Altered MENIN expression disrupts the MAFA differentiation pathway in insulinoma.

Science.gov (United States)

Hamze, Z; Vercherat, C; Bernigaud-Lacheretz, A; Bazzi, W; Bonnavion, R; Lu, J; Calender, A; Pouponnot, C; Bertolino, P; Roche, C; Stein, R; Scoazec, J Y; Zhang, C X; Cordier-Bussat, M

2013-12-01

The protein MENIN is the product of the multiple endocrine neoplasia type I (MEN1) gene. Altered MENIN expression is one of the few events that are clearly associated with foregut neuroendocrine tumours (NETs), classical oncogenes or tumour suppressors being not involved. One of the current challenges is to understand how alteration of MENIN expression contributes to the development of these tumours. We hypothesised that MENIN might regulate factors maintaining endocrine-differentiated functions. We chose the insulinoma model, a paradigmatic example of well-differentiated pancreatic NETs, to study whether MENIN interferes with the expression of v-MAF musculoaponeurotic fibrosarcoma oncogene homologue A (MAFA), a master glucose-dependent transcription factor in differentiated β-cells. Immunohistochemical analysis of a series of human insulinomas revealed a correlated decrease in both MENIN and MAFA. Decreased MAFA expression resulting from targeted Men1 ablation was also consistently observed in mouse insulinomas. In vitro analyses using insulinoma cell lines showed that MENIN regulated MAFA protein and mRNA levels, and bound to Mafa promoter sequences. MENIN knockdown concomitantly decreased mRNA expression of both Mafa and β-cell differentiation markers (Ins1/2, Gck, Slc2a2 and Pdx1) and, in parallel, increased the proliferation rate of tumours as measured by bromodeoxyuridine incorporation. Interestingly, MAFA knockdown alone also increased proliferation rate but did not affect the expression of candidate proliferation genes regulated by MENIN. Finally, MENIN variants with missense mutations detected in patients with MEN1 lost the WT MENIN properties to regulate MAFA. Together, our findings unveil a previously unsuspected MENIN/MAFA connection regarding control of the β-cell differentiation/proliferation balance, which could contribute to tumorigenesis.

Identification of possible targets of the Aspergillus fumigatus CRZ1 homologue, CrzA

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Goldman Gustavo H

2010-01-01

Full Text Available Abstract Background Calcineurin, a serine/threonine-specific protein phosphatase, plays an important role in the control of cell morphology and virulence in fungi. Calcineurin regulates localization and activity of a transcription factor called CRZ1. Recently, we characterize Aspergillus fumigatus CRZ1 homologue, AfCrzA. Here, we investigate which pathways are influenced by A. fumigatus AfCrzA during a short pulse of calcium by comparatively determining the transcriptional profile of A. fumigatus wild type and ΔAfcrzA mutant strains. Results We were able to observe 3,622 genes modulated in at least one timepoint in the mutant when compared to the wild type strain (3,211 and 411 at 10 and 30 minutes, respectively. Decreased mRNA abundance in the ΔcrzA was seen for genes encoding calcium transporters, transcription factors and genes that could be directly or indirectly involved in calcium metabolism. Increased mRNA accumulation was observed for some genes encoding proteins involved in stress response. AfCrzA overexpression in A. fumigatus increases the expression of several of these genes. The deleted strain of one of these genes, AfRcnA, belonging to a class of endogenous calcineurin regulators, calcipressins, had more calcineurin activity after exposure to calcium and was less sensitive to menadione 30 μM, hydrogen peroxide 2.5 mM, EGTA 25 mM, and MnCl2 25 mM. We constructed deletion, overexpression, and GFP fusion protein for the closely related A. nidulans AnRcnA. GFP::RcnA was mostly detected along the germling, did not accumulate in the nuclei and its location is not affected by the cellular response to calcium chloride. Conclusion We have performed a transcriptional profiling analysis of the A. fumigatus ΔAfcrzA mutant strain exposed to calcium stress. This provided an excellent opportunity to identify genes and pathways that are under the influence of AfCrzA. AfRcnA, one of these selected genes, encodes a modulator of calcineurin

Human Monocytes Accelerate Proliferation and Blunt Differentiation of Preadipocytes in Association With Suppression of C/Ebpα mRNA

Science.gov (United States)

Couturier, Jacob; Patel, Sanjeet G.; Iyer, Dinakar; Balasubramanyam, Ashok; Lewis, Dorothy E.

2015-01-01

Obesity, type 2 diabetes, and HIV-associated lipodystrophy are associated with abnormalities in adipocyte growth and differentiation. In persons with these conditions, adipose depots contain increased numbers of macrophages, but the origins of these cells and their specific effects are uncertain. Peripheral blood mononuclear cells (PBMC)-derived monocytes, but not T cells, cocultured via transwells with primary subcutaneous preadipocytes, increased proliferation (approximately twofold) and reduced differentiation (~50%) of preadipocytes. Gene expression analyses in proliferating preadipocytes (i.e., prior to hormonal induction of terminal differentiation) revealed that monocytes down-regulated mRNA levels of CCAAT/enhancer binding protein, alpha (C/EBPα) and up-regulated mRNA levels of G0/G1 switch 2 (G0S2) message, genes important for the regulation of adipogenesis and the cell cycle. These data indicate that circulating peripheral blood monocytes can disrupt adipogenesis by interfering with a critical step in C/EBPα and G0S2 transcription required for preadipocytes to make the transition from proliferation to differentiation. Interactions between preadipocytes and monocytes also increased the inflammatory cytokines IL-6 and IL-8, as well as a novel chemotactic cytokine, CXCL1. Additionally, the levels of both IL-6 and CXCL1 were highest when preadipocytes and monocytes were cultured together, compared to each cell in culture alone. Such cross-talk amplifies the production of mediators of tissue inflammation. PMID:21869759

Homologue Pairing in Flies and Mammals: Gene Regulation When Two Are Involved

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Manasi S. Apte

2012-01-01

Full Text Available Chromosome pairing is usually discussed in the context of meiosis. Association of homologues in germ cells enables chromosome segregation and is necessary for fertility. A few organisms, such as flies, also pair their entire genomes in somatic cells. Most others, including mammals, display little homologue pairing outside of the germline. Experimental evidence from both flies and mammals suggests that communication between homologues contributes to normal genome regulation. This paper will contrast the role of pairing in transmitting information between homologues in flies and mammals. In mammals, somatic homologue pairing is tightly regulated, occurring at specific loci and in a developmentally regulated fashion. Inappropriate pairing, or loss of normal pairing, is associated with gene misregulation in some disease states. While homologue pairing in flies is capable of influencing gene expression, the significance of this for normal expression remains unknown. The sex chromosomes pose a particularly interesting situation, as females are able to pair X chromosomes, but males cannot. The contribution of homologue pairing to the biology of the X chromosome will also be discussed.

Deep mRNA sequencing of the Tritonia diomedea brain transcriptome provides access to gene homologues for neuronal excitability, synaptic transmission and peptidergic signalling.

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Adriano Senatore

Full Text Available The sea slug Tritonia diomedea (Mollusca, Gastropoda, Nudibranchia, has a simple and highly accessible nervous system, making it useful for studying neuronal and synaptic mechanisms underlying behavior. Although many important contributions have been made using Tritonia, until now, a lack of genetic information has impeded exploration at the molecular level.We performed Illumina sequencing of central nervous system mRNAs from Tritonia, generating 133.1 million 100 base pair, paired-end reads. De novo reconstruction of the RNA-Seq data yielded a total of 185,546 contigs, which partitioned into 123,154 non-redundant gene clusters (unigenes. BLAST comparison with RefSeq and Swiss-Prot protein databases, as well as mRNA data from other invertebrates (gastropod molluscs: Aplysia californica, Lymnaea stagnalis and Biomphalaria glabrata; cnidarian: Nematostella vectensis revealed that up to 76,292 unigenes in the Tritonia transcriptome have putative homologues in other databases, 18,246 of which are below a more stringent E-value cut-off of 1x10-6. In silico prediction of secreted proteins from the Tritonia transcriptome shotgun assembly (TSA produced a database of 579 unique sequences of secreted proteins, which also exhibited markedly higher expression levels compared to other genes in the TSA.Our efforts greatly expand the availability of gene sequences available for Tritonia diomedea. We were able to extract full length protein sequences for most queried genes, including those involved in electrical excitability, synaptic vesicle release and neurotransmission, thus confirming that the transcriptome will serve as a useful tool for probing the molecular correlates of behavior in this species. We also generated a neurosecretome database that will serve as a useful tool for probing peptidergic signalling systems in the Tritonia brain.

Identification of Powdery Mildew Responsive Genes in Hevea brasiliensis through mRNA Differential Display

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Li, Xiang; Bi, Zhenghong; Di, Rong; Liang, Peng; He, Qiguang; Liu, Wenbo; Miao, Weiguo; Zheng, Fucong

2016-01-01

Powdery mildew is an important disease of rubber trees caused by Oidium heveae B. A. Steinmann. As far as we know, none of the resistance genes related to powdery mildew have been isolated from the rubber tree. There is little information available at the molecular level regarding how a rubber tree develops defense mechanisms against this pathogen. We have studied rubber tree mRNA transcripts from the resistant RRIC52 cultivar by differential display analysis. Leaves inoculated with the spores of O. heveae were collected from 0 to 120 hpi in order to identify pathogen-regulated genes at different infection stages. We identified 78 rubber tree genes that were differentially expressed during the plant–pathogen interaction. BLAST analysis for these 78 ESTs classified them into seven functional groups: cell wall and membrane pathways, transcription factor and regulatory proteins, transporters, signal transduction, phytoalexin biosynthesis, other metabolism functions, and unknown functions. The gene expression for eight of these genes was validated by qRT-PCR in both RRIC52 and the partially susceptible Reyan 7-33-97 cultivars, revealing the similar or differential changes of gene expressions between these two cultivars. This study has improved our overall understanding of the molecular mechanisms of rubber tree resistance to powdery mildew. PMID:26840302

[Differential expression of IGF-I and its mRNA in mandibular condylar cartilage of rat--direct evidence for servosystem theory of facial growth].

Science.gov (United States)

Zhou, Z; Luo, S

1998-05-01

It was studied the expression of IGF-I and its mRNA in the condylar cartilage of 10 7-week-old SD male rats by using in situ hybridization and immunohisto-chemistry technique. The results showed both IGF-I and its gene expressed in growing rat condyle. IGF-I peptide was abundant in germinal zone, and positive reaction of its mRNA was strongest in transitional and maturational zones. These indicate that condylar cartilage has the capability of local production and secretion of IGF-I, mediating the command effect of STH, and differential expression of IGF-I and its mRNA might establish the local feedback loop, which supply a direct evidence for servosystem theory of facial growth.

Differential trypanosome surface coat regulation by a CCCH protein that co-associates with procyclin mRNA cis-elements.

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Pegine Walrad

2009-02-01

Full Text Available The genome of Trypanosoma brucei is unusual in being regulated almost entirely at the post-transcriptional level. In terms of regulation, the best-studied genes are procyclins, which encode a family of major surface GPI-anchored glycoproteins (EP1, EP2, EP3, GPEET that show differential expression in the parasite's tsetse-fly vector. Although procyclin mRNA cis-regulatory sequences have provided the paradigm for post-transcriptional control in kinetoplastid parasites, trans-acting regulators of procyclin mRNAs are unidentified, despite intensive effort over 15 years. Here we identify the developmental regulator, TbZFP3, a CCCH-class predicted RNA binding protein, as an isoform-specific regulator of Procyclin surface coat expression in trypanosomes. We demonstrate (i that endogenous TbZFP3 shows sequence-specific co-precipitation of EP1 and GPEET, but not EP2 and EP3, procyclin mRNA isoforms, (ii that ectopic overexpression of TbZFP3 does not perturb the mRNA abundance of procyclin transcripts, but rather that (iii their protein expression is regulated in an isoform-specific manner, as evidenced by mass spectrometric analysis of the Procyclin expression signature in the transgenic cell lines. The TbZFP3 mRNA-protein complex (TbZFP3mRNP is identified as a trans-regulator of differential surface protein expression in trypanosomes. Moreover, its sequence-specific interactions with procyclin mRNAs are compatible with long-established predictions for Procyclin regulation. Combined with the known association of TbZFP3 with the translational apparatus, this study provides a long-sought missing link between surface protein cis-regulatory signals and the gene expression machinery in trypanosomes.

Differential regulation of renal cyclooxygenase mRNA by dietary salt intake

DEFF Research Database (Denmark)

Jensen, B L; Kurtz, A

1997-01-01

RNA correlated directly with salt intake. We conclude that dietary salt intake influences renal cyclooxygenase mRNAs zone-specifically with opposite responses between cortex and medulla. Cortical COX II-mediated prostaglandin formation is probably important in low salt states whereas medullary COX I......Experiments were done to investigate the influence of dietary salt intake on renal cyclooxygenase (COX) I and II mRNA levels. To this end rats were fed either a low NaCl diet (LS; 0.02% NaCl wt/wt) or a high NaCl diet (HS diet; 4% NaCl wt/wt) for 5, 10 and 20 days. After 10 days Na excretion...... differed 760-fold, plasma renin activity and renin mRNA were increased eight- and threefold in LS compared to HS animals. Total renal COX I mRNA decreased 50% following the LS diet and did not change after the HS diet. Conversely, COX II mRNA declined after HS intake and transiently increased after salt...

Homologue Pairing in Flies and Mammals: Gene Regulation When Two Are Involved

OpenAIRE

Apte, Manasi S.; Meller, Victoria H.

2011-01-01

Chromosome pairing is usually discussed in the context of meiosis. Association of homologues in germ cells enables chromosome segregation and is necessary for fertility. A few organisms, such as flies, also pair their entire genomes in somatic cells. Most others, including mammals, display little homologue pairing outside of the germline. Experimental evidence from both flies and mammals suggests that communication between homologues contributes to normal genome regulation. This paper will co...

Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation.

Science.gov (United States)

Tao, Wenjing; Sun, Lina; Shi, Hongjuan; Cheng, Yunying; Jiang, Dongneng; Fu, Beide; Conte, Matthew A; Gammerdinger, William J; Kocher, Thomas D; Wang, Deshou

2016-05-04

MicroRNAs (miRNAs) represent a second regulatory network that has important effects on gene expression and protein translation during biological process. However, the possible role of miRNAs in the early stages of fish sex differentiation is not well understood. In this study, we carried an integrated analysis of miRNA and mRNA expression profiles to explore their possibly regulatory patterns at the critical stage of sex differentiation in tilapia. We identified 279 pre-miRNA genes in tilapia genome, which were highly conserved in other fish species. Based on small RNA library sequencing, we identified 635 mature miRNAs in tilapia gonads, in which 62 and 49 miRNAs showed higher expression in XX and XY gonads, respectively. The predicted targets of these sex-biased miRNAs (e.g., miR-9, miR-21, miR-30a, miR-96, miR-200b, miR-212 and miR-7977) included genes encoding key enzymes in steroidogenic pathways (Cyp11a1, Hsd3b, Cyp19a1a, Hsd11b) and key molecules involved in vertebrate sex differentiation (Foxl2, Amh, Star1, Sf1, Dmrt1, and Gsdf). These genes also showed sex-biased expression in tilapia gonads at 5 dah. Some miRNAs (e.g., miR-96 and miR-737) targeted multiple genes involved in steroid synthesis, suggesting a complex miRNA regulatory network during early sex differentiation in this fish. The sequence and expression patterns of most miRNAs in tilapia are conserved in fishes, indicating the basic functions of vertebrate miRNAs might share a common evolutionary origin. This comprehensive analysis of miRNA and mRNA at the early stage of molecular sex differentiation in tilapia XX and XY gonads lead to the discovery of differentially expressed miRNAs and their putative targets, which will facilitate studies of the regulatory network of molecular sex determination and differentiation in fishes.

Identification of NoxD/Pro41 as the homologue of the p22phox NADPH oxidase subunit in fungi.

Science.gov (United States)

Lacaze, Isabelle; Lalucque, Hervé; Siegmund, Ulrike; Silar, Philippe; Brun, Sylvain

2015-03-01

NADPH oxidases (Nox) are membrane complexes that produce O2(-). Researches in mammals, plants and fungi highlight the involvement of Nox-generated ROS in cell proliferation, differentiation and defense. In mammals, the core enzyme gp91(phox)/Nox2 is associated with p22(phox) forming the flavocytochrome b558 ready for activation by a cytosolic complex. Intriguingly, no homologue of the p22(phox) gene has been found in fungal genomes, questioning how the flavoenzyme forms. Using whole genome sequencing combined with phylogenetic analysis and structural studies, we identify the fungal p22(phox) homologue as being mutated in the Podospora anserina mutant IDC(509). Functional studies show that the fungal p22(phox), PaNoxD, acts along PaNox1, but not PaNox2, a second fungal gp91(phox) homologue. Finally, cytological analysis of functional tagged versions of PaNox1, PaNoxD and PaNoxR shows clear co-localization of PaNoxD and PaNox1 and unravel a dynamic assembly of the complex in the endoplasmic reticulum and in the vacuolar system. © 2014 John Wiley & Sons Ltd.

Identification of a candidate CD5 homologue in the amphibian Xenopus laevis.

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Jürgens, J B; Gartland, L A; Du Pasquier, L; Horton, J D; Göbel, T W; Cooper, M D

1995-11-01

We identified a novel T cell Ag in the South African clawed toad (Xenopus laevis) by a mAb designated 2B1. This Ag is present in relatively high levels on most thymocytes, approximately 65% of splenocytes, 55% of PBL, and 65% of intestinal lymphocytes, but is rarely seen on IgM+ B cells in any of these tissues. Lymphocytes bearing the 2B1 Ag proliferate in response to stimulation with Con A or PHA, whereas the 2B1- lymphocytes are reactive to LPS. Biochemical analysis indicates that this Ag is a differentially phosphorylated glycoprotein of 71 to 82 kDa. The protein core of 64 kDa bears both N- and O-linked carbohydrate side chains. The amino-terminal protein sequence of the 2B1 Ag shares significant homology with both the macrophage scavenger receptor type 1 motif and the mammalian CD5/CD6 family. The biochemical characteristics and cellular distribution of the 2B1 Ag suggest that it represents the CD5 homologue in X. laevis. While T cells constitutively express this highly conserved molecule, Xenopus B cells acquire the CD5 homologue only when they are stimulated in the presence of T cells.

A Potato cDNA Encoding a Homologue of Mammalian Multidrug Resistant P-Glycoprotein

Science.gov (United States)

Wang, W.; Takezawa, D.; Poovaiah, B. W.

1996-01-01

A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with S-15-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.

Characterization of Translationally Controlled Tumour Protein from the Sea Anemone Anemonia viridis and Transcriptome Wide Identification of Cnidarian Homologues.

Science.gov (United States)

Nicosia, Aldo; Bennici, Carmelo; Biondo, Girolama; Costa, Salvatore; Di Natale, Marilena; Masullo, Tiziana; Monastero, Calogera; Ragusa, Maria Antonietta; Tagliavia, Marcello; Cuttitta, Angela

2018-01-11

Gene family encoding translationally controlled tumour protein (TCTP) is defined as highly conserved among organisms; however, there is limited knowledge of non-bilateria. In this study, the first TCTP homologue from anthozoan was characterised in the Mediterranean Sea anemone, Anemonia viridis . The release of the genome sequence of Acropora digitifera , Exaiptasia pallida , Nematostella vectensis and Hydra vulgaris enabled a comprehensive study of the molecular evolution of TCTP family among cnidarians. A comparison among TCTP members from Cnidaria and Bilateria showed conserved intron exon organization, evolutionary conserved TCTP signatures and 3D protein structure. The pattern of mRNA expression profile was also defined in A. viridis . These analyses revealed a constitutive mRNA expression especially in tissues with active proliferation. Additionally, the transcriptional profile of A. viridis TCTP ( AvTCTP ) after challenges with different abiotic/biotic stresses showed induction by extreme temperatures, heavy metals exposure and immune stimulation. These results suggest the involvement of AvTCTP in the sea anemone defensome taking part in environmental stress and immune responses.

Characterization of Translationally Controlled Tumour Protein from the Sea Anemone Anemonia viridis and Transcriptome Wide Identification of Cnidarian Homologues

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Aldo Nicosia

2018-01-01

Full Text Available Gene family encoding translationally controlled tumour protein (TCTP is defined as highly conserved among organisms; however, there is limited knowledge of non-bilateria. In this study, the first TCTP homologue from anthozoan was characterised in the Mediterranean Sea anemone, Anemonia viridis. The release of the genome sequence of Acropora digitifera, Exaiptasia pallida, Nematostella vectensis and Hydra vulgaris enabled a comprehensive study of the molecular evolution of TCTP family among cnidarians. A comparison among TCTP members from Cnidaria and Bilateria showed conserved intron exon organization, evolutionary conserved TCTP signatures and 3D protein structure. The pattern of mRNA expression profile was also defined in A. viridis. These analyses revealed a constitutive mRNA expression especially in tissues with active proliferation. Additionally, the transcriptional profile of A. viridis TCTP (AvTCTP after challenges with different abiotic/biotic stresses showed induction by extreme temperatures, heavy metals exposure and immune stimulation. These results suggest the involvement of AvTCTP in the sea anemone defensome taking part in environmental stress and immune responses.

Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.

Science.gov (United States)

Warren, Luigi; Manos, Philip D; Ahfeldt, Tim; Loh, Yuin-Han; Li, Hu; Lau, Frank; Ebina, Wataru; Mandal, Pankaj K; Smith, Zachary D; Meissner, Alexander; Daley, George Q; Brack, Andrew S; Collins, James J; Cowan, Chad; Schlaeger, Thorsten M; Rossi, Derrick J

2010-11-05

Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine. Copyright © 2010 Elsevier Inc. All rights reserved.

Identification of aldehyde oxidase 1 and aldehyde oxidase homologue 1 as dioxin-inducible genes

International Nuclear Information System (INIS)

Rivera, Steven P.; Choi, Hyun Ho; Chapman, Brett; Whitekus, Michael J.; Terao, Mineko; Garattini, Enrico; Hankinson, Oliver

2005-01-01

Aldehyde oxidases are a family of highly related molybdo-flavoenzymes acting upon a variety of compounds of industrial and medical importance. We have identified aldehyde oxidase 1 (AOX1) as a 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) inducible gene in the mouse hepatoma cell line Hepa-1. AOX1 mRNA levels were not increased by dioxin in mutant derivatives of the Hepa-1 cell line lacking either functional aryl hydrocarbon receptor (AHR) or aryl hydrocarbon receptor nuclear translocator (ARNT) proteins, thus demonstrating that transcriptional induction of AOX1 in response to dioxin occurs through the AHR pathway. Dioxin induction of AOX1 mRNA was also observed in mouse liver. In addition, levels of AOX1 protein as well as those of aldehyde oxidase homologue 1 (AOH1), a recently identified homolog of AOX1, were elevated in mouse liver in response to dioxin. Employing an aldehyde oxidase specific substrate, AOX1/AOH1 activity was shown to be induced by dioxin in mouse liver. This activity was inhibited by a known inhibitor of aldehyde oxidases, and eliminated by including tungstate in the mouse diet, which is known to lead to inactivation of molybdoflavoenzymes, thus confirming that the enzymatic activity was attributable to AOX1/AOH1. Our observations thus identify two additional xenobiotic metabolizing enzymes induced by dioxin

Nonparametric testing for DNA copy number induced differential mRNA gene expression

NARCIS (Netherlands)

van Wieringen, W.N.; van de Wiel, M.A.

