were observed in astrocytes at 5 days after stroke induction, and these increases were correlated with decreased astrogliosis (measured by GFAP) and increased synaptophysin immunostaining at twenty-one days after stroke in venlafaxine-treated rats. Finally, we show that venlafaxine reduced infarct volume after stroke resulting in increased functional recovery, which was measured using ladder rung motor tests, at 21 days after stroke. Our results indicate that the early oral administration of venlafaxine positively contributes to neuroprotection during the acute and late events that follow stroke. Copyright © 2016 Elsevier Ltd. All rights reserved.

The HDL receptor SR-BI is associated with human prostate cancer progression and plays a possible role in establishing androgen independence.

PubMed

Schörghofer, David; Kinslechner, Katharina; Preitschopf, Andrea; Schütz, Birgit; Röhrl, Clemens; Hengstschläger, Markus; Stangl, Herbert; Mikula, Mario

2015-08-07

Human prostate cancer represents one of the most frequently diagnosed cancers in men worldwide. Currently, diagnostic methods are insufficient to identify patients at risk for aggressive prostate cancer, which is essential for early treatment. Recent data indicate that elevated cholesterol levels in the plasma are a prerequisite for the progression of prostate cancer. Here, we analyzed clinical prostate cancer samples for the expression of receptors involved in cellular cholesterol uptake. We screened mRNA microarray files of prostate cancer samples for alterations in the expression levels of cholesterol transporters. Furthermore, we performed immunohistochemistry analysis on human primary prostate cancer tissue sections derived from patients to investigate the correlation of SR-BI with clinicopathological parameters and the mTOR target pS6. In contrast to LDLR, we identified SR-BI mRNA and protein expression to be induced in high Gleason grade primary prostate cancers. Histologic analysis of prostate biopsies revealed that 53.6 % of all cancer samples and none of the non-cancer samples showed high SR-BI staining intensity. The disease-free survival time was reduced (P = 0.02) in patients expressing high intra-tumor levels of SR-BI. SR-BI mRNA correlated with HSD17B1 and HSD3B1 and SR-BI protein staining showed correlation with active ribosomal protein S6 (RS = 0.828, P < 0.00001). We identified SR-BI to indicate human prostate cancer formation, suggesting that increased levels of SR-BI may be involved in the generation of a castration-resistant phenotype.

Substance P activates ADAM9 mRNA expression and induces α-secretase-mediated amyloid precursor protein cleavage.

PubMed

Marolda, R; Ciotti, M T; Matrone, C; Possenti, R; Calissano, P; Cavallaro, S; Severini, C

2012-04-01

Altered levels of Substance P (SP), a neuropeptide endowed with neuroprotective and anti-apoptotic properties, were found in brain areas and spinal fluid of Alzheimer's disease (AD) patients. One of the hallmarks of AD is the abnormal extracellular deposition of neurotoxic beta amyloid (Aβ) peptides, derived from the proteolytic processing of amyloid precursor protein (APP). In the present study, we confirmed, the neurotrophic action of SP in cultured rat cerebellar granule cells (CGCs) and investigated its effects on APP metabolism. Incubation with low (5 mM) potassium induced apoptotic cell death of CGCs and amyloidogenic processing of APP, whereas treatment with SP (200 nM) reverted these effects via NK1 receptors. The non-amyloidogenic effect of SP consisted of reduction of Aβ(1-42), increase of sAPPα and enhanced α-secretase activity, without a significant change in steady-state levels of cellular APP. The intracellular mechanisms whereby SP alters APP metabolism were further investigated by measuring mRNA and/or steady-state protein levels of key enzymes involved with α-, β- and γ-secretase activity. Among them, Adam9, both at the mRNA and protein level, was the only enzyme to be significantly down-regulated following the induction of apoptosis (K5) and up-regulated after SP treatment. In addition to its neuroprotective properties, this study shows that SP is able to stimulate non-amyloidogenic APP processing, thereby reducing the possibility of generation of toxic Aβ peptides in brain. Copyright © 2011 Elsevier Ltd. All rights reserved.

Gene expression during skeletal development in three osteopetrotic rat mutations. Evidence for osteoblast abnormalities.

PubMed

Shalhoub, V; Jackson, M E; Lian, J B; Stein, G S; Marks, S C

1991-05-25

Osteopetrosis is a group of metabolic bone diseases characterized by reductions in osteoclast development and/or function. These aspects of osteoclast biology are known to be influenced by osteoblasts and their products. To ascertain whether osteoblast dysfunction contributes to aberrations in the structural and functional properties of osteoclasts in osteopetrosis, we systematically examined gene expression as reflected by mRNA levels for a series of cell growth- and tissue-related genes associated with the osteoblast phenotype during skeletal development in normal and mutant rats of three different osteopetrotic stocks. We show that the methods used permit the reproducible isolation of undegraded total cellular RNA from bone and that mRNA levels can be reliably quantitated in these preparations. Each osteopetrotic mutation exhibits a distinct aberrant pattern of osteoblast gene expression that may be correlated with and explain some abnormalities in extracellular matrix composition, mineralization, osteoclast development, and effects of elevated serum levels of 1 alpha,25-dihydroxyvitamin D3, depending upon the mutation. Normal rats show minor variations in gene expression that reflect the genetic background (stock). This, the first comprehensive molecular analysis of osteoblast gene expression in osteopetrosis, suggests that some osteopetroses, particularly in the toothless rat, are associated with and potentially related to mechanisms associated with aberrations in osteoblast function. More generally, the present studies demonstrate alterations in gene expression as reflected by mRNA levels that are associated with functional properties of the osteoblast, particularly those contributing to the recruitment and/or differentiation of osteoclasts, thereby influencing skeletal modeling.

Whi3, an S. cerevisiae RNA-binding protein, is a component of stress granules that regulates levels of its target mRNAs.

PubMed

Holmes, Kristen J; Klass, Daniel M; Guiney, Evan L; Cyert, Martha S

2013-01-01

RNA binding proteins (RBPs) are vital to the regulation of mRNA transcripts, and can alter mRNA localization, degradation, translation, and storage. Whi3 was originally identified in a screen for small cell size mutants, and has since been characterized as an RBP. The identification of Whi3-interacting mRNAs involved in mediating cellular responses to stress suggested that Whi3 might be involved in stress-responsive RNA processing. We show that Whi3 localizes to stress granules in response to glucose deprivation or heat shock. The kinetics and pattern of Whi3 localization in response to a range of temperatures were subtly but distinctly different from those of known components of RNA processing granules. Deletion of Whi3 resulted in an increase in the relative abundance of Whi3 target RNAs, either in the presence or absence of heat shock. Increased levels of the CLN3 mRNA in whi3Δ cells may explain their decreased cell size. Another mRNA target of Whi3 encodes the zinc-responsive transcription factor Zap1, suggesting a role for Whi3 in response to zinc stress. Indeed, we found that whi3Δ cells have enhanced sensitivity to zinc toxicity. Together our results suggest an expanded model for Whi3 function: in addition to its role as a regulator of the cell cycle, Whi3 may have a role in stress-dependent RNA processing and responses to a variety of stress conditions.

Iron regulatory protein 1 is not required for the modulation of ferritin and transferrin receptor expression by iron in a murine pro-B lymphocyte cell line

PubMed Central

Schalinske, Kevin L.; Blemings, Kenneth P.; Steffen, Daniel W.; Chen, Opal S.; Eisenstein, Richard S.

1997-01-01

Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that are central components of a sensory and regulatory network that modulates vertebrate iron homeostasis. IRPs regulate iron metabolism by binding to iron responsive element(s) (IREs) in the 5′ or 3′ untranslated region of ferritin or transferrin receptor (TfR) mRNAs. Two IRPs, IRP1 and IRP2, have been identified previously. IRP1 exhibits two mutually exclusive functions as an RNA binding protein or as the cytosolic isoform of aconitase. We demonstrate that the Ba/F3 family of murine pro-B lymphocytes represents the first example of a mammalian cell line that fails to express IRP1 protein or mRNA. First, all of the IRE binding activity in Ba/F3-gp55 cells is attributable to IRP2. Second, synthesis of IRP2, but not of IRP1, is detectable in Ba/F3-gp55 cells. Third, the Ba/F3 family of cells express IRP2 mRNA at a level similar to other murine cell lines, but IRP1 mRNA is not detectable. In the Ba/F3 family of cells, alterations in iron status modulated ferritin biosynthesis and TfR mRNA level over as much as a 20- and 14-fold range, respectively. We conclude that IRP1 is not essential for regulation of ferritin or TfR expression by iron and that IRP2 can act as the sole IRE-dependent mediator of cellular iron homeostasis. PMID:9380695

Deregulated Expression of SRC, LYN and CKB Kinases by DNA Methylation and Its Potential Role in Gastric Cancer Invasiveness and Metastasis

PubMed Central

Rey, Juan Antonio; Pinto, Giovanny Rebouças; Lamarão, Leticia Martins; Montenegro, Raquel Carvalho; Alves, Ana Paula Negreiros Nunes; Assumpção, Paulo Pimentel; Borges, Barbara do Nascimento; Smith, Marília Cardoso; Burbano, Rommel Rodriguez

2015-01-01

Kinases are downstream modulators and effectors of several cellular signaling cascades and play key roles in the development of neoplastic disease. In this study, we aimed to evaluate SRC, LYN and CKB protein and mRNA expression, as well as their promoter methylation, in gastric cancer. We found elevated expression of SRC and LYN kinase mRNA and protein but decreased levels of CKB kinase, alterations that may have a role in the invasiveness and metastasis of gastric tumors. Expression of the three studied kinases was also associated with MYC oncogene expression, a possible biomarker for gastric cancer. To understand the mechanisms that regulate the expression of these genes, we evaluated the DNA promoter methylation of the three kinases. We found that reduced SRC and LYN methylation and increased CKB methylation was associated with gastric cancer. The reduced SRC and LYN methylation was associated with increased levels of mRNA and protein expression, suggesting that DNA methylation is involved in regulating the expression of these kinases. Conversely, reduced CKB methylation was observed in samples with reduced mRNA and protein expression, suggesting CKB expression was found to be only partly regulated by DNA methylation. Additionally, we found that alterations in the DNA methylation pattern of the three studied kinases were also associated with the gastric cancer onset, advanced gastric cancer, deeper tumor invasion and the presence of metastasis. Therefore, SRC, LYN and CKB expression or DNA methylation could be useful markers for predicting tumor progression and targeting in anti-cancer strategies. PMID:26460485

Deregulated Expression of SRC, LYN and CKB Kinases by DNA Methylation and Its Potential Role in Gastric Cancer Invasiveness and Metastasis.

PubMed

Mello, Adriano Azevedo; Leal, Mariana Ferreira; Rey, Juan Antonio; Pinto, Giovanny Rebouças; Lamarão, Leticia Martins; Montenegro, Raquel Carvalho; Alves, Ana Paula Negreiros Nunes; Assumpção, Paulo Pimentel; Borges, Barbara do Nascimento; Smith, Marília Cardoso; Burbano, Rommel Rodriguez

2015-01-01

Kinases are downstream modulators and effectors of several cellular signaling cascades and play key roles in the development of neoplastic disease. In this study, we aimed to evaluate SRC, LYN and CKB protein and mRNA expression, as well as their promoter methylation, in gastric cancer. We found elevated expression of SRC and LYN kinase mRNA and protein but decreased levels of CKB kinase, alterations that may have a role in the invasiveness and metastasis of gastric tumors. Expression of the three studied kinases was also associated with MYC oncogene expression, a possible biomarker for gastric cancer. To understand the mechanisms that regulate the expression of these genes, we evaluated the DNA promoter methylation of the three kinases. We found that reduced SRC and LYN methylation and increased CKB methylation was associated with gastric cancer. The reduced SRC and LYN methylation was associated with increased levels of mRNA and protein expression, suggesting that DNA methylation is involved in regulating the expression of these kinases. Conversely, reduced CKB methylation was observed in samples with reduced mRNA and protein expression, suggesting CKB expression was found to be only partly regulated by DNA methylation. Additionally, we found that alterations in the DNA methylation pattern of the three studied kinases were also associated with the gastric cancer onset, advanced gastric cancer, deeper tumor invasion and the presence of metastasis. Therefore, SRC, LYN and CKB expression or DNA methylation could be useful markers for predicting tumor progression and targeting in anti-cancer strategies.

Immune-Specific Expression and Estrogenic Regulation of the Four Estrogen Receptor Isoforms in Female Rainbow Trout (Oncorhynchus mykiss).

PubMed

Casanova-Nakayama, Ayako; Wernicke von Siebenthal, Elena; Kropf, Christian; Oldenberg, Elisabeth; Segner, Helmut

2018-03-21

Genomic actions of estrogens in vertebrates are exerted via two intracellular estrogen receptor (ER) subtypes, ERα and ERβ, which show cell- and tissue-specific expression profiles. Mammalian immune cells express ERs and are responsive to estrogens. More recently, evidence became available that ERs are also present in the immune organs and cells of teleost fish, suggesting that the immunomodulatory function of estrogens has been conserved throughout vertebrate evolution. For a better understanding of the sensitivity and the responsiveness of the fish immune system to estrogens, more insight is needed on the abundance of ERs in the fish immune system, the cellular ratios of the ER subtypes, and their autoregulation by estrogens. Consequently, the aims of the present study were (i) to determine the absolute mRNA copy numbers of the four ER isoforms in the immune organs and cells of rainbow trout, Oncorhynchus mykiss , and to compare them to the hepatic ER numbers; (ii) to analyse the ER mRNA isoform ratios in the immune system; and, (iii) finally, to examine the alterations of immune ER mRNA expression levels in sexually immature trout exposed to 17β-estradiol (E2), as well as the alterations of immune ER mRNA expression levels in sexually mature trout during the reproductive cycle. All four ER isoforms were present in immune organs-head kidney, spleen-and immune cells from head kidney and blood of rainbow trout, but their mRNA levels were substantially lower than in the liver. The ER isoform ratios were tissue- and cell-specific, both within the immune system, but also between the immune system and the liver. Short-term administration of E2 to juvenile female trout altered the ER mRNA levels in the liver, but the ERs of the immune organs and cells were not responsive. Changes of ER gene transcript numbers in immune organs and cells occurred during the reproductive cycle of mature female trout, but the changes in the immune ER profiles differed from those in the liver and gonads. The correlation between ER gene transcript numbers and serum E2 concentrations was only moderate to low. In conclusion, the low mRNA numbers of nuclear ER in the trout immune system, together with their limited estrogen-responsiveness, suggest that the known estrogen actions on trout immunity may be not primarily mediated through genomic actions, but may involve other mechanisms, such as non-genomic pathways or indirect effects.

Characterizing the effects of inorganic acid and alkaline shock on the Staphylococcus aureus transcriptome and messenger RNA turnover.

PubMed

Anderson, Kelsi L; Roux, Christelle M; Olson, Matthew W; Luong, Thanh T; Lee, Chia Y; Olson, Robert; Dunman, Paul M

2010-12-01

Staphylococcus aureus pathogenesis can be attributed partially to its ability to adapt to otherwise deleterious host-associated stresses. Here, Affymetrix GeneChips® were used to examine the S. aureus responses to inorganic acid and alkaline shock and to assess whether stress-dependent changes in mRNA turnover are likely to facilitate the organism's ability to tolerate a pH challenge. The results indicate that S. aureus adapts to pH shock by eliciting responses expected of cells coping with pH alteration, including neutralizing cellular pH, DNA repair, amino acid biosynthesis, and virulence factor expression. Further, the S. aureus response to alkaline conditions is strikingly similar to that of stringent response-induced cells. Indeed, we show that alkaline shock stimulates the accumulation of the stringent response activator (p)ppGpp. The results also revealed that pH shock significantly alters the mRNA properties of the cell. A comparison of the mRNA degradation properties of transcripts whose titers either increased or decreased in response to a sudden pH change revealed that alterations in mRNA degradation may, in part, account for the changes in the mRNA levels of factors predicted to mediate pH tolerance. A set of small stable RNA molecules were induced in response to acid- or alkaline-shock conditions and may mediate adaptation to pH stress. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Conceptual Modeling in Systems Biology Fosters Empirical Findings: The mRNA Lifecycle

PubMed Central

Dori, Dov; Choder, Mordechai

2007-01-01

One of the main obstacles to understanding complex biological systems is the extent and rapid evolution of information, way beyond the capacity individuals to manage and comprehend. Current modeling approaches and tools lack adequate capacity to model concurrently structure and behavior of biological systems. Here we propose Object-Process Methodology (OPM), a holistic conceptual modeling paradigm, as a means to model both diagrammatically and textually biological systems formally and intuitively at any desired number of levels of detail. OPM combines objects, e.g., proteins, and processes, e.g., transcription, in a way that is simple and easily comprehensible to researchers and scholars. As a case in point, we modeled the yeast mRNA lifecycle. The mRNA lifecycle involves mRNA synthesis in the nucleus, mRNA transport to the cytoplasm, and its subsequent translation and degradation therein. Recent studies have identified specific cytoplasmic foci, termed processing bodies that contain large complexes of mRNAs and decay factors. Our OPM model of this cellular subsystem, presented here, led to the discovery of a new constituent of these complexes, the translation termination factor eRF3. Association of eRF3 with processing bodies is observed after a long-term starvation period. We suggest that OPM can eventually serve as a comprehensive evolvable model of the entire living cell system. The model would serve as a research and communication platform, highlighting unknown and uncertain aspects that can be addressed empirically and updated consequently while maintaining consistency. PMID:17849002

The effect of Bacopa monnieri on gene expression levels in SH-SY5Y human neuroblastoma cells.

PubMed

Leung, How-Wing; Foo, Gabriel; Banumurthy, Gokulakrishna; Chai, Xiaoran; Ghosh, Sujoy; Mitra-Ganguli, Tora; VanDongen, Antonius M J

2017-01-01

Bacopa monnieri is a plant used as a nootropic in Ayurveda, a 5000-year-old system of traditional Indian medicine. Although both animal and clinical studies supported its role as a memory enhancer, the molecular and cellular mechanism underlying Bacopa's nootropic action are not understood. In this study, we used deep sequencing (RNA-Seq) to identify the transcriptome changes upon Bacopa treatment on SH-SY5Y human neuroblastoma cells. We identified several genes whose expression levels were regulated by Bacopa. Biostatistical analysis of the RNA-Seq data identified biological pathways and molecular functions that were regulated by Bacopa, including regulation of mRNA translation and transmembrane transport, responses to oxidative stress and protein misfolding. Pathway analysis using the Ingenuity platform suggested that Bacopa may protect against brain damage and improve brain development. These newly identified molecular and cellular determinants may contribute to the nootropic action of Bacopa and open up a new direction of investigation into its mechanism of action.

The effect of Bacopa monnieri on gene expression levels in SH-SY5Y human neuroblastoma cells

PubMed Central

Foo, Gabriel; Banumurthy, Gokulakrishna; Chai, Xiaoran; Ghosh, Sujoy

2017-01-01

Bacopa monnieri is a plant used as a nootropic in Ayurveda, a 5000-year-old system of traditional Indian medicine. Although both animal and clinical studies supported its role as a memory enhancer, the molecular and cellular mechanism underlying Bacopa’s nootropic action are not understood. In this study, we used deep sequencing (RNA-Seq) to identify the transcriptome changes upon Bacopa treatment on SH-SY5Y human neuroblastoma cells. We identified several genes whose expression levels were regulated by Bacopa. Biostatistical analysis of the RNA-Seq data identified biological pathways and molecular functions that were regulated by Bacopa, including regulation of mRNA translation and transmembrane transport, responses to oxidative stress and protein misfolding. Pathway analysis using the Ingenuity platform suggested that Bacopa may protect against brain damage and improve brain development. These newly identified molecular and cellular determinants may contribute to the nootropic action of Bacopa and open up a new direction of investigation into its mechanism of action. PMID:28832626

Activation of hepatic Nogo-B receptor expression—A new anti-liver steatosis mechanism of statins

PubMed Central

Zhang, Wenwen; Yang, Xiaoxiao; Chen, Yuanli; Hu, Wenquan; Liu, Lipei; Zhang, Xiaomeng; Liu, Mengyang; Sun, Lei; Liu, Ying; Yu, Miao; Li, Xiaoju; Li, Luyuan; Zhu, Yan; Miao, Qing Robert; Han, Jihong; Duan, Yajun

2017-01-01

Deficiency of hepatic Nogo-B receptor (NgBR) expression activates liver X receptor α (LXRα) in an adenosine monophosphate-activated protein kinase α (AMPKα)-dependent manner, thereby inducing severe hepatic lipid accumulation and hypertriglyceridemia. Statins have been demonstrated non-cholesterol lowering effects including anti-nonalcoholic fatty liver disease (NAFLD). Herein, we investigated if the anti-NAFLD function of statins depends on activation of NgBR expression. In vivo, atorvastatin protected apoE deficient or NgBR floxed, but not hepatic NgBR deficient mice, against Western diet (WD)-increased triglyceride levels in liver and serum. In vitro, statins reduced lipid accumulation in nonsilencing small hairpin RNA-transfected (shNSi), but not in NgBR small hairpin RNA-transfected (shNgBRi) HepG2 cells. Inhibition of cellular lipid accumulation by atorvastatin is related to activation of AMPKα, and inactivation of LXRα and lipogenic genes. Statin also inhibited expression of oxysterol producing enzymes. Associated with changes of hepatic lipid levels by WD or atorvastatin, NgBR expression was inversely regulated. At cellular levels, statins increased NgBR mRNA and protein expression, and NgBR protein stability. In contrast to reduced cellular cholesterol levels by statin or β-cyclodextrin, increased cellular cholesterol levels decreased NgBR expression suggesting cholesterol or its synthesis intermediates inhibit NgBR expression. Indeed, mevalonate, geranylgeraniol or geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate or farnesol, blocked atorvastatin-induced NgBR expression. Furthermore, we determined that induction of hepatic NgBR expression by atorvastatin mainly depended on inactivation of extracellular signal-regulated kinases 1/2 (ERK1/2) and protein kinase B (Akt). Taken together, our study demonstrates that statins inhibit NAFLD mainly through activation of NgBR expression. PMID:29217477

Prenatal arsenic exposure alters the programming of the glucocorticoid signaling system during embryonic development

PubMed Central

Caldwell, Katharine E.; Labrecque, Matthew T.; Solomon, Benjamin R.; Ali, Abdulmehdi; Allan, Andrea M.

2015-01-01

The glucocorticoid system, which plays a critical role in a host of cellular functions including mood disorders and learning and memory, has been reported to be disrupted by arsenic. In previous work we have developed and characterized a prenatal moderate arsenic exposure (50 ppb) model and identified several deficits in learning and memory and mood disorders, as well as alterations within the glucocorticoid receptor signaling system in the adolescent mouse. In these present studies we assessed the effects of arsenic on the glucocorticoid receptor (GR) pathway in both the placenta and the fetal brain in response at two critical periods, embryonic days 14 and 18. The focus of these studies was on the 11β-hydroxysteroid dehydrogenase enzymes (11β-HSD1 and 11β-HSD2) which play a key role in glucorticoid synthesis, as well as the expression and set point of the GR negative feedback regulation. Negative feedback regulation is established early in development. At E14 we found arsenic exposure significantly decreased expression of both protein and message in brain of GR and the 11β-HSD1, while 11β-HSD2 enzyme protein levels were increased but mRNA levels were decreased in the brain. These changes in brain protein continued into the E18 time point, but mRNA levels were no longer significantly altered. Placental HSD11B2 mRNA was not altered by arsenic treatment but protein levels were elevated at E14. GR placental protein levels were decreased at E18 in the arsenic exposed condition. This suggests that arsenic exposure may alter GR expression levels as a consequence of a prolonged developmental imbalance between 11β-HSD1 and 11β-HSD2 protein expression despite decreased 11HSDB2 mRNA. The suppression of GR and the failure to turn down 11β-HSD2 protein expression during fetal development may lead to an altered set point for GR signaling throughout adulthood. To our knowledge, these studies are the first to demonstrate that gestational exposure to moderate levels of arsenic results in altered fetal programming of the glucocorticoid system. PMID:25459689

CD147 regulates the expression of MCT1 and lactate export in multiple myeloma cells

PubMed Central

Walters, Denise K; Arendt, Bonnie K; Jelinek, Diane F

2013-01-01

Increased use of the glycolytic pathway, even in the presence of oxygen, has recently been recognized as a key characteristic of malignant cells. However, the glycolytic phenotype results in increased lactic acid production and, in order to prevent cellular acidosis, tumor cells must increase proton efflux via upregulation of pH regulators such as proton-pumps, sodium-proton exchangers, and/or monocarboxylate transporters (MCT) (e.g., MCT1, MCT4). Interestingly, expression of MCT1 and MCT4 has been previously shown to be dependent upon expression of the transmembrane glycoprotein CD147. Recently, we demonstrated that primary patient multiple myeloma (MM) cells and human MM cell lines (HMCLs) overexpress CD147. Therefore, the goal of the current study was to specifically determine if MCT1 and MCT4 were also overexpressed in MM cells. RT-PCR analysis demonstrated both primary patient MM cells and HMCLs overexpress MCT1 and MCT4 mRNA. Notably, primary MM cells or HMCLs were found to express variable levels of MCT1 and/or MCT4 at the protein level despite CD147 expression. In those HMCLs positive for MCT1 and/or MCT4 protein expression, MCT1 and/or MCT4 were found to be associated with CD147. Specific siRNA-mediated downregulation of MCT1 but not MCT4 resulted in decreased HMCL proliferation, decreased lactate export, and increased cellular media pH. However, western blot analysis revealed that downregulation of MCT1 also downregulated CD147 and vice versa despite no effect on mRNA levels. Taken together, these data demonstrate the association between MCT1 and CD147 proteins in MM cells and importance of their association for lactate export and proliferation in MM cells. PMID:24013424

CD147 regulates the expression of MCT1 and lactate export in multiple myeloma cells.

PubMed

Walters, Denise K; Arendt, Bonnie K; Jelinek, Diane F

2013-10-01

Increased use of the glycolytic pathway, even in the presence of oxygen, has recently been recognized as a key characteristic of malignant cells. However, the glycolytic phenotype results in increased lactic acid production and, in order to prevent cellular acidosis, tumor cells must increase proton efflux via upregulation of pH regulators such as proton-pumps, sodium-proton exchangers, and/or monocarboxylate transporters (MCT) (e.g., MCT1, MCT4). Interestingly, expression of MCT1 and MCT4 has been previously shown to be dependent upon expression of the transmembrane glycoprotein CD147. Recently, we demonstrated that primary patient multiple myeloma (MM) cells and human MM cell lines (HMCLs) overexpress CD147. Therefore, the goal of the current study was to specifically determine if MCT1 and MCT4 were also overexpressed in MM cells. RT-PCR analysis demonstrated both primary patient MM cells and HMCLs overexpress MCT1 and MCT4 mRNA. Notably, primary MM cells or HMCLs were found to express variable levels of MCT1 and/or MCT4 at the protein level despite CD147 expression. In those HMCLs positive for MCT1 and/or MCT4 protein expression, MCT1 and/or MCT4 were found to be associated with CD147. Specific siRNA-mediated downregulation of MCT1 but not MCT4 resulted in decreased HMCL proliferation, decreased lactate export, and increased cellular media pH. However, western blot analysis revealed that downregulation of MCT1 also downregulated CD147 and vice versa despite no effect on mRNA levels. Taken together, these data demonstrate the association between MCT1 and CD147 proteins in MM cells and importance of their association for lactate export and proliferation in MM cells.

Cellular Fibronectin Expression in Human Trabecular Meshwork and Induction by Transforming Growth Factor-β2

PubMed Central

Medina-Ortiz, Wanda E.; Belmares, Ricardo; Neubauer, Sandra; Wordinger, Robert J.; Clark, Abbot F.

2013-01-01

Purpose. Levels of TGF-β2 are higher in POAG aqueous humor, causing deposition of extracellular matrix (ECM) proteins, including fibronectin (FN), in the glaucomatous human trabecular meshwork (HTM) that may be responsible for elevated IOP. The purpose of this study was to identify the expression of cellular FN (cFN) isoforms (EDA and EDB) in HTM cells and tissues, and to determine whether TGF-β2 can induce cFN expression and fibril formation in cultured HTM cells. Methods. Expression of cFN mRNA isoforms and induction by recombinant TGF-β2 (5 ng/mL) were determined by quantitative RT-PCR. The TGF-β2 induction of EDA isoform protein expression and FN fibril formation were analyzed using Western immunoblots and immunocytochemistry (ICC), respectively. Immunohistochemistry (IHC) analysis was used to examine total FN and EDA isoform expression in normal (NTM) and glaucomatous (GTM) trabecular meshwork (TM) tissues. Results. Both cFN mRNA isoforms were expressed in cultured HTM cells and were induced by TGF-β2 after 2, 4, and 7 days (P < 0.05). Similarly, EDA isoform protein and fibril formation were increased after 4 and 7 days of TGF-β2 treatment. Finally, GTM tissues had significantly greater EDA isoform protein levels (1.7-fold, P < 0.05) compared to NTM tissues. Conclusions. This study demonstrated that cFN isoforms are expressed and induced in HTM cells by TGF-β2. Also, increased EDA isoform protein levels were seen in GTM tissues. Our findings suggest that induction of cFN isoform expression in the TM ECM may be a novel pathologic mechanism involved in the TM changes associated with glaucoma. PMID:24030464

Protective effects of organic acid component from Taraxacum mongolicum Hand.-Mazz. against LPS-induced inflammation: Regulating the TLR4/IKK/NF-κB signal pathway.

PubMed

Yang, Nan; Dong, Zibo; Tian, Gang; Zhu, Maomao; Li, Chao; Bu, Weiquan; Chen, Juan; Hou, Xuefeng; Liu, Ying; Wang, Gang; Jia, Xiaobin; Di, Liuqing; Feng, Liang

2016-12-24

TMHM is a type of Chinese medicine commonly used in medical practice and has multiple functions, including clearing heat, detoxification, reducing swelling, and tumor therapy. Previous research has demonstrated that the OAC of TMHM (TMHM-OAC) displays advantageous therapeutic action against respiratory inflammation. However, the effect of TMHM-OAC on inflammatory injury and its anti-inflammatory role requires further clarification. An in vitro inflammation damage model was employed using NHBE cells and 100ng/ml of (LPS). HPLC-DAD was conducted to analyze the components of TMHM-OAC. An ELISA was conducted to determine IL-1β, IL-6, TNF-α, and NO expression. An MTT assay was conducted to determine the cytotoxicity of TMHM-OAC. The levels of IL-1β, IL-6, TNF-α, caspase-3, caspase-8, iNOS, TLR4p-nuclear factor kappa-B kinase (p-IκκB), and p-NF-κB p65 in cellular protein, as well as the mRNA levels, were determined using WB, IF testing, and Q-PCR. TMHM-OAC significantly reduced LPS-induced NHBE cell inflammation, which was reflected in the reduced expression of relevant cytokines such as TNF-α, IL-1β, IL-6 and NO, caspase-3, and caspase-8. In addition, this component suppressed TLR4, p-IKKβ, and p-NF-κB p65 levels in both mRNA and cellular protein. TMHM-OAC can reduce LPS-induced inflammation in NHBE cells and this function could be linked to the regulation of the TLR4/IKK/NF-kB pathway. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Vitamin D receptor expression and potential role of vitamin D on cell proliferation and steroidogenesis in goat ovarian granulosa cells.

PubMed

Yao, Xiaolei; Zhang, Guomin; Guo, Yixuan; Ei-Samahy, Mohamed; Wang, Shuting; Wan, Yongjie; Han, Le; Liu, Zifei; Wang, Feng; Zhang, Yanli

2017-10-15

This study aimed to investigate the expression of the vitamin D receptor (VDR) in goat follicles and to determine the effects of Vit D 3 supplementation on goat granulosa cells (GCs) function linked to follicular development. The results demonstrated that VDR was prominently localized in GCs, with expression increasing with follicle diameter. Addition of Vit D 3 (1α,25-(OH) 2 VD 3 ; 10 nM) to GCs caused an increase in VDR and in steroidogenic acute regulator (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA expression. Additionally, Vit D 3 increased the cyclic adenosine monophosphate (cAMP), estradiol (E 2 ), and progesterone (P 4 ) levels, while it decreased anti-müllerian hormone receptor (AMHR) and follicle-stimulating hormone receptor (FSHR) mRNA expression (P < 0.05). Addition of FSH remarkably increased E 2, P 4 , and cAMP levels (P < 0.05), and Vit D 3 further enhanced the E 2 and cAMP levels in the presence of FSH (P < 0.05). Vit D 3 significantly induced the mRNA expression of CDK4 and CyclinD1, and downregulated P21 gene expression (P < 0.05). In addition, Vit D 3 significantly decreased reactive oxygen species (ROS) production and increased the mRNA and protein expression of superoxide dismutase 2 (SOD2) and catalase (CAT) (P < 0.05). In conclusion, VDR is expressed in GCs of the goat ovaries and Vit D 3 might play an important role in GCs proliferation by regulating cellular oxidative stress and cell cycle-related genes. Meanwhile, Vit D 3 enhances the E 2 and P 4 output of GCs by regulating the expression of 3β-HSD and StAR and the level of cAMP, which regulate steroidogenesis, supporting a potential role for Vit D 3 in follicular development. Copyright © 2017 Elsevier Inc. All rights reserved.

The ZntA-like NpunR4017 plays a key role in maintaining homeostatic levels of zinc in Nostoc punctiforme.

PubMed

Hudek, L; Bräu, L; Michalczyk, A A; Neilan, B A; Meeks, J C; Ackland, M L

2015-12-01

Analysis of cellular response to zinc exposure provides insights into how organisms maintain homeostatic levels of zinc that are essential, while avoiding potentially toxic cytosolic levels. Using the cyanobacterium Nostoc punctiforme as a model, qRT-PCR analyses established a profile of the changes in relative mRNA levels of the ZntA-like zinc efflux transporter NpunR4017 in response to extracellular zinc. In cells treated with 18 μM of zinc for 1 h, NpunR4017 mRNA levels increased by up to 1300 % above basal levels. The accumulation and retention of radiolabelled (65)Zn by NpunR4107-deficient and overexpressing strains were compared to wild-type levels. Disruption of NpunR4017 resulted in a significant increase in zinc accumulation up to 24 % greater than the wild type, while cells overexpressing NpunR4107 accumulated 22 % less than the wild type. Accumulation of (65)Zn in ZntA(-) Escherichia coli overexpressing NpunR4017 was reduced by up to 21 %, indicating the capacity for NpunR4017 to compensate for the loss of ZntA. These findings establish the newly identified NpunR4017 as a zinc efflux transporter and a key transporter for maintaining zinc homeostasis in N. punctiforme.

Chemokine-like factor 1 (CLFK1) is over-expressed in patients with atopic dermatitis.

PubMed

Yang, Gao-Yun; Chen, Xue; Sun, Ya-Chun; Ma, Chen-Li; Qian, Ge

2013-01-01

Human chemokine-like factor 1 (CKLF1), a recently discovered chemokine, has a broad spectrum of biological functions in immune-mediated diseases. It is highly expressed on Th2 lymphocytes and is a functional ligand for human CCR4. CKLF1 has a major role in the recruitment and activation of leucocytes, which plays an important role in the pathogenesis of allergic diseases. The present study was designed to determine the expression of CKLF1 in skin and serum in patients with atopic dermatitis (AD). The CKLF1 protein expression in skin lesion was analyzed by immunohistochemistry and ELISA. The mRNA expression of CKLF1 in skin lesion was detected by Real-time PCR. The serum levels of CKLF1, IgE, eotaxin, IL-4, IL-5, and IL-13 were measured by ELISA. Histopathological changes in the skin of AD patients showed local inflammation with epidermal thickening and significant inflammatory cellular infiltration. Immunohistochemistry results demonstrated that CKLF1-staining positive cells were located in the epidermal and dermis, and that the CKLF1 expression in AD patients was significantly higher than that in normal control. The CKLF1 mRNA expression in AD patients was significantly higher than that in healthy controls. Serum CKLF1 and IgE levels were significantly increased in AD patients, as were the serum levels of IL-4, IL-5, IL-13 and eotaxin. Both CKLF1 protien and mRNA levels are overexpressed in the skin lesion of AD patients, along with an increase in serum CKLF1 level, indicating that CKLF1 may play an important role in the development of atopic dermatitis.