2009-01-01

The central dogma of molecular biology relates DNA with mRNA. Array CGH measures DNA copy number and gene expression microarrays measure the amount of mRNA. Methods that integrate data from these two platforms may uncover meaningful biological relationships that further our understanding of cancer.

Isolation and characterization of differentially expressed genes in ...

African Journals Online (AJOL)

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Through reverse transcriptase-polymerase chain reaction analysis, priA homologue and AP-1 like transcription factor ... The oyster mushroom, Pleurotus ostreatus, and white button mushroom ..... differential display of RAPD. FEMS Microbiol.

Cloning of Interleukin-10 from African Clawed Frog (Xenopus tropicalis, with the Finding of IL-19/20 Homologue in the IL-10 Locus

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Zhitao Qi

2015-01-01

Full Text Available Interleukin-10 (IL-10 is a pleiotropic cytokine that plays an important role in immune system. In the present study, the IL-10 gene of African clawed frog (Xenopus tropicalis was first cloned, and its expression pattern and 3D structure were also analyzed. The frog IL-10 mRNA encoded 172 amino acids which possessed several conserved features found in IL-10s from other species, including five-exon/four-intron genomic structure, conserved four cysteine residues, IL-10 family motif, and six α-helices. Real-time PCR showed that frog IL-10 mRNA was ubiquitous expressed in all examined tissues, highly in some immune related tissues including kidney, spleen, and intestine and lowly in heart, stomach, and liver. The frog IL-10 mRNA was upregulated at 24 h after LPS stimulation, indicating that it plays a part in the host immune response to bacterial infection. Another IL, termed as IL-20, was identified from the frog IL-10 locus, which might be the homologue of mammalian IL-19/20 according to the analysis results of the phylogenetic tree and the sequence identities.

Increased mRNA Levels of Sphingosine Kinases and S1P Lyase and Reduced Levels of S1P Were Observed in Hepatocellular Carcinoma in Association with Poorer Differentiation and Earlier Recurrence.

Science.gov (United States)

Uranbileg, Baasanjav; Ikeda, Hitoshi; Kurano, Makoto; Enooku, Kenichiro; Sato, Masaya; Saigusa, Daisuke; Aoki, Junken; Ishizawa, Takeaki; Hasegawa, Kiyoshi; Kokudo, Norihiro; Yatomi, Yutaka

2016-01-01

Although sphingosine 1-phosphate (S1P) has been reported to play an important role in cancer pathophysiology, little is known about S1P and hepatocellular carcinoma (HCC). To clarify the relationship between S1P and HCC, 77 patients with HCC who underwent surgical treatment were consecutively enrolled in this study. In addition, S1P and its metabolites were quantitated by LC-MS/MS. The mRNA levels of sphingosine kinases (SKs), which phosphorylate sphingosine to generate S1P, were increased in HCC tissues compared with adjacent non-HCC tissues. Higher mRNA levels of SKs in HCC were associated with poorer differentiation and microvascular invasion, whereas a higher level of SK2 mRNA was a risk factor for intra- and extra-hepatic recurrence. S1P levels, however, were unexpectedly reduced in HCC compared with non-HCC tissues, and increased mRNA levels of S1P lyase (SPL), which degrades S1P, were observed in HCC compared with non-HCC tissues. Higher SPL mRNA levels in HCC were associated with poorer differentiation. Finally, in HCC cell lines, inhibition of the expression of SKs or SPL by siRNA led to reduced proliferation, invasion and migration, whereas overexpression of SKs or SPL enhanced proliferation. In conclusion, increased SK and SPL mRNA expression along with reduced S1P levels were more commonly observed in HCC tissues compared with adjacent non-HCC tissues and were associated with poor differentiation and early recurrence. SPL as well as SKs may be therapeutic targets for HCC treatment.

Monolayer structures of alkyl aldehydes: Odd-membered homologues

International Nuclear Information System (INIS)

Phillips, T.K.; Clarke, S.M.; Bhinde, T.; Castro, M.A.; Millan, C.; Medina, S.

2011-01-01

Crystalline monolayers of three aldehydes with an odd number of carbon atoms in the alkyl chain (C 7 , C 9 and C 11 ) at low coverages are observed by a combination of X-ray and neutron diffraction. Analysis of the diffraction data is discussed and possible monolayer crystal structures are proposed; although unique structures could not be ascertained for all molecules. We conclude that the structures are flat on the surface, with the molecules lying in the plane of the layer. The C 11 homologue is determined to have a plane group of either p2, pgb or pgg, and for the C 7 homologue the p2 plane group is preferred.

Targeting the Enhancer of Zeste Homologue 2 in Medulloblastoma

Science.gov (United States)

Alimova, Irina; Venkataraman, Sujatha; Harris, Peter; Marquez, Victor E.; Northcott, Paul A; Dubuc, Adrian; Taylor, Michael D; Foreman, Nicholas K; Vibhakar, Rajeev

2012-01-01

Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 that catalyzes the trimethylation of histone H3 on Lys 27, and represses gene transcription. EZH2 enhances cancer-cell proliferation and regulates stem cell maintenance and differentiation. Here, we demonstrate that EZH2 is highly expressed in medulloblastoma, a highly malignant brain tumor of childhood, and this altered expression is correlated with genomic gain of chromosome 7 in a subset of medulloblastoma. Inhibition of EZH2 by RNAi suppresses medulloblastoma tumor cell growth. We show that 3-deazaneplanocin A, a chemical inhibitor of EZH2, can suppress medulloblastoma cell growth partially by inducing apoptosis. Suppression of EZH2 expression diminishes the ability of tumor cells to form spheres in culture and strongly represses the ability of known oncogenes to transform neural stem cells. These findings establish a role of EZH2 in medulloblastoma and identify EZH2 as a potential therapeutic target especially in high-risk tumors. PMID:22287205

Differential gene expression in liver and small intestine from lactating rats compared to age-matched virgin controls detects increased mRNA of cholesterol biosynthetic genes

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Jungsuwadee Paiboon

2011-02-01

Full Text Available Abstract Background Lactation increases energy demands four- to five-fold, leading to a two- to three-fold increase in food consumption, requiring a proportional adjustment in the ability of the lactating dam to absorb nutrients and to synthesize critical biomolecules, such as cholesterol, to meet the dietary needs of both the offspring and the dam. The size and hydrophobicity of the bile acid pool increases during lactation, implying an increased absorption and disposition of lipids, sterols, nutrients, and xenobiotics. In order to investigate changes at the transcriptomics level, we utilized an exon array and calculated expression levels to investigate changes in gene expression in the liver, duodenum, jejunum, and ileum of lactating dams when compared against age-matched virgin controls. Results A two-way mixed models ANOVA was applied to detect differentially expressed genes. Significance calls were defined as a p Cyp7a1, which catalyzes the rate limiting step in the bile acid biosynthetic pathway, was also significantly increased in liver. In addition, decreased levels of mRNA associated with T-cell signaling were found in the jejunum and ileum. Several members of the Solute Carrier (SLC and Adenosine Triphosphate Binding Cassette (ABC superfamilies of membrane transporters were found to be differentially expressed; these genes may play a role in differences in nutrient and xenobiotic absorption and disposition. mRNA expression of SLC39a4_predicted, a zinc transporter, was increased in all tissues, suggesting that it is involved in increased zinc uptake during lactation. Microarray data are available through GEO under GSE19175. Conclusions We detected differential expression of mRNA from several pathways in lactating dams, including upregulation of the cholesterol biosynthetic pathway in liver and intestine, consistent with Srebp activation. Differential T-Cell signaling in the two most distal regions of the small intestine (ileum and

Differential regulation of proopiomelanocortin (POMC mRNA expression in hypothalamus and anterior pituitary following repeated cyanamide with ethanol administration

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Kinoshita Hiroshi

2005-01-01

Full Text Available Background/Aim. We have investigated proopiomelanocortin (POMC mRNA expression in the arcuate nucleus of the hypothalamus (ARC and the anterior lobe of the pituitary (AL following repeated cyanamide-ethanol reaction (CER. Methods. Adult male Sprague -Dawley rats (250 −290 gr were housed in a temperature and humidity controlled environment with free access to food and water. Four experimental groups were used as follows: saline (as control, cyanamide alone, ethanol alone and ethanol with cyanamide. The animals received daily intraperitoneal injections (i.p. of cyanamide (10mg/kg, 60 min before ethanol dosing with or without ethanol (1g/kg for 5 consecutive days, and were sacrificed 60 min after the last dosing of ethanol. The results were presented as the mean ± SEM for each group. All groups within each data set were compared by one-way ANOVA followed by Fisher PLSD test for multiple comparisons. A value of p<0.05 was considered significant. Results. The POMC mRNA levels in ARC were significantly decreased with cyanamide compared to the control and ethanol alone (p<0.05 and p<0.05 respectively, but increased in AL following repeated CER. Conclusion. We speculate that this differential regulation of POMC mRNA expression may be partially involved in the preventive effects on alcohol intake in response to CER.

mRNA localization mechanisms in Trypanosoma cruzi.

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Lysangela R Alves

Full Text Available Asymmetric mRNA localization is a sophisticated tool for regulating and optimizing protein synthesis and maintaining cell polarity. Molecular mechanisms involved in the regulated localization of transcripts are widespread in higher eukaryotes and fungi, but not in protozoa. Trypanosomes are ancient eukaryotes that branched off early in eukaryote evolution. We hypothesized that these organisms would have basic mechanisms of mRNA localization. FISH assays with probes against transcripts coding for proteins with restricted distributions showed a discrete localization of the mRNAs in the cytoplasm. Moreover, cruzipain mRNA was found inside reservosomes suggesting new unexpected functions for this vacuolar organelle. Individual mRNAs were also mobilized to RNA granules in response to nutritional stress. The cytoplasmic distribution of these transcripts changed with cell differentiation, suggesting that localization mechanisms might be involved in the regulation of stage-specific protein expression. Transfection assays with reporter genes showed that, as in higher eukaryotes, 3'UTRs were responsible for guiding mRNAs to their final location. Our results strongly suggest that Trypanosoma cruzi have a core, basic mechanism of mRNA localization. This kind of controlled mRNA transport is ancient, dating back to early eukaryote evolution.

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

Science.gov (United States)

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

The Mycobacterium tuberculosis homologue of the Mycobacterium ...

African Journals Online (AJOL)

With the completion of genome sequencing of Mycobacterium tuberculosis and upsurge in the incidence of M. tuberculosis infection worldwide partly as a result of HIV pandemic, there is need for rationale approach to vaccine and chemotherapy discoveries for M. tuberculosis. The homologue of mig gene of. Mycobacterium ...

Implication of C-type natriuretic peptide-3 signaling in glycosaminoglycan synthesis and chondrocyte hypertrophy during TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells.

Science.gov (United States)

Kocamaz, Erdogan; Gok, Duygu; Cetinkaya, Ayse; Tufan, A Cevik

2012-10-01

This study investigated the involvement of CNP-3, chick homologue for human C-type natriuretic peptide (CNP), in TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells (MSCs). Chondrogenic differentiation of MSCs in pellet cultures was induced by TGF-β1. Chondrogenic differentiation and glycosaminoglycan synthesis were analyzed on the basis of basic histology, collagen type II expression, and Alcian blue staining. Antibodies against CNP and NPR-B were used to block their function during these processes. Results revealed that expression of CNP-3 and NPR-B in MSCs were regulated by TGF-β1 in monolayer cultures at mRNA level. In pellet cultures of MSCs, TGF-β1 successfully induced chondrogenic differentiation and glycosaminoglycan synthesis. Addition of CNP into the TGF-β1 supplemented chondrogenic differentiation medium further induced the glycosaminoglycan synthesis and hypertrophy of differentiated chondrocytes in these pellets. Pellets induced with TGF-β1 and treated with antibodies against CNP and NPR-B, did show collagen type II expression, however, Alcian blue staining showing glycosaminoglycan synthesis was significantly suppressed. In conclusion, CNP-3/NPR-B signaling may strongly be involved in synthesis of glycosaminoglycans of the chondrogenic matrix and hypertrophy of differentiated chondrocytes during TGF-β1 induced chondrogenic differentiation of MSCs.

Detection of a Yersinia pestis gene homologue in rodent samples

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Timothy A. Giles

2016-08-01

Full Text Available A homologue to a widely used genetic marker, pla, for Yersinia pestis has been identified in tissue samples of two species of rat (Rattus rattus and Rattus norvegicus and of mice (Mus musculus and Apodemus sylvaticus using a microarray based platform to screen for zoonotic pathogens of interest. Samples were from urban locations in the UK (Liverpool and Canada (Vancouver. The results indicate the presence of an unknown bacterium that shares a homologue for the pla gene of Yersinia pestis, so caution should be taken when using this gene as a diagnostic marker.

Epigenetic mechanisms involved in differential MDR1 mRNA expression between gastric and colon cancer cell lines and rationales for clinical chemotherapy

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Kim Kyung-Jong

2008-08-01

Full Text Available Abstract Background The membrane transporters such as P-glycoprotein (Pgp, the MDR1 gene product, are one of causes of treatment failure in cancer patients. In this study, the epigenetic mechanisms involved in differential MDR1 mRNA expression were compared between 10 gastric and 9 colon cancer cell lines. Methods The MDR1 mRNA levels were determined using PCR and real-time PCR assays after reverse transcription. Cytotoxicity was performed using the MTT assay. Methylation status was explored by quantification PCR-based methylation and bisulfite DNA sequencing analyses. Results The MDR1 mRNA levels obtained by 35 cycles of RT-PCR in gastric cancer cells were just comparable to those obtained by 22 cycles of RT-PCR in colon cancer cells. Real-time RT-PCR analysis revealed that MDR1 mRNA was not detected in the 10 gastric cancer cell lines but variable MDR1 mRNA levels in 7 of 9 colon cancer cell lines except the SNU-C5 and HT-29 cells. MTT assay showed that Pgp inhibitors such as cyclosporine A, verapamil and PSC833 sensitized Colo320HSR (colon, highest MDR1 expression but not SNU-668 (gastric, highest and SNU-C5 (gastric, no expression to paclitaxel. Quantification PCR-based methylation analysis revealed that 90% of gastric cancer cells, and 33% of colon cancer cells were methylated, which were completely matched with the results obtained by bisulfite DNA sequencing analysis. 5-aza-2'-deoxcytidine (5AC, a DNA methyltransferase inhibitor increased the MDR1 mRNA levels in 60% of gastric cells, and in 11% of colon cancer cells. Trichostatin A (TSA, histone deacetylase inhibitor increased the MDR1 mRNA levels in 70% of gastric cancer cells and 55% of colon cancer cells. The combined treatment of 5AC with TSA increased the MDR1 mRNA levels additively in 20% of gastric cancer cells, but synergistically in 40% of gastric and 11% of colon cancer cells. Conclusion These results indicate that the MDR1 mRNA levels in gastric cancer cells are significantly

A systemic evaluation of cardiac differentiation from mRNA reprogrammed human induced pluripotent stem cells.

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Ashish Mehta

Full Text Available Genetically unmodified cardiomyocytes mandated for cardiac regenerative therapy is conceivable by "foot-print free" reprogramming of somatic cells to induced pluripotent stem cells (iPSC. In this study, we report generation of foot-print free hiPSC through messenger RNA (mRNA based reprograming. Subsequently, we characterize cardiomyocytes derived from these hiPSC using molecular and electrophysiological methods to characterize their applicability for regenerative medicine. Our results demonstrate that mRNA-iPSCs differentiate ontogenetically into cardiomyocytes with increased expression of early commitment markers of mesoderm, cardiac mesoderm, followed by cardiac specific transcriptional and sarcomeric structural and ion channel genes. Furthermore, these cardiomyocytes stained positively for sarcomeric and ion channel proteins. Based on multi-electrode array (MEA recordings, these mRNA-hiPSC derived cardiomyocytes responded predictably to various pharmacologically active drugs that target adrenergic, sodium, calcium and potassium channels. The cardiomyocytes responded chronotropically to isoproterenol in a dose dependent manner, inotropic activity of nifidipine decreased spontaneous contractions. Moreover, Sotalol and E-4031 prolonged QT intervals, while TTX reduced sodium influx. Our results for the first time show a systemic evaluation based on molecular, structural and functional properties of cardiomyocytes differentiated from mRNA-iPSC. These results, coupled with feasibility of generating patient-specific iPSCs hold great promise for the development of large-scale generation of clinical grade cardiomyocytes for cardiac regenerative medicine.

Phosphatase and tensin homologue deleted on chromosome 10 ...

African Journals Online (AJOL)

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor gene deleted or mutated in many human cancers such as glioblastoma, spinal tumors, prostate, bladder, adrenals, thyroid, breast, endometrium, and colon cancers. They result from loss of heterozygosity (LOH) for the PTEN ...

Differentiation of the mRNA transcripts originating from the alpha 1- and alpha 2-globin loci in normals and alpha-thalassemics.

OpenAIRE

Liebhaber, S A; Kan, Y W

1981-01-01

The alpha-globin polypeptide is encoded by two adjacent genes, alpha 1 and alpha 2. In the normal diploid state (alpha alpha/alpha alpha) all four alpha-globin genes are expressed. Loss or dysfunction of one or more of these genes leads to deficient alpha-globin production and results in alpha-thalassemia. We present a technique to differentially assess the steady-state levels of the alpha 1- and alpha-2-globin messenger RNA (mRNA) transcripts and thus delineate the relative level of expressi...

The hypoxic proteome is influenced by gene-specific changes in mRNA translation

International Nuclear Information System (INIS)

Koritzinsky, Marianne; Seigneuric, Renaud; Magagnin, Michael G.; Beucken, Twan van den; Lambin, Philippe; Wouters, Bradly G.

2005-01-01

Background and purpose: Hypoxia causes a rapid reduction in mRNA translation efficiency. This inhibition does not affect all mRNA species to the same extent and can therefore contribute significantly to hypoxia-induced differential protein expression. Our aim in this study was to characterize changes in gene expression during acute hypoxia and evaluate the contribution of regulation via mRNA translation on these changes. For each gene, the contribution of changes in mRNA abundance versus mRNA translation was determined. Materials and methods: DU145 prostate carcinoma cells were exposed to 4 h of hypoxia ( 2 ). Efficiently translated mRNAs were isolated by sedimentation through a sucrose gradient. Affymetrix microarray technology was used to evaluate both the transcriptional and translational contribution to gene expression. Results were validated by quantitative PCR. Results: One hundred and twenty genes were more than 4-fold upregulated by hypoxia in the efficiently translated fraction of mRNA, in comparison to only 76 genes at the level of transcription. Of the 50 genes demonstrating the largest changes in translation, 11 were found to be more than 2-fold over represented in the translated fraction in comparison to their overall transcriptional level. The gene with the highest translational contribution to its induction was CITED-2, which is a negative regulator of HIF-1 transcriptional activity. Conclusions: Gene-specific regulation of mRNA translation contributes significantly to differential gene expression during hypoxia

Expression of calmodulin mRNA in rat olfactory neuroepithelium.

Science.gov (United States)

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.

Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts

DEFF Research Database (Denmark)

Dabelsteen, S; Wandall, H H; Grøn, B

1997-01-01

Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. KGF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF mRNA is expres......Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. KGF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF m......RNA is expressed in periodontal ligament fibroblasts, and that the expression is increased upon serum stimulation. Fibroblasts from human periodontal ligament, from buccal mucosa, from gingiva, and from skin were established from explants. Alkaline phosphatase activity was used as an indicator of the periodontal...

Arc mRNA induction in striatal efferent neurons associated with response learning.

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Daberkow, D P; Riedy, M D; Kesner, R P; Keefe, K A

2007-07-01

The dorsal striatum is involved in motor-response learning, but the extent to which distinct populations of striatal efferent neurons are differentially involved in such learning is unknown. Activity-regulated, cytoskeleton-associated (Arc) protein is an effector immediate-early gene implicated in synaptic plasticity. We examined arc mRNA expression in striatopallidal vs. striatonigral efferent neurons in dorsomedial and dorsolateral striatum of rats engaged in reversal learning on a T-maze motor-response task. Male Sprague-Dawley rats learned to turn right or left for 3 days. Half of the rats then underwent reversal training. The remaining rats were yoked to rats undergoing reversal training, such that they ran the same number of trials but ran them as continued-acquisition trials. Brains were removed and processed using double-label fluorescent in situ hybridization for arc and preproenkephalin (PPE) mRNA. In the reversal, but not the continued-acquisition, group there was a significant relation between the overall arc mRNA signal in dorsomedial striatum and the number of trials run, with rats reaching criterion in fewer trials having higher levels of arc mRNA expression. A similar relation was seen between the numbers of PPE(+) and PPE(-) neurons in dorsomedial striatum with cytoplasmic arc mRNA expression. Interestingly, in behaviourally activated animals significantly more PPE(-) neurons had cytoplasmic arc mRNA expression. These data suggest that Arc in both striatonigral and striatopallidal efferent neurons is involved in striatal synaptic plasticity mediating motor-response learning in the T-maze and that there is differential processing of arc mRNA in distinct subpopulations of striatal efferent neurons.

Differential Expression Analysis by RNA-Seq Reveals Perturbations in the Platelet mRNA Transcriptome Triggered by Pathogen Reduction Systems.

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Abdimajid Osman

Full Text Available Platelet concentrates (PCs are prepared at blood banks for transfusion to patients in certain clinical conditions associated with a low platelet count. To prevent transfusion-transmitted infections via PCs, different pathogen reduction (PR systems have been developed that inactivate the nucleic acids of contaminating pathogens by chemical cross-linking, a mechanism that may also affect platelets' nucleic acids. We previously reported that treatment of stored platelets with the PR system Intercept significantly reduced the level of half of the microRNAs that were monitored, induced platelet activation and compromised the platelet response to physiological agonists. Using genome-wide differential expression (DE RNA sequencing (RNA-Seq, we now report that Intercept markedly perturbs the mRNA transcriptome of human platelets and alters the expression level of >800 mRNAs (P<0.05 compared to other PR systems and control platelets. Of these, 400 genes were deregulated with DE corresponding to fold changes (FC ≥ 2. At the p-value < 0.001, as many as 147 genes were deregulated by ≥ 2-fold in Intercept-treated platelets, compared to none in the other groups. Finally, integrated analysis combining expression data for microRNA (miRNA and mRNA, and involving prediction of miRNA-mRNA interactions, disclosed several positive and inverse correlations between miRNAs and mRNAs in stored platelets. In conclusion, this study demonstrates that Intercept markedly deregulates the platelet mRNA transcriptome, concomitant with reduced levels of mRNA-regulatory miRNAs. These findings should enlighten authorities worldwide when considering the implementation of PR systems, that target nucleic acids and are not specific to pathogens, for the management of blood products.

1200 nt rat liver mRNA identified by differential hybridization exhibits coordinate regulation with 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase

International Nuclear Information System (INIS)

Tanaka, R.D.; Clarke, C.F.; Fogelman, A.M.; Edwards, P.A.

1986-01-01

Differential hybridization has been used to identify genes in rat liver that encode transcripts which are increased by the drugs cholestyramine and mevinolin and are decreased by dietary cholesterol. This approach should prove useful in isolating and identifying coordinately regulated genes involved in the isoprene biosynthetic pathway. Rat liver poly (A) + RNA was isolated from animals fed diets supplemented with either cholestyramine and mevinolin or with cholesterol. Radiolabeled cDNAs generated from these two RNA preparations were used to screen a rat cDNAs library. A preliminary screen of 10,000 recombinants has led to the identification of a clone with an insert of 1200 bp that hybridizes to a mRNA species of about 1200 nt. The level of this RNA species in rat liver is elevated by the drugs cholestyramine and mevinolin and is decreased by cholesterol feeding. This RNA species is also decreased by mevalonate administration to rats. The regulation of this 1200 nt mRNA species mirrors that of HMG CoA reductase and HMG CoA synthase. It seems very likely that this 1200 nt mRNA encodes a polypeptide which is involved in the isoprene biosynthetic pathway

Differential distribution of calcineurin Aα isoenzyme mRNA's in rat brain

NARCIS (Netherlands)

Buttini, M.; Limonta, S.; Luyten, M.; Boddeke, H.

1993-01-01

Specific antisense oligonucleotide probes for the α isoforms of the catalytic subunit (A-subunit) of calcineurin were prepared and the distribution of Aα1 and Aα2 mRNA's has been studied in rat brain using in situ hybridization histochemistry. Clear regional differences have been observed for the

The ApaH-like phosphatase TbALPH1 is the major mRNA decapping enzyme of trypanosomes.

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Susanne Kramer

2017-06-01

Full Text Available 5'-3' decay is the major mRNA decay pathway in many eukaryotes, including trypanosomes. After deadenylation, mRNAs are decapped by the nudix hydrolase DCP2 of the decapping complex and finally degraded by the 5'-3' exoribonuclease. Uniquely, trypanosomes lack homologues to all subunits of the decapping complex, while deadenylation and 5'-3' degradation are conserved. Here, I show that the parasites use an ApaH-like phosphatase (ALPH1 as their major mRNA decapping enzyme. The protein was recently identified as a novel trypanosome stress granule protein and as involved in mRNA binding. A fraction of ALPH1 co-localises exclusively with the trypanosome 5'-3' exoribonuclease XRNA to a special granule at the posterior pole of the cell, indicating a connection between the two enzymes. RNAi depletion of ALPH1 is lethal and causes a massive increase in total mRNAs that are deadenylated, but have not yet started 5'-3' decay. These data suggest that ALPH1 acts downstream of deadenylation and upstream of mRNA degradation, consistent with a function in mRNA decapping. In vitro experiments show that recombinant, N-terminally truncated ALHP1 protein, but not a catalytically inactive mutant, sensitises the capped trypanosome spliced leader RNA to yeast Xrn1, but only if an RNA 5' polyphosphatase is included. This indicates that the decapping mechanism of ALPH1 differs from the decapping mechanism of Dcp2 by leaving more than one phosphate group at the mRNA's 5' end. This is the first reported function of a eukaryotic ApaH-like phosphatase, a bacterial-derived class of enzymes present in all phylogenetic super-groups of the eukaryotic kingdom. The substrates of eukaryotic ApaH-like phosphatases are unknown. However, the substrate of the related bacterial enzyme ApaH, diadenosine tetraphosphate, is highly reminiscent of a eukaryotic mRNA cap.

mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

Science.gov (United States)

Kropp, Jenna; Khatib, Hasan

2015-01-01

In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

Tissue-specific mRNA expression profiling in grape berry tissues

Science.gov (United States)

Grimplet, Jerome; Deluc, Laurent G; Tillett, Richard L; Wheatley, Matthew D; Schlauch, Karen A; Cramer, Grant R; Cushman, John C

2007-01-01

Background Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and

Tissue-specific mRNA expression profiling in grape berry tissues

Directory of Open Access Journals (Sweden)

Cramer Grant R

2007-06-01

Full Text Available Abstract Background Berries of grape (Vitis vinifera contain three major tissue types (skin, pulp and seed all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin and mesocarp (pulp, not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell

Clinical significance of LUNX mRNA, CK19 mRNA, CEA mRNA expression in detecting micrometastasis from lung cancer

International Nuclear Information System (INIS)

Zhu Guangying; Liu Delin; Chen Jie

2003-01-01

Objective: To evaluate the sensitivity, specificity and clinical significance of CK19 mRNA, CEA mRNA and LUNX mRNA for detecting micrometastasis by sampling the peripheral blood and regional lymph nodes of lung cancer patients. Methods: Reverse transcriptase chain reaction (RT-PCR) was used to detect LUNX mRNA, CK19 mRNA, CEA mRNA for micrometastasis by sampling the peripheral blood of 48 lung cancer patients and 44 regional lymph nodes of such patients treated by curative resection. Peripheral blood of 30 patients with pulmonary benign lesions and 10 normal healthy volunteers and lymph nodes of 6 patients with benign pulmonary diseases served as control. Results: 1) LUNX mRNA, CK19 mRNA, CEA mRNA were expressed in all (35/35) lung cancer tissues. 2) In the peripheral blood from 48 lung cancer patients, 30 (62.5%) were positive for LUNX mRNA, 24 (50.0%) positive for CK19 mRNA and 32(66.7%) positive for CEA mRNA. The positive detection rates of micrometastasis in 44 lymph nodes from lung cancer patients were 36.4% (16 out of 44) for LUNX mRNA, 27.3% (12 out of 44) for CK19 mRNA and 40.9% (18 out of 44) for CEA mRNA. 3) In the 30 blood samples from patients with pulmonary benign diseases, 2 (6.7%) expressed CK19 mRNA, but none expressed LUNX mRNA or CEA mRNA. All the 3 molecular markers were negative in the 10 blood samples from healthy volunteers. In 11 lymph nodes from patients with pulmonary benign lesions, none was positive for any of the three markers. 4) In 44 regional lymph nodes from lung cancer patients, 6 (13.6%) were positive for metastasis by histopathological examination, with a positive rate significantly lower than that of the RT-PCR (P<0.05). 5) The micrometastatic positive rate in the peripheral blood of 40 non-small cell lung cancer (NSCLC) patients was significantly related to TNM stage (P=0.01). Conclusions: LUNX mRNA, CK19 MRNA, CEA mRNA are all appropriate target genes for the detection of micrometastasis from lung cancer. LUNX mRNA and CEA mRNA

Functional analysis of three type-2 DGAT homologue genes for triacylglycerol production in the green microalga Chlamydomonas reinhardtii.

Science.gov (United States)

La Russa, M; Bogen, C; Uhmeyer, A; Doebbe, A; Filippone, E; Kruse, O; Mussgnug, J H

2012-11-30

Photosynthetic organisms like plants and algae can use sunlight to produce lipids as important metabolic compounds. Plant-derived triacylglycerols (TAGs) are valuable for human and animal nutrition because of their high energy content and are becoming increasingly important for the production of renewable biofuels. Acyl-CoA:diacylglycerol acyltransferases (DGATs) have been demonstrated to play an important role in the accumulation of TAG compounds in higher plants. DGAT homologue genes have been identified in the genome of the green alga Chlamydomonas reinhardtii, however their function in vivo is still unknown. In this work, the three most promising type-2 DGAT candidate genes potentially involved in TAG lipid accumulation (CrDGAT2a, b and c) were investigated by constructing overexpression strains. For each of the genes, three strains were identified which showed enhanced mRNA levels of between 1.7 and 29.1 times that of the wild type (wt). Total lipid contents, neutral lipids and fatty acid profiles were determined and showed that an enhanced mRNA expression level of the investigated DGAT genes did not boost the intracellular TAG accumulation or resulted in alterations of the fatty acid profiles compared to wild type during standard growth condition or during nitrogen or sulfur stress conditions. We conclude that biotechnological efforts to enhance cellular TAG amount in microalgae need further insights into the complex network of lipid biosynthesis to identify potential bottlenecks of neutral lipid production. Copyright © 2012 Elsevier B.V. All rights reserved.

Molecular cloning, sequence characterization and expression analysis of a CD63 homologue from the coleopteran beetle, Tenebrio molitor.

Science.gov (United States)

Patnaik, Bharat Bhusan; Kang, Seong Min; Seo, Gi Won; Lee, Hyo Jeong; Patnaik, Hongray Howrelia; Jo, Yong Hun; Tindwa, Hamisi; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung; Bang, In Seok; Han, Yeon Soo

2013-10-15

CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic "Cys-Cys-Gly" motif and "Cys188" residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%-56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

Molecular Cloning, Sequence Characterization and Expression Analysis of a CD63 Homologue from the Coleopteran Beetle, Tenebrio molitor

Directory of Open Access Journals (Sweden)

Yeon Soo Han

2013-10-01

Full Text Available CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63 was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic “Cys-Cys-Gly” motif and “Cys188” residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%–56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

Molecular Cloning, Sequence Characterization and Expression Analysis of a CD63 Homologue from the Coleopteran Beetle, Tenebrio molitor

Science.gov (United States)

Patnaik, Bharat Bhusan; Kang, Seong Min; Seo, Gi Won; Lee, Hyo Jeong; Patnaik, Hongray Howrelia; Jo, Yong Hun; Tindwa, Hamisi; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung; Bang, In Seok; Han, Yeon Soo

2013-01-01

CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic “Cys-Cys-Gly” motif and “Cys188” residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%–56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens. PMID:24132157

The ontogeny of nanos homologue expression in the oligochaete annelid Tubifex tubifex.

Science.gov (United States)

Mohri, Ki-Ichi; Nakamoto, Ayaki; Shimizu, Takashi

2016-01-01

We have cloned and characterized the expression of a nanos homologue (designated Ttu-nos) from the oligochaete annelid Tubifex tubifex. Ttu-nos mRNA is distributed broadly throughout the early cleavage stages. Ttu-nos is expressed in most if not all of the early blastomeres, in which Ttu-nos RNA associates with pole plasms. Ttu-nos transcripts are concentrated to 2d and 4d cells. Shortly after 2d(111) (derived from 2d cell) divides into a bilateral pair of NOPQ proteloblasts, Ttu-nos RNA vanishes from the embryo, which is soon followed by the resumption of Ttu-nos expression in nascent primary blast cells produced by teloblasts. The resumption of Ttu-nos expression occurs only in a subset of teloblast lineages (viz., M, N and Q). After Ttu-nos expression is retained in the germ band for a while, it disappears in anterior-to-posterior progression. At the end of embryogenesis, there is no trace of Ttu-nos expression. Thereafter, growing juveniles do not show any sign of Ttu-nos expression, either. The first sign of Ttu-nos expression is detected in oocytes in the ovary of young adults (ca 40 days after hatching), and its expression continues in growing oocytes that undergo yolk deposition and maturation in the ovisac. Copyright © 2015 Elsevier B.V. All rights reserved.

Differential Expression of Sox11 and Bdnf mRNA Isoforms in the Injured and Regenerating Nervous Systems

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Felix L. Struebing

2017-11-01

Full Text Available In both the central nervous system (CNS and the peripheral nervous system (PNS, axonal injury induces changes in neuronal gene expression. In the PNS, a relatively well-characterized alteration in transcriptional activation is known to promote axonal regeneration. This transcriptional cascade includes the neurotrophin Bdnf and the transcription factor Sox11. Although both molecules act to facilitate successful axon regeneration in the PNS, this process does not occur in the CNS. The present study examines the differential expression of Sox11 and Bdnf mRNA isoforms in the PNS and CNS using three experimental paradigms at different time points: (i the acutely injured CNS (retina after optic nerve crush and PNS (dorsal root ganglion after sciatic nerve crush, (ii a CNS regeneration model (retina after optic nerve crush and induced regeneration; and (iii the retina during a chronic form of central neurodegeneration (the DBA/2J glaucoma model. We find an initial increase of Sox11 in both PNS and CNS after injury; however, the expression of Bdnf isoforms is higher in the PNS relative to the CNS. Sustained upregulation of Sox11 is seen in the injured retina following regeneration treatment, while the expression of two Bdnf mRNA isoforms is suppressed. Furthermore, two isoforms of Sox11 with different 3′UTR lengths are present in the retina, and the long isoform is specifically upregulated in later stages of glaucoma. These results provide insight into the molecular cascades active during axonal injury and regeneration in mammalian neurons.

Rapid detection, differentiation and typing of methicillin-resistant Staphylococcus aureus harbouring either mecA or the new mecA homologue mecA(LGA251).

Science.gov (United States)

Stegger, M; Andersen, P S; Kearns, A; Pichon, B; Holmes, M A; Edwards, G; Laurent, F; Teale, C; Skov, R; Larsen, A R

2012-04-01

The recent finding of a new mecA homologue, mecA(LGA251) , with only 70% nucleotide homology to the conventional mecA gene has brought the routine testing for mecA as a confirmatory test for methicillin-resistant Staphylococcus aureus (MRSA) into question. A multiplex PCR was designed to differentiate mecA(LGA251) from the known mecA together with detection of lukF-PV and the spa gene fragments, enabling direct spa typing by sequencing of the PCR amplicons. The PCR analysis and subsequent spa typing were validated on a large collection (n=185) of contemporary MRSA and methicillin-sensitive S. aureus isolates, including 127 isolates carrying mecA(LGA251) . The mecA(LGA251) gene was situated in staphylococcal cassette chromosome mec type XI elements, and sequence variation within a 631-bp fragment of mecA(LGA251) in 79 isolates indicated a very conserved gene sequence. Following a successful validation, the multiplex PCR strategy was implemented in the routine testing of MRSA for national surveillance. Over a 2-month period, among 203 samples tested, 12 new MRSA cases caused by isolates carrying mecA(LGA251) were identified, emphasizing the clinical importance of testing for these new MRSA isolates. © 2011 STATENS SERUM INSTITUT. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

Isolation and characterization of an AGAMOUS homologue from cocoa

NARCIS (Netherlands)

Chaidamsari, T.; Sugiarit, H.; Santoso, D.; Angenent, G.C.; Maagd, de R.A.

2006-01-01

We report the cloning of a cDNA from TcAG, an AG (Arabidopsis thaliana MADS-box C-type transcription factor gene AGAMOUS) homologue from cocoa (Theobroma cacao L.). TcAG was in the cocoa flower expressed primarily in stamens and ovaries, comparable to AG in Arabidopsis. Additionally, we found that

The actin homologue MreB organizes the bacterial cell membrane

NARCIS (Netherlands)

Strahl, H.; Burmann, F.; Hamoen, L.W.

2014-01-01

The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate

Green tea increases anti-inflammatory tristetraprolin and decreases pro-inflammatory tumor necrosis factor mRNA levels in rats

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Roussel Anne M

2007-01-01

Full Text Available Abstract Background Tristetraprolin (TTP/ZFP36 family proteins have anti-inflammatory activity by binding to and destabilizing pro-inflammatory mRNAs such as Tnf mRNA, and represent a potential therapeutic target for inflammation-related diseases. Tea has anti-inflammatory properties but the molecular mechanisms have not been completely elucidated. We hypothesized that TTP and/or its homologues might contribute to the beneficial effects of tea as an anti-inflammatory product. Methods Quantitative real-time PCR was used to investigate the effects of green tea (0, 1, and 2 g solid extract/kg diet on the expression of Ttp family genes (Ttp/Tis11/Zfp36, Zfp36l1/Tis11b, Zfp36l2/Tis11d, Zfp36l3, pro-inflammatory genes (Tnf, Csf2/Gm-csf, Ptgs2/Cox2, and Elavl1/Hua/Hur and Vegf genes in liver and muscle of rats fed a high-fructose diet known to induce insulin resistance, oxidative stress, inflammation, and TNF-alpha levels. Results Ttp and Zfp36l1 mRNAs were the major forms in both liver and skeletal muscle. Ttp, Zfp36l1, and Zfp36l2 mRNA levels were more abundant in the liver than those in the muscle. Csf2/Gm-csf and Zfp36l3 mRNAs were undetectable in both tissues. Tea (1 g solid extract/kg diet increased Ttp mRNA levels by 50–140% but Tnf mRNA levels decreased by 30% in both tissues, and Ptgs2/Cox2 mRNA levels decreased by 40% in the muscle. Tea (2 g solid extract/kg diet increased Elavl1/Hua/Hur mRNA levels by 40% in the liver but did not affect any of the other mRNA levels in liver or muscle. Conclusion These results show that tea can modulate Ttp mRNA levels in animals and suggest that a post-transcriptional mechanism through TTP could partially account for tea's anti-inflammatory properties. The results also suggest that drinking adequate amounts of green tea may play a role in the prevention of inflammation-related diseases.

Toxicities of emamectin benzoate homologues and photodegradates to Lepidoptera.

Science.gov (United States)

Argentine, Joseph A; Jansson, Richard K; Starner, Van R; Halliday, W Ross

2002-12-01

The toxicity of a number of emamectin benzoate homologues and photodegradates to five species of Lepidoptera was investigated using diet and foliar bioassays. The emamectin benzoate homologues B1a and B1b were equally toxic in the diet and foliar assays to Spodoptera exigua (Hübner), Heliothis virescens (F.), Tricoplusia ni (Hübner), and Spodoptera frugiperda (J. E. Smith), within each of these species. Plutella xylostella (L.) was the most sensitive species to emamectin benzoate. The AB1a photodegradate of emamectin benzoate was as toxic as the parent compound in the diet assay. However, in the foliage assay AB1a was 4.4-fold less toxic to S. exigua than the parent compound. The MFB1a photodegradate of emamectin benzoate was as toxic as the parent compound to P. xylostella, and 3.1 to 6.2 times as toxic as the parent compound to the other species in the diet assay. The order of toxicity of the photodegradates were AB1a > MFB1a > FAB1a > 8,9-Z-MAB1a > PAB1a.

Inhibition of fatty acid synthase prevents preadipocyte differentiation

International Nuclear Information System (INIS)

Schmid, Bernhard; Rippmann, Joerg F.; Tadayyon, Moh; Hamilton, Bradford S.

2005-01-01

Inhibition of fatty acid synthase (FAS) reduces food intake in rodents. As adipose tissue expresses FAS, we sought to investigate the effect of reduced FAS activity on adipocyte differentiation. FAS activity was suppressed either pharmacologically or by siRNA during differentiation of 3T3-L1 cells. Cerulenin (10 μM), triclosan (50 μM), and C75 (50 μM) reduced dramatically visible lipid droplet accumulation, while incorporation of [1- 14 C]acetate into lipids was reduced by 75%, 70%, and 90%, respectively. Additionally, the substances reduced FAS, CEBPα, and PPARγ mRNA by up to 85% compared to that of control differentiated cells. Transient transfection with FAS siRNA suppressed FAS mRNA and FAS activity, and this was accompanied by reduction of CEBPα and PPARγ mRNA levels, and complete prevention of lipid accumulation. CD36, a late marker of differentiation, was also reduced. Together, these results suggest that FAS generated signals may be essential to support preadipocyte differentiation

Mirna biogenesis pathway is differentially regulated during adipose derived stromal/stem cell differentiation.

Science.gov (United States)

Martin, E C; Qureshi, A T; Llamas, C B; Burow, M E; King, A G; Lee, O C; Dasa, V; Freitas, M A; Forsberg, J A; Elster, E A; Davis, T A; Gimble, J M

2018-02-07

Stromal/stem cell differentiation is controlled by a vast array of regulatory mechanisms. Included within these are methods of mRNA gene regulation that occur at the level of epigenetic, transcriptional, and/or posttranscriptional modifications. Current studies that evaluate the posttranscriptional regulation of mRNA demonstrate microRNAs (miRNAs) as key mediators of stem cell differentiation through the inhibition of mRNA translation. miRNA expression is enhanced during both adipogenic and osteogenic differentiation; however, the mechanism by which miRNA expression is altered during stem cell differentiation is less understood. Here we demonstrate for the first time that adipose-derived stromal/stem cells (ASCs) induced to an adipogenic or osteogenic lineage have differences in strand preference (-3p and -5p) for miRNAs originating from the same primary transcript. Furthermore, evaluation of miRNA expression in ASCs demonstrates alterations in both miRNA strand preference and 5'seed site heterogeneity. Additionally, we show that during stem cell differentiation there are alterations in expression of genes associated with the miRNA biogenesis pathway. Quantitative RT-PCR demonstrated changes in the Argonautes (AGO1-4), Drosha, and Dicer at intervals of ASC adipogenic and osteogenic differentiation compared to untreated ASCs. Specifically, we demonstrated altered expression of the AGOs occurring during both adipogenesis and osteogenesis, with osteogenesis increasing AGO1-4 expression and adipogenesis decreasing AGO1 gene and protein expression. These data demonstrate changes to components of the miRNA biogenesis pathway during stromal/stem cell differentiation. Identifying regulatory mechanisms for miRNA processing during ASC differentiation may lead to novel mechanisms for the manipulation of lineage differentiation of the ASC through the global regulation of miRNA as opposed to singular regulatory mechanisms.

Characterization and cloning of TMV resistance gene N homologues ...

African Journals Online (AJOL)

Tobacco cultivars Nicotiana tabacum cv. Samsun NN plants carrying the N gene contain a multitude of N-related genes. We cloned a few N homologues and isolated two full-length cDNAs of NL-C26 and NL-B69 genes from N. tabacum cv. Samsun NN. Nucleotide sequence analysis showed that the coding regions of ...

Ectopic expression and knocking-down of LINE-1 mRNA in human mesenchymal stem cells: impact on in vitro osteogenic and adipogenic differentiation

KAUST Repository

Atinbayeva, Nazerke

2018-05-01

There are two classes of transposable elements: DNA transposons and retrotransposons. DNA transposons spread in the genome by “cut and paste” mechanism. In contrast, retrotransposons use copy and paste strategy involving RNA and retrotranscriptase mediated mechanism; these include long interspersed nuclear elements-1 (LINE-1, L1) and short interspersed nuclear elements (SINE). In mammals, in order to maintain genome integrity both types of transposons are tightly repressed. However, some copies of retrotransposons are still active in germ cells contributing to natural variation. Surprisingly, recent reports indicate that also somatic cells support L1 reactivation in early development, in particular in the brain leading to mosaicism. However, whether L1 retrotransposition is a part of other cell lineage developmental programs and its functional significance in the context of cell differentiation remain to be elucidated. To address this question, I investigated whether L1 retrotransposition was occurring during in vitro osteogenic and adipogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). Interestingly, clinical observations have revealed loss of bone density in HIV-infected individuals treated with nucleoside analogs that inhibit HIV retrotranscriptase, as well as the endogenous one encoded by L1s. This observation made us to hypothesize that transposable elements played a positive role in post-natal bone homeostasis. I found that while adipogenesis is “retrotransposition free”, osteogenic differentiation is a “retrotransposition-prone” process and its inhibition blocks its genetic program. Indeed, L1 DNA content does not change during adipogenic differentiation and that of retrotranscriptase does not have any effect on the acquisition of a terminally differentiated phenotype. In contrast, soon after MSCs commitment into pre-osteoblasts, L1 retrotransposable elements increase their expression and actively transpose

The monomeric orphan nuclear receptor Schistosoma mansoni Ftz-F1 dimerizes specifically and functionally with the schistosome RXR homologue, SmRXR1

International Nuclear Information System (INIS)

Bertin, Benjamin; Caby, Stephanie; Oger, Frederik; Sasorith, Souphatta; Wurtz, Jean-Marie; Pierce, Raymond J.