Effects of interferon-gamma and lipopolysaccharide on macrophage iron metabolism are mediated by nitric oxide-induced degradation of iron regulatory protein 2.

PubMed

Kim, S; Ponka, P

2000-03-03

Iron regulatory proteins (IRP-1 and IRP-2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements, which are located in the 3'-untranslated region and the 5'-untranslated region of their respective mRNAs. Cellular iron levels affect binding of IRPs to iron-responsive elements and consequently expression of TfR and ferritin. Moreover, NO(*), a redox species of nitric oxide that interacts primarily with iron, can activate IRP-1 RNA binding activity resulting in an increase in TfR mRNA levels. Recently we found that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO(+) (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA binding of IRP-2 followed by IRP-2 degradation, and these changes were associated with a decrease in TfR mRNA levels (Kim, S., and Ponka, P. (1999) J. Biol. Chem. 274, 33035-33042). In this study, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) increased IRP-1 binding activity, whereas RNA binding of IRP-2 decreased and was followed by a degradation of this protein. Moreover, the decrease of IRP-2 binding/protein levels was associated with a decrease in TfR mRNA levels in LPS/IFN-gamma-treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. Furthermore, LPS/IFN-gamma-stimulated RAW 264.7 cells showed increased rates of ferritin synthesis. These results suggest that NO(+)-mediated degradation of IRP-2 plays a major role in iron metabolism during inflammation.

Developmental Timing of the Effects of Maternal Care on Gene Expression and Epigenetic Regulation of Hormone Receptor Levels in Female Rats

PubMed Central

Peña, Catherine Jensen; Neugut, Y. Dana

2013-01-01

Maternal care experienced during postnatal development has enduring effects on neuroendocrine function and behavior. Previous studies in rats have illustrated the effect of maternal licking/grooming (LG) on hormone receptors and maternal behavior of adult female offspring associated with altered DNA methylation. However, the developmental timing of these effects, which provide insight into the cellular and molecular pathways through which early experience alters later behavior, had not been explored. Here, we demonstrate the developmental emergence of these outcomes and use cross-fostering to identify sensitive periods for these effects. Estrogen receptor (ER)α and ERβ mRNA levels within the medial preoptic area (MPOA) of the hypothalamus were increased by postnatal day (PN)21 in female offspring of high LG dams; LG-associated increases in oxytocin receptor mRNA levels were observed beyond the weaning period. Quantification of ERα-immunoreactivity indicated a high degree of neuroanatomical specificity of LG effects within the MPOA that were observed by PN6. Reduced DNA methylation and histone 3 lysine 9 tri-methylation and increased histone 3 lysine 4 tri-methylation at the ERα gene promoter (Esr1) were detected at PN21 in high LG female offspring. Latency to engage in maternal behavior toward donor pups was significantly shorter among high LG females. Cross-fostering revealed that maternal sensitization and MPOA ERα levels are sensitive to maternal care experienced before but not after PN10. Differential windows of plasticity were identified for ERβ and oxytocin receptor mRNA levels. These studies contribute significantly to our understanding of the molecular, neurobiological, and behavioral pathways through which variation in maternal behavior is transmitted from one generation to the next. PMID:24002038

Neuronal zinc-α2-glycoprotein is decreased in temporal lobe epilepsy in patients and rats.

PubMed

Liu, Ying; Wang, Teng; Liu, Xi; Wei, Xin; Xu, Tao; Yin, Maojia; Ding, Xueying; Mo, Lijuan; Chen, Lifen

2017-08-15

Zinc-α2-glycoprotein (ZAG) is a 42-kDa protein encoded by the AZGP1 gene that is known as a lipid mobilizing factor and is highly homologous to major histocompatibility complex class I family molecules. Recently, transcriptomic research has shown that AZGP1 expression is reduced in the brain tissue of epilepsy patients. However, the cellular distribution and biological role of ZAG in the brain and epilepsy are unclear. Patients with refractory temporal lobe epilepsy (TLE) and brain trauma were included in this study, and pentylenetetrazole (PTZ)-kindled rats were also used. The existence and level of ZAG in the brain were identified using immunohistochemistry, double-labeled immunofluorescence and western blot, and the expression level of AZGP1 mRNA was determined with quantitative real-time polymerase chain reaction (qrt-PCR). To explore the potential biological role of ZAG in the brain, co-immunoprecipitation (Co-IP) of phosphorylated ERK (p-ERK), TGF-β1 and ZAG was also performed. ZAG was found in the cytoplasm of neurons in brain tissue from both patients and rats. The levels of AZGP1 mRNA and ZAG were lower in refractory TLE patients and PTZ-kindled rats than in controls. In addition, the ZAG level decreased as PTZ kindling continued. Co-IP identified direct binding between p-ERK, TGF-β1 and ZAG. ZAG was found to be synthesized in neurons, and both the AZGP1 mRNA and ZAG protein levels were decreased in epilepsy patients and rat models. The reduction in ZAG may participate in the pathogenesis and pathophysiology of epilepsy by interacting with p-ERK and TGF-β1, promoting inflammation, regulating the metabolism of ketone bodies, or affecting other epilepsy-related molecules. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Expression of transcription factors during sodium phenylacetate induced erythroid differentiation in K562 cells.

PubMed

Rath, A V; Schmahl, G E; Niemeyer, C M

1997-01-01

During 15 days of treatment of K562 cells with sodium phenylacetate, we observed an increase in the cellular hemoglobin concentration with a similar increase in the expression of gamma-globin mRNA. Morphological studies demonstrated characteristic features of erythroid differentiation and maturation. At the same time there was no change in the level of expression of the cell surface antigenes CD33, CD34, CD45, CD71 and glycophorin A. Likewise, the level of expression of the erythroid transcription factors GATA-1, GATA-2, NF-E2, SCL and RBTN2, all expressed in untreated K562 cells, did not increase during sodium phenylacetate induced erythroid differentiation. The expression of the nuclear factors Evi-1 and c-myb, known to inhibit erythroid differentiation, did not decrease. We conclude that sodium phenylacetate treatment of K562 cells increases gamma-globin mRNA and induces cell maturation as judged by morphology without affecting the expression of the erythroid transcription factors, some of which are known to be involved in the regulation of beta-like globin genes.

Genome-Wide Characterization of Light-Regulated Genes in Neurospora crassa

PubMed Central

Wu, Cheng; Yang, Fei; Smith, Kristina M.; Peterson, Matthew; Dekhang, Rigzin; Zhang, Ying; Zucker, Jeremy; Bredeweg, Erin L.; Mallappa, Chandrashekara; Zhou, Xiaoying; Lyubetskaya, Anna; Townsend, Jeffrey P.; Galagan, James E.; Freitag, Michael; Dunlap, Jay C.; Bell-Pedersen, Deborah; Sachs, Matthew S.

2014-01-01

The filamentous fungus Neurospora crassa responds to light in complex ways. To thoroughly study the transcriptional response of this organism to light, RNA-seq was used to analyze capped and polyadenylated mRNA prepared from mycelium grown for 24 hr in the dark and then exposed to light for 0 (control) 15, 60, 120, and 240 min. More than three-quarters of all defined protein coding genes (79%) were expressed in these cells. The increased sensitivity of RNA-seq compared with previous microarray studies revealed that the RNA levels for 31% of expressed genes were affected two-fold or more by exposure to light. Additionally, a large class of mRNAs, enriched for transcripts specifying products involved in rRNA metabolism, showed decreased expression in response to light, indicating a heretofore undocumented effect of light on this pathway. Based on measured changes in mRNA levels, light generally increases cellular metabolism and at the same time causes significant oxidative stress to the organism. To deal with this stress, protective photopigments are made, antioxidants are produced, and genes involved in ribosome biogenesis are transiently repressed. PMID:25053707

Differential regulation of oestrogen receptor β isoforms by 5′ untranslated regions in cancer

PubMed Central

Smith, Laura; Brannan, Rebecca A; Hanby, Andrew M; Shaaban, Abeer M; Verghese, Eldo T; Peter, Mark B; Pollock, Steven; Satheesha, Sampoorna; Szynkiewicz, Marcin; Speirs, Valerie; Hughes, Thomas A

2010-01-01

Abstract Oestrogen receptors (ERs) are critical regulators of the behaviour of many cancers. Despite this, the roles and regulation of one of the two known ERs – ERβ– are poorly understood. This is partly because analyses have been confused by discrepancies between ERβ expression at mRNA and proteins levels, and because ERβ is expressed as several functionally distinct isoforms. We investigated human ERβ 5′ untranslated regions (UTRs) and their influences on ERβ expression and function. We demonstrate that two alternative ERβ 5′UTRs have potent and differential influences on expression acting at the level of translation. We show that their influences are modulated by cellular context and in carcinogenesis, and demonstrate the contributions of both upstream open reading frames and RNA secondary structure. These regulatory mechanisms offer explanations for the non-concordance of ERβ mRNA and protein. Importantly, we also demonstrate that 5′UTRs allow the first reported mechanisms for differential regulation of the expression of the ERβ isoforms 1, 2 and 5, and thereby have critical influences on ERβ function. PMID:20920096

miR-143 decreases COX-2 mRNA stability and expression in pancreatic cancer cells

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

Pham, Hung; Department of Medicine, Veterans Affair Greater Los Angeles Healthcare System, Los Angeles, CA 90073; Ekaterina Rodriguez, C.

2013-09-13

Highlights: •Pancreatic cancer cells express low miR-143 levels and elevated p-MEK, p-MAPK and RREB1. •MEK inhibitors U0126 and PD98059 increase miR-143 expression. •miR-143 decreases COX-2 mRNA stability and expression and PGE{sub 2}. •miR-143 decreases p-p38MAPK, p-MEK, p-MAPK and RREB1 expression. -- Abstract: Small non-coding RNAs, microRNAs (miRNA), inhibit the translation or accelerate the degradation of message RNA (mRNA) by targeting the 3′-untranslated region (3′-UTR) in regulating growth and survival through gene suppression. Deregulated miRNA expression contributes to disease progression in several cancers types, including pancreatic cancers (PaCa). PaCa tissues and cells exhibit decreased miRNA, elevated cyclooxygenase (COX)-2 and increased prostaglandinmore » E{sub 2} (PGE{sub 2}) resulting in increased cancer growth and metastases. Human PaCa cell lines were used to demonstrate that restoration of miRNA-143 (miR-143) regulates COX-2 and inhibits cell proliferation. miR-143 were detected at fold levels of 0.41 ± 0.06 in AsPC-1, 0.20 ± 0.05 in Capan-2 and 0.10 ± 0.02 in MIA PaCa-2. miR-143 was not detected in BxPC-3, HPAF-II and Panc-1 which correlated with elevated mitogen-activated kinase (MAPK) and MAPK kinase (MEK) activation. Treatment with 10 μM of MEK inhibitor U0126 or PD98059 increased miR-143, respectively, by 187 ± 18 and 152 ± 26-fold in BxPC-3 and 182 ± 7 and 136 ± 9-fold in HPAF-II. miR-143 transfection diminished COX-2 mRNA stability at 60 min by 2.6 ± 0.3-fold in BxPC-3 and 2.5 ± 0.2-fold in HPAF-II. COX-2 expression and cellular proliferation in BxPC-3 and HPAF-II inversely correlated with increasing miR-143. PGE{sub 2} levels decreased by 39.3 ± 5.0% in BxPC-3 and 48.0 ± 3.0% in HPAF-II transfected with miR-143. Restoration of miR-143 in PaCa cells suppressed of COX-2, PGE{sub 2}, cellular proliferation and MEK/MAPK activation, implicating this pathway in regulating miR-143 expression.« less

Role of the uridine/cytidine kinase 2 mutation in cellular sensitiveness toward 3'-ethynylcytidine treatment of human cancer cells.

PubMed

Sato, Akira; Takano, Takeshi; Hiramoto, Akiko; Naito, Tomoharu; Matsuda, Akira; Fukushima, Masakazu; Wataya, Yusuke; Kim, Hye-Sook

2017-08-01

A nucleosidic medicine, 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine [3'-ethynylcytidine (ECyd)], is a potent inhibitor of RNA polymerase I and shows anticancer activity to various human solid tumors in vitro and in vivo. ECyd is phosphorylated to 3'-ethyntlcytidine 5'-monophosphate by uridine/cytidine kinase 2 (UCK2) and subsequently further to diphosphate and triphosphate (3'-ethyntlcytidine 5'-diphosphate, 3'-ethyntlcytidine 5'-triphosphate). 3'-Ethyntlcytidine 5'-triphosphate is an active metabolite that can inhibit RNA polymerase I competitively, causing cancer cell death. Here, to identify the UCK2 mutation for detecting responder or nonresponder to ECyd, we investigated the relationship between point mutation of the UCK2 gene and response to ECyd in various human solid tumors. We identified several functional point mutations including the splice-site mutation of the UCK2 gene IVS5+5 G>A. In addition, we found that the IVS5+5 G>A variant generates an aberrant mRNA transcript, namely, truncated mRNA was produced and normal mRNA levels were markedly decreased in the ECyd-resistant cancer cell line HT1080. We concluded that these findings strongly suggest that the IVS5+5 G>A variant would affect the expression level of the UCK2 transcript, resulting in decreased sensitivity to ECyd.

Transcriptomic Analysis Reveals Wound Healing of Morus alba Root Extract by Up-Regulating Keratin Filament and CXCL12/CXCR4 Signaling.

PubMed

Kim, Kang-Hoon; Chung, Won-Seok; Kim, Yoomi; Kim, Ki-Suk; Lee, In-Seung; Park, Ji Young; Jeong, Hyeon-Soo; Na, Yun-Cheol; Lee, Chang-Hun; Jang, Hyeung-Jin

2015-08-01

Facilitation of the wound healing process is important because a prolonged wound site increases pain and the risk of infection. In oriental medicine, an extract of Morus alba root (MA) has usually been prescribed as traditional treatment for accelerating wound healing, and it has been proven to be safe for centuries. To study the molecular mechanism of MA-mediated skin wound healing, we performed a primary cell culture and a skin explant culture and observed significant difference between the groups with and without MA extract. In the cellular system, a real-time cell analysis and real-time quantitative PCR were performed. It was found that MA extract enhanced proliferation in a dose-dependent manner on Kera-308 cell line, and up-regulated keratin expression including wound-induced Krt6a. In skin explant culture, the mRNA level derived from cell outgrowth displayed a tendency toward more up-regulated mRNA associated keratin filaments and toward a more up-regulated mRNA level of C-X-C motif chemokine 12 (CXCL12) and a chemokine receptor 4 (CXCR4) axis signaling pathway downstream. In this process, we concluded that MA extract had a scientific possibility of wound repair by increasing intracellular and extracellular supports and by inducing a CXCL12/CXCR4 signaling pathway. Copyright © 2015 John Wiley & Sons, Ltd.

Mycophenolate Mofetil Treatment of Systemic Sclerosis Reduces Myeloid Cell Numbers and Attenuates the Inflammatory Gene Signature in Skin.

PubMed

Hinchcliff, Monique; Toledo, Diana M; Taroni, Jaclyn N; Wood, Tammara A; Franks, Jennifer M; Ball, Michael S; Hoffmann, Aileen; Amin, Sapna M; Tan, Ainah U; Tom, Kevin; Nesbeth, Yolanda; Lee, Jungwha; Ma, Madeleine; Aren, Kathleen; Carns, Mary A; Pioli, Patricia A; Whitfield, Michael L

2018-01-31

Fewer than half of patients with systemic sclerosis demonstrate modified Rodnan skin score improvement during mycophenolate mofetil (MMF) treatment. To understand the molecular basis for this observation, we extended our prior studies and characterized molecular and cellular changes in skin biopsies from subjects with systemic sclerosis treated with MMF. Eleven subjects completed ≥24 months of MMF therapy. Two distinct skin gene expression trajectories were observed across six of these subjects. Three of the six subjects showed attenuation of the inflammatory signature by 24 months, paralleling reductions in CCL2 mRNA expression in skin and reduced numbers of macrophages and myeloid dendritic cells in skin biopsies. MMF cessation at 24 months resulted in an increased inflammatory score, increased CCL2 mRNA and protein levels, modified Rodnan skin score rebound, and increased numbers of skin myeloid cells in these subjects. In contrast, three other subjects remained on MMF >24 months and showed a persistent decrease in inflammatory score, decreasing or stable modified Rodnan skin score, CCL2 mRNA reductions, sera CCL2 protein levels trending downward, reduction in monocyte migration, and no increase in skin myeloid cell numbers. These data summarize molecular changes during MMF therapy that suggest reduction of innate immune cell numbers, possibly by attenuating expression of chemokines, including CCL2. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Characterization of GABAA receptor ligands with automated patch-clamp using human neurons derived from pluripotent stem cells

PubMed Central

Yuan, Nina Y.; Poe, Michael M.; Witzigmann, Christopher; Cook, James M.; Stafford, Douglas; Arnold, Leggy A.

2016-01-01

Introduction Automated patch clamp is a recent but widely used technology to assess pre-clinical drug safety. With the availability of human neurons derived from pluripotent stem cells, this technology can be extended to determine CNS effects of drug candidates, especially those acting on the GABAA receptor. Methods iCell Neurons (Cellular Dynamics International, A Fujifilm Company) were cultured for ten days and analyzed by patch clamp in the presence of agonist GABA or in combination with positive allosteric GABAA receptor modulators. Both efficacy and affinity were determined. In addition, mRNA of GABAA receptor subunits were quantified by qRT-PCR. Results We have shown that iCell Neurons are compatible with the IonFlux microfluidic system of the automated patch clamp instrument. Resistance ranging from 15-25 MΩ was achieved for each trap channel of patch clamped cells in a 96-well plate format. GABA induced a robust change of current with an EC50 of 0.43 μM. Positive GABAA receptor modulators diazepam, HZ166, and CW-04-020 exhibited EC50 values of 0.42 μM, 1.56 μM, and 0.23 μM, respectively. The α2/α3/α5 selective compound HZ166-induced the highest potentiation (efficacy) of 810% of the current induced by 100 nM GABA. Quantification of GABAA receptor mRNA in iCell Neurons revealed high levels of α5 and β3 subunits and low levels of α1, which is similar to the configuration in human neonatal brain. Discussion iCell Neurons represent a new cellular model to characterize GABAergic compounds using automated patch clamp. These cells have excellent representation of cellular GABAA receptor distribution that enable determination of total small molecule efficacy and affinity as measured by cell membrane current change. PMID:27544543

Molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets

PubMed Central

Arango, Daniel; Morohashi, Kengo; Yilmaz, Alper; Kuramochi, Kouji; Parihar, Arti; Brahimaj, Bledi; Grotewold, Erich; Doseff, Andrea I.

2013-01-01

Flavonoids constitute the largest class of dietary phytochemicals, adding essential health value to our diet, and are emerging as key nutraceuticals. Cellular targets for dietary phytochemicals remain largely unknown, posing significant challenges for the regulation of dietary supplements and the understanding of how nutraceuticals provide health value. Here, we describe the identification of human cellular targets of apigenin, a flavonoid abundantly present in fruits and vegetables, using an innovative high-throughput approach that combines phage display with second generation sequencing. The 160 identified high-confidence candidate apigenin targets are significantly enriched in three main functional categories: GTPase activation, membrane transport, and mRNA metabolism/alternative splicing. This last category includes the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2), a factor involved in splicing regulation, mRNA stability, and mRNA transport. Apigenin binds to the C-terminal glycine-rich domain of hnRNPA2, preventing hnRNPA2 from forming homodimers, and therefore, it perturbs the alternative splicing of several human hnRNPA2 targets. Our results provide a framework to understand how dietary phytochemicals exert their actions by binding to many functionally diverse cellular targets. In turn, some of them may modulate the activity of a large number of downstream genes, which is exemplified here by the effects of apigenin on the alternative splicing activity of hnRNPA2. Hence, in contrast to small-molecule pharmaceuticals designed for defined target specificity, dietary phytochemicals affect a large number of cellular targets with varied affinities that, combined, result in their recognized health benefits. PMID:23697369

The Human T-Lymphotropic Virus Type 1 Tax Protein Inhibits Nonsense-Mediated mRNA Decay by Interacting with INT6/EIF3E and UPF1

PubMed Central

Mocquet, Vincent; Neusiedler, Julia; Rende, Francesca; Cluet, David; Robin, Jean-Philippe; Terme, Jean-Michel; Duc Dodon, Madeleine; Wittmann, Jürgen; Morris, Christelle; Le Hir, Hervé; Ciminale, Vincenzo

2012-01-01

In this report, we analyzed whether the degradation of mRNAs by the nonsense-mediated mRNA decay (NMD) pathway was affected in human T-lymphotropic virus type 1 (HTLV-1)-infected cells. This pathway was indeed strongly inhibited in C91PL, HUT102, and MT2 cells, and such an effect was also observed by the sole expression of the Tax protein in Jurkat and HeLa cells. In line with this activity, Tax binds INT6/EIF3E (here called INT6), which is a subunit of the translation initiation factor eukaryotic initiation factor 3 (eIF3) required for efficient NMD, as well as the NMD core factor upstream frameshift protein 1 (UPF1). It was also observed that Tax expression alters the morphology of processing bodies (P-bodies), the cytoplasmic structures which concentrate RNA degradation factors. The presence of UPF1 in these subcellular compartments was increased by Tax, whereas that of INT6 was decreased. In line with these effects, the level of the phosphorylated form of UPF1 was increased in the presence of Tax. Analysis of several mutants of the viral protein showed that the interaction with INT6 is necessary for NMD inhibition. The alteration of mRNA stability was observed to affect viral transcripts, such as that coding for the HTLV-1 basic leucine zipper factor (HBZ), and also several cellular mRNAs sensitive to the NMD pathway. Our data indicate that the effect of Tax on viral and cellular gene expression is not restricted to transcriptional control but can also involve posttranscriptional regulation. PMID:22553336

Low concentrations of alendronate increase the local invasive potential of osteoblastic sarcoma cell lines via connexin 43 activation.

PubMed

Yoshitani, Kazuhiro; Kido, Akira; Honoki, Kanya; Akahane, Manabu; Fujii, Hiromasa; Tanaka, Yasuhito

2011-07-15

Bisphosphonates (BPs) are agents used for treating disorders of excessive bone resorption. In addition, due to their cell-killing activity, BPs were potent candidates for adjuvant cancer therapy. On the other hand, low-concentrations of BPs have been reported to increase cellular viability in several types of tumor cells. Therefore, we focused on the effect of BPs on cellular aggressiveness of malignant bone tumors at low concentrations. MTS assay was performed using osteosarcoma cell lines MG63 and HOS, fibrosarcoma cell line HT1080, and prostate cancer cell line PC3. All the cell lines showed toxicity at high concentrations. On the other hand, at lower concentrations, the cellular viabilities of HOS and MG63 were rather higher than those of untreated controls. Since this tendency was most evident, HOS was used for further assays, including cellular motility, bone resorption activity, and cathepsin K activity. The low-concentration of alendronate enhanced cellular viability and motility, which correlated with the expression of connexin 43 at the mRNA and protein levels. Interestingly, oleamide, a potent connexin 43 inhibitor, had an inhibitory effect on the enhanced proliferation. Our data suggest that alendronate may enhance the proliferation of osteoblastic cell line through connexin 43 activation. Copyright © 2011 Elsevier GmbH. All rights reserved.

Quercetin affects glutathione levels and redox ratio in human aortic endothelial cells not through oxidation but formation and cellular export of quercetin-glutathione conjugates and upregulation of glutamate-cysteine ligase.

PubMed

Li, Chuan; Zhang, Wei-Jian; Choi, Jaewoo; Frei, Balz

2016-10-01

Endothelial dysfunction due to vascular inflammation and oxidative stress critically contributes to the etiology of atherosclerosis. The intracellular redox environment plays a key role in regulating endothelial cell function and is intimately linked to cellular thiol status, including and foremost glutathione (GSH). In the present study we investigated whether and how the dietary flavonoid, quercetin, affects GSH status of human aortic endothelial cells (HAEC) and their response to oxidative stress. We found that treating cells with buthionine sulfoximine to deplete cellular GSH levels significantly reduced the capacity of quercetin to inhibit lipopolysaccharide (LPS)-induced oxidant production. Furthermore, incubation of HAEC with quercetin caused a transient decrease and then full recovery of cellular GSH concentrations. The initial decline in GSH was not accompanied by a corresponding increase in glutathione disulfide (GSSG). To the contrary, GSSG levels, which were less than 0.5% of GSH levels at baseline (0.26±0.01 vs. 64.7±1.9nmol/mg protein, respectively), decreased by about 25% during incubation with quercetin. As a result, the GSH: GSSG ratio increased by about 70%, from 253±7 to 372±23. These quercetin-induced changes in GSH and GSSG levels were not affected by treating HAEC with 500µM ascorbic acid phosphate for 24h to increase intracellular ascorbate levels. Incubation of HAEC with quercetin also led to the appearance of extracellular quercetin-glutathione conjugates, which was paralleled by upregulation of the multidrug resistance protein 1 (MRP1). Furthermore, quercetin slightly but significantly increased mRNA and protein levels of glutamate-cysteine ligase (GCL) catalytic and modifier subunits. Taken together, our results suggest that quercetin causes loss of GSH in HAEC, not because of oxidation but due to formation and cellular export of quercetin-glutathione conjugates. Induction by quercetin of GCL subsequently restores GSH levels, thereby suppressing LPS-induced oxidant production. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Sub-cellular mRNA localization modulates the regulation of gene expression by small RNAs in bacteria

NASA Astrophysics Data System (ADS)

Teimouri, Hamid; Korkmazhan, Elgin; Stavans, Joel; Levine, Erel

2017-10-01

Small non-coding RNAs can exert significant regulatory activity on gene expression in bacteria. In recent years, substantial progress has been made in understanding bacterial gene expression by sRNAs. However, recent findings that demonstrate that families of mRNAs show non-trivial sub-cellular distributions raise the question of how localization may affect the regulatory activity of sRNAs. Here we address this question within a simple mathematical model. We show that the non-uniform spatial distributions of mRNA can alter the threshold-linear response that characterizes sRNAs that act stoichiometrically, and modulate the hierarchy among targets co-regulated by the same sRNA. We also identify conditions where the sub-cellular organization of cofactors in the sRNA pathway can induce spatial heterogeneity on sRNA targets. Our results suggest that under certain conditions, interpretation and modeling of natural and synthetic gene regulatory circuits need to take into account the spatial organization of the transcripts of participating genes.

The multifunctional Staufen proteins: conserved roles from neurogenesis to synaptic plasticity

PubMed Central

Heraud-Farlow, Jacki E.; Kiebler, Michael A.

2014-01-01

Staufen (Stau) proteins belong to a family of RNA-binding proteins (RBPs) that are important for RNA localisation in many organisms. In this review we discuss recent findings on the conserved role played by Stau during both the early differentiation of neurons and in the synaptic plasticity of mature neurons. Recent molecular data suggest mechanisms for how Stau2 regulates mRNA localisation, mRNA stability, translation, and ribonucleoprotein (RNP) assembly. We offer a perspective on how this multifunctional RBP has been adopted to regulate mRNA localisation under several different cellular and developmental conditions. PMID:25012293

Hsp27 binding to the 3'UTR of bim mRNA prevents neuronal death during oxidative stress-induced injury: a novel cytoprotective mechanism.

PubMed

Dávila, David; Jiménez-Mateos, Eva M; Mooney, Claire M; Velasco, Guillermo; Henshall, David C; Prehn, Jochen H M

2014-11-01

Neurons face a changeable microenvironment and therefore need mechanisms that allow rapid switch on/off of their cytoprotective and apoptosis-inducing signaling pathways. Cellular mechanisms that control apoptosis activation include the regulation of pro/antiapoptotic mRNAs through their 3'-untranslated region (UTR). This region holds binding elements for RNA-binding proteins, which can control mRNA translation. Here we demonstrate that heat shock protein 27 (Hsp27) prevents oxidative stress-induced cell death in cerebellar granule neurons by specific regulation of the mRNA for the proapoptotic BH3-only protein, Bim. Hsp27 depletion induced by oxidative stress using hydrogen peroxide (H2O2) correlated with bim gene activation and subsequent neuronal death, whereas enhanced Hsp27 expression prevented these. This effect could not be explained by proteasomal degradation of Bim or bim promoter inhibition; however, it was associated with a specific increase in the levels of bim mRNA and with its binding to Hsp27. Finally, we determined that enhanced Hsp27 expression in neurons exposed to H2O2 or glutamate prevented the translation of a reporter plasmid where bim-3'UTR mRNA sequence was cloned downstream of a luciferase gene. These results suggest that repression of bim mRNA translation through binding to the 3'UTR constitutes a novel cytoprotective mechanism of Hsp27 during stress in neurons. © 2014 Dávila et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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

Wang, Jing; Ma, Zihao; Carr, Steven A.

Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this “guilt-by-association” (GBA) approach. Recent advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC).more » Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies. Molecular & Cellular Proteomics 16: 10.1074/mcp.M116.060301, 121–134, 2017.« less

[Significant increase of glucose transport activity in breast cancer].

PubMed

Li, Juan; Yang, Shou-jing; Zhao, Xi-long; Zhang, Ya-qing; Li, Kai-nan; Cui, Ji-hong; Li, Jing

2008-02-01

To study the expression level and significance of glucose transporter 1 (Glut-1) in normal breast tissue, adenosis, adenoma and breast carcinoma. A total of 147 cases of female breast tissue samples, including 92 cases of invasive ductal carcinoma, 26 cases of breast fibroadenoma, 24 cases of breast adenosis and 5 cases of normal breast tissues, were collected for quantitative detection of the expression of Glut-1 protein by immunohistochemistry (EnVision method) and Western blot, and its mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). In normal breast tissue and benign lesions of the breast, Glut-1 was undetectable or only weakly detectable in cytoplasm of ductal and acinar epithelia. In contrast, the intensity of Glut-1 staining was significantly higher in invasive ductal carcinomas (P = 0.0002) with protein expression predominantly in cellular membrane and lesser in cytoplasm. Western blot and RT-PCR analyses showed that the expression of Glut-1 protein and mRNA were significantly increased in invasive ductal carcinoma than fibroadenoma (P =0.001 for protein; P <0.05 for mRNA) and adenosis (P =0.001 for protein; P < 0.05 for mRNA). There was a significant difference among groups (P = 0.0002 for protein; P = 0.0001 for mRNA). Glucose transport activity, as indicated by Glut-1 protein and its mRNA expression, significantly increases in breast carcinoma than non-cancerous lesions. The over-expression of Glut-1 in breast carcinoma is tightly coupled with tumor cell proliferation, invasion and metastasis, implying that Glut-1 may serve as a new marker in the early diagnosis and prognostication of breast malignancy as well as a new therapeutic target.

Mutations in FLVCR1 Cause Posterior Column Ataxia and Retinitis Pigmentosa

PubMed Central

Rajadhyaksha, Anjali M.; Elemento, Olivier; Puffenberger, Erik G.; Schierberl, Kathryn C.; Xiang, Jenny Z.; Putorti, Maria L.; Berciano, José; Poulin, Chantal; Brais, Bernard; Michaelides, Michel; Weleber, Richard G.; Higgins, Joseph J.

2010-01-01

The study of inherited retinal diseases has advanced our knowledge of the cellular and molecular mechanisms involved in sensory neural signaling. Dysfunction of two specific sensory modalities, vision and proprioception, characterizes the phenotype of the rare, autosomal-recessive disorder posterior column ataxia and retinitis pigmentosa (PCARP). Using targeted DNA capture and high-throughput sequencing, we analyzed the entire 4.2 Mb candidate sequence on chromosome 1q32 to find the gene mutated in PCARP in a single family. Employing comprehensive bioinformatic analysis and filtering, we identified a single-nucleotide coding variant in the feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), a gene encoding a heme-transporter protein. Sanger sequencing confirmed the FLVCR1 mutation in this family and identified different homozygous missense mutations located within the protein's transmembrane channel segment in two other unrelated families with PCARP. To determine whether the selective pathologic features of PCARP correlated with FLVCR1 expression, we examined wild-type mouse Flvcr1 mRNA levels in the posterior column of the spinal cord and the retina via quantitative real-time reverse-transcriptase PCR. The Flvcr1 mRNA levels were most abundant in the retina, followed by the posterior column of the spinal cord and other brain regions. These results suggest that aberrant FLVCR1 causes a selective degeneration of a subpopulation of neurons in the retina and the posterior columns of the spinal cord via dysregulation of heme or iron homeostasis. This finding broadens the molecular basis of sensory neural signaling to include common mechanisms that involve proprioception and vision. PMID:21070897

The N Terminus of Andes Virus L Protein Suppresses mRNA and Protein Expression in Mammalian Cells

PubMed Central

Heinemann, Patrick; Schmidt-Chanasit, Jonas

2013-01-01

Little is known about the structure and function of the 250-kDa L protein of hantaviruses, although it plays a central role in virus genome transcription and replication. When attempting to study Andes virus (ANDV) L protein in mammalian cells, we encountered difficulties. Even in a strong overexpression system, ANDV L protein could not be detected by immunoblotting. Deletion analysis revealed that the 534 N-terminal amino acid residues determine the low-expression phenotype. Inhibition of translation due to RNA secondary structures around the start codon, rapid proteasomal degradation, and reduced half-life time were excluded. However, ANDV L protein expression could be rescued upon mutation of the catalytic PD-E-K motif and further conserved residues of the putative endonuclease at the N terminus of the protein. In addition, wild-type ANDV L rather than expressible L mutants suppressed the level of L mRNA, as well as reporter mRNAs. Wild-type L protein also reduced the synthesis of cellular proteins in the high-molecular-weight range. Using expressible ANDV L mutants as a tool for localization studies, we show that L protein colocalizes with ANDV N and NSs but not Gc protein. A fraction of L protein also colocalized with the cellular processing (P) body component DCP1a. Overall, these data suggest that ANDV L protein possesses a highly active endonuclease at the N terminus suppressing the level of its own as well as heterologous mRNAs upon recombinant expression in mammalian cells. PMID:23576516

The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

PubMed

Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

2016-04-26

Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

Translational Control in Cancer Etiology

PubMed Central

Ruggero, Davide

2013-01-01

The link between perturbations in translational control and cancer etiology is becoming a primary focus in cancer research. It has now been established that genetic alterations in several components of the translational apparatus underlie spontaneous cancers as well as an entire class of inherited syndromes known as “ribosomopathies” associated with increased cancer susceptibility. These discoveries have illuminated the importance of deregulations in translational control to very specific cellular processes that contribute to cancer etiology. In addition, a growing body of evidence supports the view that deregulation of translational control is a common mechanism by which diverse oncogenic pathways promote cellular transformation and tumor development. Indeed, activation of these key oncogenic pathways induces rapid and dramatic translational reprogramming both by increasing overall protein synthesis and by modulating specific mRNA networks. These translational changes promote cellular transformation, impacting almost every phase of tumor development. This paradigm represents a new frontier in the multihit model of cancer formation and offers significant promise for innovative cancer therapies. Current research, in conjunction with cutting edge technologies, will further enable us to explore novel mechanisms of translational control, functionally identify translationally controlled mRNA groups, and unravel their impact on cellular transformation and tumorigenesis. PMID:22767671

Tipping the balance of RNA stability by 3' editing of the transcriptome.

PubMed

Chung, Christina Z; Seidl, Lauren E; Mann, Mitchell R; Heinemann, Ilka U

2017-11-01

The regulation of active microRNAs (miRNAs) and maturation of messenger RNAs (mRNAs) that are competent for translation is a crucial point in the control of all cellular processes, with established roles in development and differentiation. Terminal nucleotidyltransferases (TNTases) are potent regulators of RNA metabolism. TNTases promote the addition of single or multiple nucleotides to an RNA transcript that can rapidly alter transcript stability. The well-known polyadenylation promotes transcript stability while the newly discovered but ubiquitious 3'-end polyuridylation marks RNA for degradation. Monoadenylation and uridylation are essential control mechanisms balancing mRNA and miRNA homeostasis. This review discusses the multiple functions of non-canonical TNTases, focusing on their substrate range, biological functions, and evolution. TNTases directly control mRNA and miRNA levels, with diverse roles in transcriptome stabilization, maturation, silencing, or degradation. We will summarize the current state of knowledge on non-canonical nucleotidyltransferases and their function in regulating miRNA and mRNA metabolism. We will review the discovery of uridylation as an RNA degradation pathway and discuss the evolution of nucleotidyltransferases along with their use in RNA labeling and future applications as therapeutic targets. The biochemically and evolutionarily highly related adenylyl- and uridylyltransferases play antagonizing roles in the cell. In general, RNA adenylation promotes stability, while uridylation marks RNA for degradation. Uridylyltransferases evolved from adenylyltransferases in multiple independent evolutionary events by the insertion of a histidine residue into the active site, altering nucleotide, but not RNA specificity. Understanding the mechanisms regulating RNA stability in the cell and controlling the transcriptome is essential for efforts aiming to influence cellular fate. Selectively enhancing or reducing RNA stability allows for alterations in the transcriptome, proteome, and downstream cellular processes. Genetic, biochemical, and clinical data suggest TNTases are potent targets for chemotherapeutics and have been exploited for RNA labeling applications. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

Valproic acid exposure decreases Cbp/p300 protein expression and histone acetyltransferase activity in P19 cells

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

Lamparter, Christina L.