2005-01-01

In an attempt to understand development and differentiation processes of the parasitic blood fluke Schistosoma mansoni, several members of the nuclear receptor superfamily were cloned, including SmFtz-F1 (S. mansoni Fushi Tarazu-factor 1). The Ftz-F1 nuclear receptor subfamily only contains orphan receptors that bind to their response element as monomers. Whereas SmFtz-F1 displays these basic functional properties, we have identified an original and specific interaction between SmFtz-F1 and the schistosome RXR homologue, SmRXR1. The mammalian two-hybrid assay showed that the D, E, and F domains of SmFtz-F1 were capable of interacting specifically with the E domain of SmRXR1 but not with that of mouse RXRα. Using three-dimensional LBD homology modelling and structure-guided mutagenesis, we were able to demonstrate the essential role of exposed residues located in the dimerization interfaces of both receptors in the maintenance of the interaction. Cotransfection experiments with constructions encoding full-length nuclear receptors show that SmRXR1 potentiates the transcriptional activity of SmFtz-F1 from various promoters. Nevertheless, the lack of identification of a dimeric response element for this SmFtz-F1/SmRXR1 heterodimer seems to indicate a 'tethering' mechanism. Thus, our results suggest for the first time that a member of the Ftz-F1 family could heterodimerize functionally with a homologue of the universal heterodimerization partner of nuclear receptors. This unique property confirms that SmFtz-F1 may be involved in the development and differentiation of schistosome-specific structures

An Arabidopsis chloroplast-targeted Hsp101 homologue, APG6, has an essential role in chloroplast development as well as heat-stress response.

Science.gov (United States)

Myouga, Fumiyoshi; Motohashi, Reiko; Kuromori, Takashi; Nagata, Noriko; Shinozaki, Kazuo

2006-10-01

Analysis of albino or pale-green (apg) mutants is important for identifying nuclear genes responsible for chloroplast development and pigment synthesis. We have identified 38 apg mutants by screening 11 000 Arabidopsis Ds-tagged lines. One mutant, apg6, contains a Ds insertion in a gene encoding APG6 (ClpB3), a homologue of the heat-shock protein Hsp101 (ClpB1). We isolated somatic revertants and identified two Ds-tagged and one T-DNA-tagged mutant alleles of apg6. All three alleles gave the same pale-green phenotype. These results suggest that APG6 is important for chloroplast development. The APG6 protein contains a transit peptide and is localized in chloroplasts. The plastids of apg6 pale-green cells were smaller than those of the wild type, and contained undeveloped thylakoid membranes. APG6 mRNA accumulated in response to heat shock in various organs, but not in response to other abiotic stresses. Under normal conditions, APG6 is constitutively expressed in the root tips, the organ boundary region, the reproductive tissues of mature plants where plastids exist as proplastids, and slightly in the stems and leaves. In addition, constitutive overexpression of APG6 in transgenic plants inhibited chloroplast development and resulted in a mild pale-green phenotype. The amounts of chloroplast proteins related to photosynthesis were markedly decreased in apg6 mutants. These results suggest that APG6 functions as a molecular chaperone involved in plastid differentiation mediating internal thylakoid membrane formation and conferring thermotolerance to chloroplasts during heat stress. The APG6 protein is not only involved in heat-stress response in chloroplasts, but is also essential for chloroplast development.

Gene function in early mouse embryonic stem cell differentiation

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Campbell Pearl A

2007-03-01

Full Text Available Abstract Background Little is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5 undergoing undirected differentiation into embryoid bodies (EBs over a period of two weeks. Results We identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1, our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2 that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set. Conclusion Our analysis profiles for the first time gene expression at a very early stage of m

Nerve growth factor mRNA in brain: localization by in situ hybridization

International Nuclear Information System (INIS)

Rennert, P.D.; Heinrich, G.

1986-01-01

Nerve Growth Factor is a 118 amino acid polypeptide that plays an important role in the differentiation and survival of neurons. The recent discovery that a mRNA that encodes beta Nerve Growth Factor is present in brain suggests that the Nerve Growth Factor gene may not only regulate gene expression of peripheral but also of central neurons. To identify the site(s) of Nerve Growth Factor mRNA production in the brain and to determine which cells express the Nerve Growth Factor gene, the technique of in situ hybridization was employed. A 32P-labeled RNA probe complementary to Nerve Growth Factor mRNA hybridized to cells in the stratum granulosum of the dentate gyrus and the stratum pyramidale of the hippocampus. These observations identify for the first time cellular sites of Nerve Growth Factor gene expression in the central nervous system, and suggest that Nerve Growth Factor mRNA is produced by neurons

Midbrain expression of Delta-like 1 homologue is regulated by GDNF and is associated with dopaminergic differentiation

DEFF Research Database (Denmark)

Christophersen, Nicolai S.; Gronborg, Mette; Petersen, Thomas Nordahl

2007-01-01

upregulation with increased positive staining of cell bodies in the SNc and fibers in the striatum. Analysis of the developmental regulation of Dlk1 in the murine ventral midbrain showed that the upregulation of Dlk1 mRNA correlated with the generation of tyrosine hydroxylase (TH)-positive neurons. Furthermore...

MicroRNA-15a finetunes the level of Delta-like 1 homologue (DLK1) in proliferating 3T3-L1 preadipocytes

DEFF Research Database (Denmark)

Andersen, Ditte C; Jensen, Charlotte H; Schneider, Mikael

2010-01-01

Delta like 1 homologue (Dlk1) exists in both transmembrane and soluble molecular forms, and is implicated in cellular growth and plays multiple roles in development, tissue regeneration, and cancer. Thus, DLK1 levels are critical for cell function, and abnormal DLK1 expression can be lethal...... increases with cell density, and peaks at the same stage where membrane DLK1(M) and soluble DLK1(S) are found at maximum levels. Remarkably, miR-15a represses the amount of all Dlk1 variants at the mRNA level but also the level of DLK1(M) protein while it increases the amount of DLK1(S) supporting a direct...... while increasing cell numbers, scenarios that were completely rescued by addition of purified DLK1(S). Our data thus imply that miR-15a regulates cell size and proliferation by fine-tuning Dlk1 among others, and further emphasize miR-15a and DLK1 levels to play important roles in growth signaling...

Simultaneous detection of mRNA and protein stem cell markers in live cells

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Bao Gang

2009-04-01

Full Text Available Abstract Background Biological studies and medical application of stem cells often require the isolation of stem cells from a mixed cell population, including the detection of cancer stem cells in tumor tissue, and isolation of induced pluripotent stem cells after eliciting the expression of specific genes in adult cells. Here we report the detection of Oct-4 mRNA and SSEA-1 protein in live carcinoma stem cells using respectively molecular beacon and dye-labeled antibody, aiming to establish a new method for stem cells detection and isolation. Results Quantification of Oct-4 mRNA and protein in P19 mouse carcinoma stem cells using respectively RT-PCR and immunocytochemistry confirmed that their levels drastically decreased after differentiation. To visualize Oct-4 mRNA in live stem cells, molecular beacons were designed, synthesized and validated, and the detection specificity was confirmed using control studies. We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells. Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol. We demonstrated that, using a combination of Oct-4 mRNA-targeting molecular beacon with SSEA-1 antibody in flow cytometric analysis, undifferentiated stem cells can be clearly distinguished from differentiated cells. We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology. Conclusion Molecular beacons have the potential to provide a powerful tool for highly specific detection and isolation of stem cells, including cancer stem cells and induced pluripotent stem (iPS cells without disturbing cell physiology. It is advantageous to perform simultaneous detection of intracellular (mRNA and cell-surface (protein stem cell markers in flow cytometric analysis, which may lead to high detection sensitivity and efficiency.

Analysis of the intercaste transcriptional profile of Melipona scutellaris Latreille, 1811 (Hymenoptera, Apidae, Meliponini) by mRNA differential display.

Science.gov (United States)

Siquieroli, Ana Carolina S; Vieira, Carlos U; Carvalho-Zilse, Gislene A; Goulart, Luiz R; Kerr, Warwick E; Bonetti, Ana M

2009-01-01

In colonies of Melipona scutellaris Latreille, 1811 workers can be found with four ganglion nerve cells, a morphological characteristic of the queen. It is hypothesized that these workers, called intercastes, or phenocopies, are phenotypically-like workers, but genotypically identical to queens due to this specific trait. Workers with the same number of ganglion as queens seem to be intercastes between queens and workers. Our objective was to analyze the mRNA pro files of workers, queens, and intercastes of M. scutellaris through DDRT-PCR. Three hundred (300) pupae with white eyes were collected and externally identified according to the number of abdominal nerve ganglions: workers (5 ganglions), queens (4 ganglions) and intercastes (4 ganglions). The analysis identified differentially expressed transcripts that were present only in workers, but absent in intercastes and queens, confirming the hypothesis, by demonstrating the environmental effect on the queen genotype that generated phenotype-like workers.

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

Science.gov (United States)

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.

Expression of the chitinase family glycoprotein YKL-40 in undifferentiated, differentiated and trans-differentiated mesenchymal stem cells.

Directory of Open Access Journals (Sweden)

Daniel J Hoover

Full Text Available The glycoprotein YKL-40 (CHI3L1 is a secreted chitinase family protein that induces angiogenesis, cell survival, and cell proliferation, and plays roles in tissue remodeling and immune regulation. It is expressed primarily in cells of mesenchymal origin, is overexpressed in numerous aggressive carcinomas and sarcomas, but is rarely expressed in normal ectodermal tissues. Bone marrow-derived mesenchymal stem cells (MSCs can be induced to differentiate into various mesenchymal tissues and trans-differentiate into some non-mesenchymal cell types. Since YKL-40 has been used as a mesenchymal marker, we followed YKL-40 expression as undifferentiated MSCs were induced to differentiate into bone, cartilage, and neural phenotypes. Undifferentiated MSCs contain significant levels of YKL-40 mRNA but do not synthesize detectable levels of YKL-40 protein. MSCs induced to differentiate into chondrocytes and osteocytes soon began to express and secrete YKL-40 protein, as do ex vivo cultured chondrocytes and primary osteocytes. In contrast, MSCs induced to trans-differentiate into neurons did not synthesize YKL-40 protein, consistent with the general absence of YKL-40 protein in normal CNS parenchyma. However, these trans-differentiated neurons retained significant levels of YKL-40 mRNA, suggesting the mechanisms which prevented YKL-40 translation in undifferentiated MSCs remained in place, and that these trans-differentiated neurons differ in at least this way from neurons derived from neuronal stem cells. Utilization of a differentiation protocol containing β-mercaptoethanol resulted in cells that expressed significant amounts of intracellular YKL-40 protein that was not secreted, which is not seen in normal cells. Thus the synthesis of YKL-40 protein is a marker for MSC differentiation into mature mesenchymal phenotypes, and the presence of untranslated YKL-40 mRNA in non-mesenchymal cells derived from MSCs reflects differences between differentiated and

Differential regulation of amyloid-β-protein mRNA expression within hippocampal neuronal subpopulations in Alzheimer disease

International Nuclear Information System (INIS)

Higgins, G.A.; Lewis, D.A.; Bahmanyar, S.; Goldgaber, D.; Gajdusek, D.C.; Young, W.G.; Morrison, J.H.; Wilson, M.C.

1988-01-01

The authors have mapped the neuroanatomical distribution of amyloid-β-protein mRNA within neuronal subpopulations of the hippocampal formation in the cynomolgus monkey (Macaca fascicularis), normal aged human, and patients with Alzheimer disease. Amyloid-β-protein mRNA appears to be expressed in all hippocampal neurons, but at different levels of abundance. In the central nervous system of monkey and normal aged human, image analysis shows that neurons of the dentate gyrus and cornu Ammonis fields contain a 2.5-times-greater hybridization signal than is present in neurons of the subiculum and entorhinal cortex. In contrast, in the Alzheimer disease hippocampal formation, the levels of amyloid-β-protein mRNA in the cornu Ammonis field 3 and parasubiculum are equivalent. These findings suggest that within certain neuronal subpopulations cell type-specific regulation of amyloid-β-protein gene expression may be altered in Alzheimer disease

Partial functional complementation between human and mouse cytomegalovirus chemokine receptor homologues

DEFF Research Database (Denmark)

Farrell, Helen E; Abraham, Alexander M; Cardin, Rhonda D

2011-01-01

The human cytomegalovirus (CMV) proteins US28 and UL33 are homologous to chemokine receptors (CKRs). Knockout of the mouse CMV M33 protein (UL33 homologue) results in substantial attenuation of salivary gland infection/replication and reduced efficiency of reactivation from tissue explants. M33-m...

Combining miRNA and mRNA Expression Profiles in Wilms Tumor Subtypes

Directory of Open Access Journals (Sweden)

Nicole Ludwig

2016-03-01

Full Text Available Wilms tumor (WT is the most common childhood renal cancer. Recent findings of mutations in microRNA (miRNA processing proteins suggest a pivotal role of miRNAs in WT genesis. We performed miRNA expression profiling of 36 WTs of different subtypes and four normal kidney tissues using microarrays. Additionally, we determined the gene expression profile of 28 of these tumors to identify potentially correlated target genes and affected pathways. We identified 85 miRNAs and 2107 messenger RNAs (mRNA differentially expressed in blastemal WT, and 266 miRNAs and 1267 mRNAs differentially expressed in regressive subtype. The hierarchical clustering of the samples, using either the miRNA or mRNA profile, showed the clear separation of WT from normal kidney samples, but the miRNA pattern yielded better separation of WT subtypes. A correlation analysis of the deregulated miRNA and mRNAs identified 13,026 miRNA/mRNA pairs with inversely correlated expression, of which 2844 are potential interactions of miRNA and their predicted mRNA targets. We found significant upregulation of miRNAs-183, -301a/b and -335 for the blastemal subtype, and miRNAs-181b, -223 and -630 for the regressive subtype. We found marked deregulation of miRNAs regulating epithelial to mesenchymal transition, especially in the blastemal subtype, and miRNAs influencing chemosensitivity, especially in regressive subtypes. Further research is needed to assess the influence of preoperative chemotherapy and tumor infiltrating lymphocytes on the miRNA and mRNA patterns in WT.

mRNA transfection of mouse and human neural stem cell cultures.

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Samuel McLenachan

Full Text Available The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

mRNA Transfection of Mouse and Human Neural Stem Cell Cultures

Science.gov (United States)

McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J.; Chen, Fred K.

2013-01-01

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages. PMID:24386231

mRNA transfection of mouse and human neural stem cell cultures.

Science.gov (United States)

McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J; Chen, Fred K

2013-01-01

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

Myeloperoxidase mRNA detection for lineage determination of leukemic blasts: retrospective analysis.

Science.gov (United States)

Crisan, D; Anstett, M J

1995-07-01

Myeloperoxidase (MPO) mRNA is an early myeloid marker; its detection in the morphologically and immunophenotypically primitive blasts of acute undifferentiated leukemia (AUL) establishes myeloid lineage and allows reclassification as acute myelogenous leukemia with minimal differentiation (AML-MO). We have previously reported a procedure for MPO mRNA detection by RT-PCR (reverse transcription-polymerase chain reaction) and an adaptation for use of routine hematology smears. This variant procedure allows retrospective analysis of mRNA and is used in the present study to evaluate the lineage of leukemic blasts in seven cases with morphology and cytochemistry consistent with AUL. All hematology smears used in this study were air-dried, unstained or Wright-stained and stored at room temperature for periods varying between 3 days and 2 years. MPO mRNA was detected in six cases, establishing the myeloid lineage of the blasts and the diagnosis of AML-MO. In the remaining case, the blasts were MPO mRNA negative, confirming the diagnosis of AUL. The RT-PCR procedure for retrospective mRNA analysis is useful in the clinical setting, due to its high specificity and sensitivity, speed (less than 24 h), safety (no radioactivity) and convenient use of routine hematology smears; it is particularly attractive in clinical situations when fresh or frozen specimens are no longer available at the time when the need for molecular diagnostics becomes apparent.

Purification, crystallization and preliminary X-ray analysis of SGR6054, a Streptomyces homologue of the mycobacterial integration host factor mIHF

International Nuclear Information System (INIS)

Nomoto, Ryohei; Tezuka, Takeaki; Miyazono, Ken-ichi; Tanokura, Masaru; Horinouchi, Sueharu; Ohnishi, Yasuo

2012-01-01

A Streptomyces homologue of the mycobacterial integration host factor mIHF was heterologously produced, purified and crystallized in the presence of a 16-mer duplex DNA by the sitting-drop vapour-diffusion method. The best crystal diffracted X-rays to 2.22 Å resolution and belonged to space group C2. The mycobacterial integration host factor (mIHF) is a small nonspecific DNA-binding protein that is essential for the growth of Mycobacterium smegmatis. mIHF homologues are widely distributed among Actinobacteria, and a Streptomyces homologue of mIHF is involved in control of sporulation and antibiotic production in S. coelicolor A3(2). Despite their important biological functions, a structure of mIHF or its homologues has not been elucidated to date. Here, the S. griseus mIHF homologue (SGR6054) was expressed and purified from Escherichia coli and crystallized in the presence of a 16-mer duplex DNA by the sitting-drop vapour-diffusion method. The plate-shaped crystal belonged to space group C2, with unit-cell parameters a = 88.53, b = 69.35, c = 77.71 Å, β = 96.63°, and diffracted X-rays to 2.22 Å resolution

Purification, crystallization and preliminary X-ray analysis of SGR6054, a Streptomyces homologue of the mycobacterial integration host factor mIHF

Energy Technology Data Exchange (ETDEWEB)

Nomoto, Ryohei; Tezuka, Takeaki; Miyazono, Ken-ichi; Tanokura, Masaru; Horinouchi, Sueharu; Ohnishi, Yasuo [Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

2012-08-31

A Streptomyces homologue of the mycobacterial integration host factor mIHF was heterologously produced, purified and crystallized in the presence of a 16-mer duplex DNA by the sitting-drop vapour-diffusion method. The best crystal diffracted X-rays to 2.22 Å resolution and belonged to space group C2. The mycobacterial integration host factor (mIHF) is a small nonspecific DNA-binding protein that is essential for the growth of Mycobacterium smegmatis. mIHF homologues are widely distributed among Actinobacteria, and a Streptomyces homologue of mIHF is involved in control of sporulation and antibiotic production in S. coelicolor A3(2). Despite their important biological functions, a structure of mIHF or its homologues has not been elucidated to date. Here, the S. griseus mIHF homologue (SGR6054) was expressed and purified from Escherichia coli and crystallized in the presence of a 16-mer duplex DNA by the sitting-drop vapour-diffusion method. The plate-shaped crystal belonged to space group C2, with unit-cell parameters a = 88.53, b = 69.35, c = 77.71 Å, β = 96.63°, and diffracted X-rays to 2.22 Å resolution.

Analysis of the Intercaste Transcriptional Profile of Melipona scutellaris Latreille, 1811 (Hymenoptera, Apidae, Meliponini by mRNA Differential Display

Directory of Open Access Journals (Sweden)

ANA CAROLINA S SIQUIEROLI

2009-01-01

Full Text Available In colonies of Melipona scutellaris Latreille, 1811 workers can be found with four ganglion nerve cells, a morphological characteristic of the queen. It is hypothesized that these workers, called intercastes, or phenocopies, are phenotypically-like workers, but genotypically identical to queens due to this specific trait. Workers with the same number of ganglion as queens seem to be intercastes between queens and workers. Our objective was to analyze the mRNA pro files of workers, queens, and intercastes of M. scutellaris through DDRT-PCR. Three hundred (300 pupae with white eyes were collected and externally identified according to the number of abdominal nerve ganglions: workers (5 ganglions, queens (4 ganglions and intercastes (4 ganglions. The analysis identified differentially expressed transcripts that were present only in workers, but absent in intercastes and queens, confirming the hypothesis, by demonstrating the environmental effect on the queen genotype that generated phenotype-like workers.

The effect of dexamethasone and triiodothyronine on terminal differentiation of primary bovine chondrocytes and chondrogenically differentiated mesenchymal stem cells.

Science.gov (United States)

Randau, Thomas M; Schildberg, Frank A; Alini, Mauro; Wimmer, Matthias D; Haddouti, El-Mustapha; Gravius, Sascha; Ito, Keita; Stoddart, Martin J

2013-01-01

The newly evolved field of regenerative medicine is offering solutions in the treatment of bone or cartilage loss and deficiency. Mesenchymal stem cells, as well as articular chondrocytes, are potential cells for the generation of bone or cartilage. The natural mechanism of bone formation is that of endochondral ossification, regulated, among other factors, through the hormones dexamethasone and triiodothyronine. We investigated the effects of these hormones on articular chondrocytes and chondrogenically differentiated mesenchymal stem cells, hypothesizing that these hormones would induce terminal differentiation, with chondrocytes and differentiated stem cells being similar in their response. Using a 3D-alginate cell culture model, bovine chondrocytes and chondrogenically differentiated stem cells were cultured in presence of triiodothyronine or dexamethasone, and cell proliferation and extracellular matrix production were investigated. Collagen mRNA expression was measured by real-time PCR. Col X mRNA and alkaline phosphatase were monitored as markers of terminal differentiation, a prerequisite of endochondral ossification. The alginate culture system worked well, both for the culture of chondrocytes and for the chondrogenic differentiation of mesenchymal stem cells. Dexamethasone led to an increase in glycosaminoglycan production. Triiodothyronine increased the total collagen production only in chondrocytes, where it also induced signs of terminal differentiation, increasing both collagen X mRNA and alkaline phosphatase activity. Dexamethasone induced terminal differentiation in the differentiated stem cells. The immature articular chondrocytes used in this study seem to be able to undergo terminal differentiation, pointing to their possible role in the onset of degenerative osteoarthritis, as well as their potential for a cell source in bone tissue engineering. When chondrocyte-like cells, after their differentiation, can indeed be moved on towards terminal

Cloning and characterization of maize ZmSPK1, a homologue to ...

African Journals Online (AJOL)

hope&shola

2006-03-15

Mar 15, 2006 ... homologue to nonfermenting1-related protein kinase2 ... RT-PCR analysis showed that the ZmSPK1 expression was induced by mannitol, salt and ... MAPKKK in which each component is activated by .... It has been one of the main ... Protein kinase ATP-binding region signature is shown in gray box.

Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

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Daniela Toro-Ascuy

2016-11-01

Full Text Available The human immunodeficiency virus type-1 (HIV-1 unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1, Staufen double-stranded RNA binding protein 1/2 (STAU1/2, or components of miRNA-induced silencing complex (miRISC and processing bodies (PBs. More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A, allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2, an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries.

From Cell Death to Metabolism: Holin-Antiholin Homologues with New Functions

DEFF Research Database (Denmark)

van den Esker, Marielle H.; Kovács, Ákos T.; Kuipers, Oscar P.

2017-01-01

, but their functions can be different, depending on the species. Using a series of biochemical and genetic approaches, in a recent article in mBio, Charbonnier et al. (mBio 8:e00976-17, 2017, https://doi.org/10.1128/mBio.00976-17) demonstrate that the antiholin homologue in Bacillus subtilis transports pyruvate...

Regulation of elastin synthesis in developing sheep nuchal ligament by elastin mRNA levels

International Nuclear Information System (INIS)

Davidson, J.M.; Smith, K.; Shibahara, S.; Tolstoshev, P.; Crystal, R.G.

1982-01-01

Levels of elastin production in explant culture of fetal sheep nuchal ligament and corresponding levels of translatable elastin mRNA were determined in parallel studies during a period of rapid growth of the embryo. The identity of the explant culture and cell-free proucts was confirmed by peptide mapping, immunoprecipitation, and the characteristic lack of histidine and methionine. Elastin production was quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioimmune precipitation. The translation products could be labeled with methionine only when NH 2 -terminally donated as f-Met-tRNA/sup Met//sub f/. Explant cultures showed a large rise in elastin production from 70 days after conception to 150 days after conception. Cell free translation of RNA demonstrated a parallel in elastin mRNA levels and in elastin mRNA per cell. It appears, therefore, that the marked emphasis the differentiating muchal ligament places on elastin production is modulated, at least in part, by the quantities of available elastin in mRNA

Genome-wide analysis of miRNA and mRNA transcriptomes during amelogenesis.

Science.gov (United States)

Yin, Kaifeng; Hacia, Joseph G; Zhong, Zhe; Paine, Michael L

2014-11-19

In the rodent incisor during amelogenesis, as ameloblast cells transition from secretory stage to maturation stage, their morphology and transcriptome profiles change dramatically. Prior whole genome transcriptome analysis has given a broad picture of the molecular activities dominating both stages of amelogenesis, but this type of analysis has not included miRNA transcript profiling. In this study, we set out to document which miRNAs and corresponding target genes change significantly as ameloblasts transition from secretory- to maturation-stage amelogenesis. Total RNA samples from both secretory- and maturation-stage rat enamel organs were subjected to genome-wide miRNA and mRNA transcript profiling. We identified 59 miRNAs that were differentially expressed at the maturation stage relative to the secretory stage of enamel development (False Discovery Rate (FDR)<0.05, fold change (FC)≥1.8). In parallel, transcriptome profiling experiments identified 1,729 mRNA transcripts that were differentially expressed in the maturation stage compared to the secretory stage (FDR<0.05, FC≥1.8). Based on bioinformatics analyses, 5.8% (629 total) of these differentially expressed genes (DEGS) were highlighted as being the potential targets of 59 miRNAs that were differentially expressed in the opposite direction, in the same tissue samples. Although the number of predicted target DEGs was not higher than baseline expectations generated by examination of stably expressed miRNAs, Gene Ontology (GO) analysis showed that these 629 DEGS were enriched for ion transport, pH regulation, calcium handling, endocytotic, and apoptotic activities. Seven differentially expressed miRNAs (miR-21, miR-31, miR-488, miR-153, miR-135b, miR-135a and miR298) in secretory- and/or maturation-stage enamel organs were confirmed by in situ hybridization. Further, we used luciferase reporter assays to provide evidence that two of these differentially expressed miRNAs, miR-153 and miR-31, are potential

Three TFL1 homologues regulate floral initiation in the biofuel plant Jatropha curcas

Science.gov (United States)

Li, Chaoqiong; Fu, Qiantang; Niu, Longjian; Luo, Li; Chen, Jianghua; Xu, Zeng-Fu

2017-01-01

Recent research revealed that TERMINAL FLOWER 1 (TFL1) homologues are involved in the critical developmental process of floral initiation in several plant species. In this study, the functions of three putative TFL1 homologues (JcTFL1a, JcTFL1b and JcTFL1c) in the biofuel plant Jatropha curcas were analysed using the transgenic approach. JcTFL1b and JcTFL1c, but not JcTFL1a, could complement the TFL1 function and rescue early flowering and determinate inflorescence phenotype in tfl1-14 Arabidopsis mutant, thus suggesting that JcTFL1b and JcTFL1c may be homologues of TFL1. Transgenic Jatropha overexpressing JcTFL1a, JcTFL1b or JcTFL1c showed late flowering, whereas only JcTFL1b and JcTFL1c overexpression delayed flowering in transgenic Arabidopsis. JcTFL1b-RNAi transgenic Jatropha consistently exhibited moderately early flowering phenotype. JcFT and JcAP1 were significantly downregulated in transgenic Jatropha overexpressing JcTFL1a, JcTFL1b or JcTFL1c, which suggested that the late flowering phenotype of these transgenic Jatropha may result from the repressed expression of JcFT and JcAP1. Our results indicate that these three JcTFL1 genes play redundant roles in repressing flowering in Jatropha. PMID:28225036

Behaviour of the homologues of Rf and Db in complexing media

International Nuclear Information System (INIS)

Trubert, D.; Monroy Guzman, F.; Hussonnois, M.; Brillard, L.; Le Naour, C.; Servajean, V.; Constantinescu, O.; Constantinescu, M.; Ardisson, G.; Barci, V.; Weiss, B.

1999-01-01

In order to study the chemical behaviour of the trans-actinide elements, the chemical properties of their most probable homologues have been investigated by ion exchange methods in various complexing media. A new chromatographic method allowing the determination of distribution coefficients in the case o short-lived isotopes has been developed and successfully tested with the RACHEL device. (authors)

The actin homologue MreB organizes the bacterial cell membrane

OpenAIRE

Strahl, Henrik; Bürmann, Frank; Hamoen, Leendert W.

2014-01-01

The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate bacterial cell wall synthesis. We noticed that the MreB cytoskeleton influences fluorescent staining of the cytoplasmic membrane. Detailed analyses combining an array of mutants, using specific lip...

Silver-staining mRNA differential display method and cloning of tumor related genes in HepG2 cell line%银染mRNA差异显示法克隆肝癌相关基因

Institute of Scientific and Technical Information of China (English)

王新; 黄裕新; 闻勤生; 王庆莉

2001-01-01

AIM　To develop mRNA differential display polymerize chain reaction (DD-PCR) method with silver staining and to clone the liver cancer related genes in HepG2 cell line. METHODS　Total RNA was extracted from the HepG2 cells and L02 cells. Their first strains of cDNA were produced by reverse transcription (RT). The cDNA were amplified by PCR using an anchored primer 5 dT11G combined with eight arbitrary primers respectively. The products of PCR were analyzed on a denaturing 60 g*L-1 polyacrylamide gel. The differential DNA bands on gel were seen by silver-staining.RESULTS　Several liver cancer related genes were isolated and identified by the sensitive silver-staining mRNA differential display developed. CONCLUSION　The established mRNA differential display method with silver staining is a simple, efficient means which has higher value for screening and cloning differentially expressed genes.%目的建立银染mRNA差异显示方法，筛选并克隆肝癌相关基因. 方法以建系的人肝癌细胞HepG2和正常的肝细胞L02的总RNA为模板，用5-dT11G锚定引物和8条随机引物（AP1～AP8）组合进行RT-PCR扩增，PCR产物经60 g*L-1尿素变性聚丙烯酰胺凝胶电泳分离后，凝胶银盐染色显示差异的DNA片段. 结果建立了快速敏感的银染mRNA差异显示方法，分离并克隆了一些肝癌相关基因. 结论建立的银染mRNA差异显示法简单、有效，在差异表达基因的筛选中有较高的实用价值.

Identification and characterisation of the angiotensin converting enzyme-3 (ACE3) gene: a novel mammalian homologue of ACE

OpenAIRE

Rella, Monika; Elliot, Joann L; Revett, Timothy J; Lanfear, Jerry; Phelan, Anne; Jackson, Richard M; Turner, Anthony J; Hooper, Nigel M

2007-01-01

Abstract Background Mammalian angiotensin converting enzyme (ACE) plays a key role in blood pressure regulation. Although multiple ACE-like proteins exist in non-mammalian organisms, to date only one other ACE homologue, ACE2, has been identified in mammals. Results Here we report the identification and characterisation of the gene encoding a third homologue of ACE, termed ACE3, in several mammalian genomes. The ACE3 gene is located on the same chromosome downstream of the ACE gene. Multiple ...

An exploration of Glb1 Homologue AntibodyLevels in Children at Increased Risk for Type 1 Diabetes mellitus

Science.gov (United States)

Simpson, M.; Mojibian, M.; Barriga, K.; Scott, F.W.; Fasano, A.; Rewers, M.; Norris, J.M.

2010-01-01

Aims To determine whether Glb1 homologue antibodies are associated with islet autoimmunity (IA) in children at increased risk for type 1 diabetes (T1D), and to investigate their relation with putative environmental correlates of T1D. Methods We selected a sample from the Diabetes Autoimmunity Study in the Young (DAISY), a prospective study of children at increased risk for T1D. Cases were those who were positive for insulin, glutamic acid decarboxylase (GAD), or insulinoma-associated antigen-2 (IA-2) autoantibodies on two consecutive visits and either diagnosed with diabetes mellitus or still autoantibody positive when selected. Controls were from the same increased risk group, of similar age as the cases but negative for autoantibodies. Sera from 91 IA cases and 82 controls were analyzed in a blinded manner for immunoglobulin G (IgG) antibodies to Glb1 homologue by ELISA. Results Adjusting for family history of T1D and HLA-DR4 positivity, Glb1 homologue antibodies were not associated with IA case status (OR: 1.01, 95% CI: 0.99 – 1.03). Adjusting for age, family history of T1D, and HLA-DR4 positivity, Glb1 homologue antibody levels were inversely associated with breast-feeding duration (beta = −0.08, p = 0.001) and directly associated with current intake of foods containing gluten (beta = 0.24, p = 0.007) in IA cases but not in controls. Zonulin, a biomarker of gut permeability, was directly associated with Glb1 homologue antibody levels in cases (beta = 0.73, p = 0.003) but not in controls. Conclusion Differences in correlates of Glb1 antibodies in IA cases and controls suggest an underlying difference in mucosal immune response. PMID:19622083

Generation of human induced pluripotent stem cells using non-synthetic mRNA.

Science.gov (United States)

Rohani, L; Fabian, C; Holland, H; Naaldijk, Y; Dressel, R; Löffler-Wirth, H; Binder, H; Arnold, A; Stolzing, A

2016-05-01

Here we describe some of the crucial steps to generate induced pluripotent stem cells (iPSCs) using mRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribed mRNA. V. virus' 2'-O-Methyltransferase enzyme creates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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.

Biodegradation of diesel fuel by a microbial consortium in the presence of 1-alkoxymethyl-2-methyl-5-hydroxypyridinium chloride homologues

DEFF Research Database (Denmark)

Chrzanowski, L; Stasiewicz, M; Owsianiak, Mikolaj

2009-01-01

hypothesize that in the presence of diesel fuel low-water-soluble ionic liquids may become more toxic to hydrocarbon-degrading microorganisms. In this study the influence of 1-alkoxymethyl-2-methyl-5-hydroxypyridinium chloride homologues (side-chain length from C-3 to C-18) on biodegradation of diesel fuel...... by a bacterial consortium was investigated. Whereas test performed for the consortium cultivated on disodium succinate showed that toxicity of the investigated ionic liquids decreased with increase in side-chain length, only higher homologues (C-8-C-18) caused a decrease in diesel fuel biodegradation......, respectively. We conclude that in the presence of hydrocarbons acting as a solvent, the increased bioavailability of hydrophobic homologues is responsible for the decrease in biodegradation efficiency of diesel fuel....

The effects of valproic acid on the mRNA expression of Natriuretic ...

African Journals Online (AJOL)

Mona Hajikazemi

2017-04-28

Apr 28, 2017 ... Real Time RT-PCR was used to quantify differential mRNA expression of NPR-A and KCNQ1 genes. Two-way ANOVA and bonferroni post-tests were used to analyze data statistically. Results: We showed that VPA treatment inhibits the growth of SW-480 cells more efficiently compared to. HT-29. NPR-A ...

Omega-3 Polyunsaturated Fatty Acids Enhance Neuronal Differentiation in Cultured Rat Neural Stem Cells

Directory of Open Access Journals (Sweden)

Masanori Katakura

2013-01-01

Full Text Available Polyunsaturated fatty acids (PUFAs can induce neurogenesis and recovery from brain diseases. However, the exact mechanisms of the beneficial effects of PUFAs have not been conclusively described. We recently reported that docosahexaenoic acid (DHA induced neuronal differentiation by decreasing Hes1 expression and increasing p27kip1 expression, which causes cell cycle arrest in neural stem cells (NSCs. In the present study, we examined the effect of eicosapentaenoic acid (EPA and arachidonic acid (AA on differentiation, expression of basic helix-loop-helix transcription factors (Hes1, Hes6, and NeuroD, and the cell cycle of cultured NSCs. EPA also increased mRNA levels of Hes1, an inhibitor of neuronal differentiation, Hes6, an inhibitor of Hes1, NeuroD, and Map2 mRNA and Tuj-1-positive cells (a neuronal marker, indicating that EPA induced neuronal differentiation. EPA increased the mRNA levels of p21cip1 and p27kip1, a cyclin-dependent kinase inhibitor, which indicated that EPA induced cell cycle arrest. Treatment with AA decreased Hes1 mRNA but did not affect NeuroD and Map2 mRNA levels. Furthermore, AA did not affect the number of Tuj-1-positive cells or cell cycle progression. These results indicated that EPA could be involved in neuronal differentiation by mechanisms alternative to those of DHA, whereas AA did not affect neuronal differentiation in NSCs.

A Hexose Transporter Homologue Controls Glucose Repression in the Methylotrophic Yeast Hansenula polymorpha

NARCIS (Netherlands)

Stasyk, Oleh V.; Stasyk, Olena G.; Komduur, Janet; Veenhuis, Marten; Cregg, James M.; Sibirny, Andrei A.

2004-01-01

Peroxisome biogenesis and synthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha are under the strict control of glucose repression. We identified an H. polymorpha glucose catabolite repression gene (HpGCR1) that encodes a hexose transporter homologue. Deficiency in GCR1

PTEN drives Th17 cell differentiation by preventing IL-2 production.

Science.gov (United States)

Kim, Hyeong Su; Jang, Sung Woong; Lee, Wonyong; Kim, Kiwan; Sohn, Hyogon; Hwang, Soo Seok; Lee, Gap Ryol

2017-11-06

T helper 17 (Th17) cells are a CD4 + T cell subset that produces IL-17A to mediate inflammation and autoimmunity. IL-2 inhibits Th17 cell differentiation. However, the mechanism by which IL-2 is suppressed during Th17 cell differentiation remains unclear. Here, we show that phosphatase and tensin homologue (PTEN) is a key factor that regulates Th17 cell differentiation by suppressing IL-2 production. Th17-specific Pten deletion ( Pten fl/fl Il17a cre ) impairs Th17 cell differentiation in vitro and ameliorated symptoms of experimental autoimmune encephalomyelitis (EAE), a model of Th17-mediated autoimmune disease. Mechanistically, Pten deficiency up-regulates IL-2 and phosphorylation of STAT5, but reduces STAT3 phosphorylation, thereby inhibiting Th17 cell differentiation. PTEN inhibitors block Th17 cell differentiation in vitro and in the EAE model. Thus, PTEN plays a key role in Th17 cell differentiation by blocking IL-2 expression. © 2017 Kim et al.

Structural studies on a non-toxic homologue of type II RIPs from ...

Indian Academy of Sciences (India)

Structural studies on a non-toxic homologue of type II RIPs from bitter gourd: Molecular basis of non-toxicity, conformational selection and glycan structure. MS accepted http://www.ias.ac.in/jbiosci. THYAGESHWAR CHANDRAN, ALOK SHARMA and M VIJAYAN. J. Biosci. 40(5), October 2015, 929–941, © Indian Academy of ...

The role of mRNA translation in the adaptation to hypoxia

International Nuclear Information System (INIS)

Koritzinsky, M.; Wouters, B.G.; Koumenis, C.

2003-01-01

Hypoxia commonly occurs in human tumours and is associated with a poor prognosis. We and others have shown that global mRNA translation is rapidly inhibited during hypoxia. However, some mRNAs, such as those coding for HIF-1 α and VEGF, remain efficiently translated. We therefore hypothesize that the inhibition of mRNA translation serves to promote hypoxia tolerance in two ways: i) through conservation of energy and ii) through differential gene expression involved in hypoxia adaptation. We are investigating the mechanisms responsible for the down regulation of protein synthesis during hypoxia, and how specific mRNAs maintain their ability to be translated under such conditions. Our goal is to understand the significance of these regulatory mechanisms for hypoxia tolerance in vitro and tumor growth in vivo. We have previously shown that one mechanism responsible for inhibiting protein synthesis during hypoxia is the activation of PERK, which inhibits the essential translation factor eIF2 α . Here we show that PERK-/- MEFs are not able to inhibit protein synthesis efficiently during hypoxia and are significantly less tolerant to hypoxia than wt cells. We also show that other mechanisms are important for sustained low protein synthesis during chronic hypoxia. We demonstrate that the eIF4F complex is disrupted during prolonged hypoxia, and that this is mediated by 4E-BP1 and 4E-T. eIF4F is essential for translation which is dependent upon the 5'mRNA cap-structure. These studies therefore indicate a switch from the inhibition of all translation through eIF2 α during acute hypoxia, to the inhibition of only cap-dependent translation during chronic hypoxia. This model predicts the differential induction of genes that can be translated cap-independently during chronic hypoxia, which is consistent with the observed differential translation of HIF-1 α and VEGF. The functional significance of the disruption of the eIF4F complex during hypoxia is currently being addressed

Differential between Protein and mRNA Expression of CCR7 and SSTR5 Receptors in Crohn's Disease Patients

Directory of Open Access Journals (Sweden)

Nathalie Taquet

2009-01-01

Full Text Available Crohn's disease (CD is a multifactorial chronic inflammatory bowel disease of unknown cause. The aim of the present study was to explore if mRNA over-expression of SSTR5 and CCR7 found in CD patients could be correlated to respective protein expression. When compared to healthy donors, SSTR5 was over-expressed 417 ± 71 times in CD peripheral blood mononuclear cells (PBMCs. Flow cytometry experiments showed no correlation between mRNA and protein expression for SSTR5 in PBMCs. In an attempt to find a reason of such a high mRNA expression, SSTR5 present on CD PBMCs were tested and found as biologically active as on healthy cells. In biopsies of CD intestinal tissue, SSTR5 was not over-expressed but CCR7, unchanged in PBMCs, was over-expressed by 10 ± 3 times in the lamina propria. Confocal microscopy showed a good correlation of CCR7 mRNA and protein expression in CD intestinal biopsies. Our data emphasize flow and image cytometry as impossible to circumvent in complement to molecular biology so to avoid false interpretation on receptor expressions. Once confirmed by further large-scale studies, our preliminary results suggest a role for SSTR5 and CCR7 in CD pathogenesis.

The CD11a partner in Sus scrofa lymphocyte function-associated antigen-1 (LFA-1: mRNA cloning, structure analysis and comparison with mammalian homologues

Directory of Open Access Journals (Sweden)

Thomas Anne VT

2005-10-01

Full Text Available Abstract Background Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alphaLbeta2, the most abundant and widely expressed beta2-integrin, is required for many cellular adhesive interactions during the immune response. Many studies have shown that LFA-1 is centrally involved in the pathogenesis of several diseases caused by Repeats-in-toxin (RTX -producing bacteria. Results The porcine-LFA-1 CD11a (alpha subunit coding sequence was cloned, sequenced and compared with the available mammalian homologues in this study. Despite some focal differences, it shares all the main characteristics of these latter. Interestingly, as in sheep and humans, an allelic variant with a triplet insertion resulting in an additional Gln-744 was consistently identified, which suggests an allelic polymorphism that might be biologically relevant. Conclusion Together with the pig CD18-encoding cDNA, which has been available for a long time, the sequence data provided here will allow the successful expression of porcine CD11a, thus giving the first opportunity to express the Sus scrofa beta2-integrin LFA-1 in vitro as a tool to examine the specificities of inflammation in the porcine species.

Regulation of mRNA translation influences hypoxia tolerance

International Nuclear Information System (INIS)

Koritzinsky, M.; Wouters, B.G.; Koumenis, C.

2003-01-01

Hypoxia is a heterogenous but common characteristic of human tumours and poor oxygenation is associated with poor prognosis. We believe that the presence of viable hypoxic tumor cells reflects in part an adaptation and tolerance of these cells to oxygen deficiency. Since oxidative phosphorylation is compromized during hypoxia, adaptation may involve both the upregulation of glycolysis as well as downregulation of energy consumption. mRNA translation is one of the most energy costly cellular processes, and we and others have shown that global mRNA translation is rapidly inhibited during hypoxia. However, some mRNAs, including those coding for HIF-1 α and VEGF, remain efficiently translated during hypoxia. Clearly, the mechanisms responsible for the overall inhibition of translation during hypoxia does not compromize the translation of certain hypoxia-induced mRNA species. We therefore hypothesize that the inhibition of mRNA translation serves to promote hypoxia tolerance in two ways: i) through conservation of energy and ii) through differential gene expression involved in hypoxia adaptation. We have recently identified two pathways that are responsible for the global inhibition of translation during hypoxia. The phosphorylation of the eukaryotic initiation factor eIF2 α by the ER resident kinase PERK results in down-regulation of protein synthesis shortly after the onset of hypoxia. In addition, the initiation complex eIF4F is disrupted during long lasting hypoxic conditions. The identification of the molecular pathways responsible for the inhibition of overall translation during hypoxia has rendered it possible to investigate their importance for hypoxia tolerance. We have found that mouse embryo fibroblasts that are knockout for PERK and therefore not able to inhibit protein synthesis efficiently during oxygen deficiency are significantly less tolerant to hypoxia than their wildtype counterparts. We are currently also investigating the functional significance

Molecular cloning and expression of the human homologue of the murine gene encoding myeloid leukemia-inhibitory factor

International Nuclear Information System (INIS)

Gough, N.M.; Gearing, D.P.; King, J.A.; Willson, T.A.; Hilton, D.J.; Nicola, N.A.; Metcalf, D.