The teratogenicity of the antiepileptic drug valproic acid (VPA) is well established and its inhibition of histone deacetylases (HDAC) is proposed as an initiating factor. Recently, VPA-mediated HDAC inhibition was demonstrated to involve transcriptional downregulation of histone acetyltransferases (HATs), which was proposed to compensate for the increased acetylation resulting from HDAC inhibition. Cbp and p300 are HATs required for embryonic development and deficiencies in either are associated with congenital malformations and embryolethality. The objective of the present study was to characterize Cbp/p300 following VPA exposure in P19 cells. Consistent with previous studies, exposure to 5 mM VPA over 24 hmore » induced a moderate decrease in Cbp/p300 mRNA, which preceded a strong decrease in total cellular protein mediated by ubiquitin-proteasome degradation. Nuclear Cbp/p300 protein was also decreased following VPA exposure, although to a lesser extent. Total cellular and nuclear p300 HAT activity was reduced proportionately to p300 protein levels, however while total cellular HAT activity also decreased, nuclear HAT activity was unaffected. Using the Cbp/p300 HAT inhibitor C646, we demonstrated that HAT inhibition similarly affected many of the same endpoints as VPA, including increased reactive oxygen species and caspase-3 cleavage, the latter of which could be attenuated by pre-treatment with the antioxidant catalase. C646 exposure also decreased NF-κB/p65 protein, which was not due to reduced mRNA and was not attenuated with catalase pre-treatment. This study provides support for an adaptive HAT response following VPA exposure and suggests that reduced Cbp/p300 HAT activity could contribute to VPA-mediated alterations. - Highlights: • VPA exposure in vitro downregulates Cbp/p300 mRNA and induces protein degradation. • Cbp/p300 histone acetyltransferase activity is similarly reduced with VPA exposure. • Inhibition of Cbp/p300 acetyltransferase activity induces apoptosis, involving ROS. • This inhibition of activity also reduces NFκB expression independently of ROS. • Reduced Cbp/p300 acetyltransferase activity may contribute to VPA teratogenesis.« less

Nuclear imprisonment of host cellular mRNA by nsp1β protein of porcine reproductive and respiratory syndrome virus

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

Han, Mingyuan, E-mail: hanming@umich.edu; Ke, Hanz

Positive-strand RNA genomes function as mRNA for viral protein synthesis which is fully reliant on host cell translation machinery. Competing with cellular protein translation apparatus needs to ensure the production of viral proteins, but this also stifles host innate defense. In the present study, we showed that porcine reproductive and respiratory syndrome virus (PRRSV), whose replication takes place in the cytoplasm, imprisoned host cell mRNA in the nucleus, which suggests a novel mechanism to enhance translation of PRRSV genome. PRRSV nonstructural protein (nsp) 1β was identified as the nuclear protein playing the role for host mRNA nuclear retention and subversionmore » of host protein synthesis. A SAP (SAF-A/B, Acinus, and PIAS) motif was identified in nsp1β with the consensus sequence of {sub 126}-LQxxLxxxGL-{sub 135}. In situ hybridization unveiled that SAP mutants were unable to cause nuclear retention of host cell mRNAs and did not suppress host protein synthesis. In addition, these SAP mutants reverted PRRSV-nsp1β-mediated suppression of interferon (IFN) production, IFN signaling, and TNF-α production pathway. Using reverse genetics, a series of SAP mutant PRRS viruses, vK124A, vL126A, vG134A, and vL135A were generated. No mRNA nuclear retention was observed during vL126A and vL135A infections. Importantly, vL126A and vL135A did not suppress IFN production. For other arteriviruses, mRNA nuclear accumulation was also observed for LDV-nsp1β and SHFV-nsp1β. EAV-nsp1 was exceptional and did not block the host mRNA nuclear export. - Highlights: •PRRS virus blocks host mRNA nuclear export to the cytoplasm. •PRRSV nsp1β is the viral protein responsible for host mRNA nuclear retention. •SAP domain in nsp1β is essential for host mRNA nuclear retention and type I interferon suppression. •Mutation in the SAP domain of nsp1β causes the loss of function. •Host mRNA nuclear retention by nsp1β is common in the family Arteriviridae, except equine arteritis virus.« less

Cellular localization of thrombopoietin mRNA in the liver by in situ hybridization.

PubMed

Nomura, S; Ogami, K; Kawamura, K; Tsukamoto, I; Kudo, Y; Kanakura, Y; Kitamura, Y; Miyazaki, H; Kato, T

1997-07-01

The expression of thrombopoietin (TPO) mRNA is observed in several tissues, including liver, kidney, brain, skeletal muscle, intestine, spleen, and bone marrow. Among these organs, the highest expression of TPO mRNA is detected in the liver. We identified cells producing TPO by means of in situ hybridization of adult rat liver using digoxigenin-11-UTP-labeled cRNA probes. We found that the cells expressing TPO mRNA also expressed serum albumin mRNA. TPO mRNA was detected in parenchymal cells (hepatocytes) but not in non-parenchymal cells (including endothelial cells, epithelial cells, and so forth). To determine the location of TPO expression in embryogenesis, sections of fetal mice were further analyzed by in situ hybridization. TPO mRNA was detected only in hepatocytes of fetal liver, which was also the major site of hematopoiesis. The expression of TPO mRNA in fetal liver was observed from 12.5 days postcoitus. Northern blot analysis showed that mouse liver transcribed the same size of TPO mRNA in the fetus and in the adult. These results clearly demonstrate that hepatocytes are the primary site of TPO production in the liver from fetus to adult.

Regulation of RNA polymerase III transcription during transformation of human IMR90 fibroblasts with defined genetic elements.

PubMed

Durrieu-Gaillard, Stéphanie; Dumay-Odelot, Hélène; Boldina, Galina; Tourasse, Nicolas J; Allard, Delphine; André, Fabrice; Macari, Françoise; Choquet, Armelle; Lagarde, Pauline; Drutel, Guillaume; Leste-Lasserre, Thierry; Petitet, Marion; Lesluyes, Tom; Lartigue-Faustin, Lydia; Dupuy, Jean-William; Chibon, Frédéric; Roeder, Robert G; Joubert, Dominique; Vagner, Stéphan; Teichmann, Martin

2018-01-01

RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.

Programmable oligonucleotide probes design and applications for in situ and in vivo RNA imaging in cells

NASA Astrophysics Data System (ADS)

Cheglakov, Zoya

Unequal spreading of mRNA is a frequent experience observed in varied cell lines. The study of cellular processes dynamics and precise localization of mRNAs offers a vital toolbox to target specific proteins in precise cytoplasmic areas and provides a convenient instrument to uncover their mechanisms and functions. Latest methodological innovations have allowed imaging of a single mRNA molecule in situ and in vivo. Today, Fluorescent In Situ Hybridization (FISH) methods allow the studying of mRNA expression and offer a vital toolbox for accurate biological models. Studies enable analysis of the dynamics of an individual mRNA, have uncovered the multiplex RNA transport systems. With all current approaches, a single mRNA tracking in the mammalian cells is still challenging. This thesis describes mRNA detection methods based on programmable fluorophore-labeled DNA structures complimentary to native targets providing an accurate mRNA imaging in mammalian cells. First method represents beta-actin (ACTB) transcripts in situ detection in human cells, the technique strategy is based on programmable DNA probes, amplified by rolling circle amplification (RCA). The method reports precise localization of molecule of interest with an accuracy of a single-cell. Visualization and localization of specific endogenous mRNA molecules in real-time in vivo has the promising to innovate cellular biology studies, medical analysis and to provide a vital toolbox in drugs invention area. Second method described in this thesis represents miR-21 miRNA detection within a single live-cell resolution. The method using fluorophore-labeled short synthetic DNAs probes forming a stem-loop shape and generating Fluorescent Resonance Energy Transfer (FRET) as a result of target-probes hybridization. Catalytic nucleic acid (DNAzymes) probes are cooperative tool for precise detection of different mRNA targets. With assistance of a complementary fluorophore-quencher labeled substrate, the DNAzymes provide a beneficial strategy for simultaneous tracking readily accomplished by multicolor imaging with diverse fluorescent tags. The third method in this thesis will demonstrate the advantage of DNAzymes probes amplification, and offers potential strategy to monitor miRNAs in mammalian live cells.

Arc/Arg3.1 mRNA expression reveals a subcellular trace of prior sound exposure in adult primary auditory cortex.

PubMed

Ivanova, T N; Matthews, A; Gross, C; Mappus, R C; Gollnick, C; Swanson, A; Bassell, G J; Liu, R C

2011-05-05

Acquiring the behavioral significance of sound has repeatedly been shown to correlate with long term changes in response properties of neurons in the adult primary auditory cortex. However, the molecular and cellular basis for such changes is still poorly understood. To address this, we have begun examining the auditory cortical expression of an activity-dependent effector immediate early gene (IEG) with documented roles in synaptic plasticity and memory consolidation in the hippocampus: Arc/Arg3.1. For initial characterization, we applied a repeated 10 min (24 h separation) sound exposure paradigm to determine the strength and consistency of sound-evoked Arc/Arg3.1 mRNA expression in the absence of explicit behavioral contingencies for the sound. We used 3D surface reconstruction methods in conjunction with fluorescent in situ hybridization (FISH) to assess the layer-specific subcellular compartmental expression of Arc/Arg3.1 mRNA. We unexpectedly found that both the intranuclear and cytoplasmic patterns of expression depended on the prior history of sound stimulation. Specifically, the percentage of neurons with expression only in the cytoplasm increased for repeated versus singular sound exposure, while intranuclear expression decreased. In contrast, the total cellular expression did not differ, consistent with prior IEG studies of primary auditory cortex. Our results were specific for cortical layers 3-6, as there was virtually no sound driven Arc/Arg3.1 mRNA in layers 1-2 immediately after stimulation. Our results are consistent with the kinetics and/or detectability of cortical subcellular Arc/Arg3.1 mRNA expression being altered by the initial exposure to the sound, suggesting exposure-induced modifications in the cytoplasmic Arc/Arg3.1 mRNA pool. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

DAPK1 as an independent prognostic marker in liver cancer.

PubMed

Li, Ling; Guo, Libin; Wang, Qingshui; Liu, Xiaolong; Zeng, Yongyi; Wen, Qing; Zhang, Shudong; Kwok, Hang Fai; Lin, Yao; Liu, Jingfeng

2017-01-01

The death-associated protein kinase 1 (DAPK1) can act as an oncogene or a tumor suppressor gene depending on the cellular context as well as external stimuli. Our study aims to investigate the prognostic significance of DAPK1 in liver cancer in both mRNA and protein levels. The mRNA expression of DAPK1 was extracted from the Gene Expression Omnibus database in three independent liver cancer datasets while protein expression of DAPK1 was detected by immunohistochemistry in our Chinese liver cancer patient cohort. The associations between DAPK1 expression and clinical characteristics were tested. DAPK1 mRNA expression was down-regulated in liver cancer. Low levels of DAPK1 mRNA were associated with shorter survival in a liver cancer patient cohort ( n  = 115;  p  = 0.041), while negative staining of DAPK1 protein was significantly correlated with shorter time to progression ( p  = 0.002) and overall survival ( p  = 0.02). DAPK1 was an independent prognostic marker for both time to progression and overall survival by multivariate analysis. Liver cancer with the b-catenin mutation has a lower DAPK1 expression, suggesting that DAPK1 may be regulated under the b-catenin pathway. In addition, we also identified genes that are co-regulated with DAPK1. DAPK1 expression was positively correlated with IRF2, IL7R, PCOLCE and ZBTB16, and negatively correlated with SLC16A3 in both liver cancer datasets. Among these genes, PCOLCE and ZBTB16 were significantly down-regulated, while SLC16A3 was significantly upregulated in liver cancer. By using connectivity mapping of these co-regulated genes, we have identified amcinonide and sulpiride as potential small molecules that could potentially reverse DAPK1/PCOLCE/ZBTB16/SLC16A3 expression. Our study demonstrated for the first time that both DAPK1 mRNA and protein expression levels are important prognostic markers in liver cancer, and have identified genes that may contribute to DAPK1-mediated liver carcinogenesis.

Oxacillin sensitization of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius by antisense peptide nucleic acids in vitro.

PubMed

Goh, Shan; Loeffler, Anette; Lloyd, David H; Nair, Sean P; Good, Liam

2015-11-11

Antibiotic resistance genes can be targeted by antisense agents, which can reduce their expression and thus restore cellular susceptibility to existing antibiotics. Antisense inhibitors can be gene and pathogen specific, or designed to inhibit a group of bacteria having conserved sequences within resistance genes. Here, we aimed to develop antisense peptide nucleic acids (PNAs) that could be used to effectively restore susceptibility to β-lactams in methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP). Antisense PNAs specific for conserved regions of the mobilisable gene mecA, and the growth essential gene, ftsZ, were designed. Clinical MRSA and MRSP strains of high oxacillin resistance were treated with PNAs and assayed for reduction in colony forming units on oxacillin plates, reduction in target gene mRNA levels, and cell size. Anti-mecA PNA at 7.5 and 2.5 μM reduced mecA mRNA in MRSA and MRSP (p < 0.05). At these PNA concentrations, 66 % of MRSA and 92 % of MRSP cells were killed by oxacillin (p < 0.01). Anti-ftsZ PNA at 7.5 and 2.5 μM reduced ftsZ mRNA in MRSA and MRSP, respectively (p ≤ 0.05). At these PNA concentrations, 86 % of MRSA cells and 95 % of MRSP cells were killed by oxacillin (p < 0.05). Anti-ftsZ PNAs resulted in swelling of bacterial cells. Scrambled PNA controls did not affect MRSA but sensitized MRSP moderately to oxacillin without affecting mRNA levels. The antisense PNAs effects observed provide in vitro proof of concept that this approach can be used to reverse β-lactam resistance in staphylococci. Further studies are warranted as clinical treatment alternatives are needed.

Does Harvey-Ras gene expression lead to oral squamous cell carcinoma? A clinicopathological aspect

PubMed Central

Krishna, Akhilesh; Singh, Shraddha; Singh, Vineeta; Kumar, Vijay; Singh, Uma Shankar; Sankhwar, Satya Narayan

2018-01-01

Background: Harvey-Ras (H-Ras) is an important guanosine triphosphatase protein for the regulation of cellular growth and survival. Altered Ras signaling has been observed in different types of cancer either by gene amplification and/or mutation. The H-Ras oncogene mutations are well reported, but expression of the H-Ras gene is still unknown. Objective: This study aimed to examine both protein and messenger-RNA (mRNA) expressions of H-Ras in oral squamous cell carcinoma (OSCC) and analyzed the association with risk habits and the clinicopathological profile of cases. Methodology: A total of 65 tissue specimens of OSCC (case group) and equal number of normal tissues (control group) were included in this study. H-Ras protein and mRNA expressions were analyzed using immunohistochemical and quantitative real time-polymerase chain reaction techniques, respectively. Results: The H-Ras protein was significantly overexpressed in the oral carcinoma group compared to the normal group (P = 0.03). Most of the OSCC cases showed positive staining with moderate expression, while negative and moderate staining was high in the control group. The majority of H-Ras positive cases were found in individuals with multiple risk habits including tobacco chewing. The risk of H-Ras positivity was 1.46 times higher in smokers than non-smokers. H-Ras positivity increased in cases affected with buccal mucosa site and higher grade of carcinoma. Relative mRNA level of H-Ras was significantly elevated in oral carcinoma as compared with the control group (P ≤ 0.001). Protein and mRNA levels of H-Ras in case group was poorly correlated. Conclusion: H-Ras oncogene expression was markedly higher in oral carcinoma, and it can be a prognostic marker and target for an effective molecular therapy. PMID:29731559

Curcumin Attenuates Lipopolysaccharide-Induced Hepatic Lipid Metabolism Disorder by Modification of m6 A RNA Methylation in Piglets.

PubMed

Lu, Na; Li, Xingmei; Yu, Jiayao; Li, Yi; Wang, Chao; Zhang, Lili; Wang, Tian; Zhong, Xiang

2018-01-01

N 6 -methyladenosine (m 6 A) regulates gene expression and affects cellular metabolism. In this study, we checked whether the regulation of lipid metabolism by curcumin is associated with m 6 A RNA methylation. We investigated the effects of dietary curcumin supplementation on lipopolysaccharide (LPS)-induced liver injury and lipid metabolism disorder, and on m 6 A RNA methylation in weaned piglets. A total of 24 Duroc × Large White × Landrace piglets were randomly assigned to control, LPS, and CurL (LPS challenge and 200 mg/kg dietary curcumin) groups (n = 8/group). The results showed that curcumin reduced the increase in relative liver weight as well as the concentrations of aspartate aminotransferase and lactate dehydrogenase induced by LPS injection in the plasma and liver of weaning piglets (p < 0.05). The amounts of total cholesterol and triacylglycerols were decreased by curcumin compared to that by the LPS injection (p < 0.05). Additionally, curcumin reduced the expression of Bcl-2 and Bax mRNA, whereas it increased the p53 mRNA level in the liver (p < 0.05). Curcumin inhibited the enhancement of SREBP-1c and SCD-1 mRNA levels induced by LPS in the liver. Notably, dietary curcumin affected the expression of METTL3, METTL14, ALKBH5, FTO, and YTHDF2 mRNA, and increased the abundance of m 6 A in the liver of piglets. In conclusion, the protective effect of curcumin in LPS-induced liver injury and hepatic lipid metabolism disruption might be due to the increase in m 6 A RNA methylation. Our study provides mechanistic insights into the effect of curcumin in protecting against hepatic injury during inflammation and metabolic diseases. © 2018 AOCS.

Higher gamma-aminobutyric acid neuron density in the white matter of orbital frontal cortex in schizophrenia.

PubMed

Joshi, Dipesh; Fung, Samantha J; Rothwell, Alice; Weickert, Cynthia Shannon

2012-11-01

In the orbitofrontal cortex (OFC), reduced gray matter volume and reduced glutamic acid decarboxylase 67kDa isoform (GAD67) messenger (m)RNA are found in schizophrenia; however, how these alterations relate to developmental pathology of interneurons is unclear. The present study therefore aimed to determine if increased interstitial white matter neuron (IWMN) density exists in the OFC; whether gamma-aminobutyric acid (GABA)ergic neuron density in OFC white matter was altered; and how IWMN density may be related to an early-expressed inhibitory neuron marker, Dlx1, in OFC gray matter in schizophrenia. IWMN densities were determined (38 schizophrenia and 38 control subjects) for neuronal nuclear antigen (NeuN+) and 65/67 kDa isoform of glutamic acid decarboxylase immunopositive (GAD65/67+) neurons. In situ hybridization was performed to determine Dlx1 and GAD67 mRNA expression in the OFC gray matter. NeuN and GAD65/67 immunopositive cell density was significantly increased in the superficial white matter in schizophrenia. Gray matter Dlx1 and GAD67 mRNA expression were reduced in schizophrenia. Dlx1 mRNA levels were negatively correlated with GAD65/67 IWMN density. Our study provides evidence that pathology of IWMNs in schizophrenia includes GABAergic interneurons and that increased IWMN density may be related to GABAergic deficits in the overlying gray matter. These findings provide evidence at the cellular level that the OFC is a site of pathology in schizophrenia and support the hypothesis that inappropriate migration of cortical inhibitory interneurons occurs in schizophrenia. Copyright © 2012 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Tadalafil modulates aromatase activity and androgen receptor expression in a human osteoblastic cell in vitro model.

PubMed

Aversa, A; Fittipaldi, S; Bimonte, V M; Wannenes, F; Papa, V; Francomano, D; Greco, E A; Lenzi, A; Migliaccio, S

2016-02-01

Phosphodiesterase type-5 inhibitor (PDE5i) tadalafil administration in men with erectile dysfunction is associated with increased testosterone/estradiol ratio, leading to hypothesize a potential increased effect of androgen action on target tissues. We aimed to characterize, in a cellular model system in vitro, the potential modulation of aromatase and sex steroid hormone receptors upon exposure to tadalafil (TAD). Human osteoblast-like cells SAOS-2 were chosen as an in vitro model system since osteoblasts are target of steroid hormones. Cells were tested for viability upon TAD exposure, which increased cell proliferation. Then, cells were treated with/without TAD for several times to evaluate potential modulation in PDE5, aromatase (ARO), androgen (AR) and estrogen (ER) receptor expression. Osteoblasts express significant levels of both PDE5 mRNA and protein. Exposure of cells to increasing concentrations of TAD (10(-8)-10(-7) M) decreased PDE5 mRNA and protein expression. Also, TAD inhibited ARO mRNA and protein expression leading to an increase in testosterone levels in the supernatants. Interestingly, TAD increased total AR mRNA and protein expression and decreased ERα, with an increased ratio of AR/ER, suggesting preferential androgenic vs estrogenic pathway activation. Our results demonstrate for the first time that TAD decreases ARO expression and increases AR protein expression in human SAOS-2, strongly suggesting a new control of steroid hormones pathway by PDE5i. These findings might represent the first evidence of translational actions of PDE5i on AR, which leads to hypothesize a growing relevance of this molecule in men with prostate cancer long-term treated with TAD for sexual rehabilitation.

Asian and Siberian ginseng as a potential modulator of immune function: an in vitro cytokine study using mouse macrophages.

PubMed

Wang, Huamin; Actor, Jeffrey K; Indrigo, Jessica; Olsen, Margaret; Dasgupta, Amitava

2003-01-01

Ginseng is a widely used herbal product in China, other Asian countries, and in the Unites States. There is a traditional belief that ginseng stimulates immune functions. In this study, the innate effects of Asian and Siberian ginsengs on cytokines and chemokines produced by cultured macrophages were examined. The effects of Asian and Siberian ginseng on cytokines and chemokines produced by cultured macrophages were examined. Mouse macrophages (J774A.1) were incubated with Asian or Siberian ginseng at varying concentrations (1, 10, 100, and 1000 microg/ml) for 24 h and then harvested for RNA isolation. The expression levels of IL-1beta, IL-12, TNF-alpha, MIP-1 alpha, and MIP-2 mRNA were measured by quantitative PCR. Our data showed that Asian ginseng induced a statistically significant increase in IL-12 expression at both mRNA and protein levels. However, the minor twofold increase is probably biologically insignificant. No significant increase of IL-12 by Siberian ginseng was observed at any dose level studied. No significant change in IL-1beta, IL-15, TNF-alpha, or MIP-1alpha mRNA was observed by either Asian or Siberian ginseng treatment. Our data showed statistically significant differential regulation of IL-12 by Asian ginseng. Siberian ginseng did not show a statistically significant increase. We conclude that both Asian ginseng and Siberian ginseng cannot significantly stimulate innate macrophage immune functions that influence cellular immune responses. Therefore, contrary to the popular belief, Asian and Siberian ginseng may not stimulate immune function.

Iron regulatory proteins and their role in controlling iron metabolism.

PubMed

Kühn, Lukas C

2015-02-01

Cellular iron homeostasis is regulated by post-transcriptional feedback mechanisms, which control the expression of proteins involved in iron uptake, release and storage. Two cytoplasmic proteins with mRNA-binding properties, iron regulatory proteins 1 and 2 (IRP1 and IRP2) play a central role in this regulation. Foremost, IRPs regulate ferritin H and ferritin L translation and thus iron storage, as well as transferrin receptor 1 (TfR1) mRNA stability, thereby adjusting receptor expression and iron uptake via receptor-mediated endocytosis of iron-loaded transferrin. In addition splice variants of iron transporters for import and export at the plasma-membrane, divalent metal transporter 1 (DMT1) and ferroportin are regulated by IRPs. These mechanisms have probably evolved to maintain the cytoplasmic labile iron pool (LIP) at an appropriate level. In certain tissues, the regulation exerted by IRPs influences iron homeostasis and utilization of the entire organism. In intestine, the control of ferritin expression limits intestinal iron absorption and, thus, whole body iron levels. In bone marrow, erythroid heme biosynthesis is coordinated with iron availability through IRP-mediated translational control of erythroid 5-aminolevulinate synthase mRNA. Moreover, the translational control of HIF2α mRNA in kidney by IRP1 coordinates erythropoietin synthesis with iron and oxygen supply. Besides IRPs, body iron absorption is negatively regulated by hepcidin. This peptide hormone, synthesized and secreted by the liver in response to high serum iron, downregulates ferroportin at the protein level and thereby limits iron absorption from the diet. Hepcidin will not be discussed in further detail here.

Selenium status affects selenoprotein expression, reproduction, and F₁ generation locomotor activity in zebrafish (Danio rerio).

PubMed

Penglase, Sam; Hamre, Kristin; Rasinger, Josef D; Ellingsen, Staale

2014-06-14

Se is an essential trace element, and is incorporated into selenoproteins which play important roles in human health. Mammalian selenoprotein-coding genes are often present as paralogues in teleost fish, and it is unclear whether the expression patterns or functions of these fish paralogues reflect their mammalian orthologues. Using the model species zebrafish (Danio rerio; ZF), we aimed to assess how dietary Se affects key parameters in Se metabolism and utilisation including glutathione peroxidase (GPX) activity, the mRNA expression of key Se-dependent proteins (gpx1a, gpx1b, sepp1a and sepp1b), oxidative status, reproductive success and F1 generation locomotor activity. From 27 d until 254 d post-fertilisation, ZF were fed diets with graded levels of Se ranging from deficient ( < 0·10 mg/kg) to toxic (30 mg/kg). The mRNA expression of gpx1a and gpx1b and GPX activity responded in a similar manner to changes in Se status. GPX activity and mRNA levels were lowest when dietary Se levels (0·3 mg/kg) resulted in the maximum growth of ZF, and a proposed bimodal mechanism in response to Se status below and above this dietary Se level was identified. The expression of the sepp1 paralogues differed, with only sepp1a responding to Se status. High dietary Se supplementation (30 mg/kg) decreased reproductive success, while the offspring of ZF fed above 0·3 mg Se/kg diet had lower locomotor activity than the other groups. Overall, the novel finding of low selenoprotein expression and activity coinciding with maximum body growth suggests that even small Se-induced variations in redox status may influence cellular growth rates.

Large scale real-time PCR analysis of mRNA abundance in rainbow trout eggs in relationship with egg quality and post-ovulatory ageing.

PubMed

Aegerter, Sandrine; Jalabert, Bernard; Bobe, Julien

2005-11-01

The mRNA levels of 39 target genes were monitored in unfertilized eggs of 14 rainbow trout sampled the day of ovulation and again 5, 14, and 21 days later. For all 56 collected egg batches, an egg sample was fertilized to estimate egg quality by monitoring embryonic development. Remaining eggs were used for RNA extraction and subsequent real-time PCR analysis. A significant drop of egg quality was observed when eggs were held in the body cavity for 14 or 21 days post-ovulation (dpo). During the same period, eight transcripts (nucleoplasmin or Npm2, ferritin H, tubulin beta, JNK1, cyclin A1, cyclin A2, cathepsin Z, and IGF2) exhibited a differential abundance at one or several collection time(s). Interestingly, we observed higher levels of cyclins A1 and A2 mRNAs in eggs taken 5 days post-ovulation than in eggs taken, from the same females, at the time of ovulation. In addition, seven transcripts exhibited a differential abundance between low quality and high quality eggs. Low quality eggs were characterized by lower levels of Npm2, tubulin beta, and IGF1 transcripts. In contrast, keratins 8 and 18, cathepsin Z, and prostaglandin synthase 2 were more abundant in low quality eggs than in high quality eggs. In this study, we have demonstrated differences in mRNA levels in the rainbow trout egg that are reflective of developmental competence differences induced by post-ovulatory ageing. The putative role of these transcripts in post-ovulatory ageing-induced egg quality defects is discussed with special attention for corresponding cellular functions.

Immunotoxicity of surface waters contaminated by municipal effluents to the snail Lymnaea stagnalis.

PubMed

Gust, M; Fortier, M; Garric, J; Fournier, M; Gagné, F

2013-01-15

The immunotoxic effects of surface waters contaminated by a municipal effluent dispersion plume were examined in the snail Lymnaea stagnalis. Snails were exposed to surface waters where changes in hemocyte counts, viability, levels of reactive oxygen species (ROS), reduced thiols and phagocytic activity were tracked following exposure periods of 3h and 3 and 7d. Changes in mRNA expression of some genes in the hemocytes were also assessed after 7d of exposure, as follows: genes coding for catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GSR), selenium-dependent glutathione peroxidase (SeGPX), two isoforms of the nitric oxide synthetase (NOS1 and NOS2), molluscan defensive molecule (MDM), toll-like receptor 4 (TLR4), allograft inflammatory factor-1 (AIF), and heat-shock protein 70 (HSP70). At the sites closest to the discharge point, exposure led to impaired hemocyte viability and intracellular thiol levels and also an increase of hemocyte count, ROS levels and phagocytosis. Phagocytosis and ROS levels in hemocytes were correlated with heterotrophic bacterial counts in snails. We found four genes with increased mRNA expression as a response to exposure of municipal wastewaters: TLR4 (6-fold), HSP70 (2-fold), SeGPx (4-fold) and CAT (2-fold). Immunocompetence responses were analyzed by canonical analysis to seek out relationships with mRNA expression of the genes involved in stress, pattern recognition, cellular and humoral responses. The data revealed that genes involved in oxidative stress were strongly involved with immunocompetence and that the resulting immune responses were influenced both by the bacterial and pollutant loadings of the effluent. Copyright © 2012 Elsevier B.V. All rights reserved.

Genome-wide expression profiling of in vivo-derived bloodstream parasite stages and dynamic analysis of mRNA alterations during synchronous differentiation in Trypanosoma brucei

PubMed Central

Kabani, Sarah; Fenn, Katelyn; Ross, Alan; Ivens, Al; Smith, Terry K; Ghazal, Peter; Matthews, Keith

2009-01-01

Background Trypanosomes undergo extensive developmental changes during their complex life cycle. Crucial among these is the transition between slender and stumpy bloodstream forms and, thereafter, the differentiation from stumpy to tsetse-midgut procyclic forms. These developmental events are highly regulated, temporally reproducible and accompanied by expression changes mediated almost exclusively at the post-transcriptional level. Results In this study we have examined, by whole-genome microarray analysis, the mRNA abundance of genes in slender and stumpy forms of T.brucei AnTat1.1 cells, and also during their synchronous differentiation to procyclic forms. In total, five biological replicates representing the differentiation of matched parasite populations derived from five individual mouse infections were assayed, with RNAs being derived at key biological time points during the time course of their synchronous differentiation to procyclic forms. Importantly, the biological context of these mRNA profiles was established by assaying the coincident cellular events in each population (surface antigen exchange, morphological restructuring, cell cycle re-entry), thereby linking the observed gene expression changes to the well-established framework of trypanosome differentiation. Conclusion Using stringent statistical analysis and validation of the derived profiles against experimentally-predicted gene expression and phenotypic changes, we have established the profile of regulated gene expression during these important life-cycle transitions. The highly synchronous nature of differentiation between stumpy and procyclic forms also means that these studies of mRNA profiles are directly relevant to the changes in mRNA abundance within individual cells during this well-characterised developmental transition. PMID:19747379

The Structure and Function of the Rous Sarcoma virus RNA Stability Element

PubMed Central

Withers, Johanna B.; Beemon, Karen L.

2013-01-01

For simple retroviruses, such as the Rous sarcoma virus (RSV), post-transcriptional control elements regulate viral RNA splicing, export, stability, and packaging into virions. These RNA sequences interact with cellular host proteins to regulate and facilitate productive viral infections. One such element, known as the RSV stability element (RSE), is required for maintaining stability of the full-length unspliced RNA. This viral RNA serves as the mRNA for the Gag and Pol proteins and also as the genome packaged in progeny virions. When the RSE is deleted from the viral RNA, the unspliced RNA becomes unstable and is degraded in a Upf1-dependent manner. Current evidence suggests that the RSE inhibits recognition of the viral gag termination codon by the nonsense-mediated mRNA decay (NMD) pathway. We believe that the RSE acts as an insulator to NMD, thereby preventing at least one of the required functional steps that target an mRNA for degradation. Here, we discuss the history of the RSE and the current model of how the RSE is interacting with cellular NMD factors. PMID:21769913

Lipofuscin accumulation and autophagy in glaucomatous human lamina cribrosa cells.

PubMed

McElnea, Elizabeth M; Hughes, Emily; McGoldrick, Aloysius; McCann, Amanda; Quill, Barry; Docherty, Neil; Irnaten, Mustapha; Farrell, Michael; Clark, Abbot F; O'Brien, Colm J; Wallace, Deborah M

2014-12-02

Disease associated alterations in the phenotype of lamina cribrosa (LC) cells are implicated in changes occurring at the optic nerve head (ONH) in glaucoma. Lipofuscin, the formation of which is driven by reactive oxygen species (ROS), is an intralysosomal, non-degradable, auto-fluorescent macromolecule which accumulates with age and can affect autophagy - the lysosomal degradation of a cell's constituents. We aimed to compare the content of lipofuscin-like material and markers of autophagy in LC cells from normal and glaucoma donor eyes. The number and size of peri-nuclear lysosomes were examined by transmission electron microscopy (TEM). Cellular auto-fluorescence was quantified by flow cytometry. Cathepsin K mRNA levels were assessed by PCR. Autophagy protein 5 (Atg5) mRNA and protein levels were analysed by PCR and Western blot. Protein levels of subunits of the microtubule associated proteins (MAP) 1A and 1B, light chain 3 (LC3) I and II were analysed by Western blot. Immunohistochemical staining of LC3-II in ONH sections from normal and glaucomatous donor eyes was performed. A significant increase in the number of peri-nuclear lysosomes [4.1 × 10,000 per high power field (h.p.f.) ± 1.9 vs. 2.0 × 10,000 per h.p.f. ± 1.3, p = 0.002, n = 3] and whole cell auto-fluorescence (83.62 ± 45.1 v 41.01 ± 3.9, p = 0.02, n = 3) was found in glaucomatous LC cells relative to normal LC cells. Glaucomatous LC cells possessed significantly higher levels of Cathepsin K mRNA and Atg5 mRNA and protein. Enhanced levels of LC3-II were found in both LC cells and optic nerve head sections from glaucoma donors. Increased lipofuscin formation is characteristic of LC cells from donors with glaucoma. This finding confirms the importance of oxidative stress in glaucoma pathogenesis. Intracellular lipofuscin accumulation may have important effects on autophagy the modification of which could form the basis for future novel glaucoma treatments.

Effects of Two Types of Melatonin-Loaded Nanocapsules with Distinct Supramolecular Structures: Polymeric (NC) and Lipid-Core Nanocapsules (LNC) on Bovine Embryo Culture Model.

PubMed

Komninou, Eliza Rossi; Remião, Mariana Härter; Lucas, Caroline Gomes; Domingues, William Borges; Basso, Andrea Cristina; Jornada, Denise Soledade; Deschamps, João Carlos; Beck, Ruy Carlos Ruver; Pohlmann, Adriana Raffin; Bordignon, Vilceu; Seixas, Fabiana Kömmling; Campos, Vinicius Farias; Guterres, Silvia Stanisçuaski; Collares, Tiago

2016-01-01

Melatonin has been used as a supplement in culture medium to improve the efficiency of in vitro produced mammalian embryos. Through its ability to scavenge toxic oxygen derivatives and regulate cellular mRNA levels for antioxidant enzymes, this molecule has been shown to play a protective role against damage by free radicals, to which in vitro cultured embryos are exposed during early development. In vivo and in vitro studies have been performed showing that the use of nanocapsules as active substances carriers increases stability, bioavailability and biodistribution of drugs, such as melatonin, to the cells and tissues, improving their antioxidant properties. These properties can be modulated through the manipulation of formula composition, especially in relation to the supramolecular structures of the nanocapsule core and the surface area that greatly influences drug release mechanisms in biological environments. This study aimed to evaluate the effects of two types of melatonin-loaded nanocapsules with distinct supramolecular structures, polymeric (NC) and lipid-core (LNC) nanocapsules, on in vitro cultured bovine embryos. Embryonic development, apoptosis, reactive oxygen species (ROS) production, and mRNA levels of genes involved in cell apoptosis, ROS and cell pluripotency were evaluated after supplementation of culture medium with non-encapsulated melatonin (Mel), melatonin-loaded polymeric nanocapsules (Mel-NC) and melatonin-loaded lipid-core nanocapsules (Mel-LNC) at 10-6, 10-9, and 10-12 M drug concentrations. The highest hatching rate was observed in embryos treated with 10-9 M Mel-LNC. When compared to Mel and Mel-NC treatments at the same concentration (10-9 M), Mel-LNC increased embryo cell number, decreased cell apoptosis and ROS levels, down-regulated mRNA levels of BAX, CASP3, and SHC1 genes, and up-regulated mRNA levels of CAT and SOD2 genes. These findings indicate that nanoencapsulation with LNC increases the protective effects of melatonin against oxidative stress and cell apoptosis during in vitro embryo culture in bovine species.

Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen.

PubMed

Lee, H J; Lee, S B; Chung, J S; Han, S U; Han, O; Guh, J O; Jeon, J S; An, G; Back, K

2000-06-01

Protoporphyrinogen oxidase (Protox), the penultimate step enzyme of the branch point for the biosynthetic pathway of Chl and hemes, is the target site of action of diphenyl ether (DPE) herbicides. However, Bacillus subtilis Protox is known to be resistant to the herbicides. In order to develop the herbicide-resistant plants, the transgenic rice plants were generated via expression of B. subtilis Protox gene under ubiquitin promoter targeted to the cytoplasm or to the plastid using Agrobacterium-mediated gene transformation. The integration and expression of the transgene were investigated at T0 generation by DNA and RNA blots. Most transgenic rice plants revealed one copy transgene insertion into the rice genome, but some with 3 copies. The expression levels of B. subtilis Protox mRNA appeared to correlate with the copy number. Furthermore, the plastidal transgenic lines exhibited much higher expression of the Protox mRNA than the cytoplasmic transgenic lines. The transgenic plants expressing the B. subtilis Protox gene at T0 generation were found to be resistant to oxyfluorfen when judged by cellular damage with respect to cellular leakage, Chl loss, and lipid peroxidation. The transgenic rice plants targeted to the plastid exhibited higher resistance to the herbicide than the transgenic plants targeted to the cytoplasm. In addition, possible resistance mechanisms in the transgenic plants to DPE herbicides are discussed.

Alterations of overused supraspinatus tendon: a possible role of glycosaminoglycans and HARP/pleiotrophin in early tendon pathology.

PubMed

Attia, Mohamed; Scott, Alexander; Duchesnay, Arlette; Carpentier, Gilles; Soslowsky, Louis J; Huynh, Minh Bao; Van Kuppevelt, Toin H; Gossard, Camille; Courty, José; Tassoni, Marie-Claude; Martelly, Isabelle

2012-01-01

Supraspinatus tendon overuse injuries lead to significant pain and disability in athletes and workers. Despite the prevalence and high social cost of these injuries, the early pathological events are not well known. We analyzed the potential relation between glycosaminoglycan (GAG) composition and phenotypic cellular alteration using a rat model of rotator cuff overuse. Total sulfated GAGs increased after 4 weeks of overuse and remained elevated up to 16 weeks. GAG accumulation was preceded by up-regulation of decorin, versican, and aggrecan proteoglycans (PGs) mRNAs and proteins and biglycan PG mRNA after 2 weeks. At 2 weeks, collagen 1 transcript decreased whereas mRNAs for collagen 2, collagen 3, collagen 6, and the transcription factor Sox9 were increased. Protein levels of heparin affine regulatory peptide (HARP)/pleiotrophin, a cytokine known to regulate developmental chondrocyte formation, were enhanced especially at 4 weeks, without up-regulation of HARP/pleiotrophin mRNA. Further results suggest that the increased GAGs present in early lesions may sequester HARP/pleiotrophin, which could contribute to a loss of tenocyte's phenotype. All these modifications are characteristic of a shift towards the chondrocyte phenotype. Identification of these early changes in the extra-cellular matrix may help to prevent the progression of the pathology to more disabling, degenerative alterations. Copyright © 2011 Orthopaedic Research Society.

Arsenite and its metabolites, MMA(III) and DMA(III), modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice.

PubMed

Medina-Díaz, I M; Estrada-Muñiz, E; Reyes-Hernández, O D; Ramírez, P; Vega, L; Elizondo, G

2009-09-01

Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half the drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA(III) induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA(III) increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA(III) induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.

Arsenite and its metabolites, MMA{sup III} and DMA{sup III}, modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice

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

Medina-Diaz, I.M.; Estrada-Muniz, E.; Reyes-Hernandez, O.D.

Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half themore » drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA{sup III} induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA{sup III} increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA{sup III} induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.« less

Resveratrol-induced transcriptional up-regulation of ASMase (SMPD1) of human leukemia and cancer cells

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

Mizutani, Naoki; College of Life and Health Sciences, Chubu University, Kasugai; Omori, Yukari

2016-02-19

Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increasedmore » by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5′-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment. - Highlights: • Resveratrol inhibited cell proliferation of K562 and HCT116 cells. • Resveratrol increased cellular ceramide and decreased sphingomyelin and S1P. • ASMase mRNA and activity were increased with resveratrol. • ASMase inhibition suppressed RSV-induced ceramide accumulation. • Increased ASMase transcription was at least partially due to EGR family proteins.« less

Bombyx mori nucleopolyhedrovirus nucleic acid binding proteins BRO-B and BRO-E associate with host T-cell intracellular antigen 1 homologue BmTRN-1 to influence protein synthesis during infection.

PubMed

Kotani, Eiji; Muto, Sayaka; Ijiri, Hiroshi; Mori, Hajime

2015-07-01

Previous reports have indicated that the Bombyx mori nucleopolyhedrovirus (BmNPV) nucleic acid binding proteins BRO-B and BRO-E are expressed during the early stage of infection and that the BRO family likely supports the regulation of mRNA; however, no study has directly examined the function of BRO family proteins in virus-permissive cells. Here, we show that BRO-B and BRO-E associate with cellular T-cell intracellular antigen 1 homologue (BmTRN-1), a translational regulator, and other cellular translation-related proteins in silkworm cells during viral infection. We created BM-N cells that expressed BRO-B/E to study molecular interactions between BmTRN-1 and BRO-B/E and how they influenced protein synthesis. Fluorescent microscopy revealed that BmTRN-1 was localized in cytoplasmic foci during BmNPV infection. Immunofluorescence studies confirmed that BmTRN-1 and BRO-B/E were colocalized in the amorphous conspicuous cytoplasmic foci. Reporter gene studies revealed that co-expression of BRO-B/E synergistically led to a significant decrease in protein synthesis from a designed transcript carrying the 5'untranslated region of a cellular mRNA with no significant change of transcript abundance. Additionally, RNA interference-mediated knockdown of BmTRN-1 resulted in a marked inhibition of the ability of BRO-B/E to regulate the transcript. These results suggested that the association of BmTRN-1 with BRO-B/E is responsible for the inhibitory regulation of certain mRNAs at the post-transcriptional level and add an additional mechanism for how baculoviruses control protein synthesis during infection.

Development, food intake, and ethinylestradiol influence hepatic triglyceride lipase and LDL-receptor mRNA levels in rats.

PubMed

Staels, B; Jansen, H; van Tol, A; Stahnke, G; Will, H; Verhoeven, G; Auwerx, J

1990-07-01

The influence of development and ethinylestradiol on low density lipoprotein (LDL)-receptor mRNA and hepatic triglyceride lipase (HTGL) activity and mRNA levels was studied in rat liver and intestine. Intestinal LDL-receptor mRNA levels are maximal in the perinatal period, whereas liver LDL-receptor and HTGL mRNA levels are highest after weaning in adult life. All mRNA levels reach a maximum between day 15 and 20 when rats still consume a lipid-rich diet, and increase twofold during weaning. Liver and intestinal LDL-receptor mRNA levels are not influenced by ovariectomy, but increase after ethinylestradiol treatment. Liver LDL-receptor mRNA shows a dose-dependent increase after ethinylestradiol and a sevenfold rise in liver LDL-receptor mRNA is attained with a dose of 2000 micrograms/day. Intestinal LDL-receptor mRNA increases slightly more than twofold after ethinylestradiol and this increase is not dose-dependent. Changes in LDL-receptor mRNA are independent of changes in food intake induced by ethinylestradiol treatment, since they are still observed after pair-feeding. The ethinylestradiol-induced increases in LDL-receptor mRNA levels are reflected by decreased serum apoB levels. HTGL mRNA levels increase after ovariectomy and show a dose-dependent decrease after ethinylestradiol. Pair-feeding abolishes the increase seen after ovariectomy, while the estrogen-mediated decrease is attenuated. These alterations in HTGL mRNA are reflected by similar changes in liver HTGL activity.(ABSTRACT TRUNCATED AT 250 WORDS)

A HIF-1alpha-related gene involved in cell protection from hypoxia by suppression of mitochondrial function.

PubMed

Kakinuma, Yoshihiko; Katare, Rajesh G; Arikawa, Mikihiko; Muramoto, Kazuyo; Yamasaki, Fumiyasu; Sato, Takayuki

2008-01-23

Recently, we reported that acetylcholine-induced hypoxia-inducible factor-1alpha protects cardiomyocytes from hypoxia; however, the downstream factors reducing hypoxic stress are unknown. We identified apoptosis inhibitor (AI) gene as being differentially expressed between von Hippel Lindau (VHL) protein-positive cells with high levels of GRP78 expression and VHL-negative cells with lower GRP levels, using cDNA subtraction. AI decreased GRP78 level, suppressed mitochondrial function, reduced oxygen consumption and, ultimately, suppressed hypoxia-induced apoptosis. By contrast, knockdown of the AI gene increased mitochondrial function. Hypoxic cardiomyocytes and ischemic myocardium showed increased AI mRNA expression. These findings suggest that AI is involved in suppressing mitochondrial function, thereby leading to cellular stress eradication and consequently to protection during hypoxia.

PCB 126 toxicity is modulated by cross-talk between caveolae and Nrf2 signaling

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

Petriello, Michael C.; University of Kentucky Superfund Research Center, Lexington, KY 40536; Han, Sung Gu

2014-06-01

Environmental toxicants such as polychlorinated biphenyls (PCBs) have been implicated in the promotion of multiple inflammatory disorders including cardiovascular disease, but information regarding mechanisms of toxicity and cross-talk between relevant cell signaling pathways is lacking. To examine the hypothesis that cross-talk between membrane domains called caveolae and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways alters PCB-induced inflammation, caveolin-1 was silenced in vascular endothelial cells, resulting in a decreased PCB-induced inflammatory response. Cav-1 silencing (siRNA treatment) also increased levels of Nrf2-ARE transcriptional binding, resulting in higher mRNA levels of the antioxidant genes glutathione s-transferase and NADPH dehydrogenase quinone-1 in both vehiclemore » and PCB-treated systems. Along with this upregulated antioxidant response, Cav-1 siRNA treated cells exhibited decreased mRNA levels of the Nrf2 inhibitory protein Keap1 in both vehicle and PCB-treated samples. Silencing Cav-1 also decreased protein levels of Nrf2 inhibitory proteins Keap1 and Fyn kinase, especially in PCB-treated cells. Further, endothelial cells from wildtype and Cav-1 −/− mice were isolated and treated with PCB to better elucidate the role of functional caveolae in PCB-induced endothelial inflammation. Cav-1 −/− endothelial cells were protected from PCB-induced cellular dysfunction as evidenced by decreased vascular cell adhesion molecule (VCAM-1) protein induction. Compared to wildtype cells, Cav-1 −/− endothelial cells also allowed for a more effective antioxidant response, as observed by higher levels of the antioxidant genes. These data demonstrate novel cross-talk mechanisms between Cav-1 and Nrf2 and implicate the reduction of Cav-1 as a protective mechanism for PCB-induced cellular dysfunction and inflammation. - Highlights: • Reduction of caveolin-1 protein protects against polychlorinated biphenyl toxicity. • Decreasing caveolin-1 levels increases the Nrf2 antioxidant response. • Reducing caveolin-1 levels decreases expression of Nrf2 inhibitory proteins. • Caveolin-1/Nrf2 cross-talk is evident in mouse, human, and porcine endothelial cells.« less

Nutrient sensing and insulin signaling in neuropeptide-expressing immortalized, hypothalamic neurons: A cellular model of insulin resistance.

PubMed

Fick, Laura J; Belsham, Denise D

2010-08-15

Obesity and type 2 diabetes mellitus represent a significant global health crisis. These two interrelated diseases are typified by perturbed insulin signaling in the hypothalamus. Using novel hypothalamic cell lines, we have begun to elucidate the molecular and intracellular mechanisms involved in the hypothalamic control of energy homeostasis and insulin resistance. In this review, we present evidence of insulin and glucose signaling pathways that lead to changes in neuropeptide gene expression. We have identified some of the molecular mechanisms involved in the control of de novo hypothalamic insulin mRNA expression. And finally, we have defined key mechanisms involved in the etiology of cellular insulin resistance in hypothalamic neurons that may play a fundamental role in cases of high levels of insulin or saturated fatty acids, often linked to the exacerbation of obesity and diabetes.

Role of the DNA Damage Response in Human Papillomavirus RNA Splicing and Polyadenylation.

PubMed

Nilsson, Kersti; Wu, Chengjun; Schwartz, Stefan

2018-06-12

Human papillomaviruses (HPVs) have evolved to use the DNA repair machinery to replicate its DNA genome in differentiated cells. HPV activates the DNA damage response (DDR) in infected cells. Cellular DDR factors are recruited to the HPV DNA genome and position the cellular DNA polymerase on the HPV DNA and progeny genomes are synthesized. Following HPV DNA replication, HPV late gene expression is activated. Recent research has shown that the DDR factors also interact with RNA binding proteins and affects RNA processing. DDR factors activated by DNA damage and that associate with HPV DNA can recruit splicing factors and RNA binding proteins to the HPV DNA and induce HPV late gene expression. This induction is the result of altered alternative polyadenylation and splicing of HPV messenger RNA (mRNA). HPV uses the DDR machinery to replicate its DNA genome and to activate HPV late gene expression at the level of RNA processing.

Expression of different functional isoforms in haematopoiesis.

PubMed

Grech, Godfrey; Pollacco, Joel; Portelli, Mark; Sacco, Keith; Baldacchino, Shawn; Grixti, Justine; Saliba, Christian

2014-01-01

Haematopoiesis is a complex process regulated at various levels facilitating rapid responses to external factors including stress, modulation of lineage commitment and terminal differentiation of progenitors. Although the transcription program determines the RNA pool of a cell, various mRNA strands can be obtained from the same template, giving rise to multiple protein isoforms. The majority of variants and isoforms co-occur in normal haematopoietic cells or are differentially expressed at various maturity stages of progenitor maturation and cellular differentiation within the same lineage or across lineages. Genetic aberrations or specific cellular states result in the predominant expression of abnormal isoforms leading to deregulation and disease. The presence of upstream open reading frames (uORF) in 5' untranslated regions (UTRs) of a transcript, couples the utilization of start codons with the cellular status and availability of translation initiation factors (eIFs). In addition, tissue-specific and cell lineage-specific alternative promoter use, regulates several transcription factors producing transcript variants with variable 5' exons. In this review, we propose to give a detailed account of the differential isoform formation, causing haematological malignancies.

A platelet-activating factor (PAF) receptor deficiency exacerbates diet-induced obesity but PAF/PAF receptor signaling does not contribute to the development of obesity-induced chronic inflammation.

PubMed

Yamaguchi, Masahiko; Matsui, Masakazu; Higa, Ryoko; Yamazaki, Yasuhiro; Ikari, Akira; Miyake, Masaki; Miwa, Masao; Ishii, Satoshi; Sugatani, Junko; Shimizu, Takao

2015-02-15

Platelet-activating factor (PAF) is a well-known phospholipid that mediates acute inflammatory responses. In the present study, we investigated whether PAF/PAF receptor signaling contributed to chronic inflammation in the white adipose tissue (WAT) of PAF receptor-knockout (PAFR-KO) mice. Body and epididymal WAT weights were higher in PAFR-KO mice fed a high-fat diet (HFD) than in wild-type (WT) mice. TNF-α mRNA expression levels in epididymal WAT and the infiltration of CD11c-positive macrophages into epididymal WAT, which led to chronic inflammation, were also elevated in HFD-fed PAFR-KO mice. HFD-fed PAFR-KO mice had higher levels of fasting serum glucose than HFD-fed WT mice as well as impaired glucose tolerance. Although PAF receptor signaling up-regulated the expression of TNF-α and lipopolysaccharide induced the expression of acyl-CoA:lysophosphatidylcholine acyltransferase 2 (LPCAT2) mRNA in bone marrow-derived macrophages, no significant differences were observed in the expression of LPCAT2 mRNA and PAF levels in epididymal WAT between HFD-fed mice and normal diet-fed mice. In addition to our previous finding in which energy expenditure in PAF receptor (PAFR)-deficient mice was low due to impaired brown adipose tissue function, the present study demonstrated that PAF/PAF receptor signaling up-regulated the expression of Ucp1 mRNA, which is essential for cellular thermogenesis, in 3T3-L1 adipocytes. We concluded that the marked accumulation of abdominal fat due to HFD feeding led to more severe chronic inflammation in WAT, which is associated with glucose metabolism disorders, in PAFR-KO mice than in WT mice, and PAF/PAF receptor signaling may regulate energy expenditure and adiposity. Copyright © 2015 Elsevier Inc. All rights reserved.

Altered regulation of Nur77 nuclear receptor gene expression in the mesocorticolimbic regions of rat brain by amphetamine sensitization.

PubMed

Bhardwaj, Sanjeev K; Dodat, Fatéma; Lévesque, Daniel; Srivastava, Lalit K

2018-05-08

The mechanisms underlying psychostimulant drug-induced sensitization include long-term cellular and molecular adaptations in dopaminergic circuits. Nur77, a member of the Nur family of transcription factors, is expressed in brain regions receiving dopamine inputs and plays a role in activity-induced synaptic modification. Here we evaluated changes in Nur77 mRNA levels in the medial prefrontal cortex (mPFC), dorsal striatum (Str) and nucleus accumbens (NAc) of rats receiving a repeated, sensitizing regimen of amphetamine (AMPH). Results were compared to two groups of controls - animals receiving repeated injections of saline (Rp-SAL) or with no treatment (CON). Two weeks after the last injection, the effect of an acute challenge dose of AMPH on Nur77 expression was evaluated using in-situ hybridization. Repeated AMPH treatment (Rp-AMPH) increased the levels of Nur77 mRNA in the mPFC, NAc core and shell regions. However, the effects of an acute injection of AMPH in each of the three groups of animals was distinct. Whereas an acute AMPH led to a significant increase of Nur77 in all brain regions of the CON animals, it had no significant effect in Rp-SAL animals. Interestingly, in acute AMPH-injected Rp-AMPH animals, Nur77 mRNA levels in the mPFC, Str and NAc regions were significantly lower compared to CON and Rp-SAL animals treated with acute AMPH. There was a positive correlation between AMPH -induced locomotor activity and Nur77 mRNA expression in CON animals; however, this relationship was absent in Rp-SAL and Rp-AMPH animals. The data suggest that Nur77 is a part of neuroadaptive changes caused by either mild stress of repeated injections as well as AMPH-sensitization and may play a role in abnormal behaviors induced by the drug. Copyright © 2018. Published by Elsevier B.V.

The multifunctional Staufen proteins: conserved roles from neurogenesis to synaptic plasticity.

PubMed

Heraud-Farlow, Jacki E; Kiebler, Michael A

2014-09-01

Staufen (Stau) proteins belong to a family of RNA-binding proteins (RBPs) that are important for RNA localisation in many organisms. In this review we discuss recent findings on the conserved role played by Stau during both the early differentiation of neurons and in the synaptic plasticity of mature neurons. Recent molecular data suggest mechanisms for how Stau2 regulates mRNA localisation, mRNA stability, translation, and ribonucleoprotein (RNP) assembly. We offer a perspective on how this multifunctional RBP has been adopted to regulate mRNA localisation under several different cellular and developmental conditions. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Glucocorticoid receptor interacts with PNRC2 in a ligand-dependent manner to recruit UPF1 for rapid mRNA degradation.

PubMed

Cho, Hana; Park, Ok Hyun; Park, Joori; Ryu, Incheol; Kim, Jeonghan; Ko, Jesang; Kim, Yoon Ki

2015-03-31

Glucocorticoid receptor (GR), which was originally known to function as a nuclear receptor, plays a role in rapid mRNA degradation by acting as an RNA-binding protein. The mechanism by which this process occurs remains unknown. Here, we demonstrate that GR, preloaded onto the 5'UTR of a target mRNA, recruits UPF1 through proline-rich nuclear receptor coregulatory protein 2 (PNRC2) in a ligand-dependent manner, so as to elicit rapid mRNA degradation. We call this process GR-mediated mRNA decay (GMD). Although GMD, nonsense-mediated mRNA decay (NMD), and staufen-mediated mRNA decay (SMD) share upstream frameshift 1 (UPF1) and PNRC2, we find that GMD is mechanistically distinct from NMD and SMD. We also identify de novo cellular GMD substrates using microarray analysis. Intriguingly, GMD functions in the chemotaxis of human monocytes by targeting chemokine (C-C motif) ligand 2 (CCL2) mRNA. Thus, our data provide molecular evidence of a posttranscriptional role of the well-studied nuclear hormone receptor, GR, which is traditionally considered a transcription factor.

EphA2 Is a Potential Player of Malignant Cellular Behavior in Non-Metastatic Renal Cell Carcinoma Cells but Not in Metastatic Renal Cell Carcinoma Cells.

PubMed

Cho, Min Chul; Cho, Sung Yong; Yoon, Cheol Yong; Lee, Seung Bae; Kwak, Cheol; Kim, Hyeon Hoe; Jeong, Hyeon

2015-01-01

To investigate the role of EphA2 in malignant cellular behavior in renal cell carcinoma (RCC) cells and whether FAK/RhoA signaling can act as downstream effectors of EphA2 on RCC cells. Expression of EphA2 protein in non-metastatic RCC (Caki-2 and A498), metastatic RCC cells (Caki-1 and ACHN), HEK-293 cells and prostate cancer cells (PC-3 and DU-145; positive controls of EphA2 expression) was evaluated by Western blot. Changes in mRNA or protein expression of EphA2, FAK or membrane-bound RhoA following EphA2, FAK or RhoA small interfering RNA (siRNA) transfection were determined by reverse transcription polymerase chain reaction or Western blot. The effect of siRNA treatment on cellular viability, apoptosis and invasion was analyzed by cell counting kit-8, Annexin-V and modified Matrigel-Boyden assays, respectively. In all RCC cell lines, the expression of EphA2 protein was detectable at variable levels; however, in HEK-293 cells, EphA2 expression was very low. Treatment with EphA2 siRNA significantly reduced the expression of EphA2 mRNA and protein in all RCC cell lines. For non-metastatic RCC cells (Caki-2 and A498) but not metastatic RCC cells (Caki-1 and ACHN), cellular viability, invasiveness, resistance to apoptosis, expression of membrane-bound RhoA protein and FAK phosphorylation were significantly decreased in EphA2 siRNA-treated cells compared to the control. In non-metastatic RCC cells, FAK siRNA significantly attenuated the invasiveness, resistance to apoptosis, as well as expression of membrane-bound RhoA protein without changing protein expression of EphA2. RhoA siRNA significantly decreased the malignant cellular behavior and expression of membrane-bound RhoA protein without changing EphA2 protein expression or FAK phosphorylation. Our data provide the first functional evidence that the EphA2/FAK/RhoA signaling pathway plays a critical role in the malignant cellular behavior of RCC and appears to be functional particularly in the early stage of malignant progression of non-metastatic RCC.

TM9/Phg1 and SadA proteins control surface expression and stability of SibA adhesion molecules in Dictyostelium.

PubMed

Froquet, Romain; le Coadic, Marion; Perrin, Jackie; Cherix, Nathalie; Cornillon, Sophie; Cosson, Pierre

2012-02-01

TM9 proteins form a family of conserved proteins with nine transmembrane domains essential for cellular adhesion in many biological systems, but their exact role in this process remains unknown. In this study, we found that genetic inactivation of the TM9 protein Phg1A dramatically decreases the surface levels of the SibA adhesion molecule in Dictyostelium amoebae. This is due to a decrease in sibA mRNA levels, in SibA protein stability, and in SibA targeting to the cell surface. A similar phenotype was observed in cells devoid of SadA, a protein that does not belong to the TM9 family but also exhibits nine transmembrane domains and is essential for cellular adhesion. A contact site A (csA)-SibA chimeric protein comprising only the transmembrane and cytosolic domains of SibA and the extracellular domain of the Dictyostelium surface protein csA also showed reduced stability and relocalization to endocytic compartments in phg1A knockout cells. These results indicate that TM9 proteins participate in cell adhesion by controlling the levels of adhesion proteins present at the cell surface.

Vulnerability of Gastric Mucosa in Diabetic Rats, Its Pathogenesis and Amelioration by Cuminum cyminum

PubMed Central

Vador, N.; Jagtap, Aarti G.; Damle, Archana

2012-01-01

Various studies have indicated that peptic ulcers occurring during the course of diabetic state are more severe and often associated with complications such as gastrointestinal bleeding. This study is the first attempt to understand the pathogenesis of gastric ulcers occurring during the diabetic state considering alternate biochemical pathways using suitable markers and its amelioration by Cuminum cyminum. In this study, diabetic rats showed a progressive increase in the stomach advanced glycated end products formation, gastric mucosal tumour necrosis factor-α and Thiobarbituric acid reactive substances levels as compared to normal control (nondiabetic) rats. There was decrease in gastric mucosal content, antioxidant enzymes and cellular ATPase enzyme levels of diabetic gastric mucosa when compared to the normal control group. mRNA expression of epidermal growth factor was found to be significantly higher as compared to normal control animals. Further methanol extract of Cuminum cyminum treatment to diabetic animals caused a reduction in blood glucose, and ulcer score when compared to diabetic control rats. It significantly increased gastric mucus content, antioxidant status and cellular ATPase enzyme levels as compared to diabetic control animals. Methanol extract of Cuminum cyminum inhibited advanced glycated end products formation in vitro as well as in vivo. PMID:23716866

Fluoride reduced the immune privileged function of mouse Sertoli cells via the regulation of Fas/FasL system.

PubMed

Sun, Zilong; Nie, Qingli; Zhang, Lianjie; Niu, Ruiyan; Wang, Jundong; Wang, Shaolin

2017-02-01

Previous investigations have demonstrated the adverse impacts of fluoride on Sertoli cells (SCs), such as oxidative stress and apoptosis. SCs are the crucial cellular components that can create the immune privileged environment in testis. However, the effect of fluoride on SCs immune privilege is unknown. In this study, mouse SCs were exposed to sodium fluoride with varying concentrations of 10 -5 , 10 -4 , and 10 -3  mol/L to establish the model of fluoride-treated SCs (F-SCs) in vitro. After 48 h of incubation, F-SCs were transplanted underneath the kidney capsule of mice for 21 days, or cocultured with spleen lymphocytes for another 48 h. Immunohistochemical analysis of GATA4 in SCs grafts underneath kidney capsule presented less SCs distribution and obvious immune cell infiltration in F-SCs groups. In addition, the levels of FasL protein and mRNA in non-cocultured F-SCs decreased with the increase of fluoride concentration. When cocultured with F-SCs, lymphocytes presented significantly high cell viability and low apoptosis in F-SCs groups. Protein and mRNA expressions of FasL in cocultured F-SCs and Fas in lymphocytes were reduced, and the caspase 8 and caspase 3 mRNA levels were also decreased in fluoride groups in a dose-dependent manner. These findings indicated that fluoride influenced the testicular immune privilege through disturbing the Fas/FasL system. Copyright © 2016 Elsevier Ltd. All rights reserved.

The role of EMMPRIN expression in ovarian epithelial carcinomas.

PubMed

Zhao, Yang; Chen, Shuo; Gou, Wen-feng; Niu, Zhe-feng; Zhao, Shuang; Xiao, Li-jun; Takano, Yasuo; Zheng, Hua-chuan

2013-09-01

Extracellular matrix metalloproteinase inducer (EMMPRIN) was reported to involve in the invasion and metastasis of malignancies by regulating the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) in stromal and cancer cells. The study aimed to clarify the role of EMMPRIN expression in tumorigenesis and progression of ovarian epithelial carcinomas. EMMPRIN siRNA were transfected into ovarian carcinoma cells with the phenotypes and their related molecules examined. EMMPRIN expression was determined in ovarian normal tissue, benign and borderline tumors, and epithelial carcinomas by real-time PCR, western blot, and immunohistochemistry. EMMPRIN siRNA treatment resulted in a lower growth, G 1 arrest, apoptotic induction, decreased migration, and invasion. The transfectants showed reduced expression of Wnt5a, Akt, p70s6k, Bcl-xL, survivin, VEGF, and MMP-9 than mock and control cells at both mRNA and protein levels. According to real-time PCR and western blot, EMMPRIN mRNA or protein level was higher in ovarian borderline tumor and carcinoma than normal ovary and benign tumors (P<0.05), and positively correlated with dedifferentiation and FIGO staging (P<0.05). Immunohistochemically, EMMPRIN expression was positively correlated with FIGO staging, dedifferentiation, Ki-67 expression, the lower cumulative and relapse-free survival rate (P<0.05). Upregulated expression of EMMPRIN protein and mRNA might be involved in the pathogenesis, differentiation, and progression of ovarian carcinomas, possibly by modulating cellular events, such as proliferation, cell cycle, apoptosis, migration, and invasion.

The role of EMMPRIN expression in ovarian epithelial carcinomas

PubMed Central

Zhao, Yang; Chen, Shuo; Gou, Wen-feng; Niu, Zhe-feng; Zhao, Shuang; Xiao, Li-jun; Takano, Yasuo; Zheng, Hua-chuan

2013-01-01

Purpose: Extracellular matrix metalloproteinase inducer (EMMPRIN) was reported to involve in the invasion and metastasis of malignancies by regulating the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) in stromal and cancer cells. The study aimed to clarify the role of EMMPRIN expression in tumorigenesis and progression of ovarian epithelial carcinomas. Methods: EMMPRIN siRNA were transfected into ovarian carcinoma cells with the phenotypes and their related molecules examined. EMMPRIN expression was determined in ovarian normal tissue, benign and borderline tumors, and epithelial carcinomas by real-time PCR, western blot, and immunohistochemisty. Results: EMMPRIN siRNA treatment resulted in a lower growth, G1 arrest, apoptotic induction, decreased migration, and invasion. The transfectants showed reduced expression of Wnt5a, Akt, p70s6k, Bcl-xL, survivin, VEGF, and MMP-9 than mock and control cells at both mRNA and protein levels. According to real-time PCR and western blot, EMMPRIN mRNA or protein level was higher in ovarian borderline tumor and carcinoma than normal ovary and benign tumors (P < 0.05), and positively correlated with dedifferentiation and FIGO staging (P < 0.05). Immuhistochemically, EMMPRIN expression was positively correlated with FIGO staging, dedifferentiation, Ki-67 expression, the lower cumulative and relapse-free survival rate (P < 0.05). Conclusions: Upregulated expression of EMMPRIN protein and mRNA might be involved in the pathogenesis, differentiation, and progression of ovarian carcinomas, possibly by modulating cellular events, such as proliferation, cell cycle, apoptosis, migration, and invasion. PMID:23966157

Localization of PPARdelta in murine central nervous system: expression in oligodendrocytes and neurons.

PubMed

Woods, John W; Tanen, Michael; Figueroa, David J; Biswas, Chhabi; Zycband, Emanuel; Moller, David E; Austin, Christopher P; Berger, Joel P

2003-06-13

The peroxisome proliferator-activated receptors (PPARs), PPARdelta, PPARgamma and PPARalpha, comprise a subclass of the supergene family of nuclear receptors. As such they are ligand-regulated transcription factors whose major effects are mediated by altering expression of target genes. PPARdelta has been shown to be ubiquitously expressed in mammals. However, its primary biological role(s) has yet to be defined. Several recent studies have demonstrated that PPARdelta is the most highly expressed PPAR isoform in the central nervous system, but ambiguity still exists as to the specific brain sub-regions and cells in which it is expressed. Here, utilizing novel, isoform-selective PPARdelta riboprobes and an anti-peptide antibody, we performed a series of in situ hybridization and immunolocalization studies to determine the distribution of PPARdelta in the central nervous system (CNS) of mice. We found that PPARdelta mRNA and protein is expressed throughout the brain, with particularly high levels in the entorhinal cortex, hypothalamus and hippocampus, and lower levels in the corpus callosum and caudate putamen. At the cellular level, PPARdelta mRNA and protein were found to be expressed in oligodendrocytes and neurons but not astrocytes. Such results suggest a role for PPARdelta in both myelination and neuronal functioning within the CNS.

Increased Cellular Proliferation And Inflammatory Cytokines In Tonsils Derived From Children With Obstructive Sleep Apnea

PubMed Central

Kim, Jinkwan; Bhattacharjee, Rakesh; Dayyat, Ehab; Snow, Ayelet B.; Kheirandish-Gozal, Leila; Goldman, Julie L.; Li, Richard C.; Serpero, Laura D.; Clair, Heather B.; Gozal, David

2009-01-01

Adenotonsillar hypertrophy is the major pathophysiological mechanism underlying obstructive sleep apnea (OSA) and recurrent tonsillitis (RI) in children. The increased expression of various mediators of the inflammatory response in tonsils of OSA patients prompted our hypothesis that the enhanced local and systemic inflammation in OSA children would promote tonsillar proliferation. Mixed cell cultures from tonsils recovered during adenotonsillectomy in children with OSA and RI were established, and proliferative rates were assessed. Cells were also cultured to determine levels of pro-inflammatory cytokines and anti-oxidant protein levels and mRNA expression. Global cell proliferative rates from OSA tonsils were significantly higher than RI (P<0.01), with CD3+, CD4+, and CD8+ cell proliferation being higher in OSA (P<0.05). Moreover, pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1α were highly expressed in OSA-derived tonsils. Furthermore, thioredoxin (TRX), an anti-oxidant protein, was also highly expressed in OSA tonsils at the mRNA and protein levels (p<0.01). Thus, T-cells are in a highly proliferative state in the tonsils of children with OSA, and are associated with increased production of proinflammatory cytokines and TRX, when compared to children with RI. PMID:19581829

Substance P enhances collagen remodeling and MMP-3 expression by human tenocytes.

PubMed

Fong, Gloria; Backman, Ludvig J; Hart, David A; Danielson, Patrik; McCormack, Bob; Scott, Alex

2013-01-01

The loss of collagen organization is considered a hallmark histopathologic feature of tendinosis. At the cellular level, tenocytes have been shown to produce signal substances that were once thought to be restricted to neurons. One of the main neuropeptides implicated in tendinosis, substance P (SP), is known to influence collagen organization, particularly after injury. The aim of this study was to examine the influence of SP on collagen remodeling by primary human tendon cells cultured in vitro in three-dimensional collagen lattices. We found that SP stimulation led to an increased rate of collagen remodeling mediated via the neurokinin-1 receptor (NK-1 R), the preferred cell receptor for SP. Gene expression analysis showed that SP stimulation resulted in significant increases in MMP3, COL3A1 and ACTA2 mRNA levels in the collagen lattices. Furthermore, cyclic tensile loading of tendon cell cultures along with the administration of exogenous SP had an additive effect on MMP3 expression. Immunoblotting confirmed that SP increased MMP3 protein levels via the NK-1 R. This study indicates that SP, mediated via NK-1 R, increases collagen remodeling and leads to increased MMP3 mRNA and protein expression that is further enhanced by cyclic mechanical loading. Copyright © 2012 Orthopaedic Research Society.

Secretion of Galectin-9 as a DAMP during Dengue Virus Infection in THP-1 Cells.