1988-01-01

A human homologue of the recently cloned murine leukemia-inhibitory factor (LIF) gene was isolated from a genomic library by using the marine cDNA as a hybridization probe. The nucleotide sequence of the human gene indicated that human LIF has 78% amino acid sequence identity with murine LIF, with no insertions or deletions, and that the region of the human gene encoding the mature protein has one intervening sequence. After oligonucleotide-mediated mutagenesis, the mature protein-coding region of the LIF gene was introduced into the yeast expression vector YEpsec1. Yeast cells transformed with the resulting recombinant could be induced with galactose to produce high levels of a factor that induced the differentiation of murine M1 leukemic cells in a manner analogous to murine LIF. This factor competed with 125 I-labeled native murine LIF for binding to specific cellular receptors on murine cells, compatible with a high degree of structural similarity between the murine and human factors

Novel subtractive transcription-based amplification of mRNA (STAR method and its application in search of rare and differentially expressed genes in AD brains

Directory of Open Access Journals (Sweden)

Walker P Roy

2006-11-01

Full Text Available Abstract Background Alzheimer's disease (AD is a complex disorder that involves multiple biological processes. Many genes implicated in these processes may be present in low abundance in the human brain. DNA microarray analysis identifies changed genes that are expressed at high or moderate levels. Complementary to this approach, we described here a novel technology designed specifically to isolate rare and novel genes previously undetectable by other methods. We have used this method to identify differentially expressed genes in brains affected by AD. Our method, termed Subtractive Transcription-based Amplification of mRNA (STAR, is a combination of subtractive RNA/DNA hybridization and RNA amplification, which allows the removal of non-differentially expressed transcripts and the linear amplification of the differentially expressed genes. Results Using the STAR technology we have identified over 800 differentially expressed sequences in AD brains, both up- and down- regulated, compared to age-matched controls. Over 55% of the sequences represent genes of unknown function and roughly half of them were novel and rare discoveries in the human brain. The expression changes of nearly 80 unique genes were further confirmed by qRT-PCR and the association of additional genes with AD and/or neurodegeneration was established using an in-house literature mining tool (LitMiner. Conclusion The STAR process significantly amplifies unique and rare sequences relative to abundant housekeeping genes and, as a consequence, identifies genes not previously linked to AD. This method also offers new opportunities to study the subtle changes in gene expression that potentially contribute to the development and/or progression of AD.

Gametophyte differentiation and imprinting control in plants: Crosstalk between RBR and chromatin.

Science.gov (United States)

Johnston, Amal J; Gruissem, Wilhelm

2009-01-01

The Retinoblastoma (pRb) pathway has been implicated as a convergent regulatory unit in the control of cell cycle and disease. We have shown that a crosstalk between RETINOBLASTOMA RELATED (RBR), the Arabidopsis homologue of pRb, and the genes encoding proteins of the chromatin complexes involved in DNA or histone methylation, controls gametophytic and post-fertilization differentiation events and a subset of imprinting effects. We describe here a plausible model that incorporates several components of the plant Retinoblastoma pathway, thus offering a novel paradigm that merges the traditional cell cycle and the chromatin components in the control of cell differentiation and imprinting.

TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast

OpenAIRE

Helliwell, S. B.; Wagner, P.; Kunz, J.; Deuter-Reinhard, M.; Henriquez, R.; Hall, M. N.

1994-01-01

The Saccharomyces cerevisiae genes TOR1 and TOR2 were originally identified by mutations that confer resistance to the immunosuppressant rapamycin. TOR2 was previously shown to encode an essential 282-kDa phosphatidylinositol kinase (PI kinase) homologue. The TOR1 gene product is also a large (281 kDa) PI kinase homologue, with 67% identity to TOR2. TOR1 is not essential, but a TOR1 TOR2 double disruption uniquely confers a cell cycle (G1) arrest as does exposure to rapamycin; disruption of T...

Hypophyseal corticosteroids stimulate somatotrope differentiation in the embryonic chicken pituitary gland.

Science.gov (United States)

Zheng, Jun; Takagi, Hiroyasu; Tsutsui, Chihiro; Adachi, Akihito; Sakai, Takafumi

2008-03-01

Although it is known that glucocorticoids induce differentiation of growth hormone (GH)-producing cells in rodents and birds, the effect of mineralocorticoids on GH mRNA expression and the origin of corticosteroids affecting somatotrope differentiation have not been elucidated. In this study, we therefore carried out experiments to determine the effect of mineralocorticoids on GH mRNA expression in the chicken anterior pituitary gland in vitro and to determine whether corticosteroids are synthesized in the chicken embryonic pituitary gland. In a pituitary culture experiment with E11 embryos, both corticosterone and aldosterone stimulated GH mRNA expression and increased the number of GH cells in both lobes of the pituitary gland in a dose-dependent manner. These effects of the corticosteroids were significantly reversed by pretreatment with mifepristone, a glucocorticoid receptor (GR) antagonist, or spironolactone, a mineralocorticoid receptor (MR) antagonist. Interestingly, an in vitro serum-free culture experiment with an E11 pituitary gland showed that the GH mRNA level spontaneously increased during cultivation for 2 days without any extra stimulation, and this increase in GH mRNA level was completely suppressed by metyrapone, a corticosterone-producing enzyme P450C11 inhibitor. Moreover, progesterone, the corticosterone precursor, also stimulated GH mRNA expression in the cultured chicken pituitary gland, and this effect was blocked by pretreatment with metyrapone. We also detected mRNA expression of enzymes of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase1 (3beta-HSD1) in the developmental chicken pituitary gland from E14 and E18, respectively. These results suggest that mineralocorticoids as well as glucocorticoids can stimulate GH mRNA expression and that corticosteroids generated in the embryonic pituitary gland by intrinsic steroidogenic enzymes stimulate somatotrope differentiation.

Crystal structure of myotoxin-II: a myotoxic phospholipase A2 - homologue from Bothrops moojeni venom

International Nuclear Information System (INIS)

Azevedo, W.F.; Ward, R.J.; Lombardi, F.R.; Arni, R.K.; Soares, A.M.; Giglio, J.R.; Fontes, M.R.M.

1997-01-01

Full text. Phospho lipases A2 (PLA 2 ; E C 3.1.1.4, phosphatides s n-2 acyl hydrolases) hydrolysis the s n-2 ester bond of phospholipids showing enhanced activity at lamellar or membrane surfaces. Intracellular PLA 2 s are involved at phospholipid metabolism and signal transduction, whereas extracellular PLA 2 s are found in mammalian pancreatic juices, the venoms of snakes, lizards and insects. Based on their high primary sequence similarity, extracellular PLA 2 s are separated into Classes I, II and III. Class II PLA 2 s are found in snake venoms of Crotalidae an Viperidae species, and include the sub-family of Lys PLA 2 s homologue. he coordination of the Ca 2+ ion in the PLA 2 calcium-binding loop includes and aspartate at position 49. In the catalytically active PLA 2 s, this calcium ion plays a critical role in the stabilization of the tetrahedral transition state intermediate in the catalytic mechanism. The conservative substitution Asp49-Lys results in a decreased calcium affinity with a concomitant loss of catalytic activity, and naturally occurring PLA 2 s-homologues showing the same substitution are catalytically inactive. However, the Lys PLA 2 s possess cytolytic and myotoxic activities and furthermore retain the ability to disrupt the integrity of both plasma membranes and model lipid layers by a ca 2+ -independent mechanism for which there is no evidence of lipid hydrolysis. Lys 49 PLA 2 homologues have been isolated from several Bothrops spp. venoms including B. moojeni. Therefore, in order to improve our understanding of the molecular basis of the myotoxic and Ca 2+ independent membrane damaging activities we have determined the crystal structure of MjTX-II, a Lys 49 homologue from the venom of B. moojeni. The model presented has been determined at 2.0 A resolution and refined to a crystallographic residual of 19.7% (R f ree=28.1%). (author)

Changes in the expression of collagen genes show two stages in chondrocyte differentiation in vitro

OpenAIRE

1988-01-01

This report deals with the quantitation of both mRNA and transcription activity of type I collagen gene and of three cartilage-specific collagens (types II, IX, and X) during in vitro differentiation of chick chondrocytes. Differentiation was obtained by transferal to suspension culture of dedifferentiated cells passaged for 3 wk as adherent cells. The type I collagen mRNA, highly represented in the dedifferentiated cells, rapidly decreased during chondrocyte differentiation. On the contrary,...

Differential expression of ozone-induced gene during exposures to ...

African Journals Online (AJOL)

Differential expression of ozone-induced gene during exposures to salt stress in Polygonum sibiricum Laxm leaves, stem and underground stem. ... PcOZI-1 mRNA in untreated plants was detected at low levels in underground stem, leaves and at higher levels in stem. PcOZI-1 mRNA accumulation was transiently induced ...

Structure of HLA-A*1101 in complex with a hepatitis B peptide homologue

DEFF Research Database (Denmark)

Blicher, Thomas; Kastrup, Jette Sandholm; Pedersen, Lars Østergaard

2006-01-01

A high-resolution structure of the human MHC-I molecule HLA-A*1101 is presented in which it forms a complex with a sequence homologue of a peptide that occurs naturally in hepatitis B virus DNA polymerase. The sequence of the bound peptide is AIMPARFYPK, while that of the corresponding natural...

Characterization of Two 20kDa-Cement Protein (cp20k) Homologues in Amphibalanus amphitrite

KAUST Repository

He, Li-Sheng

2013-05-22

The barnacle, Amphibalanus amphitrite, is a common marine fouling organism. Understanding the mechanism of barnacle adhesion will be helpful in resolving the fouling problem. Barnacle cement is thought to play a key role in barnacle attachment. Although several adult barnacle cement proteins have been identified in Megabalanus rosa, little is known about their function in barnacle settlement. In this study, two homologous 20k-cement proteins (cp20k) in Amphibalanus amphitrite, named Bamcp20k-1 and Bamcp20k-2, were characterized. The two homologues share primary sequence structure with proteins from other species including Megabalanus rosa and Fistulobalanus albicostatus. The conserved structure included repeated Cys domains and abundant charged amino acids, such as histidine. In this study we demonstrated that Bamcp20k-1 localized at the α secretory cells in the cyprid cement gland, while Bamcp20k-2 localized to the β secretory cells. The differential localizations suggest differential regulation for secretion from the secretory cells. Both Bamcp20k-1 and Bamcp20k-2 from cyprids dissolved in PBS. However, adult Bamcp20k-2, which was dominant in the basal shell of adult barnacles, was largely insoluble in PBS. Solubility increased in the presence of the reducing reagent Dithiothreitol (DTT), suggesting that the formation of disulfide bonds plays a role in Bamcp20k-2 function. In comparison, Bamcp20k-1, which was enriched in soft tissue, could not be easily detected in the shell and base by Western blot and easily dissolved in PBS. These differential solubilities and localizations indicate that Bamcp20k-1 and Bamcp20k-2 have distinct functions in barnacle cementing. © 2013 He et al.

Characterization of Two 20kDa-Cement Protein (cp20k) Homologues in Amphibalanus amphitrite

KAUST Repository

He, Li-Sheng; Zhang, Gen; Qian, Pei-Yuan

2013-01-01

The barnacle, Amphibalanus amphitrite, is a common marine fouling organism. Understanding the mechanism of barnacle adhesion will be helpful in resolving the fouling problem. Barnacle cement is thought to play a key role in barnacle attachment. Although several adult barnacle cement proteins have been identified in Megabalanus rosa, little is known about their function in barnacle settlement. In this study, two homologous 20k-cement proteins (cp20k) in Amphibalanus amphitrite, named Bamcp20k-1 and Bamcp20k-2, were characterized. The two homologues share primary sequence structure with proteins from other species including Megabalanus rosa and Fistulobalanus albicostatus. The conserved structure included repeated Cys domains and abundant charged amino acids, such as histidine. In this study we demonstrated that Bamcp20k-1 localized at the α secretory cells in the cyprid cement gland, while Bamcp20k-2 localized to the β secretory cells. The differential localizations suggest differential regulation for secretion from the secretory cells. Both Bamcp20k-1 and Bamcp20k-2 from cyprids dissolved in PBS. However, adult Bamcp20k-2, which was dominant in the basal shell of adult barnacles, was largely insoluble in PBS. Solubility increased in the presence of the reducing reagent Dithiothreitol (DTT), suggesting that the formation of disulfide bonds plays a role in Bamcp20k-2 function. In comparison, Bamcp20k-1, which was enriched in soft tissue, could not be easily detected in the shell and base by Western blot and easily dissolved in PBS. These differential solubilities and localizations indicate that Bamcp20k-1 and Bamcp20k-2 have distinct functions in barnacle cementing. © 2013 He et al.

The utility of peripheral thyrotropin receptor mRNA in the management of differentiated thyroid cancer.

Science.gov (United States)

Aliyev, Altay; Soundararajan, Saranya; Bucak, Emre; Gupta, Manjula; Hatipoglu, Betul; Nasr, Christian; Siperstein, Allan; Berber, Eren

2015-10-01

Our aim was to analyze the utility of peripheral thyrotropin receptor (TSHR) messenger RNA (mRNA) in predicting and detecting the recurrence of differentiated thyroid cancer. Peripheral blood TSHR-mRNA was obtained in 103 patients before and after total thyroidectomy. An analysis was performed to correlate peripheral blood TSHR-mRNA concentration with oncologic outcomes. Tumor types were papillary (n = 92), follicular (n = 9) and Hürthle cell (n = 2) cancer. Preoperative TSHR-mRNA was ≥1.02 ng/μg in 85% (88/103). On follow-up (median 48 months), 10 patients (10 %) developed recurrence. Recurrence rate in patients with a preoperative TSHR-mRNA ≥ 1.02 ng/μg was 11% versus 0% in those with a lesser concentration. TSHR-mRNA correctly diagnosed 7 (70%) of 10 recurrences. Of 19 patients with positive thyroglobulin (Tg) antibodies, TSHR-mRNA confirmed disease-free status in 12 (63%) and recurrence in 1 (5%). For Tg, TSHR-mRNA and whole-body radioactive iodine scan, sensitivity was 70%, 70%, and 75%; specificity 94%, 76%, 97%; PPV 54%, 24%, and 67%; and NPV 97%, 96%, and 98%, respectively, in detecting recurrent disease. This study shows that patients with preoperative TSHR-mRNA ≥1.02 ng/μg may be at a greater risk for recurrence compared with those with a lesser concentration. In the presence of Tg antibodies, TSHR-mRNA accurately predicted disease status in 68% of patients. Its overall performance in detecting recurrence was similar to Tg and whole-body radioactive iodine scan, albeit with lower specificity and PPV. Copyright © 2015 Elsevier Inc. All rights reserved.

Bropirimine inhibits osteoclast differentiation through production of interferon-β

International Nuclear Information System (INIS)

Suzuki, Hiroaki; Mochizuki, Ayako; Yoshimura, Kentaro; Miyamoto, Yoichi; Kaneko, Kotaro; Inoue, Tomio; Chikazu, Daichi; Takami, Masamichi; Kamijo, Ryutaro

2015-01-01

Bropirimine is a synthetic agonist for toll-like receptor 7 (TLR7). In this study, we investigated the effects of bropirimine on differentiation and bone-resorbing activity of osteoclasts in vitro. Bropirimine inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) induced by receptor activator of nuclear factor κB ligand (RANKL) in a concentration-dependent manner. Furthermore, it suppressed the mRNA expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), a master transcription factor for osteoclast differentiation, without affecting BMM viability. Bropirimine also inhibited osteoclast differentiation induced in co-cultures of mouse bone marrow cells (BMCs) and mouse osteoblastic UAMS-32 cells in the presence of activated vitamin D_3. Bropirimine partially suppressed the expression of RANKL mRNA in UAMS-32 cells induced by activated vitamin D_3. Finally, the anti-interferon-β (IFN-β) antibody restored RANKL-dependent differentiation of BMMs into osteoclasts suppressed by bropirimine. These results suggest that bropirimine inhibits differentiation of osteoclast precursor cells into osteoclasts via TLR7-mediated production of IFN-β.

Bropirimine inhibits osteoclast differentiation through production of interferon-β

Energy Technology Data Exchange (ETDEWEB)

Suzuki, Hiroaki [Department of Biochemistry, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Mochizuki, Ayako [Department of Oral Physiology, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Yoshimura, Kentaro; Miyamoto, Yoichi [Department of Biochemistry, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Kaneko, Kotaro [Department of Biochemistry, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Department of Oral and Maxillofacial Surgery, Tokyo Medical University, Tokyo 160-0023 (Japan); Inoue, Tomio [Department of Oral Physiology, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Chikazu, Daichi [Department of Oral and Maxillofacial Surgery, Tokyo Medical University, Tokyo 160-0023 (Japan); Takami, Masamichi [Department of Pharmacology, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Kamijo, Ryutaro, E-mail: kamijor@dent.showa-u.ac.jp [Department of Biochemistry, Showa University School of Dentistry, Tokyo 142-8555 (Japan)

2015-11-06

Bropirimine is a synthetic agonist for toll-like receptor 7 (TLR7). In this study, we investigated the effects of bropirimine on differentiation and bone-resorbing activity of osteoclasts in vitro. Bropirimine inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) induced by receptor activator of nuclear factor κB ligand (RANKL) in a concentration-dependent manner. Furthermore, it suppressed the mRNA expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), a master transcription factor for osteoclast differentiation, without affecting BMM viability. Bropirimine also inhibited osteoclast differentiation induced in co-cultures of mouse bone marrow cells (BMCs) and mouse osteoblastic UAMS-32 cells in the presence of activated vitamin D{sub 3}. Bropirimine partially suppressed the expression of RANKL mRNA in UAMS-32 cells induced by activated vitamin D{sub 3}. Finally, the anti-interferon-β (IFN-β) antibody restored RANKL-dependent differentiation of BMMs into osteoclasts suppressed by bropirimine. These results suggest that bropirimine inhibits differentiation of osteoclast precursor cells into osteoclasts via TLR7-mediated production of IFN-β.

Crystal structure of a bacterial homologue of glucose transporters GLUT1-4.

Science.gov (United States)

Sun, Linfeng; Zeng, Xin; Yan, Chuangye; Sun, Xiuyun; Gong, Xinqi; Rao, Yu; Yan, Nieng

2012-10-18

Glucose transporters are essential for metabolism of glucose in cells of diverse organisms from microbes to humans, exemplified by the disease-related human proteins GLUT1, 2, 3 and 4. Despite rigorous efforts, the structural information for GLUT1-4 or their homologues remains largely unknown. Here we report three related crystal structures of XylE, an Escherichia coli homologue of GLUT1-4, in complex with d-xylose, d-glucose and 6-bromo-6-deoxy-D-glucose, at resolutions of 2.8, 2.9 and 2.6 Å, respectively. The structure consists of a typical major facilitator superfamily fold of 12 transmembrane segments and a unique intracellular four-helix domain. XylE was captured in an outward-facing, partly occluded conformation. Most of the important amino acids responsible for recognition of D-xylose or d-glucose are invariant in GLUT1-4, suggesting functional and mechanistic conservations. Structure-based modelling of GLUT1-4 allows mapping and interpretation of disease-related mutations. The structural and biochemical information reported here constitutes an important framework for mechanistic understanding of glucose transporters and sugar porters in general.

Full-length mRNA sequencing uncovers a widespread coupling between transcription initiation and mRNA processing.

Science.gov (United States)

Anvar, Seyed Yahya; Allard, Guy; Tseng, Elizabeth; Sheynkman, Gloria M; de Klerk, Eleonora; Vermaat, Martijn; Yin, Raymund H; Johansson, Hans E; Ariyurek, Yavuz; den Dunnen, Johan T; Turner, Stephen W; 't Hoen, Peter A C

2018-03-29

The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. In MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells. Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.

Expression and significance of cyclooxygenase-2 mRNA in benign and malignant ascites

Science.gov (United States)

Lu, Jing; Li, Xiao-Feng; Kong, Li-Xia; Ma, Lin; Liao, Su-Huan; Jiang, Chang-You

2013-01-01

AIM: To investigate the mRNA expression of cyclooxygensae-2 (COX-2) in benign and malignant ascites, and to explore the difference in COX-2 mRNA expression among different diseases. METHODS: A total of 36 samples were collected from the Fifth Affiliated Hospital of Sun Yat-Sen University and divided into two experimental groups: benign ascites (n = 21) and malignant ascites (n = 15). Benign ascites included cirrhotic ascites (n = 10) and tuberculous ascites (n = 5). Malignant ascites included oophoroma (n = 7), cancer of colon (n = 5), cancer of the liver (n = 6), gastric cancer (n = 2), and bladder carcinoma (n = 1). The mRNA expression of COX-2 in ascites was examined with reverse transcriptase polymerase chain reaction (RT-PCR) technology, and the positive rate of COX-2 mRNA was compared between different diseases. RESULTS: The positive rate of COX-2 mRNA in malignant ascites was 42.9% (9/21), which was significantly higher than in benign ascites, 6.7% (1/15), difference being significant between these two groups (χ2 = 4.051, P = 0.044). The proportion of the positive rate in the malignant ascites was as follows: ovarian cancers 57.1% (4/7), colon cancer 40.0% (2/5), liver cancer 33.3% (2/6), gastric cancer 50.0% (1/2), and bladder cancer 0.00% (0/1). However, there was no significant difference in COX-2 mRNA expression among various tumors with malignant ascites (χ2 = 1.614, P = 0.806). Among the benign ascites, COX-2 mRNA levels were different between the tuberculous ascites (0/5) and cirrhotic ascites (1/10), but there was no significant difference (P = 1.000). CONCLUSION: COX-2 mRNA, detected by RT-PCR, is useful in the differential diagnosis of benign and malignant ascites, which also has potential value in the clinical diagnosis of tumors. PMID:24187465

Short- and medium-chain chlorinated paraffins in biota from the European Arctic -- differences in homologue group patterns.

Science.gov (United States)

Reth, Margot; Ciric, Anita; Christensen, Guttorm N; Heimstad, Eldbjørg S; Oehme, Michael

2006-08-15

Congener and homologue group patterns of chlorinated paraffins (CPs) in biota can be influenced by different processes, but these are not well studied yet. Short- (SCCPs) and medium-chain chlorinated paraffins (MCCPs) were quantified in liver from Arctic char and seabirds (little auk and kittiwake) collected at Bear Island (European Arctic) as well as in cod from Iceland and Norway. CP concentrations were between 5 and 88 ng/g wet weight (ww) for SCCPs and between 5 and 55 ng/g ww for MCCPs with one exception of 370 ng/g measured in a liver sample from little auk. The SCCP homologue group patterns were compared with those of technical mixtures and of SCCPs present in cod liver from the Baltic Sea. The latter showed a more common SCCP homologue distribution (sum of C(11) and C(12)>60%) in contrast to cod liver from the Northwest of Europe, which had a high abundance of C(10) and C(12) congeners. Seabirds from Bear Island contained an equally distributed SCCP homologue group pattern. In Arctic char, the SCCP distribution was closer to technical products, but with a high proportion (average of 18.9%) of C(10) congeners. A comparison of C(10)/C(12) ratios confirmed the higher abundance of C(10) congeners in samples from higher latitudes. For the first time, MCCPs could be detected in Arctic samples. The average proportion of C(14) congeners was 65.8%. The C(14)/C(15) abundance ratio was similar to technical mixtures. High-chlorinated CPs (Cl(>7)) were also detectable. The average chlorine content of the SCCPs was 61.9% (59.0-63.3%), and that of the MCCPs 55.8% (54.5-57.4%).

mRNA Cancer Vaccines-Messages that Prevail.