PubMed

Dapat, Isolde C; Pascapurnama, Dyshelly Nurkartika; Iwasaki, Hiroko; Labayo, Hannah Karen; Chagan-Yasutan, Haorile; Egawa, Shinichi; Hattori, Toshio

2017-07-28

Damage-associated molecular patterns (DAMPs) are endogenous cellular molecules released to the extracellular environment in response to stress conditions such as virus infection. Galectins are β-galactoside-binding proteins that are widely expressed in cells and tissues of the immune system, are localized in the cell cytoplasm, and have roles in inflammatory responses and immune responses against infection. Elevated levels of galectin-9 (Gal-9) in natural human infections have been documented in numerous reports. To investigate the effect of dengue virus (DENV) infection on expression of endogenous Gal-9, monocytic THP-1 cells were infected with varying doses of DENV-3 (multiplicity of infection (MOI) 0.01, 0.03 and 0.1) and incubated at varying time points (Day 1, Day 2, Day 3). Results showed augmentation of Gal-9 levels in the supernatant, reduction of Gal-9 levels in the cells and decreased expression of LGALS9 mRNA, while DENV-3 mRNA copies for all three doses remained stable through time. Dengue virus induced the secretion of Gal-9 as a danger response; in turn, Gal-9 and other inflammatory factors, and stimulated effector responses may have limited further viral replication. The results in this pilot experiment add to the evidence of Gal-9 as a potential DAMP.

Dietary proanthocyanidins boost hepatic NAD(+) metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats.

PubMed

Aragonès, Gerard; Suárez, Manuel; Ardid-Ruiz, Andrea; Vinaixa, Maria; Rodríguez, Miguel A; Correig, Xavier; Arola, Lluís; Bladé, Cinta

2016-04-22

Proanthocyanidins (PACs) have been reported to modulate multiple targets by simultaneously controlling many pivotal metabolic pathways in the liver. However, the precise mechanism of PAC action on the regulation of the genes that control hepatic metabolism remains to be clarified. Accordingly, we used a metabolomic approach combining both nuclear magnetic resonance and mass spectrometry analysis to evaluate the changes induced by different doses of grape-seed PACs in the liver of healthy rats. Here, we report that PACs significantly increased the hepatic nicotinamide adenine dinucleotide (NAD(+)) content in a dose-dependent manner by specifically modulating the hepatic concentrations of the major NAD(+) precursors as well as the mRNA levels of the genes that encode the enzymes involved in the cellular metabolism of NAD(+). Notably, Sirtuin 1 (Sirt1) gene expression was also significantly up-regulated in a dose-response pattern. The increase in both the NAD(+) availability and Sirt1 mRNA levels, in turn, resulted in the hepatic activation of SIRT1, which was significantly associated with improved protection against hepatic triglyceride accumulation. Our data clearly indicates that PAC consumption could be a valid tool to enhance hepatic SIRT1 activity through the modulation of NAD(+) levels.

Dietary proanthocyanidins boost hepatic NAD+ metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats

PubMed Central

Aragonès, Gerard; Suárez, Manuel; Ardid-Ruiz, Andrea; Vinaixa, Maria; Rodríguez, Miguel A.; Correig, Xavier; Arola, Lluís; Bladé, Cinta

2016-01-01

Proanthocyanidins (PACs) have been reported to modulate multiple targets by simultaneously controlling many pivotal metabolic pathways in the liver. However, the precise mechanism of PAC action on the regulation of the genes that control hepatic metabolism remains to be clarified. Accordingly, we used a metabolomic approach combining both nuclear magnetic resonance and mass spectrometry analysis to evaluate the changes induced by different doses of grape-seed PACs in the liver of healthy rats. Here, we report that PACs significantly increased the hepatic nicotinamide adenine dinucleotide (NAD+) content in a dose-dependent manner by specifically modulating the hepatic concentrations of the major NAD+ precursors as well as the mRNA levels of the genes that encode the enzymes involved in the cellular metabolism of NAD+. Notably, Sirtuin 1 (Sirt1) gene expression was also significantly up-regulated in a dose-response pattern. The increase in both the NAD+ availability and Sirt1 mRNA levels, in turn, resulted in the hepatic activation of SIRT1, which was significantly associated with improved protection against hepatic triglyceride accumulation. Our data clearly indicates that PAC consumption could be a valid tool to enhance hepatic SIRT1 activity through the modulation of NAD+ levels. PMID:27102823

Analysis of antigen-specific B-cell memory directly ex vivo.

PubMed

McHeyzer-Williams, Louise J; McHeyzer-Williams, Michael G

2004-01-01

Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.

Translational Control of Viral Gene Expression in Eukaryotes

PubMed Central

Gale, Michael; Tan, Seng-Lai; Katze, Michael G.

2000-01-01

As obligate intracellular parasites, viruses rely exclusively on the translational machinery of the host cell for the synthesis of viral proteins. This relationship has imposed numerous challenges on both the infecting virus and the host cell. Importantly, viruses must compete with the endogenous transcripts of the host cell for the translation of viral mRNA. Eukaryotic viruses have thus evolved diverse mechanisms to ensure translational efficiency of viral mRNA above and beyond that of cellular mRNA. Mechanisms that facilitate the efficient and selective translation of viral mRNA may be inherent in the structure of the viral nucleic acid itself and can involve the recruitment and/or modification of specific host factors. These processes serve to redirect the translation apparatus to favor viral transcripts, and they often come at the expense of the host cell. Accordingly, eukaryotic cells have developed antiviral countermeasures to target the translational machinery and disrupt protein synthesis during the course of virus infection. Not to be outdone, many viruses have answered these countermeasures with their own mechanisms to disrupt cellular antiviral pathways, thereby ensuring the uncompromised translation of virion proteins. Here we review the varied and complex translational programs employed by eukaryotic viruses. We discuss how these translational strategies have been incorporated into the virus life cycle and examine how such programming contributes to the pathogenesis of the host cell. PMID:10839817

Rapid, High-Throughput, and Direct Molecular Beacon Delivery to Human Cancer Cells Using a Nanowire-Incorporated and Pneumatic Pressure-Driven Microdevice.

PubMed

Kim, Kyung Hoon; Kim, Jung; Choi, Jong Seob; Bae, Sunwoong; Kwon, Donguk; Park, Inkyu; Kim, Do Hyun; Seo, Tae Seok

2015-12-01

Tracking and monitoring the intracellular behavior of mRNA is of paramount importance for understanding real-time gene expression in cell biology. To detect specific mRNA sequences, molecular beacons (MBs) have been widely employed as sensing probes. Although numerous strategies for MB delivery into the target cells have been reported, many issues such as the cytotoxicity of the carriers, dependence on the random probability of MB transfer, and critical cellular damage still need to be overcome. Herein, we have developed a nanowire-incorporated and pneumatic pressure-driven microdevice for rapid, high-throughput, and direct MB delivery to human breast cancer MCF-7 cells to monitor survivin mRNA expression. The proposed microdevice is composed of three layers: a pump-associated glass manifold layer, a monolithic polydimethylsiloxane (PDMS) membrane, and a ZnO nanowire-patterned microchannel layer. The MB is immobilized on the ZnO nanowires by disulfide bonding, and the glass manifold and PDMS membrane serve as a microvalve, so that the cellular attachment and detachment on the MB-coated nanowire array can be manipulated. The combination of the nanowire-mediated MB delivery and the microvalve function enable the transfer of MB into the cells in a controllable way with high cell viability and to detect survivin mRNA expression quantitatively after docetaxel treatment. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Beyond the known functions of the CCR4-NOT complex in gene expression regulatory mechanisms: New structural insights to unravel CCR4-NOT mRNA processing machinery.

PubMed

Ukleja, Marta; Valpuesta, José María; Dziembowski, Andrzej; Cuellar, Jorge

2016-10-01

Large protein assemblies are usually the effectors of major cellular processes. The intricate cell homeostasis network is divided into numerous interconnected pathways, each controlled by a set of protein machines. One of these master regulators is the CCR4-NOT complex, which ultimately controls protein expression levels. This multisubunit complex assembles around a scaffold platform, which enables a wide variety of well-studied functions from mRNA synthesis to transcript decay, as well as other tasks still being identified. Solving the structure of the entire CCR4-NOT complex will help to define the distribution of its functions. The recently published three-dimensional reconstruction of the complex, in combination with the known crystal structures of some of the components, has begun to address this. Methodological improvements in structural biology, especially in cryoelectron microscopy, encourage further structural and protein-protein interaction studies, which will advance our comprehension of the gene expression machinery. © 2016 WILEY Periodicals, Inc.

Casein Kinase II Regulation of the Hot1 Transcription Factor Promotes Stochastic Gene Expression*

PubMed Central

Burns, Laura T.; Wente, Susan R.

2014-01-01

In Saccharomyces cerevisiae, Hog1 MAPK is activated and induces a transcriptional program in response to hyperosmotic stress. Several Hog1-responsive genes exhibit stochastic transcription, resulting in cell-to-cell variability in mRNA and protein levels. However, the mechanisms governing stochastic gene activity are not fully defined. Here we uncover a novel role for casein kinase II (CK2) in the cellular response to hyperosmotic stress. CK2 interacts with and phosphorylates the Hot1 transcription factor; however, Hot1 phosphorylation is not sufficient for controlling the stochastic response. The CK2 protein itself is required to negatively regulate mRNA expression of Hot1-responsive genes and Hot1 enrichment at target promoters. Single-cell gene expression analysis reveals altered activation of Hot1-targeted STL1 in ck2 mutants, resulting in a bimodal to unimodal shift in expression. Together, this work reveals a novel CK2 function during the hyperosmotic stress response that promotes cell-to-cell variability in gene expression. PMID:24817120

Aldo-keto reductases AKR1C1, AKR1C2 and AKR1C3 may enhance progesterone metabolism in ovarian endometriosis.

PubMed

Hevir, N; Vouk, K; Sinkovec, J; Ribič-Pucelj, M; Rižner, T Lanišnik

2011-05-30

Endometriosis is a very common disease that is characterized by increased formation of estradiol and disturbed progesterone action. This latter is usually explained by a lack of progesterone receptor B (PR-B) expression, while the role of pre-receptor metabolism of progesterone is not yet fully understood. In normal endometrium, progesterone is metabolized by reductive 20α-hydroxysteroid dehydrogenases (20α-HSDs), 3α/β-HSDs and 5α/β-reductases. The aldo-keto reductases 1C1 and 1C3 (AKR1C1 and AKR1C3) are the major reductive 20α-HSDs, while the oxidative reaction is catalyzed by 17β-HSD type 2 (HSD17B2). Also, 3α-HSD and 3β-HSD activities have been associated with the AKR1C isozymes. Additionally, 5α-reductase types 1 and 2 (SRD5A1, SRD5A2) and 5β-reductase (AKR1D1) are responsible for the formation of 5α- and 5β-reduced pregnanes. In this study, we examined the expression of PR-AB and the progesterone metabolizing enzymes in 31 specimens of ovarian endometriosis and 28 specimens of normal endometrium. Real-time PCR analysis revealed significantly decreased mRNA levels of PR-AB, HSD17B2 and SRD5A2, significantly increased mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1, and negligible mRNA levels of AKR1D1. Immunohistochemistry staining of endometriotic tissue compared to control endometrium showed significantly lower PR-B levels in epithelial cells and no significant differences in stromal cells, there were no significant differences in the expression of AKR1C3 and significantly higher AKR1C2 levels were seen only in stromal cells. Our expression analysis data at the mRNA level and partially at the cellular level thus suggest enhanced metabolism of progesterone by SRD5A1 and the 20α-HSD and 3α/β-HSD activities of AKR1C1, AKR1C2 and AKR1C3. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Fructose-rich diet differently affects angiotensin II receptor content in the nucleus and a plasma membrane fraction of visceral adipose tissue.

PubMed

Bundalo, Maja; Djordjevic, Ana; Bursac, Biljana; Zivkovic, Maja; Koricanac, Goran; Stanković, Aleksandra

2017-12-01

The adipose tissue renin-angiotensin system (RAS) is proposed to be a pathophysiological link between adipose tissue dysregulation and metabolic disorders induced by a fructose-rich diet (FRD). RAS can act intracellularly. We hypothesized that adipocyte nuclear membranes possess angiotensin receptor types 1 and 2 (AT1R and AT2R), which couple to nuclear signaling pathways and regulate oxidative gene expression under FRD conditions. We analyzed the effect of consumption of 10% fructose solution for 9 weeks on biochemical parameters, adipocyte morphology, and expression of AT1R, AT2R, AT1R-associated protein (ATRAP), NADPH oxidase 4 (NOX4), matrix metalloproteinase-9 (MMP-9), and manganese superoxide dismutase (MnSOD) in adipose tissue of Wistar rats. We detected AT1R and AT2R in the nuclear fraction. FRD reduced the level of angiotensin receptors in the nucleus, while increased AT1R and decreased AT2R levels were observed in the plasma membrane. FRD increased the ATRAP mRNA level and decreased MnSOD mRNA and protein levels. No significant differences were observed for MMP-9 and NOX4 mRNA levels. These findings coincided with hyperleptinemia, elevated blood pressure and triglycerides, and unchanged visceral adipose tissue mass and morphology in FRD rats. Besides providing evidence for nuclear localization of angiotensin receptors in visceral adipose tissue, this study demonstrates the different effects of FRD on AT1R expression in different cellular compartments. Elevated blood pressure and decreased antioxidant capacity in visceral fat of fructose-fed rats were accompanied by an increased AT1R level in the plasma membrane, while upregulation of ATRAP and a decrease of nuclear membrane AT1R suggest an increased capacity for attenuation of excessive AT1R signaling and visceral adiposity.

Nucleolin overexpression in breast cancer cell sub-populations with different stem-like phenotype enables targeted intracellular delivery of synergistic drug combination.

PubMed

Fonseca, Nuno A; Rodrigues, Ana S; Rodrigues-Santos, Paulo; Alves, Vera; Gregório, Ana C; Valério-Fernandes, Ângela; Gomes-da-Silva, Lígia C; Rosa, Manuel Santos; Moura, Vera; Ramalho-Santos, João; Simões, Sérgio; Moreira, João Nuno

2015-11-01

Breast cancer stem cells (CSC) are thought responsible for tumor growth and relapse, metastization and active evasion to standard chemotherapy. The recognition that CSC may originate from non-stem cancer cells (non-SCC) through plastic epithelial-to-mesenchymal transition turned these into relevant cell targets. Of crucial importance for successful therapeutic intervention is the identification of surface receptors overexpressed in both CSC and non-SCC. Cell surface nucleolin has been described as overexpressed in cancer cells as well as a tumor angiogenic marker. Herein we have addressed the questions on whether nucleolin was a common receptor among breast CSC and non-SCC and whether it could be exploited for targeting purposes. Liposomes functionalized with the nucleolin-binding F3 peptide, targeted simultaneously, nucleolin-overexpressing putative breast CSC and non-SCC, which was paralleled by OCT4 and NANOG mRNA levels in cells from triple negative breast cancer (TNBC) origin. In murine embryonic stem cells, both nucleolin mRNA levels and F3 peptide-targeted liposomes cellular association were dependent on the stemness status. An in vivo tumorigenic assay suggested that surface nucleolin overexpression per se, could be associated with the identification of highly tumorigenic TNBC cells. This proposed link between nucleolin expression and the stem-like phenotype in TNBC, enabled 100% cell death mediated by F3 peptide-targeted synergistic drug combination, suggesting the potential to abrogate the plasticity and adaptability associated with CSC and non-SCC. Ultimately, nucleolin-specific therapeutic tools capable of simultaneous debulk multiple cellular compartments of the tumor microenvironment may pave the way towards a specific treatment for TNBC patient care. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression and cellular localization of the transcription factor NeuroD1 in the developing and adult rat pineal gland.

PubMed

Castro, Analía E; Benitez, Sergio G; Farias Altamirano, Luz E; Savastano, Luis E; Patterson, Sean I; Muñoz, Estela M

2015-05-01

Circadian rhythms govern many aspects of mammalian physiology. The daily pattern of melatonin synthesis and secretion is one of the classic examples of circadian oscillations. It is mediated by a class of neuroendocrine cells known as pinealocytes which are not yet fully defined. An established method to evaluate functional and cytological characters is through the expression of lineage-specific transcriptional regulators. NeuroD1 is a basic helix-loop-helix transcription factor involved in the specification and maintenance of both endocrine and neuronal phenotypes. We have previously described developmental and adult regulation of NeuroD1 mRNA in the rodent pineal gland. However, the transcript levels were not influenced by the elimination of sympathetic input, suggesting that any rhythmicity of NeuroD1 might be found downstream of transcription. Here, we describe NeuroD1 protein expression and cellular localization in the rat pineal gland during development and the daily cycle. In embryonic and perinatal stages, protein expression follows the mRNA pattern and is predominantly nuclear. Thereafter, NeuroD1 is mostly found in pinealocyte nuclei in the early part of the night and in cytoplasm during the day, a rhythm maintained into adulthood. Additionally, nocturnal nuclear NeuroD1 levels are reduced after sympathetic disruption, an effect mimicked by the in vivo administration of α- and β-adrenoceptor blockers. NeuroD1 phosphorylation at two sites, Ser(274) and Ser(336) , associates with nuclear localization in pinealocytes. These data suggest that NeuroD1 influences pineal phenotype both during development and adulthood, in an autonomic and phosphorylation-dependent manner. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Resveratrol protects primary rat hepatocytes against oxidative stress damage: activation of the Nrf2 transcription factor and augmented activities of antioxidant enzymes.

PubMed

Rubiolo, Juan Andrés; Mithieux, Gilles; Vega, Félix Victor

2008-09-04

Oxidative stress is recognized as an important factor in the development of liver pathologies. The reactive oxygen species endogenously generated or as a consequence of xenobiotic metabolism are eliminated by enzymatic and nonenzymatic cellular systems. Besides endogen defences, the antioxidant consumption in the diet has an important role in the protection against the development of diseases product of oxidative damage. Resveratrol is a naturally occurring compound which is part of the human diet. This molecule has been shown to have many biological properties, including antioxidant activity. We decided to test if resveratrol could protect primary hepatocytes in culture from oxidative stress damage and if so, to determine if this compound affects the cellular detoxifying systems and their regulation through the Nrf2 transcription factor that regulates the expression of antioxidant and phase II detoxifying enzymes. Cell death by necrosis was detected by measuring the activity of lactate dehydrogenase liberated to the medium. The activities of antioxidant and phase II enzymes were measured using previously described methods. Activation of the Nrf2 transcription factor was studied by confocal microscopy and the Nrf2 and its coding mRNA levels were determined by western blot and quantitative PCR respectively. Resveratrol pre-treatment effectively protected hepatocytes in culture exposed to oxidative stress, increasing the activities of catalase, superoxide dismutase, glutathione peroxidase, NADPH quinone oxidoreductase and glutathione-S-transferase. Resveratrol increases the level of Nrf2 and induces its translocation to the nucleus. Also, it increases the concentration of the coding mRNA for Nrf2. In this work we show that resveratrol could be a useful drug for the protection of liver cells from oxidative stress induced damage.

Trigeminal Pain Molecules, Allodynia, and Photosensitivity Are Pharmacologically and Genetically Modulated in a Model of Traumatic Brain Injury

PubMed Central

Daiutolo, Brittany V.; Tyburski, Ashley; Clark, Shannon W.

2016-01-01

Abstract The pain-signaling molecules, nitric oxide synthase (NOS) and calcitonin gene-related peptide (CGRP), are implicated in the pathophysiology of post-traumatic headache (PTH) as they are for migraine. This study assessed the changes of inducible NOS (iNOS) and its cellular source in the trigeminal pain circuit, as well as the relationship between iNOS and CGRP after controlled cortical impact (CCI) injury in mice. The effects of a CGRP antagonist (MK8825) and sumatriptan on iNOS messenger RNA (mRNA) and protein were compared to vehicle at 2 weeks postinjury. Changes in CGRP levels in the trigeminal nucleus caudalis (TNC) in iNOS knockouts with CCI were compared to wild-type (WT) mice at 3 days and 2 weeks post injury. Trigeminal allodynia and photosensitivity were measured. MK8825 and sumatriptan increased allodynic thresholds in CCI groups compared to vehicle (p < 0.01), whereas iNOS knockouts were not different from WT. Photosensitivity was attenuated in MK8825 mice and iNOS knockouts compared to WT (p < 0.05). MK8825 and sumatriptan reduced levels of iNOS mRNA and iNOS immunoreactivity in the TNC and ganglia (p < 0.01). Differences in iNOS cellular localization were found between the trigeminal ganglia and TNC. Although the knockout of iNOS attenuated CGRP at 3 days (p < 0.05), it did not reduce CGRP at 2 weeks. CGRP immunoreactivity was found in the meningeal layers post-CCI, while negligible in controls. Findings support the importance of interactions between CGRP and iNOS in mediating allodynia, as well as the individual roles in photosensitivity. Mitigating prolonged increases in CGRP may be a promising intervention for treating acute PTH. PMID:26472135

Unbiased compound screening with a reporter gene assay highlights the role of p13 in the cardiac cellular stress response.

PubMed

Inoue, Naoki; Hirouchi, Taisei; Kasai, Atsushi; Higashi, Shintaro; Hiraki, Natsumi; Tanaka, Shota; Nakazawa, Takanobu; Nunomura, Kazuto; Lin, Bangzhong; Omori, Akiko; Hayata-Takano, Atsuko; Kim, Yoon-Jeong; Doi, Takefumi; Baba, Akemichi; Hashimoto, Hitoshi; Shintani, Norihito

2018-01-08

We recently showed that a 13-kDa protein (p13), the homolog protein of formation of mitochondrial complex V assembly factor 1 in yeast, acts as a potential protective factor in pancreatic islets under diabetes. Here, we aimed to identify known compounds regulating p13 mRNA expression to obtain therapeutic insight into the cellular stress response. A luciferase reporter system was developed using the putative promoter region of the human p13 gene. Overexpression of peroxisome proliferator-activated receptor gamma coactivator 1α, a master player regulating mitochondrial metabolism, increased both reporter activity and p13 expression. Following unbiased screening with 2320 known compounds in HeLa cells, 12 pharmacological agents (including 8 cardiotonics and 2 anthracyclines) that elicited >2-fold changes in p13 mRNA expression were identified. Among them, four cardiac glycosides decreased p13 expression and concomitantly elevated cellular oxidative stress. Additional database analyses showed highest p13 expression in heart, with typically decreased expression in cardiac disease. Accordingly, our results illustrate the usefulness of unbiased compound screening as a method for identifying novel functional roles of unfamiliar genes. Our findings also highlight the importance of p13 in the cellular stress response in heart. Copyright © 2017. Published by Elsevier Inc.

Human HOXA5 homeodomain enhances protein transduction and its application to vascular inflammation

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

Lee, Ji Young; Park, Kyoung sook; Cho, Eun Jung

2011-07-01

Highlights: {yields} We have developed an E. coli protein expression vector including human specific gene sequences for protein cellular delivery. {yields} The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence. {yields} HOXA5-APE1/Ref-1 inhibited TNF-alpha-induced monocyte adhesion to endothelial cells. {yields} Human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins. -- Abstract: Cellular protein delivery is an emerging technique by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an Escherichia coli expression vector including humanmore » specific gene sequences for protein cellular delivery. The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence which was matched with protein transduction domain (PTD) of homeodomain protein A5 (HOXA5) into pET expression vector. The cellular uptake of HOXA5-PTD-EGFP was detected in 1 min and its transduction reached a maximum at 1 h within cell lysates. The cellular uptake of HOXA5-EGFP at 37 {sup o}C was greater than in 4 {sup o}C. For study for the functional role of human HOXA5-PTD, we purified HOXA5-APE1/Ref-1 and applied it on monocyte adhesion. Pretreatment with HOXA5-APE1/Ref-1 (100 nM) inhibited TNF-{alpha}-induced monocyte adhesion to endothelial cells, compared with HOXA5-EGFP. Taken together, our data suggested that human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins.« less

Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins

PubMed Central

Harden, Mallory E.; Prasad, Nripesh; Griffiths, Anthony

2017-01-01

ABSTRACT The E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs). While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR) levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs) with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation. PMID:28049151

Nonsense-mediated mRNA decay factor Upf2 exists in both the nucleoplasm and the cytoplasm

PubMed Central

TATSUNO, TAKANORI; NAKAMURA, YUKA; MA, SHAOFU; TOMOSUGI, NAOHISA; ISHIGAKI, YASUHITO

2016-01-01

Upf2 protein predominantly localizes to the cytoplasmic fraction, and binds to the exon junction complex (EJC) on spliced mRNA. The present study aimed to determine the cellular site where the interaction between Upf2 and EJC occurs. First, the cell lysate was fractionated into the cytoplasm and nucleoplasm, and western blotting to detect levels of Upf2 protein was performed. Upf2 was clearly detected in the cytoplasm and in the nucleoplasm. Secondly, immunostaining was performed, and the majority of Upf2 was detected in the cytoplasmic perinuclear region; a small quantity of Upf2 was detected in the intranuclear region. RNase treatment of the cells reduced the Upf2 immunostained signal. The immune-purified fractions containing nuclear and cytoplasmic Upf2 also contained one of the EJC core factors, RBM8A. These results implied the existence of Upf2 in the nucleoplasm and the cytoplasm, and it appeared to be involved in the construction of the mRNA complex. In order to verify the construction of Upf2-binding EJC in the nucleoplasm, an in situ proximity ligation assay was performed with anti-Upf2 and anti-RBM8A antibodies. These results demonstrated that their interaction occurred not only in the cytoplasmic region, but also in the intranuclear region. Taken together, these results suggested that Upf2 combines with EJC in both the cytoplasmic and the intranuclear fractions, and that it is involved in mRNA metabolism in human cells. PMID:27221324

Region and task-specific activation of Arc in primary motor cortex of rats following motor skill learning.

PubMed

Hosp, J A; Mann, S; Wegenast-Braun, B M; Calhoun, M E; Luft, A R

2013-10-10

Motor learning requires protein synthesis within the primary motor cortex (M1). Here, we show that the immediate early gene Arc/Arg3.1 is specifically induced in M1 by learning a motor skill. Arc mRNA was quantified using a fluorescent in situ hybridization assay in adult Long-Evans rats learning a skilled reaching task (SRT), in rats performing reaching-like forelimb movement without learning (ACT) and in rats that were trained in the operant but not the motor elements of the task (controls). Apart from M1, Arc expression was assessed within the rostral motor area (RMA), primary somatosensory cortex (S1), striatum (ST) and cerebellum. In SRT animals, Arc mRNA levels in M1 contralateral to the trained limb were 31% higher than ipsilateral (p<0.001), 31% higher than in the contralateral M1 of ACT animals (p<0.001) and 48% higher than in controls (p<0.001). Arc mRNA expression in SRT was positively correlated with learning success between two sessions (r=0.52; p=0.026). For RMA, S1, ST or cerebellum no significant differences in Arc mRNA expression were found between hemispheres or across behaviors. As Arc expression has been related to different forms of cellular plasticity, these findings suggest a link between M1 Arc expression and motor skill learning in rats. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Sirt6 mRNA-incorporated endothelial microparticles (EMPs) attenuates DM patient-derived EMP-induced endothelial dysfunction.

PubMed

Jing, Tong; Ya-Shu, Kuang; Xue-Jun, Wang; Han-Jing, Hou; Yan, Lai; Yi-An, Yao; Fei, Chen; Xue-Bo, Liu

2017-12-26

Endothelial microparticles (EMPs) are small vesicles released by endothelial cells (ECs); they are considered biomarkers for endothelial dysfunction and therapeutic targets in diabetes-related vascular disease. Sirtuins have also been shown to play important roles in diabetes by regulating endothelial dysfunction. However, the effect of sirtuin-incorporated EMPs on their parental ECs remains unknown. The present study aims to investigate the diagnostic value of EMPs in diabetes and detect the protective effects of sirtuin 6 ( Sirt6 ) mRNA -incorporated EMPs on endothelial dysfunction. EMPs were prepared from cultured HUVECs and venous blood from patients with diabetes (n=10) and from healthy volunteers (n=6) after sequential centrifugation. Adv- Sirt6 or Sirt6 siRNA was used to alter Sirt6 expression. EC angiogenesis, inflammatory phenotypes, nitric oxide (NO) formation and eNOS phosphorylation were used to evaluate endothelial dysfunction. The levels of EMPs in diabetic patients and high glucose-cultured HUVECs are high, whereas Sirt6 expression in plasma and EMPs is low. EMPs generated from diabetic patients or high glucose-cultured HUVECs increase inflammatory chemokine release and blunt EC angiogenesis. Furthermore, EMPs enriched with Sirt6 mRNA induces EC angiogenesis, increases eNOS phosphorylation and impedes inflammatory chemokine release. Inhibition of Sirt6 mRNA expression in EMPs by siRNA hinders angiogenesis and eNOS phosphorylation but increases cellular inflammation. The Sirt6 mRNA -carrying EMPs may ameliorate endothelial dysfunction in diabetic patients.

Cellular Insulin Resistance Disrupts Leptin-Mediated Control of Neuronal Signaling and Transcription

PubMed Central

Nazarians-Armavil, Anaies; Menchella, Jonathan A.

2013-01-01

Central resistance to the actions of insulin and leptin is associated with the onset of obesity and type 2 diabetes mellitus, whereas leptin and insulin signaling is essential for both glucose and energy homeostasis. Although it is known that leptin resistance can lead to attenuated insulin signaling, whether insulin resistance can lead to or exacerbate leptin resistance is unknown. To investigate the molecular events underlying crosstalk between these signaling pathways, immortalized hypothalamic neuronal models, rHypoE-19 and mHypoA-2/10, were used. Prolonged insulin exposure was used to induce cellular insulin resistance, and thereafter leptin-mediated regulation of signal transduction and gene expression was assessed. Leptin directly repressed agouti-related peptide mRNA levels but induced urocortin-2, insulin receptor substrate (IRS)-1, IRS2, and IR transcription, through leptin-mediated phosphatidylinositol 3-kinase/Akt activation. Neuronal insulin resistance, as assessed by attenuated Akt phosphorylation, blocked leptin-mediated signal transduction and agouti-related peptide, urocortin-2, IRS1, IRS2, and insulin receptor synthesis. Insulin resistance caused a substantial decrease in insulin receptor protein levels, forkhead box protein 1 phosphorylation, and an increase in suppressor of cytokine signaling 3 protein levels. Cellular insulin resistance may cause or exacerbate neuronal leptin resistance and, by extension, obesity. It is essential to unravel the effects of neuronal insulin resistance given that both peripheral, as well as the less widely studied central insulin resistance, may contribute to the development of metabolic, reproductive, and cardiovascular disorders. This study provides improved understanding of the complex cellular crosstalk between insulin-leptin signal transduction that is disrupted during neuronal insulin resistance. PMID:23579487

Dietary salecan reverts partially the metabolic gene expressions and NMR-based metabolomic profiles from high-fat-diet-induced obese rats.

PubMed

Sun, Qi; Li, Minghui; Yang, Xiao; Xu, Xi; Wang, Junsong; Zhang, Jianfa

2017-09-01

Previous studies suggest that dietary salecan (a water-soluble β-glucan) effectively reduces high-fat-diet-induced adiposity through disturbing bile-acid-promoted emulsification in mice. However, the effects of salecan on metabolic genes and metabolites involved in lipid accumulation are mostly unknown. Here, we confirmed that dietary 3% and 6% salecan for 4 weeks markedly decreased fat accumulation in liver and adipose tissue in high-fat-diet rats, displaying a decrease in mRNA levels of SREBP1-C, FAS, SCD1 and ACC1 involved in de novo lipogenesis and a reduction of levels of GPAT1, DGAT1 and DGAT2 related to triglyceride synthesis. Dietary salecan also increased the mRNA levels of PPARα and CYP7A1, which are related to fatty acid oxidation and cholesterol decomposition, respectively. In the 1 H nuclear magnetic resonance metabolomic analysis, both the serum and liver metabolite profiles differed among the control groups, and the metabolic profiles of the salecan groups were shifted toward that of the low-fat-diet group. Metabolites analysis showed that salecan significantly increased hepatic glutathione and betaine levels which are related to regulation of cellular reactive oxygen species. These data demonstrate that dietary salecan not only disturbed fat digestion and absorption but also influenced lipid accumulation and metabolism in diet-induced obesity. Copyright © 2017 Elsevier Inc. All rights reserved.

Single-Cell Analysis Reveals Early Manifestation of Cancerous Phenotype in Pre-Malignant Esophageal Cells

PubMed Central

Wang, Jiangxin; Shi, Xu; Johnson, Roger H.; Kelbauskas, Laimonas; Zhang, Weiwen; Meldrum, Deirdre R.

2013-01-01

Cellular heterogeneity plays a pivotal role in a variety of functional processes in vivo including carcinogenesis. However, our knowledge about cell-to-cell diversity and how differences in individual cells manifest in alterations at the population level remains very limited mainly due to the lack of appropriate tools enabling studies at the single-cell level. We present a study on changes in cellular heterogeneity in the context of pre-malignant progression in response to hypoxic stress. Utilizing pre-malignant progression of Barrett’s esophagus (BE) as a disease model system we studied molecular mechanisms underlying the progression from metaplastic to dysplastic (pre-cancerous) stage. We used newly developed methods enabling measurements of cell-to-cell differences in copy numbers of mitochondrial DNA, expression levels of a set of mitochondrial and nuclear genes involved in hypoxia response pathways, and mitochondrial membrane potential. In contrast to bulk cell studies reported earlier, our study shows significant differences between metaplastic and dysplastic BE cells in both average values and single-cell parameter distributions of mtDNA copy numbers, mitochondrial function, and mRNA expression levels of studied genes. Based on single-cell data analysis, we propose that mitochondria may be one of the key factors in pre-malignant progression in BE. PMID:24116039

Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine.

PubMed

O'Sullivan, Finbarr; Keenan, Joanne; Aherne, Sinead; O'Neill, Fiona; Clarke, Colin; Henry, Michael; Meleady, Paula; Breen, Laura; Barron, Niall; Clynes, Martin; Horgan, Karina; Doolan, Padraig; Murphy, Richard

2017-11-07

To identify miRNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine. Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for mRNA, miRNA and proteomic profiling. mRNA microarray profiling analysis was carried out using the Affymetrix GeneChip Human Gene 1.0 ST array. miRNA microarray profiling analysis was carried out using the Affymetrix Genechip miRNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear ion trap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets (miRNA, proteomics, mRNA) was conducted in the R software environment using the Euclidean distance measure and Ward's clustering algorithm. The prediction of miRNA and oppositely correlated protein/mRNA interactions was performed using TargetScan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE miRNA, protein and mRNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database. Differential expression (DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 miRNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the miRNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology (GO) analysis of the DE mRNA identified cell adhesion, migration and ECM organization, cellular lipid and cholesterol metabolic processes, small molecule transport and a range of responses to external stimuli, while similar analysis of the DE protein list identified gene expression/transcription, epigenetic mechanisms, DNA replication, differentiation and translation ontology categories. The DE protein and gene lists were found to share 15 biological processes including for example epithelial cell differentiation [ P value ≤ 1.81613E-08 (protein list); P ≤ 0.000434311 (gene list)] and actin filament bundle assembly [ P value ≤ 0.001582797 (protein list); P ≤ 0.002733714 (gene list)]. Analysis was conducted on the three data streams acquired in parallel to identify targets undergoing potential miRNA translational repression identified 34 proteins, whose respective mRNAs were detected but no change in expression was observed. Of these 34 proteins, 27 proteins downregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 19 unique anti-correlated/upregulated microRNAs and 7 proteins upregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 15 unique anti-correlated/downregulated microRNAs. This first study providing "tri-omics" analysis of the principal intestinal cell line models Caco-2 and HT-29 has identified 34 proteins potentially undergoing miRNA translational repression.

Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine

PubMed Central

O’Sullivan, Finbarr; Keenan, Joanne; Aherne, Sinead; O’Neill, Fiona; Clarke, Colin; Henry, Michael; Meleady, Paula; Breen, Laura; Barron, Niall; Clynes, Martin; Horgan, Karina; Doolan, Padraig; Murphy, Richard

2017-01-01

AIM To identify miRNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine. METHODS Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for mRNA, miRNA and proteomic profiling. mRNA microarray profiling analysis was carried out using the Affymetrix GeneChip Human Gene 1.0 ST array. miRNA microarray profiling analysis was carried out using the Affymetrix Genechip miRNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear ion trap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets (miRNA, proteomics, mRNA) was conducted in the R software environment using the Euclidean distance measure and Ward’s clustering algorithm. The prediction of miRNA and oppositely correlated protein/mRNA interactions was performed using TargetScan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE miRNA, protein and mRNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database. RESULTS Differential expression (DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 miRNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the miRNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology (GO) analysis of the DE mRNA identified cell adhesion, migration and ECM organization, cellular lipid and cholesterol metabolic processes, small molecule transport and a range of responses to external stimuli, while similar analysis of the DE protein list identified gene expression/transcription, epigenetic mechanisms, DNA replication, differentiation and translation ontology categories. The DE protein and gene lists were found to share 15 biological processes including for example epithelial cell differentiation [P value ≤ 1.81613E-08 (protein list); P ≤ 0.000434311 (gene list)] and actin filament bundle assembly [P value ≤ 0.001582797 (protein list); P ≤ 0.002733714 (gene list)]. Analysis was conducted on the three data streams acquired in parallel to identify targets undergoing potential miRNA translational repression identified 34 proteins, whose respective mRNAs were detected but no change in expression was observed. Of these 34 proteins, 27 proteins downregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 19 unique anti-correlated/upregulated microRNAs and 7 proteins upregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 15 unique anti-correlated/downregulated microRNAs. CONCLUSION This first study providing “tri-omics” analysis of the principal intestinal cell line models Caco-2 and HT-29 has identified 34 proteins potentially undergoing miRNA translational repression. PMID:29151691

Inhibition of Macrophage CD36 Expression and Cellular Oxidized Low Density Lipoprotein (oxLDL) Accumulation by Tamoxifen: A PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR)γ-DEPENDENT MECHANISM.

PubMed

Yu, Miao; Jiang, Meixiu; Chen, Yuanli; Zhang, Shuang; Zhang, Wenwen; Yang, Xiaoxiao; Li, Xiaoju; Li, Yan; Duan, Shengzhong; Han, Jihong; Duan, Yajun

2016-08-12

Macrophage CD36 binds and internalizes oxidized low density lipoprotein (oxLDL) to facilitate foam cell formation. CD36 expression is activated by peroxisome proliferator-activated receptor γ (PPARγ). Tamoxifen, an anti-breast cancer medicine, has demonstrated pleiotropic functions including cardioprotection with unfully elucidated mechanisms. In this study, we determined that treatment of ApoE-deficient mice with tamoxifen reduced atherosclerosis, which was associated with decreased CD36 and PPARγ expression in lesion areas. At the cellular level, we observed that tamoxifen inhibited CD36 protein expression in human THP-1 monocytes, THP-1/PMA macrophages, and human blood monocyte-derived macrophages. Associated with decreased CD36 protein expression, tamoxifen reduced cellular oxLDL accumulation in a CD36-dependent manner. At the transcriptional level, tamoxifen decreased CD36 mRNA expression, promoter activity, and the binding of the PPARγ response element in CD36 promoter to PPARγ protein. Tamoxifen blocked ligand-induced PPARγ nuclear translocation and CD36 expression, but it increased PPARγ phosphorylation, which was due to that tamoxifen-activated ERK1/2. Furthermore, deficiency of PPARγ expression in macrophages abolished the inhibitory effect of tamoxifen on CD36 expression or cellular oxLDL accumulation both in vitro and in vivo Taken together, our study demonstrates that tamoxifen inhibits CD36 expression and cellular oxLDL accumulation by inactivating the PPARγ signaling pathway, and the inhibition of macrophage CD36 expression can be attributed to the anti-atherogenic properties of tamoxifen. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Transient Overexpression of adh8a Increases Allyl Alcohol Toxicity in Zebrafish Embryos

PubMed Central

Klüver, Nils; Ortmann, Julia; Paschke, Heidrun; Renner, Patrick; Ritter, Axel P.; Scholz, Stefan

2014-01-01

Fish embryos are widely used as an alternative model to study toxicity in vertebrates. Due to their complexity, embryos are believed to more resemble an adult organism than in vitro cellular models. However, concerns have been raised with respect to the embryo's metabolic capacity. We recently identified allyl alcohol, an industrial chemical, to be several orders of magnitude less toxic to zebrafish embryo than to adult zebrafish (embryo LC50 = 478 mg/L vs. fish LC50 = 0.28 mg/L). Reports on mammals have indicated that allyl alcohol requires activation by alcohol dehydrogenases (Adh) to form the highly reactive and toxic metabolite acrolein, which shows similar toxicity in zebrafish embryos and adults. To identify if a limited metabolic capacity of embryos indeed can explain the low allyl alcohol sensitivity of zebrafish embryos, we compared the mRNA expression levels of Adh isoenzymes (adh5, adh8a, adh8b and adhfe1) during embryo development to that in adult fish. The greatest difference between embryo and adult fish was found for adh8a and adh8b expression. Therefore, we hypothesized that these genes might be required for allyl alcohol activation. Microinjection of adh8a, but not adh8b mRNA led to a significant increase of allyl alcohol toxicity in embryos similar to levels reported for adults (LC50 = 0.42 mg/L in adh8a mRNA-injected embryos). Furthermore, GC/MS analysis of adh8a-injected embryos indicated a significant decline of internal allyl alcohol concentrations from 0.23-58 ng/embryo to levels below the limit of detection (< 4.6 µg/L). Injection of neither adh8b nor gfp mRNA had an impact on internal allyl alcohol levels supporting that the increased allyl alcohol toxicity was mediated by an increase in its metabolization. These results underline the necessity to critically consider metabolic activation in the zebrafish embryo. As demonstrated here, mRNA injection is one useful approach to study the role of candidate enzymes involved in metabolization. PMID:24594943

Multiple protocadherins are expressed in brain microvascular endothelial cells and might play a role in tight junction protein regulation.

PubMed

Dilling, Christina; Roewer, Norbert; Förster, Carola Y; Burek, Malgorzata

2017-10-01

Protocadherins (Pcdhs) are a large family of cadherin-related molecules. They play a role in cell adhesion, cellular interactions, and development of the central nervous system. However, their expression and role in endothelial cells has not yet been characterized. Here, we examined the expression of selected clustered Pcdhs in endothelial cells from several vascular beds. We analyzed human and mouse brain microvascular endothelial cell (BMEC) lines and primary cells, mouse myocardial microvascular endothelial cell line, and human umbilical vein endothelial cells. We examined the mRNA and protein expression of selected Pcdhs using RT-PCR, Western blot, and immunostaining. A strong mRNA expression of Pcdhs was observed in all endothelial cells tested. At the protein level, Pcdhs-gamma were detected using an antibody against the conserved C-terminal domain of Pcdhs-gamma or an antibody against PcdhgC3. Deletion of highly expressed PcdhgC3 led to differences in the tight junction protein expression and mRNA expression of Wnt/mTOR (mechanistic target of rapamycin) pathway genes as well as lower transendothelial electrical resistance. Staining of PcdhgC3 showed diffused cytoplasmic localization in mouse BMEC. Our results suggest that Pcdhs may play a critical role in the barrier-stabilizing pathways at the blood-brain barrier.

Iron induction of ferritin synthesis in soybean cell suspensions.

PubMed

Proudhon, D; Briat, J F; Lescure, A M

1989-06-01

In animal cells specialized for iron storage, iron-induced accumulation of ferritin is known to result from a shift of stored mRNA from the ribonucleoprotein fraction to polysomes. Previous reports with bean leaves suggested that in plants iron induction of ferritin synthesis would result from a regulation at the transcriptional level (F van der Mark, F Bienfait, H van der Ende [1983] Biochem Biophys Res Commun 115:463-469). Soybean (Glycine max, cv Mandarin) cell suspension cultures have been used here to support these findings. Ferritin induction is obtained by addition of Fe-citrate to the culture medium. A good correlation is found between cellular iron content and the amount of ferritin accumulation. This protein accumulation corresponds to an increase of in vitro translatable ferritin mRNA. Addition of 4 micrograms actinomycin D per milliliter to the cultures inhibits completely in vivo RNA synthesis, whereas protein synthesis was poorly affected, at least for 24 hours. During the same time, this concentration of actinomycin D strongly inhibits the iron-induced synthesis of ferritin. These results show that in soybean cell cultures, the mechanism of regulation of ferritin synthesis in response to iron does not result from recruitment of preexisting mRNA. They confirm that in plant systems, ferritin synthesis results from increased transcription of the corresponding genes.

Dynamic Changes of Smooth Muscle and Endothelial Markers in the Early Healing Process of Dacron Vascular Grafts in the Dog, Using RT-PCR.

PubMed

Ishida; Wu; Shi; Fujita; Sauvage; Hammond; Wijelath

2000-03-01

Previous studies of neointima formation on Dacron vascular grafts mainly focused on the late stages using immunohistochemistry staining for von Willebrand factor (vWF) and smooth muscle (SM) alpha-actin. However, it is impossible to use immunohistochemistry to study the early events of neointima formation, because graft samples lack sufficient cellular material. Therefore, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to demonstrate dynamic changes of SM and endothelial markers during the early stages of neointima formation. Preclotted Dacron grafts were implanted in the descending thoracic aorta of 14 mongrel dogs. Specimens were retrieved at 1-4 weeks. Total RNAs were extracted from mid-portion of graft flow surfaces, and RT-PCR for vWF, SM myosin heavy chain (MHC), and SM alpha-actin were performed and expressed as a ratio to the ribosome s17 signal. SM MHC and vWF mRNA expression was low at 1-2 weeks but elevated at 3-4 weeks (P < 0.05). However, SM alpha-actin mRNA levels were expressed consistently throughout the study period. At 3-4 weeks, vWF mRNA expression was inversely correlated to thrombus formation on the graft flow surface. Increased expressions of SM MHC and vWF mRNA corresponded to the formation of neointima and an endothelial layer at the later stages. However, SM alpha-actin mRNA expression did not vary during the healing process. The application of RT-PCR should permit further studies of gene regulation in the early vascular graft healing process in vivo. This model can also be used to study the molecular events that are involved in SM cell differentiation.

Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture.

PubMed Central

Giannini, C D; Roth, W K; Piiper, A; Zeuzem, S

1999-01-01

Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme. A 2[prime]-O-allyl modified hammerhead ribozyme was designed to cleave specifically the mutated form of the Ki- ras mRNA (GUU motif in codon 12). The activity was monitored by RT-PCR on Ki- ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2[prime]-deoxy-uridine incorporation on cell proliferation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation. A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control. The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme. The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells. In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme. Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene. PMID:10373591

Multiple Export Mechanisms for mRNAs

PubMed Central

Delaleau, Mildred; Borden, Katherine L. B.

2015-01-01

Nuclear mRNA export plays an important role in gene expression. We describe the mechanisms of mRNA export including the importance of mRNP assembly, docking with the nuclear basket of the nuclear pore complex (NPC), transit through the central channel of the NPC and cytoplasmic release. We describe multiple mechanisms of mRNA export including NXF1 and CRM1 mediated pathways. Selective groups of mRNAs can be preferentially transported in order to respond to cellular stimuli. RNAs can be selected based on the presence of specific cis-acting RNA elements and binding of specific adaptor proteins. The role that dysregulation of this process plays in human disease is also discussed. PMID:26343730

Selenium-enriched Saccharomyces cerevisiae improves growth, antioxidant status and selenoprotein gene expression in Arbor Acres broilers.

PubMed

Chen, F; Zhu, L; Qiu, H; Qin, S

2017-04-01

One hundred and fifty 7-day-old Arbor Acres broilers were randomly assigned into five groups: group 1 served as a control that was fed a basal diet without selenium (Se) supplementation; groups 2, 3 and 4 were fed the basal diet supplemented with 0.15, 0.5 and 1.5 mg Se as Se-enriched Saccharomyces cerevisiae (SSC) per kg of diet; and group 5 was fed the basal diet supplemented with 0.15 mg per kg of Se as sodium selenite (SS). Growth performance, glutathione peroxidase (GP X ) and superoxide dismutase (SOD) activities, total antioxidant capacity (T-AOC), and malondialdehyde (MDA) content in plasma and liver, and cellular glutathione peroxidase (GP X -1) and phospholipid hydroperoxide glutathione peroxidase (GP X -4) mRNA levels in liver were determined. Compared with group 1, groups 2-4 exhibited higher body weights (p < 0.05), lower feed/gain ratios, and higher GP X activities in plasma (p < 0.05) and GP X and SOD activities and GP X -1 and GP X -4 mRNA levels in liver (p < 0.05). Compared with group 5, group 2 exhibited higher GP X activity in plasma on day 21 (p < 0.05). Compared with group 2 and 5, group 3 exhibited lower MDA content in plasma on day 7 (p < 0.05), higher GP X activity in plasma, SOD activity and GP X -1 mRNA levels in liver on day 14 and 21 (p < 0.05), and higher GP X -4 mRNA levels on day 14 (p < 0.05). Compared with group 4, group 3 exhibited lower MDA contents in plasma on day 14 (p < 0.05) and in liver on day 21 (p < 0.05), higher T-AOC in plasma and higher GP X -1 mRNA levels on day 14 and 21 (p < 0.05), and higher SOD activity in plasma and higher SOD and GP X activities in liver on day 21 (p < 0.05). Thus, SSC improves growth and antioxidant status of broilers; the short-term bioavailability of SS was faster than that of SSC, but the long-term bioavailability of SSC was greater than SS. Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.

Alternative Ways to Think about Cellular Internal Ribosome Entry*

PubMed Central

Gilbert, Wendy V.

2010-01-01

Internal ribosome entry sites (IRESs) are specialized mRNA elements that allow recruitment of eukaryotic ribosomes to naturally uncapped mRNAs or to capped mRNAs under conditions in which cap-dependent translation is inhibited. Putative cellular IRESs have been proposed to play crucial roles in stress responses, development, apoptosis, cell cycle control, and neuronal function. However, most of the evidence for cellular IRES activity rests on bicistronic reporter assays, the reliability of which has been questioned. Here, the mechanisms underlying cap-independent translation of cellular mRNAs and the contributions of such translation to cellular protein synthesis are discussed. I suggest that the division of cellular mRNAs into mutually exclusive categories of “cap-dependent” and “IRES-dependent” should be reconsidered and that the implications of cellular IRES activity need to be incorporated into our models of cap-dependent initiation. PMID:20576611

Recruitment of Staufen2 Enhances Dendritic Localization of an Intron-Containing CaMKIIα mRNA.

PubMed

Ortiz, Raúl; Georgieva, Maya V; Gutiérrez, Sara; Pedraza, Neus; Fernández-Moya, Sandra M; Gallego, Carme

2017-07-05

Regulation of mRNA localization is a conserved cellular process observed in many types of cells and organisms. Asymmetrical mRNA distribution plays a particularly important role in the nervous system, where local translation of localized mRNA represents a key mechanism in synaptic plasticity. CaMKIIα is a very abundant mRNA detected in neurites, consistent with its crucial role at glutamatergic synapses. Here, we report the presence of CaMKIIα mRNA isoforms that contain intron i16 in dendrites, RNA granules, and synaptoneurosomes from primary neurons and brain. This subpopulation of unspliced mRNA preferentially localizes to distal dendrites in a synaptic-activity-dependent manner. Staufen2, a well-established marker of RNA transport in dendrites, interacts with intron i16 sequences and enhances its distal dendritic localization, pointing to the existence of intron-mediated mechanisms in the molecular pathways that modulate dendritic transport and localization of synaptic mRNAs. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Different patterns of 5{alpha}-reductase expression, cellular distribution, and testosterone metabolism in human follicular dermal papilla cells

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

Liu, Shicheng; Yamauchi, Hitoshi

Androgens regulate hair growth, and 5{alpha}-reductase (5{alpha}R) plays a pivotal role in the action of androgens on target organs. To clarify the molecular mechanisms responsible for controlling hair growth, the present study presents evidence that the human follicular dermal papilla cells (DPCs) from either beard (bDPCs) or scalp hair (sDPCs) possess endogenous 5{alpha}R activity. Real-time RT-PCR revealed that the highest level of 5{alpha}R1 mRNA was found in bDPCs, followed by sDPCs, and a low but detectable level of 5{alpha}R1 mRNA was observed in fibroblasts. Minimally detectable levels of 5{alpha}R2 mRNA were found in all three cell types. A weak bandmore » at 26 kDa corresponding to the human 5{alpha}R1 protein was detected by Western blot in both DPCs, but not in fibroblasts. Immuonofluorescence analysis confirmed that 5{alpha}R1 was localized to the cytoplasm rather than in the nuclei in both DPCs Furthermore, a 5{alpha}R assay using [{sup 14}C]testosterone labeling in intact cells revealed that testosterone was transformed primarily into androstenedione, and in small amounts, into DHT. Our results demonstrate that the 5{alpha}R activities of either bDPCs or sDPCs are stronger than that of dermal fibroblasts, despite the fact that the major steroidogenic activity is attributed to 17{beta}-HSD rather than 5{alpha}R among the three cell types. The 5{alpha}R1 inhibitor MK386 exhibited a more potent inhibitory effect on 5{alpha}R activity than finasteride (5{alpha}R2 inhibitor) in bDPCs.« less

Mechanisms of bradykinin-induced expression of connective tissue growth factor and nephrin in podocytes

PubMed Central

Msallem, J. Abou; Chalhoub, H.; Al-Hariri, M.; Saad, L.; Jaffa, M. A.; Ziyadeh, F. N.

2015-01-01

Diabetic nephropathy (DN) is the main cause of morbidity and mortality in diabetes and is characterized by mesangial matrix deposition and podocytopathy, including podocyte loss. The risk factors and mechanisms involved in the pathogenesis of DN are still not completely defined. In the present study, we aimed to understand the cellular mechanisms through which activation of B2 kinin receptors contribute to the initiation and progression of DN. Stimulation of cultured rat podocytes with bradykinin (BK) resulted in a significant increase in ROS generation, and this was associated with a significant increase in NADPH oxidase (NOX)1 and NOX4 protein and mRNA levels. BK stimulation also resulted in a signicant increase in the phosphorylation of ERK1/2 and Akt, and this effect was inhibited in the presence of NOX1 and Nox4 small interfering (si)RNA. Furthermore, podocytes stimulated with BK resulted in a significant increase in protein and mRNA levels of connective tissue growth factor (CTGF) and, at the same time, a significant decrease in protein and mRNA levels of nephrin. siRNA targeted against NOX1 and NOX4 significantly inhibited the BK-induced increase in CTGF. Nephrin expression was increased in response to BK in the presence of NOX1 and NOX4 siRNA, thus implicating a role for NOXs in modulating the BK response in podocytes. Moreover, nephrin expression in response to BK was also significantly increased in the presence of siRNA targeted against CTGF. These findings provide novel aspects of BK signal transduction pathways in pathogenesis of DN and identify novel targets for interventional strategies. PMID:26447218

Influence of different intensities of vibration on proliferation and differentiation of human periodontal ligament stem cells.

PubMed

Zhang, Chunxiang; Lu, Yanqin; Zhang, Linkun; Liu, Yang; Zhou, Yi; Chen, Yangxi; Yu, Haiyang

2015-06-19

To understand the effects of low-magnitude, high-frequency (LMHF) mechanical vibration at different intensities on human periodontal ligament stem cell (hPDLSC) proliferation and osteogenic differentiation. The effect of vibration on hPDLSC proliferation, osteogenic differentiation, tenogenic differentiation and cytoskeleton was assessed at the cellular, genetic and protein level. The PDLSC proliferation was decreased after different magnitudes of mechanical vibration; however, there were no obvious senescent cells in the experimental and the static control group. Expression of osteogenesis markers was increased. The expression of alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA was up-regulated at 0.1 g, 0.3 g, 0.6 g and 0.9 g magnitude, with the peak at 0.3 g. The type I collagen (Col-I) level was increased after vibration exposure at 0.1 g, 0.3 g, and 0.6 g, peaking at 0.3 g. The expression levels of both mRNA and protein of Runx2 and osterix (OSX) significantly increased at a magnitude of 0.1 g to 0.9 g, reached a peak at 0.3 g and then decreased slowly. The scleraxis, tenogenic markers, and mRNA expression decreased at 0.05 g, 0.1 g, and 0.3 g, and significantly increased at 0.6 g and 0.9 g. Compared with the static group, the F-actin stress fibers of hPDLSCs became thicker and clearer following vibration. The LMHF mechanical vibration promotes PDLSC osteogenic differentiation and implies the existence of a magnitude-dependent effect of vibration on determining PDLSC commitment to the osteoblast lineage. Changes in the cytoskeleton of hPDLSCs after vibration may be one of the mechanisms of the biological effects.

Influence of different intensities of vibration on proliferation and differentiation of human periodontal ligament stem cells

PubMed Central

Zhang, Chunxiang; Lu, Yanqin; Zhang, Linkun; Liu, Yang; Zhou, Yi; Chen, Yangxi

2015-01-01

Introduction To understand the effects of low-magnitude, high-frequency (LMHF) mechanical vibration at different intensities on human periodontal ligament stem cell (hPDLSC) proliferation and osteogenic differentiation. Material and methods The effect of vibration on hPDLSC proliferation, osteogenic differentiation, tenogenic differentiation and cytoskeleton was assessed at the cellular, genetic and protein level. Results The PDLSC proliferation was decreased after different magnitudes of mechanical vibration; however, there were no obvious senescent cells in the experimental and the static control group. Expression of osteogenesis markers was increased. The expression of alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA was up-regulated at 0.1 g, 0.3 g, 0.6 g and 0.9 g magnitude, with the peak at 0.3 g. The type I collagen (Col-I) level was increased after vibration exposure at 0.1 g, 0.3 g, and 0.6 g, peaking at 0.3 g. The expression levels of both mRNA and protein of Runx2 and osterix (OSX) significantly increased at a magnitude of 0.1 g to 0.9 g, reached a peak at 0.3 g and then decreased slowly. The scleraxis, tenogenic markers, and mRNA expression decreased at 0.05 g, 0.1 g, and 0.3 g, and significantly increased at 0.6 g and 0.9 g. Compared with the static group, the F-actin stress fibers of hPDLSCs became thicker and clearer following vibration. Conclusions The LMHF mechanical vibration promotes PDLSC osteogenic differentiation and implies the existence of a magnitude-dependent effect of vibration on determining PDLSC commitment to the osteoblast lineage. Changes in the cytoskeleton of hPDLSCs after vibration may be one of the mechanisms of the biological effects. PMID:26170859

Polyphenol-rich black chokeberry (Aronia melanocarpa) extract regulates the expression of genes critical for intestinal cholesterol flux in Caco-2 cells.

PubMed

Kim, Bohkyung; Park, Youngki; Wegner, Casey J; Bolling, Bradley W; Lee, Jiyoung

2013-09-01

Black chokeberry (Aronia melanocarpa) is a rich source of polyphenols. The hypolipidemic effects of polyphenol-rich black chokeberry extract (CBE) have been reported, but underlying mechanisms have not been well characterized. We investigated the effect of CBE on the expression of genes involved in intestinal lipid metabolism. Caco-2 cells were incubated with 50 or 100 μg/ml of CBE for 24 h for quantitative realtime polymerase chain reaction analysis. Expression of genes for cholesterol synthesis (3-hydroxy-3-methylglutaryl coenzyme A reductase and sterol regulatory element binding protein 2), apical cholesterol uptake (Niemann-Pick C1 Like 1 and scavenger receptor class B Type 1) and basolateral cholesterol efflux [ATP-binding cassette transporter A1 (ABCA1)] was significantly decreased by CBE compared with control. Western blot analysis confirmed that CBE inhibited expression of these proteins. In contrast, CBE markedly induced mRNA and/or protein levels of ABCG5 and ABCG8 that mediate apical cholesterol efflux to the intestinal lumen. Furthermore, CBE significantly increased mRNA and protein levels of low-density lipoprotein (LDL) receptor, and cellular LDL uptake. Expression of genes involved in lipid metabolism and lipoprotein assembly, including sterol regulatory element-binding protein 1c, fatty acid synthase and acyl-CoA oxidase 1, was significantly decreased by CBE in a dose-dependent manner. Concomitantly, CBE significantly increased sirtuin 1, 3 and 5 mRNA levels, while it decreased SIRT-2. Our data suggest that hypolipidemic effects of CBE may be attributed, at least in part, to increased apical efflux of LDL-derived cholesterol and to decreased chylomicron formation in the intestine; and specific isoforms of SIRT may play an important role in this process. Copyright © 2013 Elsevier Inc. All rights reserved.

Quantitative imaging of single mRNA splice variants in living cells

NASA Astrophysics Data System (ADS)

Lee, Kyuwan; Cui, Yi; Lee, Luke P.; Irudayaraj, Joseph

2014-06-01

Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.

A murine retrovirus co-Opts YB-1, a translational regulator and stress granule-associated protein, to facilitate virus assembly.

PubMed

Bann, Darrin V; Beyer, Andrea R; Parent, Leslie J

2014-04-01

The Gag protein of the murine retrovirus mouse mammary tumor virus (MMTV) orchestrates the assembly of immature virus particles in the cytoplasm which are subsequently transported to the plasma membrane for release from the cell. The morphogenetic pathway of MMTV assembly is similar to that of Saccharomyces cerevisiae retrotransposons Ty1 and Ty3, which assemble virus-like particles (VLPs) in intracytoplasmic ribonucleoprotein (RNP) complexes. Assembly of Ty1 and Ty3 VLPs depends upon cellular mRNA processing factors, prompting us to examine whether MMTV utilizes a similar set of host proteins to facilitate viral capsid assembly. Our data revealed that MMTV Gag colocalized with YB-1, a translational regulator found in stress granules and P bodies, in intracytoplasmic foci. The association of MMTV Gag and YB-1 in cytoplasmic granules was not disrupted by cycloheximide treatment, suggesting that these sites were not typical stress granules. However, the association of MMTV Gag and YB-1 was RNA dependent, and an MMTV RNA reporter construct colocalized with Gag and YB-1 in cytoplasmic RNP complexes. Knockdown of YB-1 resulted in a significant decrease in MMTV particle production, indicating that YB-1 plays a role in MMTV capsid formation. Analysis by live-cell imaging with fluorescence recovery after photobleaching (FRAP) revealed that the population of Gag proteins localized within YB-1 complexes was relatively immobile, suggesting that Gag forms stable complexes in association with YB-1. Together, our data imply that the formation of intracytoplasmic Gag-RNA complexes is facilitated by YB-1, which promotes MMTV virus assembly. Cellular mRNA processing factors regulate the posttranscriptional fates of mRNAs, affecting localization and utilization of mRNAs under normal conditions and in response to stress. RNA viruses such as retroviruses interact with cellular mRNA processing factors that accumulate in ribonucleoprotein complexes known as P bodies and stress granules. This report shows for the first time that mouse mammary tumor virus (MMTV), a mammalian retrovirus that assembles intracytoplasmic virus particles, commandeers the cellular factor YB-1, a key regulator of translation involved in the cellular stress response. YB-1 is essential for the efficient production of MMTV particles, a process directed by the viral Gag protein. We found that Gag and YB-1 localize together in cytoplasmic granules. Functional studies of Gag/YB-1 granules suggest that they may be sites where virus particles assemble. These studies provide significant insights into the interplay between mRNA processing factors and retroviruses.

Gene expression of regulatory enzymes of glycolysis/gluconeogenesis in regenerating rat liver.

PubMed Central

Rosa, J L; Bartrons, R; Tauler, A

1992-01-01

Levels of mRNA for glucokinase, L-pyruvate kinase, fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase were analysed during liver regeneration. Levels of mRNA for glycolytic enzymes (glucokinase and L-pyruvate kinase) decreased rapidly after partial hepatectomy. Glucokinase mRNA increased at 16-24 h, returning to normal values after this time. L-pyruvate kinase mRNA recovered control levels at 168 h. In contrast, phosphoenolpyruvate carboxykinase mRNA increased rapidly after liver resection and remained high during the regenerative process. However, the levels of fructose-1,6-bisphosphatase mRNA were not modified significantly. These results correlate with the reported increased rate of gluconeogenesis and changes in enzyme levels after partial hepatectomy. The effect of stress on the mRNA levels was also studied. All enzymes showed variations in their mRNA levels after the surgical stress. In general, the differences were more pronounced in regenerating liver than in sham-operated animals, being practically normalized at 24 h. Images Fig. 2. Fig. 3. PMID:1329724

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues.

PubMed

Molenaar, Remco J; Khurshed, Mohammed; Hira, Vashendriya V V; Van Noorden, Cornelis J F

2018-05-26

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation. We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P) + and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase's activity. The formazan's absorbance is therefore a direct measure of the dehydrogenase's activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays. Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

A Novel mouse model of enhanced proteostasis: Full-length human heat shock factor 1 transgenic mice

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

Pierce, Anson, E-mail: piercea2@uthscsa.edu; Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229; The Department of Veteran's Affairs, South Texas Veterans Health Care System, San Antonio, Texas, 78284

2010-11-05

Research highlights: {yields} Development of mouse overexpressing native human HSF1 in all tissues including CNS. {yields} HSF1 overexpression enhances heat shock response at whole-animal and cellular level. {yields} HSF1 overexpression protects from polyglutamine toxicity and favors aggresomes. {yields} HSF1 overexpression enhances proteostasis at the whole-animal and cellular level. -- Abstract: The heat shock response (HSR) is controlled by the master transcriptional regulator heat shock factor 1 (HSF1). HSF1 maintains proteostasis and resistance to stress through production of heat shock proteins (HSPs). No transgenic model exists that overexpresses HSF1 in tissues of the central nervous system (CNS). We generated a transgenicmore » mouse overexpressing full-length non-mutant HSF1 and observed a 2-4-fold increase in HSF1 mRNA and protein expression in all tissues studied of HSF1 transgenic (HSF1{sup +/0}) mice compared to wild type (WT) littermates, including several regions of the CNS. Basal expression of HSP70 and 90 showed only mild tissue-specific changes; however, in response to forced exercise, the skeletal muscle HSR was more elevated in HSF1{sup +/0} mice compared to WT littermates and in fibroblasts following heat shock, as indicated by levels of inducible HSP70 mRNA and protein. HSF1{sup +/0} cells elicited a significantly more robust HSR in response to expression of the 82 repeat polyglutamine-YFP fusion construct (Q82YFP) and maintained proteasome-dependent processing of Q82YFP compared to WT fibroblasts. Overexpression of HSF1 was associated with fewer, but larger Q82YFP aggregates resembling aggresomes in HSF1{sup +/0} cells, and increased viability. Therefore, our data demonstrate that tissues and cells from mice overexpressing full-length non-mutant HSF1 exhibit enhanced proteostasis.« less

Synergistic effects of acyclic retinoid and OSI-461 on growth inhibition and gene expression in human hepatoma cells.

PubMed

Shimizu, Masahito; Suzui, Masumi; Deguchi, Atsuko; Lim, Jin T E; Xiao, Danhua; Hayes, Julia H; Papadopoulos, Kyriakos P; Weinstein, I Bernard

2004-10-01

Hepatoma is one of the most frequently occurring cancers worldwide. However, effective chemotherapeutic agents for this disease have not been developed. Acyclic retinoid, a novel synthetic retinoid, can reduce the incidence of postsurgical recurrence of hepatoma and improve the survival rate. OSI-461, a potent derivative of exisulind, can increase intracellular levels of cyclic GMP, which leads to activation of protein kinase G and induction of apoptosis in cancer cells. In the present study, we examined the combined effects of acyclic retinoid plus OSI-461 in the HepG2 human hepatoma cell line. We found that the combination of as little as 1.0 micromol/L acyclic retinoid and 0.01 micromol/L OSI-461 exerted synergistic inhibition of the growth of HepG2 cells. Combined treatment with low concentrations of these two agents also acted synergistically to induce apoptosis in HepG2 cells through induction of Bax and Apaf-1, reduction of Bcl-2 and Bcl-xL, and activation of caspase-3, -8, and -9. OSI-461 enhanced the G0-G1 arrest caused by acyclic retinoid, and the combination of these agents caused a synergistic decrease in the levels of expression of cyclin D1 protein and mRNA, inhibited cyclin D1 promoter activity, decreased the level of hyperphosphorylated forms of the Rb protein, induced increased cellular levels of the p21(CIP1) protein and mRNA, and stimulated p21(CIP1) promoter activity. Moreover, OSI-461 enhanced the ability of acyclic retinoid to induce increased cellular levels of retinoic acid receptor beta and to stimulate retinoic acid response element-chloramphenicol acetyltransferase activity. A hypothetical model involving concerted effects on p21(CIP1) and retinoic acid receptor beta expression is proposed to explain these synergistic effects. Our results suggest that the combination of acyclic retinoid plus OSI-461 might be an effective regimen for the chemoprevention and chemotherapy of human hepatoma and possibly other malignancies.

Efficacy of peptide nucleic acid and selected conjugates against specific cellular pathologies of amyotrophic lateral sclerosis.

PubMed

Browne, Elisse C; Parakh, Sonam; Duncan, Luke F; Langford, Steven J; Atkin, Julie D; Abbott, Belinda M

2016-04-01

Cellular studies have been undertaken on a nonamer peptide nucleic acid (PNA) sequence, which binds to mRNA encoding superoxide dismutase 1, and a series of peptide nucleic acids conjugated to synthetic lipophilic vitamin analogs including a recently prepared menadione (vitamin K) analog. Reduction of both mutant superoxide dismutase 1 inclusion formation and endoplasmic reticulum stress, two of the key cellular pathological hallmarks in amyotrophic lateral sclerosis, by two of the prepared PNA oligomers is reported for the first time. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis.

PubMed

Fox, Bridget C; Devonshire, Alison S; Baradez, Marc-Olivier; Marshall, Damian; Foy, Carole A

2012-08-15

Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT-qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis. Copyright © 2012 Elsevier Inc. All rights reserved.

Differential senescence capacities in meibomian gland carcinoma and basal cell carcinoma.

PubMed

Zhang, Leilei; Huang, Xiaolin; Zhu, Xiaowei; Ge, Shengfang; Gilson, Eric; Jia, Renbing; Ye, Jing; Fan, Xianqun

2016-03-15

Meibomian gland carcinoma (MGC) and basal cell carcinoma (BCC) are common eyelid carcinomas that exhibit highly dissimilar degrees of proliferation and prognoses. We address here the question of the differential mechanisms between these two eyelid cancers that explain their different outcome. A total of 102 confirmed MGC and 175 diagnosed BCC cases were analyzed. Twenty confirmed MGC and twenty diagnosed BCC cases were collected to determine the telomere length, the presence of senescent cells, and the expression levels of the telomere capping shelterin complex, P53, and the E3 ubiquitin ligase Siah1. Decreased protein levels of the shelterin subunits, shortened telomere length, over-expressed Ki-67, and Bcl2 as well as mutations in P53 were detected both in MGC and BCC. It suggests that the decreased protein levels of the shelterin complex and the shortened telomere length contribute to the tumorigenesis of MGC and BCC. However, several parameters distinguish MGC from BCC samples: (i) the mRNA level of the shelterin subunits decreased in MGC but it increased in BCC; (ii) P53 was more highly mutated in MGC; (iii) Siah1 mRNA was over-expressed in BCC; (iv) BCC samples contain a higher level of senescent cells; (v) Ki-67 and Bcl2 expression were lower in BCC. These results support a model where a preserved P53 checkpoint in BCC leads to cellular senescence and reduced tumor proliferation as compared to MGC. © 2015 UICC.

Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons

PubMed Central

2014-01-01

Background Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Results Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. Conclusions We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission. PMID:24898526

Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons.

PubMed

Pfeiffer-Guglielmi, Brigitte; Dombert, Benjamin; Jablonka, Sibylle; Hausherr, Vanessa; van Thriel, Christoph; Schöbel, Nicole; Jansen, Ralf-Peter

2014-06-04

Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission.

Th17 Cells and Activated Dendritic Cells Are Increased in Vitiligo Lesions

PubMed Central

Fuentes-Duculan, Judilyn; Moussai, Dariush; Gulati, Nicholas; Sullivan-Whalen, Mary; Gilleaudeau, Patricia; Cohen, Jules A.; Krueger, James G.

2011-01-01

Background Vitiligo is a common skin disorder, characterized by progressive skin de-pigmentation due to the loss of cutaneous melanocytes. The exact cause of melanocyte loss remains unclear, but a large number of observations have pointed to the important role of cellular immunity in vitiligo pathogenesis. Methodology/Principal Findings In this study, we characterized T cell and inflammation-related dermal dendritic cell (DC) subsets in pigmented non-lesional, leading edge and depigmented lesional vitiligo skin. By immunohistochemistry staining, we observed enhanced populations of CD11c+ myeloid dermal DCs and CD207+ Langerhans cells in leading edge vitiligo biopsies. DC-LAMP+ and CD1c+ sub-populations of dermal DCs expanded significantly in leading edge and lesional vitiligo skin. We also detected elevated tissue mRNA levels of IL-17A in leading edge skin biopsies of vitiligo patients, as well as IL-17A positive T cells by immunohistochemistry and immunofluorescence. Langerhans cells with activated inflammasomes were also noted in lesional vitiligo skin, along with increased IL-1ß mRNA, which suggest the potential of Langerhans cells to drive Th17 activation in vitiligo. Conclusions/Significance These studies provided direct tissue evidence that implicates active Th17 cells in vitiligo skin lesions. We characterized new cellular immune elements, in the active margins of vitiligo lesions (e.g. populations of epidermal and dermal dendritic cells subsets), which could potentially drive the inflammatory responses. PMID:21541348

Exosomal particles secreted by prostate cancer cells are potent mRNA and protein vehicles for the interference of tumor and tumor environment.

PubMed

Rauschenberger, Lisa; Staar, Doreen; Thom, Kathleen; Scharf, Christian; Venz, Simone; Homuth, Georg; Schlüter, Rabea; Brandenburg, Lars-Ove; Ziegler, Patrick; Zimmermann, Uwe; Weitschies, Werner; Völker, Uwe; Lendeckel, Uwe; Walther, Reinhard; Burchardt, Martin; Stope, Matthias B

2016-03-01

Remodeling of the tumor environment and the modulation of tumor associated non-malignant cells are essential events in tumor progression. Exosomes are small membranous vesicles of 50-150 nm in diameter, which are secreted into the extracellular space and supposedly serve as vehicles for signal and effector molecules to modulate adjacent target cells. We characterized the mRNA and protein composition as well as cellular functions of prostate cancer cell-derived exosomes. Exosomes were prepared from prostate cancer cell culture supernatant by ultracentrifugation and subsequently characterized by dynamic light scattering and electron microscopy. Exosomal mRNA and protein composition were analyzed by DNA microarrays and gel electrophoresis coupled with mass spectrometry. Physiological effects of exosomes were studied by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release cell assays. Using a SILAC approach, putative uptake of exosomal human proteins in canine cells and canine de novo synthesis of proteins specified by exosome-transferred human mRNA was analyzed in MDCK cells via mass spectrometry. Preparations of exosomes revealed typical cup shaped particles of 150 nm in diameter. Analysis of mRNA and protein composition of exosomes exhibited a wide range of mRNA and protein species. Interestingly, the packaging of at least small proteins into exosomes was apparently unspecific, as shown with the example of two model proteins. In cell culture incubation experiments exosomal preparations of prostate cancer cells caused anti-proliferative effects. MS analysis revealed the uptake of exosomal human proteins into canine cells after 6 hr of incubation. The results reveal a distinct exosomal functionality in the modulation of the prostatic tumor adjacent environment. The multitude of translocated factors implies the induction of numerous effects in tumor-associated target cells, including impact on cellular growth. © 2015 Wiley Periodicals, Inc.

Alterations of polyunsaturated fatty acid metabolism in ovarian tissues of polycystic ovary syndrome rats.

PubMed

Huang, Rong; Xue, Xinli; Li, Shengxian; Wang, Yuying; Sun, Yun; Liu, Wei; Yin, Huiyong; Tao, Tao

2018-03-30

The metabolism of polyunsaturated fatty acids (PUFAs) remains poorly characterized in ovarian tissues of patients with polycystic ovary syndrome (PCOS). This study aimed to explore alterations in the levels of PUFAs and their metabolites in serum and ovarian tissues in a PCOS rat model treated with a high-fat diet and andronate. Levels of PUFAs and their metabolites were measured using gas/liquid chromatography-mass spectrometry after the establishment of a PCOS rat model. Only 3 kinds of PUFAs [linoleic acid, arachidonic acid (AA) and docosahexaenoic acid] were detected in both the circulation and ovarian tissues of the rats, and their concentrations were lower in ovarian tissues than in serum. Moreover, significant differences in the ovarian levels of AA were observed between control, high-fat diet-fed and PCOS rats. The levels of prostaglandins, AA metabolites via the cyclooxygenase (COX) pathway, in ovarian tissues of the PCOS group were significantly increased compared to those in the controls. Further studies on the mechanism underlying this phenomenon showed a correlation between decreased expression of phosphorylated cytosolic phospholipase A2 (p-cPLA2) and increased mRNA and protein expression of COX2, potentially leading to a deeper understanding of altered AA and prostaglandin levels in ovarian tissues of PCOS rats. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

The Central Nervous System (CNS)-independent Anti-bone-resorptive Activity of Muscle Contraction and the Underlying Molecular and Cellular Signatures*

PubMed Central

Qin, Weiping; Sun, Li; Cao, Jay; Peng, Yuanzhen; Collier, Lauren; Wu, Yong; Creasey, Graham; Li, Jianhua; Qin, Yiwen; Jarvis, Jonathan; Bauman, William A.; Zaidi, Mone; Cardozo, Christopher

2013-01-01

Muscle and bone work as a functional unit. Cellular and molecular mechanisms underlying effects of muscle activity on bone mass are largely unknown. Spinal cord injury (SCI) causes muscle paralysis and extensive sublesional bone loss and disrupts neural connections between the central nervous system (CNS) and bone. Muscle contraction elicited by electrical stimulation (ES) of nerves partially protects against SCI-related bone loss. Thus, application of ES after SCI provides an opportunity to study the effects of muscle activity on bone and roles of the CNS in this interaction, as well as the underlying mechanisms. Using a rat model of SCI, the effects on bone of ES-induced muscle contraction were characterized. The SCI-mediated increase in serum C-terminal telopeptide of type I collagen (CTX) was completely reversed by ES. In ex vivo bone marrow cell cultures, SCI increased the number of osteoclasts and their expression of mRNA for several osteoclast differentiation markers, whereas ES significantly reduced these changes; SCI decreased osteoblast numbers, but increased expression in these cells of receptor activator of NF-κB ligand (RANKL) mRNA, whereas ES increased expression of osteoprotegerin (OPG) and the OPG/RANKL ratio. A microarray analysis revealed that ES partially reversed SCI-induced alterations in expression of genes involved in signaling through Wnt, FSH, parathyroid hormone (PTH), oxytocin, and calcineurin/nuclear factor of activated T-cells (NFAT) pathways. ES mitigated SCI-mediated increases in mRNA levels for the Wnt inhibitors DKK1, sFRP2, and sclerostin in ex vivo cultured osteoblasts. Our results demonstrate an anti-bone-resorptive activity of muscle contraction by ES that develops rapidly and is independent of the CNS. The pathways involved, particularly Wnt signaling, suggest future strategies to minimize bone loss after immobilization. PMID:23530032

Methylation-reprogrammed Wnt/β-catenin signalling mediated prenatal hypoxia-induced brain injury in foetal and offspring rats.

PubMed

Zhang, Yingying; Zhang, Mengshu; Li, Lingjun; Wei, Bin; He, Axin; Lu, Likui; Li, Xiang; Zhang, Lubo; Xu, Zhice; Sun, Miao

2018-05-28

Prenatal hypoxia (PH) is a common pregnancy complication, harmful to brain development. This study investigated whether and how PH affected Wnt pathway in the brain. Pregnant rats were exposed to hypoxia (10.5% O 2 ) or normoxia (21% O 2 ; Control). Foetal brain weight and body weight were decreased in the PH group, the ratio of brain weight to body weight was increased significantly. Prenatal hypoxia increased mRNA expression of Wnt3a, Wnt7a, Wnt7b and Fzd4, but not Lrp6. Activated β-catenin protein and Fosl1 expression were also significantly up-regulated. Increased Hif1a expression was found in the PH group associated with the higher Wnt signalling. Among 5 members of the Sfrp family, Sfrp4 was down-regulated. In the methylation-regulating genes, higher mRNA expressions of Dnmt1 and Dnmt3b were found in the PH group. Sodium bisulphite and sequencing revealed hyper-methylation in the promoter region of Sfrp4 gene in the foetal brain, accounting for its decreased expression and contributing to the activation of the Wnt-Catenin signalling. The study of PC12 cells treated with 5-aza further approved that decreased methylation could result in the higher Sfrp4 expression. In the offspring hippocampus, protein levels of Hif1a and mRNA expression of Sfrp4 were unchanged, whereas Wnt signal pathway was inhibited. The data demonstrated that PH activated the Wnt pathway in the foetal brain, related to the hyper-methylation of Sfrp4 as well as Hif1a signalling. Activated Wnt signalling might play acute protective roles to the foetal brain in response to hypoxia, also would result in disadvantageous influence on the offspring in long-term. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

mRNA interactome capture in mammalian cells.

PubMed

Kastelic, Nicolai; Landthaler, Markus

2017-08-15

Throughout their entire life cycle, mRNAs are associated with RNA-binding proteins (RBPs), forming ribonucleoprotein (RNP) complexes with highly dynamic compositions. Their interplay is one key to control gene regulatory mechanisms from mRNA synthesis to decay. To assay the global scope of RNA-protein interactions, we and others have published a method combining crosslinking with highly stringent oligo(dT) affinity purification to enrich proteins associated with polyadenylated RNA (poly(A)+ RNA). Identification of the poly(A)+ RNA-bound proteome (also: mRNA interactome capture) has by now been applied to a diversity of cell lines and model organisms, uncovering comprehensive repertoires of RBPs and hundreds of novel RBP candidates. In addition to determining the RBP catalog in a given biological system, mRNA interactome capture allows the examination of changes in protein-mRNA interactions in response to internal and external stimuli, altered cellular programs and disease. Copyright © 2017. Published by Elsevier Inc.

Amicoumacin A inhibits translation by stabilizing mRNA interaction with the ribosome

PubMed Central

Polikanov, Yury S.; Osterman, Ilya A.; Szal, Teresa; Tashlitsky, Vadim N.; Serebryakova, Marina V.; Kusochek, Pavel; Bulkley, David; Malanicheva, Irina A.; Efimenko, Tatyana A.; Efremenkova, Olga V.; Konevega, Andrey L.; Shaw, Karen J.; Bogdanov, Alexey A.; Rodnina, Marina V.; Dontsova, Olga A.; Mankin, Alexander S.; Steitz, Thomas A.; Sergiev, Petr V.

2014-01-01

SUMMARY We demonstrate that the antibiotic amicoumacin A (AMI) whose cellular target was unknown, is a potent inhibitor of protein synthesis. Resistance mutations in helix 24 of the 16S rRNA mapped the AMI binding site to the small ribosomal subunit. The crystal structure of bacterial ribosome in complex with AMI solved at 2.4 Å resolution revealed that the antibiotic makes contacts with universally conserved nucleotides of 16S rRNA in the E site and the mRNA backbone. Simultaneous interactions of AMI with 16S rRNA and mRNA and the in vivo experimental evidence suggest that it may inhibit the progression of the ribosome along mRNA. Consistent with this proposal, binding of AMI interferes with translocation in vitro. The inhibitory action of AMI can be partly compensated by mutations in the translation elongation factor G. PMID:25306919

Increased IMP dehydrogenase gene expression in solid tumor tissues and tumor cell lines

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

Collart, F.R.; Chubb, C.B.; Mirkin, B.L.

1992-07-10

IMP dehydrogenase, a regulatory enzyme of guanine nucleotide biosynthesis, may play a role in cell proliferation and malignancy. To assess this possibility, we examined IMP dehydrogenase expression in a series of human solid tumor tissues and tumor cell lines in comparison with their normal counterparts. Increased IMP dehydrogenase gene expression was observed in brain tumors relative to normal brain tissue and in sarcoma cells relative to normal fibroblasts. Similarly, in several B- and T-lymphoid leukemia cell lines, elevated levels of IMP dehydrogenase mRNA and cellular enzyme were observed in comparison with the levels in peripheral blood lymphocytes. These results aremore » consistent with an association between increased IMP dehydrogenase expression and either enhanced cell proliferation or malignant transformation.« less

Small Molecule Inhibitors of Staphylococcus aureus RnpA Alter Cellular mRNA Turnover, Exhibit Antimicrobial Activity, and Attenuate Pathogenesis

PubMed Central

Olson, Patrick D.; Kuechenmeister, Lisa J.; Anderson, Kelsi L.; Daily, Sonja; Beenken, Karen E.; Roux, Christelle M.; Reniere, Michelle L.; Lewis, Tami L.; Weiss, William J.; Pulse, Mark; Nguyen, Phung; Simecka, Jerry W.; Morrison, John M.; Sayood, Khalid; Asojo, Oluwatoyin A.; Smeltzer, Mark S.; Skaar, Eric P.; Dunman, Paul M.

2011-01-01

Methicillin-resistant Staphylococcus aureus is estimated to cause more U.S. deaths annually than HIV/AIDS. The emergence of hypervirulent and multidrug-resistant strains has further amplified public health concern and accentuated the need for new classes of antibiotics. RNA degradation is a required cellular process that could be exploited for novel antimicrobial drug development. However, such discovery efforts have been hindered because components of the Gram-positive RNA turnover machinery are incompletely defined. In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro. Exploiting this activity, high through-put and secondary screening assays identified a small molecule inhibitor of RnpA-mediated in vitro RNA degradation. This agent was shown to limit cellular mRNA degradation and exhibited antimicrobial activity against predominant methicillin-resistant S. aureus (MRSA) lineages circulating throughout the U.S., vancomycin intermediate susceptible S. aureus (VISA), vancomycin resistant S. aureus (VRSA) and other Gram-positive bacterial pathogens with high RnpA amino acid conservation. We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus. Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy. PMID:21347352

[Relationship between endothelial damage and p120-catenin in paraquat intoxication and the protective effect of mangiferin].

PubMed

He, Qing; Ai, Jiao; Huang, Yu

2014-06-01

To investigate the relationship between endothelial damage and p120-catenin (p120-ctn) in a model of paraquat intoxication, and the modulatory effect of mangiferin on p120-ctn. Human umbilical vein endothelial cells (HUVECs) were cultured in two compartment spreading apparatus in vitro. The endothelial cells were divided into three groups: control group (cultured in DMEM with 10% fetal bovine serum), paraquat group (paraquat was added to the medium with final concentration of 0.05 μmol/L) and mangiferin group (cultured in medium with addition of paraquat for 30 minutes, then mangiferin was added in a final concentration of 20 μmol/L). The cellular permeability at 6, 12, 24, 48, 72 hours after culture in the three groups was measured. The expressions of p120-ctn 1A, p120-ctn 3A mRNA and p120-ctn protein were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot analysis. The distribution of p120-ctn protein was observed by immunofluorescence. Compared with control group, cellular permeability in paraquat and mangiferin groups were increased with prolongation of time, and peaked at 72 hours [(29.86 ± 3.98)%, (24.39 ± 2.79)% vs. (11.71 ± 1.67)%, both P<0.05]. The cellular permeability was significantly lower in mangiferin group than that in paraquat group at different time points (all P<0.05). At 6 hours after intoxication, the expressions of p120-ctn 1A, p120-ctn 3A mRNA (gray value) and p120-ctn protein (gray value) were significantly lower in paraquat group than those in control group (p120-ctn 1A mRNA: 0.150 ± 0.024 vs. 0.433 ± 0.024, p120-ctn 3A mRNA: 0.316 ± 0.043 vs. 0.701 ± 0.020, p120-ctn protein: 0.485 ± 0.031 vs. 0.763 ± 0.038, all P<0.01). The expressions of p120-ctn 1A, p120-ctn 3A mRNA and p120-ctn protein were significantly higher in mangiferin group than those in paraquat group from 6 hours on (p120-ctn 1A mRNA: 0.281 ± 0.021 vs. 0.150 ± 0.024, p120-ctn 3A mRNA: 0.602 ± 0.042 vs. 0.316 ± 0.043, p120-ctn protein: 0.675 ± 0.031 vs. 0.485 ± 0.031, all P<0.01), and they were gradually increased with prolongation of time, and peaked at 72 hours (p120-ctn 1A mRNA: 1.376 ± 0.128 vs. 0.150 ± 0.024, p120-ctn 3A mRNA: 1.251 ± 0.059 vs. 0.316 ± 0.043, p120-ctn protein: 0.844 ± 0.050 vs. 0.485±0.031, all P<0.01). Under upright fluorescence microscope, p120-ctn was mainly distributed in the cell membrane in control group, with a slight expression in cytoplasm, and no expression in the nuclei. With prolongation of time, p120-ctn expression in the cell membrane was gradually decreased in paraquat group, while it was increased in the cytoplasm and nuclei, with blurring of cell membrane and widening of cellular gap. p120-ctn expression was improved on the cell membrane in mangiferin group at corresponding time points, with decreased in expression in nuclei and cytoplasm. The p120-ctn protein plays an important role in the enhancement of endothelial permeability in paraquat intoxication, and mangiferin may attenuate endothelial injury in paraquat intoxication possibly through modulation of p120-ctn protein.

Lassa and Mopeia virus replication in human monocytes/macrophages and in endothelial cells: different effects on IL-8 and TNF-alpha gene expression.

PubMed

Lukashevich, I S; Maryankova, R; Vladyko, A S; Nashkevich, N; Koleda, S; Djavani, M; Horejsh, D; Voitenok, N N; Salvato, M S

1999-12-01

Cells of the mononuclear and endothelial lineages are targets for viruses which cause hemorrhagic fevers (HF) such as the filoviruses Marburg and Ebola, and the arenaviruses Lassa and Junin. A recent model of Marburg HF pathogenesis proposes that virus directly causes endothelial cell damage and macrophage release of TNF-alpha which increases the permeability of endothelial monolayers [Feldmann et al. , 1996]. We show that Lassa virus replicates in human monocytes/macrophages and endothelial cells without damaging them. Human endothelial cells (HUVEC) are highly susceptible to infection by both Lassa and Mopeia (a non-pathogenic Lassa-related arenavirus). Whereas monocytes must differentiate into macrophages before supporting even low level production of these viruses, the virus yields in the culture medium of infected HUVEC cells reach more than 7 log10 PFU/ml without cellular damage. In contrast to filovirus, Lassa virus replication in monocytes/macrophages fails to stimulate TNF-alpha gene expression and even down-regulates LPS-stimulated TNF-alpha mRNA synthesis. The expression of IL-8, a prototypic proinflammatory CXC chemokine, was also suppressed in Lassa virus infected monocytes/macrophages and HUVEC on both the protein and mRNA levels. This contrasts with Mopeia virus infection of HUVEC in which neither IL-8 mRNA nor protein are reduced. The cumulative down-regulation of TNF-alpha and IL-8 expression could explain the absence of inflammatory and effective immune responses in severe cases of Lassa HF. Copyright 1999 Wiley-Liss, Inc.

Elevated GRIA1 mRNA expression in Layer II/III and V pyramidal cells of the DLPFC in schizophrenia

PubMed Central

O’Connor, J.A.; Hemby, S.E.

2012-01-01

The functional integrity of the dorsolateral prefrontal cortex (DLPFC) is altered in schizophrenia leading to profound deficits in working memory and cognition. Growing evidence indicates that dysregulation of glutamate signaling may be a significant contributor to the pathophysiology mediating these effects; however, the contribution of NMDA and AMPA receptors in the mediation of this deficit remains unclear. The equivocality of data regarding ionotropic glutamate receptor alterations of subunit expression in the DLPFC of schizophrenics is likely reflective of subtle alterations in the cellular and molecular composition of specific neuronal populations within the region. Given previous evidence of Layer II/III and V pyramidal cell alterations in schizophrenia and the significant influence of subunit composition on NMDA and AMPA receptor function, laser capture microdissection combined with quantitative PCR was used to examine the expression of AMPA (GRIA1-4) and NMDA (GRIN1, 2A and 2B) subunit mRNA levels in Layer II/III and Layer V pyramidal cells in the DLPFC. Comparisons were made between individuals diagnosed with schizophrenia, bipolar disorder, major depressive disorder and controls (n=15/group). All subunits were expressed at detectable levels in both cell populations for all diseases as well as for the control group. Interestingly, GRIA1 mRNA was significantly increased in both cell types in the schizophrenia group compare to controls, while similar trends were observed in major depressive disorder (Layers II/III and V) and bipolar disorder (Layer V). These data suggest that increased GRIA1 subunit expression may contribute to schizophrenia pathology. PMID:17942280

MicroRNA Profiling of Sendai Virus-Infected A549 Cells Identifies miR-203 as an Interferon-Inducible Regulator of IFIT1/ISG56

PubMed Central

Buggele, William A.

2013-01-01

The mammalian type I interferon (IFN) response is a primary barrier for virus infection and is essential for complete innate and adaptive immunity. Both IFN production and IFN-mediated antiviral signaling are the result of differential cellular gene expression, a process that is tightly controlled at transcriptional and translational levels. To determine the potential for microRNA (miRNA)-mediated regulation of the antiviral response, small-RNA profiling was used to analyze the miRNA content of human A549 cells at steady state and following infection with the Cantell strain of Sendai virus, a potent inducer of IFN and cellular antiviral responses. While the miRNA content of the cells was largely unaltered by infection, specific changes in miRNA abundance were identified during Sendai virus infection. One miRNA, miR-203, was found to accumulate in infected cells and in response to IFN treatment. Results indicate that miR-203 is an IFN-inducible miRNA that can negatively regulate a number of cellular mRNAs, including an IFN-stimulated gene target, IFIT1/ISG56, by destabilizing its mRNA transcript. PMID:23785202

A Conserved Developmental Patterning Network Produces Quantitatively Different Output in Multiple Species of Drosophila

PubMed Central

Meyer, Miriah; Wunderlich, Zeba; Simirenko, Lisa; Luengo Hendriks, Cris L.; Keränen, Soile V. E.; Henriquez, Clara; Knowles, David W.; Biggin, Mark D.; Eisen, Michael B.; DePace, Angela H.

2011-01-01

Differences in the level, timing, or location of gene expression can contribute to alternative phenotypes at the molecular and organismal level. Understanding the origins of expression differences is complicated by the fact that organismal morphology and gene regulatory networks could potentially vary even between closely related species. To assess the scope of such changes, we used high-resolution imaging methods to measure mRNA expression in blastoderm embryos of Drosophila yakuba and Drosophila pseudoobscura and assembled these data into cellular resolution atlases, where expression levels for 13 genes in the segmentation network are averaged into species-specific, cellular resolution morphological frameworks. We demonstrate that the blastoderm embryos of these species differ in their morphology in terms of size, shape, and number of nuclei. We present an approach to compare cellular gene expression patterns between species, while accounting for varying embryo morphology, and apply it to our data and an equivalent dataset for Drosophila melanogaster. Our analysis reveals that all individual genes differ quantitatively in their spatio-temporal expression patterns between these species, primarily in terms of their relative position and dynamics. Despite many small quantitative differences, cellular gene expression profiles for the whole set of genes examined are largely similar. This suggests that cell types at this stage of development are conserved, though they can differ in their relative position by up to 3–4 cell widths and in their relative proportion between species by as much as 5-fold. Quantitative differences in the dynamics and relative level of a subset of genes between corresponding cell types may reflect altered regulatory functions between species. Our results emphasize that transcriptional networks can diverge over short evolutionary timescales and that even small changes can lead to distinct output in terms of the placement and number of equivalent cells. PMID:22046143

ZKSCAN1 gene and its related circular RNA (circZKSCAN1) both inhibit hepatocellular carcinoma cell growth, migration, and invasion but through different signaling pathways.

PubMed

Yao, Zhicheng; Luo, Jingyan; Hu, Kunpeng; Lin, Jizong; Huang, He; Wang, Qiangliang; Zhang, Peng; Xiong, Zhiyong; He, Chonghua; Huang, Zejian; Liu, Bo; Yang, Yang

2017-04-01

There is increasing evidence that circular RNA (circRNA) are involved in cancer development, but the regulation and function of human circRNA remain largely unknown. In this study, we demonstrated that ZKSCAN1, a zinc finger family gene, is expressed in both linear and circular (circZKSCAN1) forms of RNA in human hepatocellular carcinoma (HCC) tissues and cell lines. Here, we analyzed a cohort of 102 patients and found that expression of both ZKSCAN1mRNA and circZKSCAN1 was significantly lower (P < 0.05) in the HCC samples compared with that in matched adjacent nontumorous tissues by reverse transcription PCR (RT-PCR). The low expression level of ZKSCAN1 was only associated with tumor size (P = 0.032), while the cirZKSCAN1 levels varied in patients with different tumor numbers (P < 0.01), cirrhosis (P = 0.031), vascular invasion (P = 0.002), or microscopic vascular invasion (P = 0.002), as well as with the tumor grade (P < 0.001). Silencing both ZKSCAN1mRNA and circZKSCAN1 promoted cell proliferation, migration, and invasion. In contrast, overexpression of both forms of RNA repressed HCC progression in vivo and in vitro. Silencing or overexpression of both forms of RNA did not interfere with each other. RNA-seq revealed a very different molecular basis for the observed effects; ZKSCAN1mRNA mainly regulated cellular metabolism, while circZKSCAN1 mediated several cancer-related signaling pathways, suggesting a nonredundant role for ZKSCAN1mRNA and circRNA. In conclusion, our results revealed two post-translational products (ZKSCAN1mRNA and circZKSCAN1) that cooperated closely with one another to inhibit growth, migration, and invasion of HCC. cirZKSCAN1 might be a useful marker for the diagnosis of HCC. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells

PubMed Central

Zekavati, Anna; Nasir, Asghar; Alcaraz, Amor; Aldrovandi, Maceler; Marsh, Phil; Norton, John D.; Murphy, John J.

2014-01-01

The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells. PMID:25014217

Constitutive mRNA expression and protein activity levels of nine ABC efflux transporters in seven permanent cell lines derived from different tissues of rainbow trout (Oncorhynchus mykiss).

PubMed

Fischer, Stephan; Loncar, Jovica; Zaja, Roko; Schnell, Sabine; Schirmer, Kristin; Smital, Tvrtko; Luckenbach, Till

2011-01-25

Permanent fish cell lines have become common model systems for determining ecotoxicological effects of pollutants. For these cell lines little is known on the cellular active transport mechanisms that control the amount of a compound entering the cell, such as the MXR (multixenobiotic resistance) system mediated by ATP binding cassette (ABC) transport proteins. Therefore, for toxic evaluation of chemicals with those cells information on MXR is important. We here present data on constitutive mRNA expression and protein activity levels of a series of ABC efflux transporters in seven permanent cell lines derived from liver (RTL-W1; R1) and liver hepatoma (RTH-149), gill (RTgill-W1), gonad (RTG-2), gut (RTgutGC) and brain (RTbrain) of rainbow trout (Oncorhynchus mykiss). In addition to known transporters abcb1 (designated here abcb1a), abcb11, abcc1-3, abcc5 and abcg2, we quantified expression levels of a newly identified abcb1 isoform (abcb1b) and abcc4, previously unknown in trout. Quantitative real time PCR (qPCR) indicated that mRNA of the examined ABC transporters was constitutively expressed in all cell lines. Transporter mRNA expression patterns were similar in all cell lines, with expression levels of abcc transporters being 80 to over 1000 fold higher than for abcg2, abcb1a/b and abcb11 (abcc1-5>abcg2>abcb1a/b, 11). Transporter activity in the cell lines was determined by measuring uptake of transporter type specific fluorescent substrates in the presence of activity inhibitors. The combination of the ABCB1 and ABCC transporter substrate calcein-AM with inhibitors cyclosporine A, PSC833 and MK571 resulted in a concentration-dependent fluorescence increase of up to 3-fold, whereas reversin 205 caused a slight, but not concentration-dependent fluorescence increase. Accumulation of the dyes Hoechst 33342 and 2',7'-dichlorodihydrofluorescein diacetate was basically unchanged in the presence of Ko134 and taurocholate, respectively, indicating low Abcg2 and Abcb11 activities, in accordance with low abcg2 and abcb11 transcript levels. Our data indicate that transporter expression and activity patterns in the different trout cell lines are irrespective of the tissue of origin, but are determined by factors of cell cultivation. 2010 Elsevier B.V. All rights reserved.

Modulatory effects of levamisole and garlic oil on the immune response of Wistar rats: Biochemical, immunohistochemical, molecular and immunological study.

PubMed

Mohamed, Essam Hassan; Baiomy, Ahmed Abdel-Aziz; Ibrahim, Zein Shaban; Soliman, Mohamed Mohamed

2016-09-01

Levamisole (LEVA) and garlic are prevalent immunomodulators in humans and animals. Therefore, the present study aimed to examine the immunomodulatory effects of LEVA and garlic oil (GO) alone or in combination on the immune response of Wistar rats. A total of 24 male Wistar rats were allocated into four equal groups: Control group, which was given ad libitum access to food and water; and groups 2‑4, which were orally administered LEVA [2.5 mg/kg body weight (BW) every 2 days], GO, (5 ml/kg BW daily), or LEVA plus GO, respectively for 4 consecutive weeks. Serum immunoglobulin (Ig)G and IgM levels were measured using a radial immunodiffusion assay. Serum cytokine levels, including interferon (IFN)-γ, interleukin (IL)-5 and tumor necrosis factor (TNF)-α, were measured using enzyme‑linked immunosorbent assay kits. Total blood counts were measured automatically using a cell counter. Serum lysozyme enzymatic activity was determined by measuring the diameters of the zones of clearance relative to lysozyme. Immunohistochemical detection of CD4 and CD8 was carried out using the streptavidin-biotin-peroxidase method. Furthermore, the mRNA expression levels of IL‑4, IL‑5 and IL‑12 were measured in the leukocytes and thymus gland by semi-quantitative polymerase chain reaction. The results revealed that LEVA increased serum levels of IFN‑γ, IL‑5 and TNF‑α cytokines, whereas co‑administration of LEVA and GO decreased the stimulatory action of LEVA alone. LEVA and GO alone increased the serum levels of IgG, IgM and total blood cell counts, and co‑administration of GO and LEVA inhibited the effects of LEVA. At the cellular level, in the spleen, LEVA increased immunoreactivity of CD4 and CD8, whereas co‑administration of GO with LEVA decreased this strong expression. At the molecular level, in leukocytes, LEVA upregulated the mRNA expression levels of IL‑2, IL‑4 and IL‑5, whereas GO alone downregulated mRNA expression. Co‑administration of GO with LEVA inhibited the LEVA‑induced upregulation of IL‑2, IL‑4 and IL‑5 mRNA expression. In the thymus, both LEVA and GO upregulated the mRNA expression levels of IL‑4 and IL‑5, whereas LEVA alone did not affect IL‑12 mRNA expression. Co‑administration of GO with LEVA inhibited LEVA‑induced upregulation of IL‑4 and GO‑induced upregulation of IL‑12 expression, and had an additive upregulatory effect on IL‑5 expression. In conclusion, LEVA stimulated T‑helper (Th)1 cytokines, whereas GO stimulated a Th2 response, and co‑administration of GO with LEVA inhibited the stimulatory effects of LEVA and balanced the Th1/Th2 response.

RNA epitranscriptomics: Regulation of infection of RNA and DNA viruses by N6 -methyladenosine (m6 A).

PubMed

Tan, Brandon; Gao, Shou-Jiang

2018-04-26

N 6 -methyladenosine (m 6 A) was discovered 4 decades ago. However, the functions of m 6 A and the cellular machinery that regulates its changes have just been revealed in the last few years. m 6 A is an abundant internal mRNA modification on cellular RNA and is implicated in diverse cellular functions. Recent works have demonstrated the presence of m 6 A in the genomes of RNA viruses and transcripts of a DNA virus with either a proviral or antiviral role. Here, we first summarize what is known about the m 6 A "writers," "erasers," "readers," and "antireaders" as well as the role of m 6 A in mRNA metabolism. We then review how the replications of numerous viruses are enhanced and restricted by m 6 A with emphasis on the oncogenic DNA virus, Kaposi sarcoma-associated herpesvirus (KSHV), whose m 6 A epitranscriptome was recently mapped. In the context of KSHV, m 6 A and the reader protein YTHDF2 acts as an antiviral mechanism during viral lytic replication. During viral latency, KSHV alters m 6 A on genes that are implicated in cellular transformation and viral latency. Lastly, we discuss future studies that are important to further delineate the functions of m 6 A in KSHV latent and lytic replication and KSHV-induced oncogenesis. Copyright © 2018 John Wiley & Sons, Ltd.

A genome-wide approach identifies distinct but overlapping subsets of cellular mRNAs associated with Staufen1- and Staufen2-containing ribonucleoprotein complexes

PubMed Central

Furic, Luc; Maher-Laporte, Marjolaine; DesGroseillers, Luc

2008-01-01

Messenger RNAs are associated with multiple RNA-binding proteins to form ribonucleoprotein (mRNP) complexes. These proteins are important regulators of the fate of their target mRNAs. In human cells, Staufen1 and Staufen2 proteins, coded by two different genes, are double-stranded RNA-binding proteins involved in several cellular functions including mRNA localization, translation, and decay. Although 51% identical, these proteins are nevertheless found in different RNA particles. In addition, differential splicing events generate Staufen2 isoforms that only differ at their N-terminal extremities. In this paper, we used a genome-wide approach to identify and compare the mRNA targets of mammalian Staufen proteins. The mRNA content of Staufen mRNPs was identified by probing DNA microarrays with probes derived from mRNAs isolated from immunopurified Staufen-containing complexes following transfection of HEK293T cells with Stau155-HA, Stau259-HA, or Stau262-HA expressors. Our results indicate that 7% and 11% of the cellular RNAs expressed in HEK293T cells are found in Stau1- and in Stau2-containing mRNPs, respectively. A comparison of Stau1- and Stau2-containing mRNAs identifies a relatively low percentage of common mRNAs; the percentage of common mRNAs highly increases when mRNAs in Stau259-HA- and Stau262-containing mRNPs are compared. There is a predominance of mRNAs involved in cell metabolism, transport, transcription, regulation of cell processes, and catalytic activity. All these subsets of mRNAs are mostly distinct from those associated with FMRP or IMP, although some mRNAs overlap. Consistent with a model of post-transcriptionnal gene regulation, our results show that Stau1- and Stau2-mRNPs associate with distinct but overlapping sets of cellular mRNAs. PMID:18094122

A genome-wide approach identifies distinct but overlapping subsets of cellular mRNAs associated with Staufen1- and Staufen2-containing ribonucleoprotein complexes.

PubMed

Furic, Luc; Maher-Laporte, Marjolaine; DesGroseillers, Luc

2008-02-01

Messenger RNAs are associated with multiple RNA-binding proteins to form ribonucleoprotein (mRNP) complexes. These proteins are important regulators of the fate of their target mRNAs. In human cells, Staufen1 and Staufen2 proteins, coded by two different genes, are double-stranded RNA-binding proteins involved in several cellular functions including mRNA localization, translation, and decay. Although 51% identical, these proteins are nevertheless found in different RNA particles. In addition, differential splicing events generate Staufen2 isoforms that only differ at their N-terminal extremities. In this paper, we used a genome-wide approach to identify and compare the mRNA targets of mammalian Staufen proteins. The mRNA content of Staufen mRNPs was identified by probing DNA microarrays with probes derived from mRNAs isolated from immunopurified Staufen-containing complexes following transfection of HEK293T cells with Stau1(55)-HA, Stau2(59)-HA, or Stau2(62)-HA expressors. Our results indicate that 7% and 11% of the cellular RNAs expressed in HEK293T cells are found in Stau1- and in Stau2-containing mRNPs, respectively. A comparison of Stau1- and Stau2-containing mRNAs identifies a relatively low percentage of common mRNAs; the percentage of common mRNAs highly increases when mRNAs in Stau2(59)-HA- and Stau2(62)-containing mRNPs are compared. There is a predominance of mRNAs involved in cell metabolism, transport, transcription, regulation of cell processes, and catalytic activity. All these subsets of mRNAs are mostly distinct from those associated with FMRP or IMP, although some mRNAs overlap. Consistent with a model of post-transcriptional gene regulation, our results show that Stau1- and Stau2-mRNPs associate with distinct but overlapping sets of cellular mRNAs.

Hamp1 mRNA and plasma hepcidin levels are influenced by sex and strain but do not predict tissue iron levels in inbred mice.