Science.gov (United States)

Grunwitz, Christian; Kranz, Lena M

2017-01-01

During the last decade, mRNA became increasingly recognized as a versatile tool for the development of new innovative therapeutics. Especially for vaccine development, mRNA is of outstanding interest and numerous clinical trials have been initiated. Strikingly, all of these studies have proven that large-scale GMP production of mRNA is feasible and concordantly report a favorable safety profile of mRNA vaccines. Induction of T-cell immunity is a multi-faceted process comprising antigen acquisition, antigen processing and presentation, as well as immune stimulation. The effectiveness of mRNA vaccines is critically dependent on making the antigen(s) of interest available to professional antigen-presenting cells, especially DCs. Efficient delivery of mRNA into DCs in vivo remains a major challenge in the mRNA vaccine field. This review summarizes the principles of mRNA vaccines and highlights the importance of in vivo mRNA delivery and recent advances in harnessing their therapeutic potential.

Principles of mRNA transport in yeast.

Science.gov (United States)

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.

Assessment of flhDC mRNA levels in Serratia liquefaciens swarm cells

DEFF Research Database (Denmark)

Tolker-Nielsen, Tim; Christensen, Allan Beck; Holmstrøm, K.

2000-01-01

We reported previously that artificial overexpression of the flhDC operon in liquid-grown Serratia liquefaciens resulted in the formation of filamentous, multinucleated, and hyperflagellated cells that were indistinguishable from surface-induced swarm cells (L. Eberl, G. Christiansen, S. Molin, a......, vegetative cells. This suggests that surface-induced S. liquefaciens swarm cell differentiation, although dependent on flhDC gene expression, does not occur through elevated flhDC mRNA levels....

Genetic Modification of Human Pancreatic Progenitor Cells Through Modified mRNA.

Science.gov (United States)

Lu, Song; Chow, Christie C; Zhou, Junwei; Leung, Po Sing; Tsui, Stephen K; Lui, Kathy O

2016-01-01

In this chapter, we describe a highly efficient genetic modification strategy for human pancreatic progenitor cells using modified mRNA-encoding GFP and Neurogenin-3. The properties of modified mRNA offer an invaluable platform to drive protein expression, which has broad applicability in pathway regulation, directed differentiation, and lineage specification. This approach can also be used to regulate expression of other pivotal transcription factors during pancreas development and might have potential therapeutic values in regenerative medicine.

Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis: interferon-beta treatment increases IL-10 mRNA expression while reducing IL-23 mRNA expression

DEFF Research Database (Denmark)

Krakauer, M.; Sorensen, P.; Khademi, M.

2008-01-01

volunteers served to confirm initial findings. mRNA was analyzed by real-time reverse transcriptase polymerase chain reaction (PCR). RESULTS: We found elevated expression of interleukin (IL)-23 and IL-10 in untreated MS patients. IFN-beta therapy increased IL-10 and decreased IL-23 expression independently...... of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

Serotonin receptor, SERT mRNA and correlations with symptoms in males with alcohol dependence and suicide.

Science.gov (United States)

Thompson, P M; Cruz, D A; Olukotun, D Y; Delgado, P L

2012-09-01

This study tested the hypothesis that abnormalities in components of the serotonin (5HT) system in the prefrontal cortex are associated with suicide in alcohol-dependent subjects. Second, we assessed the relationship of lifetime impulsivity and mood symptoms with prefrontal cortex 5-HT measures. Tissue was obtained from Brodmann's areas (BA) 9 and 24 in postmortem samples of individuals who were alcohol dependent with suicide (n = 5), alcohol dependent without suicide (n = 9) and normal controls (n = 5). Serotonin receptor (5HT) and serotonin reuptake transporter (SERT) mRNA were measured. Interviews with next of kin estimated lifetime impulsivity and mood symptoms in the last week of life. Serotonin receptor 1A (5HT1A) mRNA in BA 9 was elevated in the alcohol dependence without suicide group compared with controls. In the alcohol dependence with suicide group, anxiety symptoms were associated with decreased BA 24 SERT mRNA and depressive symptoms with BA 9 5HT1A mRNA expression. In the alcohol dependent only group impulsivity is correlated with increased BA 9, and BA 24 serotonin receptor 2A mRNA. Our data suggest region-specific change, rather than global serotonin blunting is involved in alcohol dependence and suicide. It also suggests that symptoms are differentially influenced by prefrontal cortex serotonin receptor mRNA levels. © 2011 John Wiley & Sons A/S.

Hypoxia inhibits hypertrophic differentiation and endochondral ossification in explanted tibiae.

Directory of Open Access Journals (Sweden)

Jeroen C H Leijten

Full Text Available Hypertrophic differentiation of growth plate chondrocytes induces angiogenesis which alleviates hypoxia normally present in cartilage. In the current study, we aim to determine whether alleviation of hypoxia is merely a downstream effect of hypertrophic differentiation as previously described or whether alleviation of hypoxia and consequent changes in oxygen tension mediated signaling events also plays an active role in regulating the hypertrophic differentiation process itself.Fetal mouse tibiae (E17.5 explants were cultured up to 21 days under normoxic or hypoxic conditions (21% and 2.5% oxygen respectively. Tibiae were analyzed on growth kinetics, histology, gene expression and protein secretion.The oxygen level had a strong influence on the development of explanted fetal tibiae. Compared to hypoxia, normoxia increased the length of the tibiae, length of the hypertrophic zone, calcification of the cartilage and mRNA levels of hypertrophic differentiation-related genes e.g. MMP9, MMP13, RUNX2, COL10A1 and ALPL. Compared to normoxia, hypoxia increased the size of the cartilaginous epiphysis, length of the resting zone, calcification of the bone and mRNA levels of hyaline cartilage-related genes e.g. ACAN, COL2A1 and SOX9. Additionally, hypoxia enhanced the mRNA and protein expression of the secreted articular cartilage markers GREM1, FRZB and DKK1, which are able to inhibit hypertrophic differentiation.Collectively our data suggests that oxygen levels play an active role in the regulation of hypertrophic differentiation of hyaline chondrocytes. Normoxia stimulates hypertrophic differentiation evidenced by the expression of hypertrophic differentiation related genes. In contrast, hypoxia suppresses hypertrophic differentiation of chondrocytes, which might be at least partially explained by the induction of GREM1, FRZB and DKK1 expression.

mRNA expression of genes regulating lipid metabolism in ringed seals (Pusa hispida) from differently polluted areas

International Nuclear Information System (INIS)

Castelli, Martina Galatea; Rusten, Marte; Goksøyr, Anders; Routti, Heli

2014-01-01

Highlights: •Genes regulating lipid metabolism were studied in ringed seals. •We compared highly contaminated Baltic seals and less contaminated Svalbard seals. •mRNA expression of hepatic PPARγ was higher in the Baltic seals. •mRNA expression of adipose PPARγ target genes was higher in the Baltic seals. •Contaminant exposure may affect lipid metabolism in the Baltic ringed seals. -- Abstract: There is a growing concern about the ability of persistent organic pollutants (POPs) to influence lipid metabolism. Although POPs are found at high concentrations in some populations of marine mammals, for example in the ringed seal (Pusa hispida) from the Baltic Sea, little is known about the effects of POPs on their lipid metabolism. An optimal regulation of lipid metabolism is crucial for ringed seals during the fasting/molting season. This is a physiologically stressful period, during which they rely on the energy stored in their fat reserves. The mRNA expression levels for seven genes involved in lipid metabolism were analyzed in liver and/or blubber tissue from molting ringed seals from the polluted Baltic Sea and a less polluted reference location, Svalbard (Norway). mRNA expression of genes encoding peroxisome proliferator-activated receptors (PPAR) α and γ and their target genes acyl-coenzyme A oxidase 1 (ACOX1) and cluster of differentiation 36 (CD36) were analyzed in liver. mRNA expression level of genes encoding PPARβ, PPARγ and their target genes encoding fatty acid binding protein 4 (FABP4) and adiponectin (ADIPOQ) were measured in inner and middle blubber layers. In addition, we evaluated the influence of molting status on hepatic mRNA expression of genes encoding PPARs and their target genes in ringed seals from Svalbard. Our results show higher mRNA expression of genes encoding hepatic PPARγ and adipose PPARβ, FABP4, and ADIPOQ in the Baltic seals compared to the Svalbard seals. A positive relationship between mRNA expressions of genes

mRNA expression of genes regulating lipid metabolism in ringed seals (Pusa hispida) from differently polluted areas

Energy Technology Data Exchange (ETDEWEB)

Castelli, Martina Galatea [Norwegian Polar Institute, Fram Centre, 9296 Tromsø (Norway); University of Bergen, Department of Biology, 5020 Bergen (Norway); Rusten, Marte; Goksøyr, Anders [University of Bergen, Department of Biology, 5020 Bergen (Norway); Routti, Heli, E-mail: heli.routti@npolar.no [Norwegian Polar Institute, Fram Centre, 9296 Tromsø (Norway)

2014-01-15

Highlights: •Genes regulating lipid metabolism were studied in ringed seals. •We compared highly contaminated Baltic seals and less contaminated Svalbard seals. •mRNA expression of hepatic PPARγ was higher in the Baltic seals. •mRNA expression of adipose PPARγ target genes was higher in the Baltic seals. •Contaminant exposure may affect lipid metabolism in the Baltic ringed seals. -- Abstract: There is a growing concern about the ability of persistent organic pollutants (POPs) to influence lipid metabolism. Although POPs are found at high concentrations in some populations of marine mammals, for example in the ringed seal (Pusa hispida) from the Baltic Sea, little is known about the effects of POPs on their lipid metabolism. An optimal regulation of lipid metabolism is crucial for ringed seals during the fasting/molting season. This is a physiologically stressful period, during which they rely on the energy stored in their fat reserves. The mRNA expression levels for seven genes involved in lipid metabolism were analyzed in liver and/or blubber tissue from molting ringed seals from the polluted Baltic Sea and a less polluted reference location, Svalbard (Norway). mRNA expression of genes encoding peroxisome proliferator-activated receptors (PPAR) α and γ and their target genes acyl-coenzyme A oxidase 1 (ACOX1) and cluster of differentiation 36 (CD36) were analyzed in liver. mRNA expression level of genes encoding PPARβ, PPARγ and their target genes encoding fatty acid binding protein 4 (FABP4) and adiponectin (ADIPOQ) were measured in inner and middle blubber layers. In addition, we evaluated the influence of molting status on hepatic mRNA expression of genes encoding PPARs and their target genes in ringed seals from Svalbard. Our results show higher mRNA expression of genes encoding hepatic PPARγ and adipose PPARβ, FABP4, and ADIPOQ in the Baltic seals compared to the Svalbard seals. A positive relationship between mRNA expressions of genes

Towards structural studies of the old yellow enzyme homologue SYE4 from Shewanella oneidensis and its complexes at atomic resolution

International Nuclear Information System (INIS)

Elegheert, Jonathan; Hemel, Debbie van den; Dix, Ina; Stout, Jan; Van Beeumen, Jozef; Brigé, Ann; Savvides, Savvas N.

2009-01-01

Of the four old yellow enzyme homologues found in S. oneidensis, SYE4 is the homologue most implicated in resistance to oxidative stress. SYE4 was recombinantly expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Shewanella oneidensis is an environmentally versatile Gram-negative γ-proteobacterium that is endowed with an unusually large proteome of redox proteins. Of the four old yellow enzyme (OYE) homologues found in S. oneidensis, SYE4 is the homologue most implicated in resistance to oxidative stress. SYE4 was recombinantly expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the orthorhombic space group P2 1 2 1 2 1 and were moderately pseudo-merohedrally twinned, emulating a P422 metric symmetry. The native crystals of SYE4 were of exceptional diffraction quality and provided complete data to 1.10 Å resolution using synchrotron radiation, while crystals of the reduced enzyme and of the enzyme in complex with a wide range of ligands typically led to high-quality complete data sets to 1.30–1.60 Å resolution, thus providing a rare opportunity to dissect the structure–function relationships of a good-sized enzyme (40 kDa) at true atomic resolution. Here, the attainment of a number of experimental milestones in the crystallographic studies of SYE4 and its complexes are reported, including isolation of the elusive hydride–Meisenheimer complex

Adipose cell differentiation: evidence for a two-step process in the polyamine-dependent Ob1754 clonal line.

Science.gov (United States)

Amri, E Z; Dani, C; Doglio, A; Etienne, J; Grimaldi, P; Ailhaud, G

1986-01-01

A subclone of preadipocyte Ob17 cells has been isolated (Ob1754 clonal line). Confluent Ob1754 cells treated with an inhibitor of spermidine and spermine synthesis, methylglyoxal bis(guanylhydrazone), were totally dependent upon putrescine addition for the expression of glycerol-3-phosphate dehydrogenase which behaved as a late marker of adipose conversion. Under these conditions, the early expression of lipoprotein lipase during growth arrest remained unchanged. Studies at the mRNA level showed that the expression of unidentified pOb24 and pGH3 mRNAs, which was parallel to that of lipoprotein lipase, is independent of polyamine addition whereas the late emergence of glycerol-3-phosphate dehydrogenase mRNA was putrescine-dependent and co-ordinated with the expression of pAL422 mRNA encoding for a myelin-P2 homologue [Bernlohr, Angus, Lane, Bolanowski & Kelly (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5468-5472]. The appearance of lipoprotein lipase preceded DNA synthesis and post-confluent mitoses which were both putrescine-dependent and which took place before the appearance of glycerol-3-phosphate dehydrogenase. Thus the adipose conversion of Ob1754 cells involves the expression of at least two separate sets of markers which are differently regulated. Images Fig. 3. Fig. 6. PMID:3800927

VDR mRNA overexpression is associated with worse prognostic factors in papillary thyroid carcinoma

Directory of Open Access Journals (Sweden)

June Young Choi

2017-03-01

Full Text Available The purpose of this study was to assess the relationship between vitamin D receptor gene (VDR expression and prognostic factors in papillary thyroid cancer (PTC. mRNA sequencing and somatic mutation data from The Cancer Genome Atlas (TCGA were analyzed. VDR mRNA expression was compared to clinicopathologic variables by linear regression. Tree-based classification was applied to find cutoff and patients were split into low and high VDR group. Logistic regression, Kaplan–Meier analysis, differentially expressed gene (DEG test and pathway analysis were performed to assess the differences between two VDR groups. VDR mRNA expression was elevated in PTC than that in normal thyroid tissue. VDR expressions were high in classic and tall-cell variant PTC and lateral neck node metastasis was present. High VDR group was also associated with classic and tall cell subtype, AJCC stage IV and lower recurrence-free survival. DEG test reveals that 545 genes were upregulated in high VDR group. Thyroid cancer-related pathways were enriched in high VDR group in pathway analyses. VDR mRNA overexpression was correlated with worse prognostic factors such as subtypes of papillary thyroid carcinoma that are known to be worse prognosis, lateral neck node metastasis, advanced stage and recurrence-free survival.

Effect of Phosphatase and Tensin Homologue on Chromosome 10 on Angiotensin II-Mediated Proliferation, Collagen Synthesis, and Akt/P27 Signaling in Neonatal Rat Cardiac Fibroblasts

Directory of Open Access Journals (Sweden)

Ling Nie

2016-01-01

Full Text Available Cardiac fibroblasts (CFs play a key role in cardiac fibrosis by regulating the balance between extracellular matrix synthesis and breakdown. Although phosphatase and tensin homologue on chromosome 10 (PTEN has been found to play an important role in cardiovascular disease, it is not clear whether PTEN is involved in functional regulation of CFs. In the present study, PTEN was overexpressed in neonatal rat CFs via recombinant adenovirus-mediated gene transfer. The effects of PTEN overexpression on cell-cycle progression and angiotensin II- (Ang II- mediated regulation of collagen metabolism, synthesis of matrix metalloproteinases, and Akt/P27 signaling were investigated. Compared with uninfected cells and cells infected with green fluorescent protein-expressing adenovirus (Ad-GFP, cells infected with PTEN-expressing adenovirus (Ad-PTEN significantly increased PTEN protein and mRNA levels in CFs (P<0.05. The proportion of CFs in the G1/S cell-cycle phase was significantly higher for PTEN-overexpressing cells. In addition, Ad-PTEN decreased mRNA expression and the protein synthesis rate of collagen types I and III and antagonized Ang II-induced collagen synthesis. Overexpression of PTEN also decreased Ang II-induced matrix metalloproteinase-2 (MMP-2 and tissue inhibitor of metalloproteinase-1 (TIMP-1 production as well as gelatinase activity. Moreover, Ad-PTEN decreased Akt expression and increased P27 expression independent of Ang II stimulation. These results suggest that PTEN could regulate its functional effects in neonatal rat CFs partially via the Akt/P27 signaling pathway.

Permissive effect of dexamethasone on the increase of proenkephalin mRNA induced by depolarization of chromaffin cells

International Nuclear Information System (INIS)

Naranjo, J.R.; Mocchetti, I.; Schwartz, J.P.; Costa, E.

1986-01-01

In cultured bovine chromaffin cells, changes in the dynamic state of enkephalin stores elicited experimentally were studied by measuring cellular proenkephalin mRNA, as well as enkephalin precursors and authentic enkephalin content of cells and culture media. In parallel, tyrosine hydroxylase mRNA and catecholamine cell content were also determined. Low concentrations (0.5-100 pM) of dexamethasone increased the cell contents of proenkephalin mRNA and enkephalin-containing peptides. High concentrations of the hormone(1 μM) were required to increase the cell contents of tyrosine hydroxylase mRNA and catecholamines. Depolarization of the cells with 10 μM veratridine resulted in a depletion of enkephalin and catecholamine stores after 24 hr. The enkephalin, but not the catecholamine, content was restored by 48 hr. An increase in proenkephalin mRNA content might account for the recovery; this increase was curtailed by tetrodotoxin and enhanced by 10 pM dexamethasone. Tyrosine hydroxylase mRNA content was not significantly modified by depolarization, even in the presence of 1 μM dexamethasone. Aldosterone, progesterone, testosterone, or estradiol (1 μM) failed to change proenkephalin mRNA. Hence, dexamethasone appears to exert a specific permissive action on the stimulation of the proenkephalin gene elicited by depolarization. Though the catecholamines and enkephalins are localized in the same chromaffin granules and are coreleased by depolarization, the genes coding for the processes that are rate limiting in the production of these neuromodulators can be differentially regulated

Presence of albumin mRNA precursors in nuclei of analbuminemic rat liver lacking cytoplasmic albumin mRNA.

OpenAIRE

Esumi, H; Takahashi, Y; Sekiya, T; Sato, S; Nagase, S; Sugimura, T

1982-01-01

Analbuminemic rats, which lack serum albumin, were previously found to have no albumin mRNA in the cytoplasm of the liver. In the present study, the existence of nuclear albumin mRNA precursors in the liver of analbuminemic rats was examined by RNA X cDNA hybridization kinetics. Albumin mRNA precursors were present in the nuclei of analbuminemic rat liver at almost normal levels, despite the absence of albumin mRNA from the cytoplasm. Nuclear RNA of analbuminemic rat liver was subjected to el...

A mRNA and cognate microRNAs localize in the nucleolus.

Science.gov (United States)

Reyes-Gutierrez, Pablo; Ritland Politz, Joan C; Pederson, Thoru

2014-01-01

We previously discovered that a set of 5 microRNAs are concentrated in the nucleolus of rat myoblasts. We now report that several mRNAs are also localized in the nucleoli of these cells as determined by microarray analysis of RNA from purified nucleoli. Among the most abundant of these nucleolus-localized mRNAs is that encoding insulin-like growth factor 2 (IGF2), a regulator of myoblast proliferation and differentiation. The presence of IGF2 mRNA in nucleoli was confirmed by fluorescence in situ hybridization, and RT-PCR experiments demonstrated that these nucleolar transcripts are spliced, thus arriving from the nucleoplasm. Bioinformatics analysis predicted canonically structured, highly thermodynamically stable interactions between IGF2 mRNA and all 5 of the nucleolus-localized microRNAs. These results raise the possibility that the nucleolus is a staging site for setting up particular mRNA-microRNA interactions prior to export to the cytoplasm.

Self-assembly of diphenylalanine backbone homologues and their combination with functionalized carbon nanotubes.

Science.gov (United States)

Dinesh, Bhimareddy; Squillaci, Marco A; Ménard-Moyon, Cécilia; Samorì, Paolo; Bianco, Alberto

2015-10-14

The integration of carbon nanotubes (CNTs) into organized nanostructures is of great interest for applications in materials science and biomedicine. In this work we studied the self-assembly of β and γ homologues of diphenylalanine peptides under different solvent and pH conditions. We aimed to investigate the role of peptide backbone in tuning the formation of different types of nanostructures alone or in combination with carbon nanotubes. In spite of having the same side chain, β and γ peptides formed distinctively different nanofibers, a clear indication of the role played by the backbone homologation on the self-assembly. The variation of the pH allowed to transform the nanofibers into spherical structures. Moreover, the co-assembly of β and γ peptides with carbon nanotubes covalently functionalized with the same peptide generated unique dendritic assemblies. This comparative study on self-assembly using diphenylalanine backbone homologues and of the co-assembly with CNT covalent conjugates is the first example exploring the capacity of β and γ peptides to adopt precise nanostructures, particularly in combination with carbon nanotubes. The dendritic organization obtained by mixing carbon nanotubes and peptides might find interesting applications in tissue engineering and neuronal interfacing.

Is the Prosthetic Homologue Necessary for Embodiment?

Science.gov (United States)

Dornfeld, Chelsea; Swanston, Michelle; Cassella, Joseph; Beasley, Casey; Green, Jacob; Moshayev, Yonatan; Wininger, Michael

2016-01-01

Embodiment is the process by which patients with limb loss come to accept their peripheral device as a natural extension of self. However, there is little guidance as to how exacting the prosthesis must be in order for embodiment to take place: is it necessary for the prosthetic hand to look just like the absent hand? Here, we describe a protocol for testing whether an individual would select a hand that looks like their own from among a selection of five hands, and whether the hand selection (regardless of homology) is consistent across multiple exposures to the same (but reordered) set of candidate hands. Pilot results using healthy volunteers reveals that hand selection is only modestly consistent, and that selection of the prosthetic homologue is atypical (61 of 192 total exposures). Our protocol can be executed in minutes, and makes use of readily available equipment and softwares. We present both a face-to-face and a virtual protocol, for maximum flexibility of implementation.

Differential display-mediated identification of three drought

Indian Academy of Sciences (India)

L.) O. Kuntze], a woody and perennial plant of commercial importance. Using differential display of mRNA, three drought-modulated expressed sequence tags (ESTs) were identified. Northern and BLAST analysis revealed that clone dr1 ...

Inhibition of hydroxyapatite growth by casein, a potential salivary phosphoprotein homologue.