PubMed

McLachlan, Stela; Page, Kathryn E; Lee, Seung-Min; Loguinov, Alex; Valore, Erika; Hui, Simon T; Jung, Grace; Zhou, Jie; Lusis, Aldons J; Fuqua, Brie; Ganz, Tomas; Nemeth, Elizabeta; Vulpe, Chris D

2017-11-01

Iron homeostasis is tightly regulated, and the peptide hormone hepcidin is considered to be a principal regulator of iron metabolism. Previous studies in a limited number of mouse strains found equivocal sex- and strain-dependent differences in mRNA and serum levels of hepcidin and reported conflicting data on the relationship between hepcidin ( Hamp1 ) mRNA levels and iron status. Our aim was to clarify the relationships between strain, sex, and hepcidin expression by examining multiple tissues and the effects of different dietary conditions in multiple inbred strains. Two studies were done: first, Hamp1 mRNA, liver iron, and plasma diferric transferrin levels were measured in 14 inbred strains on a control diet; and second, Hamp1 mRNA and plasma hepcidin levels in both sexes and iron levels in the heart, kidneys, liver, pancreas, and spleen in males were measured in nine inbred/recombinant inbred strains raised on an iron-sufficient or high-iron diet. Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). However, liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice fed iron-sufficient or high-iron diets, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males. We also measured plasma erythroferrone, performed RNA-sequencing analysis of liver samples from six inbred strains fed the iron-sufficient, low-iron, or high-iron diets, and explored differences in gene expression between the strains with the highest and lowest hepcidin levels. NEW & NOTEWORTHY Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). Liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males. Copyright © 2017 the American Physiological Society.

UPF1 silenced cellular model systems for screening of read-through agents active on β039 thalassemia point mutation.

PubMed

Salvatori, Francesca; Pappadà, Mariangela; Breveglieri, Giulia; D'Aversa, Elisabetta; Finotti, Alessia; Lampronti, Ilaria; Gambari, Roberto; Borgatti, Monica

2018-05-15

Nonsense mutations promote premature translational termination, introducing stop codons within the coding region of mRNAs and causing inherited diseases, including thalassemia. For instance, in β 0 39 thalassemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, ribosomal read-through molecules, such as aminoglycoside antibiotics, have been tested on mRNAs carrying premature stop codons. These findings have introduced new hopes for the development of a pharmacological approach to the β 0 39 thalassemia therapy. While several strategies, designed to enhance translational read-through, have been reported to inhibit NMD efficiency concomitantly, experimental tools for systematic analysis of mammalian NMD inhibition by translational read-through are lacking. We developed a human cellular model of the β 0 39 thalassemia mutation with UPF-1 suppressed and showing a partial NMD suppression. This novel cellular model could be used for the screening of molecules exhibiting preferential read-through activity allowing a great rescue of the mutated transcripts.

Antiallergic effect of fisetin on IgE-mediated mast cell activation in vitro and on passive cutaneous anaphylaxis (PCA).

PubMed

Jo, Woo-Ri; Park, Hye-Jin

2017-10-01

Fisetin (3,7,3',4'-tetrahydroxyflavone), a naturally occurring bioactive flavonoid, has been shown to inhibit inflammation. However, little is known about the effect of fisetin on immunoglobulin E (IgE)-mediated allergic responses. In this study, the effect of fisetin on rat basophilic leukemia (RBL-2H3) cell-mediated allergic reactions was investigated. Fisetin inhibited β-hexosaminidase release and decreased the level of interleukin-4 and tumor necrosis factor-α mRNA in IgE/antigen (IgE/Ag)-stimulated RBL-2H3 cells. To elucidate the antiallergic mechanism, we examined the levels of signaling molecules responsible for degranulation and release of inflammatory cytokines. Fisetin decreased the levels of activated spleen tyrosine kinase, Gab2 proteins, linker of activated T cells, extracellular signal-related kinase 1/2 in the IgE/Ag-stimulated RBL2H3 cells, and NFκB and STAT3 proteins activated in the ear tissue of mice with passive cutaneous anaphylaxis (PCA). In addition, fisetin significantly lowered of FcɛRI α-subunit mRNA expression. Consistent with the cellular data, fisetin markedly suppressed RBL-2H3 cell-dependent PCA in IgE/Ag-sensitized mice. These results suggest that fisetin may have potential as a therapeutic agent for the treatment of allergic diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

The Ubiquitin-Conjugating Enzyme E2-EPF Is Overexpressed in Primary Breast Cancer and Modulates Sensitivity to Topoisomerase II Inhibition1

PubMed Central

Tedesco, Donato; Zhang, Jianhuan; Trinh, Lan; Lalehzadeh, Guita; Meisner, Rene; Yamaguchi, Ken D; Ruderman, Daniel L; Dinter, Harald; Zajchowski, Deborah A

2007-01-01

We identified the ubiquitin-conjugating enzyme E2-EPF mRNA as differentially expressed in breast tumors relative to normal tissues and performed studies to elucidate its putative role in cancer. We demonstrated that overexpression of E2-EPF protein correlated with estrogen receptor (ER) negativity in breast cancer specimens and that its expression is cell cycle-regulated, suggesting a potential function for E2-EPF in cell cycle progression. However, reduction of E2-EPF protein levels by > 80% using RNAi had no significant effects on the proliferation of HeLa cervical cancer cells or ER- MDA-MB-231 or MDA-MB-453 breast cancer cells. Because E2-EPF protein levels were elevated during the G2/M phase of the cell cycle and because E2-EPF mRNA in tumor specimens was frequently coexpressed with genes involved in cell cycle control, spindle assembly, and mitotic surveillance, the possibility that E2-EPF might have a function in the cellular response to agents that induce a G2 checkpoint or an M checkpoint was investigated. E2-EPF knockdown sensitized HeLa cells to the topoisomerase (topo) II inhibitors etoposide and doxorubicin and also increased topo IIα protein levels. These data suggest that combined administration of topo II-directed drugs and E2-EPF inhibitors may enhance their clinical effectiveness. PMID:17710163

The ubiquitin-conjugating enzyme E2-EPF is overexpressed in primary breast cancer and modulates sensitivity to topoisomerase II inhibition.

PubMed

Tedesco, Donato; Zhang, Jianhuan; Trinh, Lan; Lalehzadeh, Guita; Meisner, Rene; Yamaguchi, Ken D; Ruderman, Daniel L; Dinter, Harald; Zajchowski, Deborah A

2007-07-01

We identified the ubiquitin-conjugating enzyme E2-EPF mRNA as differentially expressed in breast tumors relative to normal tissues and performed studies to elucidate its putative role in cancer. We demonstrated that overexpression of E2-EPF protein correlated with estrogen receptor (ER) negativity in breast cancer specimens and that its expression is cell cycle-regulated, suggesting a potential function for E2-EPF in cell cycle progression. However, reduction of E2-EPF protein levels by > 80% using RNAi had no significant effects on the proliferation of HeLa cervical cancer cells or ER(-) MDA-MB-231 or MDA-MB-453 breast cancer cells. Because E2-EPF protein levels were elevated during the G(2)/M phase of the cell cycle and because E2-EPF mRNA in tumor specimens was frequently coexpressed with genes involved in cell cycle control, spindle assembly, and mitotic surveillance, the possibility that E2-EPF might have a function in the cellular response to agents that induce a G(2) checkpoint or an M checkpoint was investigated. E2-EPF knockdown sensitized HeLa cells to the topoisomerase (topo) II inhibitors etoposide and doxorubicin and also increased topo IIalpha protein levels. These data suggest that combined administration of topo II-directed drugs and E2-EPF inhibitors may enhance their clinical effectiveness.

Mechanical Loading of Articular Cartilage Reduces IL-1-Induced Enzyme Expression

PubMed Central

Torzilli, P. A.; Bhargava, M.; Chen, C. T.

2011-01-01

Objective: Exposure of articular cartilage to interleukin-1 (IL-1) results in increased synthesis of matrix degrading enzymes. Previously mechanical load applied together with IL-1 stimulation was found to reduce aggrecan cleavage by ADAMTS-4 and 5 and MMP-1, -3, -9, and -13 and reduce proteoglycan loss from the extracellular matrix. To further delineate the inhibition mechanism the gene expression of ADAMTS-4 and 5; MMP-1, -3, -9, and -13; and TIMP-1, -2, and -3 were measured. Design: Mature bovine articular cartilage was stimulated with a 0.5 MPa compressive stress and 10 ng/ml of IL-1α for 3 days and then allowed to recover without stimulation for 1 additional day. The media was assayed for proteoglycan content on a daily basis, while chondrocyte gene expression (mRNA) was measured during stimulation and 1 day of recovery. Results: Mechanical load alone did not change the gene expression for ADAMTS, MMP, or TIMP. IL-1 caused an increase in gene expression for all enzymes after 1 day of stimulation while not affecting the TIMP levels. Load applied together with IL-1 decreased the expression levels of ADAMTS-4 and -5 and MMP-1 and -3 and increased TIMP-3 expression. Conclusions: A mechanical load appears to modify cartilage degradation by IL-1 at the cellular level by reducing mRNA. PMID:22039566

A family of cellular proteins related to snake venom disintegrins.

PubMed

Weskamp, G; Blobel, C P

1994-03-29

Disintegrins are short soluble integrin ligands that were initially identified in snake venom. A previously recognized cellular protein with a disintegrin domain was the guinea pig sperm protein PH-30, a protein implicated in sperm-egg membrane binding and fusion. Here we present peptide sequences that are characteristic for several cellular disintegrin-domain proteins. These peptide sequences were deduced from cDNA sequence tags that were generated by polymerase chain reaction from various mouse tissue and a mouse muscle cell line. Northern blot analysis with four sequence tags revealed distinct mRNA expression patterns. Evidently, cellular proteins containing a disintegrin domain define a superfamily of potential integrin ligands that are likely to function in important cell-cell and cell-matrix interactions.

Autotaxin Expression Is Regulated at the Post-transcriptional Level by the RNA-binding Proteins HuR and AUF1.

PubMed

Sun, Shuhong; Zhang, Xiaotian; Lyu, Lin; Li, Xixi; Yao, Siliang; Zhang, Junjie

2016-12-09

Autotaxin (ATX) is a key enzyme that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a lysophospholipid mediator that regulates cellular activities through its specific G protein-coupled receptors. The ATX-LPA axis plays an important role in various physiological and pathological processes, especially in inflammation and cancer development. Although the transcriptional regulation of ATX has been widely studied, the post-transcriptional regulation of ATX is largely unknown. In this study, we identified conserved adenylate-uridylate (AU)-rich elements in the ATX mRNA 3'-untranslated region (3'UTR). The RNA-binding proteins HuR and AUF1 directly bound to the ATX mRNA 3'UTR and had antagonistic functions in ATX expression. HuR enhanced ATX expression by increasing ATX mRNA stability, whereas AUF1 suppressed ATX expression by promoting ATX mRNA decay. HuR and AUF1 were involved in ATX regulation in Colo320 human colon cancer cells and the LPS-stimulated human monocytic THP-1 cells. HuR knockdown suppressed ATX expression in B16 mouse melanoma cells, leading to inhibition of cell migration. This effect was reversed by AUF1 knockdown to recover ATX expression or by the addition of LPA. These results suggest that the post-transcriptional regulation of ATX expression by HuR and AUF1 modulates cancer cell migration. In summary, we identified HuR and AUF1 as novel post-transcriptional regulators of ATX expression, thereby elucidating a novel mechanism regulating the ATX-LPA axis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of orbital spaceflight on human osteoblastic cell physiology and gene expression

NASA Technical Reports Server (NTRS)

Harris, S. A.; Zhang, M.; Kidder, L. S.; Evans, G. L.; Spelsberg, T. C.; Turner, R. T.

2000-01-01

During long-term spaceflight, astronauts lose bone, in part due to a reduction in bone formation. It is not clear, however, whether the force imparted by gravity has direct effects on bone cells. To examine the response of bone forming cells to weightlessness, human fetal osteoblastic (hFOB) cells were cultured during the 17 day STS-80 space shuttle mission. Fractions of conditioned media were collected during flight and shortly after landing for analyses of glucose utilization and accumulation of type I collagen and prostaglandin E(2) (PGE(2)). Total cellular RNA was isolated from flight and ground control cultures after landing. Measurement of glucose levels in conditioned media indicated that glucose utilization occurred at a similar rate in flight and ground control cultures. Furthermore, the levels of type I collagen and PGE(2) accumulation in the flight and control conditioned media were indistinguishable. The steady-state levels of osteonectin, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) were not significantly changed following spaceflight. Gene-specific reductions in mRNA levels for cytokines and skeletal growth factors were detected in the flight cultures using RNase protection assays. Steady-state mRNA levels for interleukin (IL)-1alpha and IL-6 were decreased 8 h following the flight and returned to control levels at 24 h postflight. Also, transforming growth factor (TGF)-beta(2) and TGF-beta(1) message levels were modestly reduced at 8 h and 24 h postflight, although the change was not statistically significant at 8 h. These data suggest that spaceflight did not significantly affect hFOB cell proliferation, expression of type I collagen, or PGE(2) production, further suggesting that the removal of osteoblastic cells from the context of the bone tissue results in a reduced ability to respond to weightlessness. However, spaceflight followed by return to earth significantly impacted the expression of cytokines and skeletal growth factors, which have been implicated as mediators of the bone remodeling cycle. It is not yet clear whether these latter changes were due to weightlessness or to the transient increase in loading resulting from reentry.

Static pressure accelerates ox-LDL-induced cholesterol accumulation via SREBP-1-mediated caveolin-1 downregulation in cultured vascular smooth muscle cells

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

Luo, Di-xian, E-mail: luodixian_2@163.com; Institute of Pharmacy and Pharmacology, College of Science and Technology, University of South China, Hengyang 421001, Hunan; First People's Hospital of Chenzhou City, Chenzhou 423000, Hunan

Research highlights: {yields} Vertical static pressure accelerates ox-LDL-induced cholesterol accumulation in cultured vascular smooth muscle cells. {yields} Static pressure induces SREBP-1 activation. {yields} Static pressure downregulates the expressions of caveolin-1 by activating SREBP-1. {yields} Static pressure also downregulates the transcription of ABCA1 by activating SREBP-1. {yields} Static pressure increases ox-LDL-induced cholesterol accumulation by SREBP-1-mediated caveolin-1 downregulation in vascular smooth muscle cells cultured in vitro. -- Abstract: Objective: To investigate the effect of static pressure on cholesterol accumulation in vascular smooth muscle cells (VSMCs) and its mechanism. Methods: Rat-derived VSMC cell line A10 treated with 50 mg/L ox-LDL and different staticmore » pressures (0, 60, 90, 120, 150, 180 mm Hg) in a custom-made pressure incubator for 48 h. Intracellular lipid droplets and lipid levels were assayed by oil red O staining and HPLC; The mRNA levels of caveolin-1 and ABCA1, the protein levels of caveolin-1 SREBP-1 and mature SREBP-1 were respectively detected by RT-PCR or western blot. ALLN, an inhibitor of SREBP metabolism, was used to elevate SREBP-1 protein level in VSMCs treated with static pressure. Results: Static pressures significantly not only increase intracellular lipid droplets in VSMCs, but also elevate cellular lipid content in a pressure-dependent manner. Intracellular free cholesterol (FC), cholesterol ester (CE), total cholesterol (TC) were respectively increased from 60.5 {+-} 2.8 mg/g, 31.8 {+-} 0.7 mg/g, 92.3 {+-} 2.1 mg/g at atmosphere pressure (ATM, 0 mm Hg) to 150.8 {+-} 9.4 mg/g, 235.9 {+-} 3.0 mg/g, 386.7 {+-} 6.4 mg/g at 180 mm Hg. At the same time, static pressures decrease the mRNA and protein levels of caveolin-1, and induce the activation and nuclear translocation of SREBP-1. ALLN increases the protein level of mature SREBP-1 and decreases caveolin-1 expression, so that cellular lipid levels were upregulated. Conclusion: Static pressures stimulate ox-LDL-induced cholesterol accumulation in cultured VSMCs through decreasing caveolin-1 expression via inducing the maturation and nuclear translocation of SREBP-1.« less

The role of IL-1beta in reduced IL-7 production by stromal and epithelial cells: a model for impaired T-cell numbers in the gut during HIV-1 infection.

PubMed

Thang, P H; Ruffin, N; Brodin, D; Rethi, B; Cam, P D; Hien, N T; Lopalco, L; Vivar, N; Chiodi, F

2010-08-01

Interleukin (IL)-7 is a key cytokine in T-cell homeostasis. Stromal cells, intestinal epithelial cells and keratinocytes are known to produce this cytokine. The mechanisms and cellular factors regulating IL-7 production are still unclear. We assessed whether IL-1beta and interferon (IFN)-gamma, cytokines produced during inflammatory conditions, may impact on IL-7 production. We used human intestinal epithelial cells (DLD-1 cell line) and bone marrow stromal cells (HS27 cell line), known to produce IL-7; IL-7 production was evaluated at the mRNA and protein levels. To assess whether treatment of HS27 cells with IL-1beta and/or IFN-gamma leads to changes in the gene expression of cytokines, Toll-like receptors (TLRs) and chemokines, we analysed gene expression profiles using the whole-genome microarray Human Gene 1.0 ST. We found that IFN-gamma enhanced the expression of IL-7 mRNA (P < 0.001) in both cell lines. IL-1beta treatment led to a significant down-regulation (P < 0.001) of IL-7 mRNA expression in both cell lines. The IL-7 concentration in supernatants collected from treated DLD-1 and HS27 cell cultures reflected the trend of IL-7 mRNA levels. The gene profiles revealed dramatic changes in expression of cytokines and their receptors (IL-7/IL-7R alpha; IL-1alpha,IL-1beta/IL-1R1; IFN-gamma/IFN-gammaR1), of IFN regulatory factors (IRF-1 and 2), of TLRs and of important chemo-attractants for T cells. The microarray results were verified by additional methods. Our results are discussed in the setting of inflammation and T-cell survival in the gut compartment during HIV-1 infection where stromal and epithelial cells may produce factors that contribute to impaired IL-7 homeostasis and homing of T cells.

Endocrine systems in juvenile anadromous and landlocked Atlantic salmon (Salmo salar): Seasonal development and seawater acclimation

USGS Publications Warehouse

Nilsen, Tom O.; Ebbesson, Lars O.E.; Kiilerich, P.; Bjornsson, B. Th; Madsen, Steffen S.; McCormick, S.D.; Stefansson, S.O.

2008-01-01

The present study compares developmental changes in plasma levels of growth hormone (GH), insulin-like growth factor I (IGF-I) and cortisol, and mRNA levels of their receptors and the prolactin receptor (PRLR) in the gill of anadromous and landlocked Atlantic salmon during the spring parr-smolt transformation (smoltification) period and following four days and one month seawater (SW) acclimation. Plasma GH and gill GH receptor (GHR) mRNA levels increased continuously during the spring smoltification period in the anadromous, but not in landlocked salmon. There were no differences in plasma IGF-I levels between strains, or any increase during smoltification. Gill IGF-I and IGF-I receptor (IGF-IR) mRNA levels increased in anadromous salmon during smoltification, with no changes observed in landlocked fish. Gill PRLR mRNA levels remained stable in both strains during spring. Plasma cortisol levels in anadromous salmon increased 5-fold in May and June, but not in landlocked salmon. Gill glucocorticoid receptor (GR) mRNA levels were elevated in both strains at the time of peak smoltification in anadromous salmon, while mineralocorticoid receptor (MR) mRNA levels remained stable. Only anadromous salmon showed an increase of gill 11??-hydroxysteroid dehydrogenase type-2 (11??-HSD2) mRNA levels in May. GH and gill GHR mRNA levels increased in both strains following four days of SW exposure in mid-May, whereas only the anadromous salmon displayed elevated plasma GH and GHR mRNA after one month in SW. Plasma IGF-I increased after four days in SW in both strains, decreasing in both strains after one month in SW. Gill IGF-I mRNA levels were only increased in landlocked salmon after 4 days in SW. Gill IGF-IR mRNA levels in SW did not differ from FW levels in either strain. Gill PRLR mRNA did not change after four days of SW exposure, and decreased in both strains after one month in SW. Plasma cortisol levels did not change following SW exposure in either strain. Gill GR, 11??-HSD2 and MR mRNA levels increased after four days in SW in both strains, whereas only the anadromous strain maintained elevated gill GR and 11??-HSD2 mRNA levels after one month in SW. The results indicate that hormones and receptors of the GH and cortisol axes are present at significantly lower levels during spring development and SW acclimation in landlocked relative to anadromous salmon. These findings suggest that attenuation of GH and cortisol axes may, at least partially, result in reduced preparatory upregulation of key gill ion-secretory proteins, possibly a result of reduced selection pressure for marine adaptations in landlocked salmon. ?? 2007 Elsevier Inc. All rights reserved.

Th22 cells are associated with hepatocellular carcinoma development and progression

PubMed Central

Qin, Shanyu; Ma, Shijia; Huang, Xiaoli; Lu, Donghong

2014-01-01

Objective IL-22-producing CD4+ T helper cells (Th22 cells) have been identified as major inducers of tissue inflammation and immune responses. Currently, no previous study explored the role of Th22 cells in the pathogenesis of hepatocellular carcinoma (HCC). The study aimed to determine the biological function of Th22 cells and its effector IL-22 in HCC patients. Methods Forty-five HCC patients and 19 healthy controls were recruited and their peripheral blood was collected. The fresh HCC tissues, adjacent HCC tissues and ten normal liver tissues were also collected. Flow cytometry analysis was used to determine the frequencies of circulating Th22 cells and Th17 cells. Serum IL-22 levels were tested by enzyme-linked immunosorbent assay (ELISA). Immunohistochemical staining and real-time polymerase chain reaction (PCR) were used to detect IL-22 protein and mRNA in tissues specimens, respectively. Results Circulating Th22 cells, Th17 cells and serum IL-22 levels were significantly elevated in HCC patients compared with those of healthy controls (P<0.001). Th22 cells were showed to be positively correlated with IL-22 in HCC patients (P<0.05), but not in healthy controls. No significant differences were found in HCC patients with HBeAg positivity or negativity in term of Th22 cells and serum IL-22 levels. The expression of IL-22 protein and mRNA was highest in HCC tissues, followed by adjacent HCC tissues and normal liver tissues. Furthermore, Th22 cells, serum IL-22 levels and IL-22 mRNA were elevated at stage III-IV compared with stage I-II of HCC (P<0.05). Conclusions Elevation of circulating Th22 cells and IL-22 may be implicated in the pathogenesis of HCC, and potentially be cellular targets for therapeutic intervention. PMID:24826053

Elevated utero/placental GR/NR3C1 is not required for the induction of parturition in the dog.

PubMed

Gram, Aykut; Trachsel, Alexandra; Boos, Alois; Kowalewski, Mariusz P

2016-10-01

The endocrine mechanisms that lead to initiation of parturition in dogs are still not fully understood. The prepartum luteolysis is associated with increased prostaglandin (PG) F2α secretion; however, there is no pregnancy- or parturition-related increase in estrogens. Moreover, unlike in other mammalian species, in the dog, increased peripartum levels of cortisol measured sporadically in maternal peripheral blood are not mandatory for normal parturition. Nevertheless, auto/paracrine effects of cortisol at the placental feto-maternal level cannot be excluded. Therefore, the aim of this study was to investigate the expression and localization of glucocorticoid receptor (GR/NR3C1) in canine utero/placental (Ut/Pl) units and uterine interplacental sites at selected time points during pregnancy (pre-implantation, post-implantation and mid-gestation), and at normal and antigestagen-induced parturition. The Ut/Pl expression of GR/NR3C1 did not change significantly from pre-implantation until mid-gestation; however, it was strongly induced during the prepartum luteolysis. Within the interplacental samples, expression of GR/NR3C1-mRNA was greater post-implantation than pre-implantation and did not change afterward, i.e. toward mid-gestation. Compartmentalization studies within the Ut/Pl units, involving placenta, endometrium and myometrium separately, performed at the prepartum luteolysis revealed the highest GR/NR3C1-mRNA levels in placenta compared with endometrium and myometrium. Interestingly, in antigestagen-treated mid-pregnancy dogs, Ut/Pl and interplacental GR/NR3C1-mRNA expression remained unaffected. At the cellular level, placental GR/NR3C1 was clearly detectable in placenta fetalis, i.e. in trophoblast cells. In conclusion, increased expression of GR/NR3C1 during normal parturition, but not following antigestagen-treatment, suggest that it is not required for initiating the signaling cascade of PG synthesis leading to the induction of parturition in the dog. © 2016 Society for Reproduction and Fertility.

Notch signaling proteins HES-1 and Hey-1 bind to insulin degrading enzyme (IDE) proximal promoter and repress its transcription and activity: Implications for cellular Aβ metabolism

PubMed Central

Leal, María C.; Surace, Ezequiel I.; Holgado, María P.; Ferrari, Carina C.; Tarelli, Rodolfo; Pitossi, Fernando; Wisniewski, Thomas; Castaño, Eduardo M.; Morelli, Laura

2012-01-01

Cerebral amyloid β (Aβ) accumulation is pathogenically associated with sporadic Alzheimer’s disease (SAD). BACE-1 is involved in Aβ generation while insulin-degrading enzyme (IDE) partakes in Aβ proteolytic clearance. Vulnerable regions in AD brains show increased BACE-1 protein levels and enzymatic activity while the opposite occurs with IDE. Another common feature in SAD brains is Notch1 overexpression. Here we demonstrate an increase in mRNA levels of Hey-1, a Notch target gene, and a decrease of IDE transcripts in the hippocampus of SAD brains as compared to controls. Transient transfection of Notch intracellular domain (NICD) in N2aSW cells, mouse neuroblastoma cells (N2a) stably expressing human amyloid precursor protein (APP) Swedish mutation, reduce IDE mRNA levels, promoting extracellular Aβ accumulation. Also, NICD, HES-1 and Hey-1 overexpression result in decreased IDE proximal promoter activity. This effect was mediated by 2 functional sites located at −379/−372 and −310 −303 from the first translation start site in the −575/−19 (556 bp) fragment of IDE proximal promoter. By site-directed mutagenesis of the IDE promoter region we reverted the inhibitory effect mediated by NICD transfection suggesting that these sites are indeed responsible for the Notch-mediated inhibition of the IDE gene expression. Intracranial injection of the Notch ligand JAG-1 in Tg2576 mice, expressing the Swedish mutation in human APP, induced overexpression of HES-1 and Hey-1 and reduction of IDE mRNA levels, respectively. Our results support our theory that a Notch-dependent IDE transcriptional modulation may impact on Aβ metabolism providing a functional link between Notch signaling and the amyloidogenic pathway in SAD. PMID:22036964

The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion.

PubMed

Hollerer, Ina; Curk, Tomaz; Haase, Bettina; Benes, Vladimir; Hauer, Christian; Neu-Yilik, Gabriele; Bhuvanagiri, Madhuri; Hentze, Matthias W; Kulozik, Andreas E

2016-09-01

Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. © 2016 Hollerer et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Reversible methylation of m6Am in the 5′ cap controls mRNA stability

PubMed Central

Mauer, Jan; Luo, Xiaobing; Blanjoie, Alexandre; Jiao, Xinfu; Grozhik, Anya V.; Patil, Deepak P.; Linder, Bastian; Pickering, Brian F.; Vasseur, Jean-Jacques; Chen, Qiuying; Gross, Steven S.; Elemento, Olivier; Debart, Françoise; Kiledjian, Megerditch; Jaffrey, Samie R.

2017-01-01

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5′ end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2′-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5′ cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability. PMID:28002401

Possible mechanisms for arsenic-induced proliferative diseases

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

Wetterhahn, K.E.; Dudek, E.J.; Shumilla, J.A.

1996-12-31

Possible mechanisms for cardiovascular diseases and cancers which have been observed on chronic exposure to arsenic have been investigated. We tested the hypothesis that nonlethal levels of arsenic are mitogenic, cause oxidative stress, increase nuclear translocation of trans-acting factors, and increase expression of genes involved in proliferation. Cultured porcine vascular (from aorta) endothelial cells were used as a model cell system to study the effects of arsenic on the target cells for cardiovascular diseases. Treatment of postconfluent cell cultures with nonovertly toxic concentrations of arsenite increased DNA synthesis, similar to the mitogenic response observed with hydrogen peroxide. Within 1 hourmore » of adding noncytotoxic concentrations of arsenite, cellular levels of oxidants increased relative to control levels, indicating that arsenite promotes cellular oxidations. Arsenite treatment increased nuclear translocation of NF-{kappa}B, an oxidative stress-responsive transcription factor, in a manner similar to that observed with hydrogen peroxide. Pretreatment of intact cells with the antioxidants N-acetylcysteine and dimethylfumarate prevented the arsenite-induced increases in cellular oxidant formation and NF-KB translocation. Arsenite had little or no effect on binding of NF-KB to its DNA recognition sequence in vitro, indicating that it is unlikely that arsenite directly affects NF-KB. The steady-state mRNA levels of intracellular adhesion molecule and urokinase-like plasminogen activator, genes associated with the active endothelial phenotype in arteriosclerosis and cancer metastasis, were increased by nontoxic concentrations of arsenite. These data suggest that arsenite promotes proliferative diseases like heart disease and cancer by activating oxidant-sensitive endothelial cell signaling and gene expression. It is possible that antioxidant therapy would be useful in preventing arsenic-induced cardiovascular disease and cancer.« less

Regulation of porphyrin synthesis and photodynamic therapy in heavy metal intoxication.

PubMed

Grinblat, Borislava; Pour, Nir; Malik, Zvi

2006-01-01

Protoporphyrin IX (PpIX) synthesis by malignant cells is successfully exploited for photodynamic therapy (PDT) following administration of 5-aminolevulinic acid (ALA) and light irradiation. The influence of two environmental heavy metal poisons, lead and gallium, on PpIX-synthesis and ALA-PDT was studied in two neu-ronal cell lines, SH-SY5Y neuroblastoma and PC12 pheochromocytoma. The heavy metal intoxication affected two of the heme-synthesis enzymes, ALA-dehydratase (ALAD) and porphobilinogen deaminase (PBGD). The present results show that lead poisoning significantly decreased the PBGD cellular level and inhibited its enzymatic activity, whereas the effects of gallium were less prominent. Although, the protein levels were reduced, the mRNA levels of PBGD remained unchanged during metal intoxication. These findings show additional inhibitory activity of lead on top of its classical effect on ALAD. Proteasome activity was enhanced during lead treatment, as measured by the AMC fluorigenic proteasome assay. The reduction in PBGD levels was not a consequence of PBGD mRNA reduced synthesis, which remained unchanged as shown by RT-PCR analysis. As a result of the lead poisoning, marked alterations in the cell cycle were observed, including a decreased G1 phase and an increased number of S phase cells. The efficacy of ALA-PDT was reduced in correlation with decreased activities of the enzymes during lead intoxication. We may conclude that lead poisoning adversely affects the outcome of ALA photodynamic therapy of cancer.

PRMT5 regulates IRES-dependent translation via methylation of hnRNP A1

PubMed Central

Gao, Guozhen; Dhar, Surbhi

2017-01-01

Abstract The type II arginine methyltransferase PRMT5 is responsible for the symmetric dimethylation of histone to generate the H3R8me2s and H4R3me2s marks, which correlate with the repression of transcription. However, the protein level of a number of genes (MEP50, CCND1, MYC, HIF1a, MTIF and CDKN1B) are reported to be downregulated by the loss of PRMT5, while their mRNA levels remain unchanged, which is counterintuitive for PRMT5's proposed role as a transcription repressor. We noticed that the majority of the genes regulated by PRMT5, at the posttranscriptional level, express mRNA containing an internal ribosome entry site (IRES). Using an IRES-dependent reporter system, we established that PRMT5 facilitates the translation of a subset of IRES-containing genes. The heterogeneous nuclear ribonucleoprotein, hnRNP A1, is an IRES transacting factor (ITAF) that regulates the IRES-dependent translation of Cyclin D1 and c-Myc. We showed that hnRNP A1 is methylated by PRMT5 on two residues, R218 and R225, and that this methylation facilitates the interaction of hnRNP A1 with IRES RNA to promote IRES-dependent translation. This study defines a new role for PRMT5 regulation of cellular protein levels, which goes beyond the known functions of PRMT5 as a transcription and splicing regulator. PMID:28115626

Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma.

PubMed

Suzuki, Shigeki; Hoshino, Hiroaki; Yoshida, Kazuma; Nakanishi, Jun; Tsuchiya-Hirata, Shizu; Kobuke, Seiji; Haruyama, Naoto; Nishimura, Fusanori; Shiba, Hideki

2018-01-15

Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC. Copyright © 2017 Elsevier Inc. All rights reserved.

Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

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

Chung, Eric; Jakinovich, Paul; Bae, Aekyung

Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1}more » knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is a suppressor of PKC activity.« less

Impact of ionizing radiation exposure on in vitro differentiation of preosteoblastic cell lines

NASA Astrophysics Data System (ADS)

Hu, Yueyuan; Lau, Patrick; Hellweg, Christine; Baumstark-Khan, Christa; Reitz, Guenther

Bone demineralization of astronauts during residence in microgravity is a well known phe-nomenon during space travel. Besides altered gravity conditions, radiation risk is considered to be one of the major health hazards for astronauts in both orbital and interplanetary space. Un-til know, little is known about the effects of space radiation on the skeletal system especially on the bone forming osteoblasts. Accelerator facilities are used to simulate parts of the radiation environment in space. We examined the effects of heavy ion exposure on osteoblastic differ-entiation of murine preosteoblastic cell lines to gain insight into potential cellular mechanisms involved in bone cellular response after exposure to heavy ions. Therefore, we examined gene expression modulation of bone specific transcription factors, osteoblast specific marker genes as well as genes function as coupling factors that link bone resorption to bone formation. mRNA levels were determined using quantitative real time reverse transcriptase PCR (qRT-PCR). Expression of a target gene was standardized to unregulated reference genes. We investigated the transcriptional regulation of Osteocalcin (OCN) as well as TGF-β1, p21(CDKN1A) and the bone specific transcription factor Runx2 (cbfa1). We investigated gene expression modula-tions after exposure to energetic carbon ions (35 MeV/u, 73 keV/µm), iron ions (1000 MeV/u, 150 keV/µm) and lead ions (29 MeV/u, 9600 keV/µm) versus low LET X-rays. X-irradiation dose-dependently increased the mRNA levels of p21(CDKN1A) and Runx2 (cbfa1) whereas expression of OCN and TGF-β1 were elevated at later time points. Exposure to heavy ions provoked a more pronounced effect on osteoblastic specific gene expression within the dif-ferentiation process. Collectively, our results indicate that heavy ions facilitate osteoblastic differentiation more effectively than X-ray. Using the proposed in vitro model we confirmed that exposure to ionizing radiation significantly modulates gene expression levels of marker genes involved in the differentiation of osteoblasts. The data presented allow us to suggest that exposure to ionizing radiation interferes with bone formation at the level of cell differentiation.

Mono-allelic expression of variegating transgene locus in the mouse.

PubMed

Opsahl, Margaret L; Springbett, Anthea; Lathe, Richard; Colman, Alan; McClenaghan, Margaret; Whitelaw, C Bruce A

2003-12-01

We have generated transgenic mice which express an ovine beta-lactoglobulin transgene during lactation. In two transgenic lines, BLG/7 and BLG/45, beta-lactoglobulin protein levels vary between siblings, reflected at the cellular level by a mosaic transgene expression pattern in the mammary tissue that is reminiscent of position effect variegation. To investigate whether this variegating expression profile can be affected by the introduction of an identical variegating locus on the homologous chromosome, we compared the beta-lactoglobulin expression profiles in mice hemizygous or homozygous for the transgene locus. In BLG/45 mice, milk protein analysis revealed that transgene expression was effectively doubled in homozygous compared to hemizygous mice. In contrast, beta-lactoglobulin protein in hemizygous and homozygous BLG/7 mice displayed a similar range; although minimum expression levels were doubled in the homozygous population, the maximum level of expression was indistinguishable between the two populations. Fluorescent in situ hybridisation (FISH) for transgene mRNA indicated that for a given protein level, the extent of cellular expression is similar in both BLG/7 populations. In homozygous mice genomic DNA and nuclear RNA FISH demonstrated that only one of the two BLG/7 loci is active in expressing cells, while two transcription foci were present in BLG/45 homozygous mice. This mono-allelic transgene expression pattern is not inherited through the germline, as hemizygous mice bred from homozygous parents expressed at the expected hemizygous population level. We discuss these observations in the context of known epigenetic events such as imprinting and trans-inactivation.