Science.gov (United States)

Romero, Maria J R H; Nakashima, Syozi; Nikaido, Toru; Ichinose, Shizuko; Sadr, Alireza; Tagami, Junji

2015-08-01

Salivary phosphoproteins are essential in tooth mineral regulation but are often overlooked in vitro. This study aimed to evaluate the effect of casein, as a salivary phosphoprotein homologue, on the deposition and growth of hydroxyapatite (HA) on tooth surfaces. Hydroxyapatite growth was quantified using seeded crystal systems. Artificial saliva (AS) containing HA powder and 0, 10, 20, 50, or 100 μg ml(-1) of casein, or 100 μg ml(-1) of dephosphorylated casein (Dcasein), was incubated for 0-8 h at 37°C, pH 7.2. Calcium concentrations were measured using atomic absorption spectroscopy (AAS). Surface precipitation of HA on bovine enamel and dentine blocks, incubated in similar conditions for 7 d, was examined using field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) with selected area electron diffraction (SAED). Casein adsorption was assessed using modified Lowry assays and zeta-potential measurements. The AAS results revealed a concentration-dependent inhibition of calcium consumption. Hydroxyapatite precipitation occurred when no casein was present, whereas precipitation of HA was apparently completely inhibited in casein-containing groups. Adsorption data demonstrated increasingly negative zeta-potential with increased casein concentration and an affinity constant similar to proline-rich proteins with Langmuir modelling. Casein inhibited the deposition and growth of HA primarily through the binding of esterized phosphate to HA active sites, indicating its potential as a mineral-regulating salivary phosphoprotein homologue in vitro. © 2015 Eur J Oral Sci.

Voltage-gated sodium channel expression and action potential generation in differentiated NG108-15 cells.

Science.gov (United States)

Liu, Jinxu; Tu, Huiyin; Zhang, Dongze; Zheng, Hong; Li, Yu-Long

2012-10-25

The generation of action potential is required for stimulus-evoked neurotransmitter release in most neurons. Although various voltage-gated ion channels are involved in action potential production, the initiation of the action potential is mainly mediated by voltage-gated Na+ channels. In the present study, differentiation-induced changes of mRNA and protein expression of Na+ channels, Na+ currents, and cell membrane excitability were investigated in NG108-15 cells. Whole-cell patch-clamp results showed that differentiation (9 days) didn't change cell membrane excitability, compared to undifferentiated state. But differentiation (21 days) induced the action potential generation in 45.5% of NG108-15 cells (25/55 cells). In 9-day-differentiated cells, Na+ currents were mildly increased, which was also found in 21-day differentiated cells without action potential. In 21-day differentiated cells with action potential, Na+ currents were significantly enhanced. Western blot data showed that the expression of Na+ channels was increased with differentiated-time dependent manner. Single-cell real-time PCR data demonstrated that the expression of Na+ channel mRNA was increased by 21 days of differentiation in NG108-15 cells. More importantly, the mRNA level of Na+ channels in cells with action potential was higher than that in cells without action potential. Differentiation induces expression of voltage-gated Na+ channels and action potential generation in NG108-15 cells. A high level of the Na+ channel density is required for differentiation-triggered action potential generation.

The role of the Saccharomyces cerevisiae lipoate protein ligase homologue, Lip3, in lipoic acid synthesis.

Science.gov (United States)

Hermes, Fatemah A; Cronan, John E

2013-10-01

The covalent attachment of lipoate to the lipoyl domains (LDs) of the central metabolism enzymes pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) is essential for their activation and thus for respiratory growth in Saccharomyces cerevisiae. A third lipoate-dependent enzyme system, the glycine cleavage system (GCV), is required for utilization of glycine as a nitrogen source. Lipoate is synthesized by extraction of its precursor, octanoyl-acyl carrier protein (ACP), from the pool of fatty acid biosynthetic intermediates. Alternatively, lipoate is salvaged from previously modified proteins or from growth medium by lipoate protein ligases (Lpls). The first Lpl to be characterized, LplA of Escherichia coli, catalyses two partial reactions: activation of the acyl chain by formation of acyl-AMP, followed by transfer of the acyl chain to lipoyl domains (LDs). There is a surprising diversity within the Lpl family of enzymes, several of which catalyse reactions other than ligation reactions. For example, the Bacillus subtilis Lpl homologue LipM is an octanoyltransferase that transfers the octanoyl moiety from octanoyl-ACP to GCV. Another B. subtilis Lpl homologue, LipL, transfers octanoate from octanoyl-GCV to other LDs in an amido-transfer reaction. Study of eukaryotic Lpls has lagged behind studies of the bacterial enzymes. We report that the Lip3 Lpl homologue of the yeast S. cerevisiae has octanoyl-CoA-protein transferase activity, and discuss implications of this activity on the physiological role of Lip3 in lipoate synthesis. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

A selection of reference genes and early-warning mRNA biomarkers for environmental monitoring using Mytilus spp. as sentinel species.

Science.gov (United States)

Lacroix, C; Coquillé, V; Guyomarch, J; Auffret, M; Moraga, D

2014-09-15

mRNA biomarkers are promising tools for environmental health assessment and reference genes are needed to perform relevant qPCR analyses in tissue samples of sentinel species. In the present study, potential reference genes and mRNA biomarkers were tested in the gills and digestive glands of native and caged mussels (Mytilus spp.) exposed to harbor pollution. Results highlighted the difficulty to find stable reference genes in wild, non-model species and suggested the use of normalization indices instead of single genes as they exhibit a higher stability. Several target genes were found differentially expressed between mussel groups, especially in gills where cyp32, π-gst and CuZn-sod mRNA levels could be biomarker candidates. Multivariate analyses confirmed the ability of mRNA levels to highlight site-effects and suggested the use of several combined markers instead of individual ones. These findings support the use of qPCR technology and mRNA levels as early-warning biomarkers in marine monitoring programs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Research Article Molecular cloning and mRNA expression pattern of ...

Indian Academy of Sciences (India)

SAMSUNG

homologue from the brain of Misgurnus anguillicaudatus using homologous cloning and ... developmental processes, including sex determination, embryonic stem cell ..... anguillicaudatus, possibly aiding in unravelling reproductive biology ... significant difference (p < 0.05) between female and male in the same tissue.

Novel lncRNAs in myogenesis: a miR-31 overlapping transcript controls myoblast differentiation.

KAUST Repository

Ballarino, Monica; Cazzella, Valentina; D'Andrea, Daniel; Grassi, Luigi; Bisceglie, Lavinia; Cipriano, Andrea; Santini, Tiziana; Pinnarò , Chiara; Morlando, Mariangela; Tramontano, Anna; Bozzoni, Irene

2014-01-01

Transcriptome analysis allowed the identification of new long noncoding RNAs differentially expressed during murine myoblast differentiation. These transcripts were classified on the basis of their expression under proliferating versus differentiated conditions, muscle-restricted activation, and subcellular localization. Several species displayed preferential expression in dystrophic (mdx) versus wild-type muscles, indicating their possible link with regenerative processes. One of the identified transcripts, lnc-31, even if originating from the same nuclear precursor of miR-31, is produced by a pathway mutually exclusive. We show that lnc-31 and its human homologue hsa-lnc-31 are expressed in proliferating myoblasts, where they counteract differentiation. In line with this, both species are more abundant in mdx muscles and in human Duchenne muscular dystrophy (DMD) myoblasts, than in their normal counterparts. Altogether, these data suggest a crucial role for lnc-31 in controlling the differentiation commitment of precursor myoblasts and indicate that its function is maintained in evolution despite the poor sequence conservation with the human counterpart.

Novel lncRNAs in myogenesis: a miR-31 overlapping transcript controls myoblast differentiation.

KAUST Repository

Ballarino, Monica

2014-12-15

Transcriptome analysis allowed the identification of new long noncoding RNAs differentially expressed during murine myoblast differentiation. These transcripts were classified on the basis of their expression under proliferating versus differentiated conditions, muscle-restricted activation, and subcellular localization. Several species displayed preferential expression in dystrophic (mdx) versus wild-type muscles, indicating their possible link with regenerative processes. One of the identified transcripts, lnc-31, even if originating from the same nuclear precursor of miR-31, is produced by a pathway mutually exclusive. We show that lnc-31 and its human homologue hsa-lnc-31 are expressed in proliferating myoblasts, where they counteract differentiation. In line with this, both species are more abundant in mdx muscles and in human Duchenne muscular dystrophy (DMD) myoblasts, than in their normal counterparts. Altogether, these data suggest a crucial role for lnc-31 in controlling the differentiation commitment of precursor myoblasts and indicate that its function is maintained in evolution despite the poor sequence conservation with the human counterpart.

Structural characterization and expression analysis of a beta-thymosin homologue (Tβ) in disk abalone, Haliotis discus discus.

Science.gov (United States)

Kasthuri, Saranya Revathy; Premachandra, H K A; Umasuthan, Navaneethaiyer; Whang, Ilson; Lee, Jehee

2013-09-15

Repertoires of proteins and small peptides play numerous physiological roles as hormones, antimicrobial peptides, and cellular signaling factors. The beta-thymosins are a group of small acidic peptides involved in processes such as actin sequestration, neuronal development, wound healing, tissue repair, and angiogenesis. Recent characterization of the beta thymosins as immunological regulators in invertebrates led to our identification and characterization of a beta-thymosin homologue (Tβ) from Haliotis discus discus. The cDNA possessed an ORF of 132 bp encoding a protein of 44 amino acids with a molecular mass of 4977 Da. The amino acid sequence shows high identity with another molluskan beta-thymosin and has a characteristic actin binding motif (LKKTET) and glutamyl donors. Phylogenetic analysis showed a close relationship with molluskan homologues, as well as its distinct identity and common ancestral origin. Genomic analysis revealed a 3 exon-2 intron structure similar to the other homologues. In silico promoter analysis also revealed significant transcription factor binding sites, providing evidence for the expression of this gene under different cellular conditions, including stress or pathogenic attack. Tissue distribution profiling revealed a ubiquitous presence in all the examined tissues, but with the highest expression in mantle and hemocyte. Immune challenge with lipopolysaccharide, poly I:C and Vibrio parahemolyticus induced beta-thymosin expression in gill and hemocytes, affirming an immune-related role in invertebrates. Copyright © 2013 Elsevier B.V. All rights reserved.

Efficient mRNA delivery with graphene oxide-polyethylenimine for generation of footprint-free human induced pluripotent stem cells.

Science.gov (United States)

Choi, Hye Yeon; Lee, Tae-Jin; Yang, Gwang-Mo; Oh, Jaesur; Won, Jihye; Han, Jihae; Jeong, Gun-Jae; Kim, Jongpil; Kim, Jin-Hoi; Kim, Byung-Soo; Cho, Ssang-Goo

2016-08-10

Clinical applications of induced pluripotent stem cells (iPSCs) require development of technologies for the production of "footprint-free" (gene integration-free) iPSCs, which avoid the potential risk of insertional mutagenesis in humans. Previously, several studies have shown that mRNA transfer can generate "footprint-free" iPSCs, but these studies did not use a delivery vehicle and thus repetitive daily transfection was required because of mRNA degradation. Here, we report an mRNA delivery system employing graphene oxide (GO)-polyethylenimine (PEI) complexes for the efficient generation of "footprint-free" iPSCs. GO-PEI complexes were found to be very effective for loading mRNA of reprogramming transcription factors and protection from mRNA degradation by RNase. Dynamic suspension cultures of GO-PEI/RNA complexes-treated cells dramatically increased the reprogramming efficiency and successfully generated rat and human iPSCs from adult adipose tissue-derived fibroblasts without repetitive daily transfection. The iPSCs showed all the hallmarks of pluripotent stem cells including expression of pluripotency genes, epigenetic reprogramming, and differentiation into the three germ layers. These results demonstrate that mRNA delivery using GO-PEI-RNA complexes can efficiently generate "footprint-free" iPSCs, which may advance the translation of iPSC technology into the clinical settings. Copyright © 2016. Published by Elsevier B.V.

A lesion mimic phenotype in tomato obtained by isolating and silencing an Lls1 homologue

NARCIS (Netherlands)

Spassieva, S; Hille, J

Lesion mimic phenotypes serve as a tool to study the regulation of cell death in plants. In order to obtain a tomato lesion mimic phenotype, we used the conservation of the lethal leaf spot 1 (Lls1) genes between plant species. The tomato Lls1 homologue was cloned, sequenced and analyzed. It showed

Self-amplifying mRNA vaccines.

Science.gov (United States)

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.

Stem cells. m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation.

Science.gov (United States)

Geula, Shay; Moshitch-Moshkovitz, Sharon; Dominissini, Dan; Mansour, Abed AlFatah; Kol, Nitzan; Salmon-Divon, Mali; Hershkovitz, Vera; Peer, Eyal; Mor, Nofar; Manor, Yair S; Ben-Haim, Moshe Shay; Eyal, Eran; Yunger, Sharon; Pinto, Yishay; Jaitin, Diego Adhemar; Viukov, Sergey; Rais, Yoach; Krupalnik, Vladislav; Chomsky, Elad; Zerbib, Mirie; Maza, Itay; Rechavi, Yoav; Massarwa, Rada; Hanna, Suhair; Amit, Ido; Levanon, Erez Y; Amariglio, Ninette; Stern-Ginossar, Noam; Novershtern, Noa; Rechavi, Gideon; Hanna, Jacob H

2015-02-27

Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N(6)-methyladenosine (m(6)A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m(6)A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m(6)A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner. Copyright © 2015, American Association for the Advancement of Science.

Cloning of cDNA sequences of a progestin-regulated mRNA from MCF7 human breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Chalbos, D; Westley, B; Alibert, C; Rochefort, H

1986-01-24

A cDNA clone corresponding to an mRNA regulated by the progestin R5020, has been isolated by differential screening of a cDNA library from the MCF7 breast cancer cell line, which contains estrogen and progesterone receptors. This probe hybridized with a single species of poly A + RNA of 8-kb molecular weight as shown by Northern blot analysis and could also be used to total RNA preparation. This recombinant cone hybridized specifically to an mRNA coding for a 250,000 daltons protein when translated in vitro. This protein was identical to the 250 kDa progestin-regulated protein that the authors previously described as shown by immunoprecipitation with specific rabbit polyclonal antibodies. Dose-response curve and specificity studies show that the accumulation of the Pg8 mRNA and that of the 250-kDa protein was increased by 5 to 30-fold following progestin treatment and that this effect was mediated by the progesterone receptor. Time course of induction indicated that the accumulation of mRNA was rapid and preceded that of the protein. This is the first report on a cloned cDNA probe of progestin-regulated mRNA in human cell lines.

Integrated Analysis of Long Noncoding RNA and mRNA Expression Profile in Advanced Laryngeal Squamous Cell Carcinoma.

Directory of Open Access Journals (Sweden)

Ling Feng

Full Text Available Long non-coding RNA (lncRNA plays an important role in tumorigenesis. However, the expression pattern and function of lncRNAs in laryngeal squamous cell carcinoma (LSCC are still unclear. To investigate the aberrantly expressed lncRNAs and mRNAs in advanced LSCC, we screened lncRNA and mRNA expression profiles in 9 pairs of primary Stage IVA LSCC tissues and adjacent non-neoplastic tissues by lncRNA and mRNA integrated microarrays. Gene Ontology and pathway analysis were performed to find out the significant function and pathway of the differentially expressed mRNAs, gene-gene functional interaction network and ceRNA network were constructed to select core mRNAs, and lncRNA-mRNA expression correlation network was built to identify the interactions between lncRNA and mRNA. qRT-PCR was performed to further validate the expressions of selected lncRNAs and mRNAs in advanced LSCC. We found 1459 differentially expressed lncRNAs and 2381 differentially expressed mRNAs, including 846 up-regulated lncRNAs and 613 down-regulated lncRNAs, 1542 up-regulated mRNAs and 839 down-regulated mRNAs. The mRNAs ITGB1, HIF1A, and DDIT4 were selected as core mRNAs, which are mainly involved in biological processes, such as matrix organization, cell cycle, adhesion, and metabolic pathway. LncRNA-mRNA expression correlation network showed LncRNA NR_027340, MIR31HG were positively correlated with ITGB1, HIF1A respectively. LncRNA SOX2-OT was negatively correlated with DDIT4. qRT-PCR further validated the expression of these lncRNAs and mRNAs. The work provides convincing evidence that the identified lncRNAs and mRNAs are potential biomarkers in advanced LSCC for further future studies.

Identification of a differentially expressed mRNA in axenic Leishmania panamensis amastigotes

Directory of Open Access Journals (Sweden)

José Arturo Gutiérrez

2001-08-01

Full Text Available Differential display technique was applied in order to identify transcripts which are present in axenic amastigotes but not in promastigotes of the Leishmania panamensis parasites. One of them was cloned and the sequence reveals an open reading frame of 364 amino acids (aprox. 40 kDa. The deduced protein is homologous to the serine/threonine protein kinases and specially to the mitogen activates protein kinases from eukaryotic species. Southern blot analysis suggest that this transcript, named lpmkh, is present in the genome of the parasite as a single copy gene. These results could imply that lpmkh could be involved in the differentiation process or the preservation of amastigotes in axenic conditions.

Detection of mRNA by reverse-transcription PCR as an indicator of viability in Phytophthora ramorum

Science.gov (United States)

A. Chimento; S.O. Cacciola; M. Garbelotto

2011-01-01

In the last few decades, the use of molecular tools has greatly improved the efficiency of plant disease diagnosis. However, one of the major setbacks of most molecular diagnostic approaches is their inability to differentiate between dead and viable pathogens. We propose a new strategy for the detection of plant pathogens, based on the use of mRNA as a viability...

The pokeweed leaf mRNA transcriptome and its regulation by jasmonic acid.

Directory of Open Access Journals (Sweden)

Kira C.M. Neller

2016-03-01

Full Text Available The American pokeweed plant, Phytolacca americana, is recognized for synthesizing pokeweed antiviral protein (PAP, a ribosome inactivating protein (RIP that inhibits the replication of several plant and animal viruses. The plant is also a heavy metal accumulator with applications in soil remediation. However, little is known about pokeweed stress responses, as large-scale sequencing projects have not been performed for this species. Here, we sequenced the mRNA transcriptome of pokeweed in the presence and absence of jasmonic acid (JA, a hormone mediating plant defense. Trinity-based de novo assembly of mRNA from leaf tissue and BLASTx homology searches against public sequence databases resulted in the annotation of 59 096 transcripts. Differential expression analysis identified JA-responsive genes that may be involved in defense against pathogen infection and herbivory. We confirmed the existence of several PAP isoforms and cloned a potentially novel isoform of PAP. Expression analysis indicated that PAP isoforms are differentially responsive to JA, perhaps indicating specialized roles within the plant. Finally, we identified 52 305 natural antisense transcript pairs, four of which comprised PAP isoforms, suggesting a novel form of RIP gene regulation. This transcriptome-wide study of a Phytolaccaceae family member provides a source of new genes that may be involved in stress tolerance in this plant. The sequences generated in our study have been deposited in the SRA database under project # SRP069141.

Intrauterine growth restriction and differential patterns of hepatic growth and expression of IGF1, PCK2, and HSDL1 mRNA in the sheep fetus in late gestation.

Science.gov (United States)

Gentili, Sheridan; Morrison, Janna L; McMillen, I Caroline

2009-06-01

Fetal adaptations to periods of substrate deprivation can result in the programming of glucose intolerance, insulin resistance, and metabolic dysfunction in later life. Placental insufficiency can be associated with either sparing or sacrifice of fetal liver growth, and these different responses may have different metabolic consequences. It is unclear what intrahepatic mechanisms determine the differential responses of the fetal liver to substrate restriction. We investigated the effects of placental restriction (PR) on liver growth and the hepatic expression of SLC2A1, IGF1, IGF2, IGF1R, IGF2R, PPARGC1A, PPARA, PRKAA1, PRKAA2, PCK2, and HSDL1 mRNA in fetal sheep at 140-145 days of gestation. A mean gestational arterial partial pressure of oxygen less than 17 mmHg was defined as hypoxic, and a relative liver of weight more than 2 SD below the mean liver weight of controls was defined as reduced liver growth. Fetuses therefore were defined as control-normoxic (C-N; n = 9), PR-normoxic (PR-N; n = 7), PR-hypoxic (PR-H; n = 8), or PR-hypoxic reduced liver growth (PR-H RLG; n = 4). Hepatic SLC2A1 mRNA expression was highest (P fetal substrate restriction may exist that protect the liver from decreased growth and, potentially, from a decreased responsiveness to the actions of insulin in postnatal life.

Periodontal status of teeth restored with crowns and its contralateral homologue, Valdivia- Chile.

OpenAIRE

Israel Antonio Juárez; Sofía Larroulet; Makarena Ojeda; Cristian Rosas

2015-01-01

ABSTRACT Aim: To determine periodontal status of fixes single prostheses (FSP) made during the year 2013 in Austral University of Chile, and its contralateral homologue (CH).Methods: All patients with FSP made during 2013, that met the selection criteria and agreed to participate were evaluated. During the year 2014 was measured: probing depth, attachment level; bleeding on probing and dental plaque index for each FSP and CH; and consigned biological width invasion. Were measured one FSP...

Expression of IL-1β mRNA in mice after whole body X-irradiation

International Nuclear Information System (INIS)

Nemoto, Kumie; Ishihara, Hiroshi; Tanaka, Izumi; Suzuki, Gen; Tsuneoka, Kazuko; Yoshida, Kazuko; Ohtsu, Hiroshi

1995-01-01

IL-1β is a stimulator of hematopoietic and inflammatory systems, and also acts as a radioprotector. After whole-body exposure to sublethal doses of ionizing radiation, the IL-1β mRNA level in spleen cells increases for a short time prior to regeneration of the spleen. We analyzed spleen cells of C3H/He mice after whole-body irradiation with 3 Gy x-rays to determine the cause of this short-term increase in the transcription level. An increase in the level of the message in spleen cells, found by Northern blot hybridization, reached its peak 5 to 7 days after irradiation. There was a low correlation between the curves of the mRNA level and the ratio of monocyte/macrophage lineage cells; a typical source of the message. Spleen macrophages that produce a large amount of the message were found 7 days after irradiation in an in situ hybridization experiment in which heterogeneous spleen cell populations were used. In contrast, spleen cells had no detectable levels of macrophages rich in IL-1β mRNA before and 17 days after irradiation. Additionally, the population of message-rich cells was 9.4% of the total number of monocytes/macrophages in the spleen. These results suggest that the short-term increase in IL-1β mRNA is a result of the heterogeneous differentiation of a subpopulation of spleen macrophages before regeneration of the spleen. (author)

Species-specific flight styles of flies are reflected in the response dynamics of a homologue motion sensitive neuron

Directory of Open Access Journals (Sweden)

Bart eGeurten

2012-03-01

Full Text Available Hoverflies and blowflies have distinctly different flight styles. Yet, both species have been shown to structure their flight behaviour in a way that facilitates extraction of 3D information from the image flow on the retina (optic flow. Neuronal candidates to analyse the optic flow are the tangential cells in the third optical ganglion – the lobula complex. These neurons are directionally selective and integrate the optic flow over large parts of the visual field. Homologue tangential cells in hoverflies and blowflies have a similar morphology. Because blowflies and hoverflies have similar neuronal layout but distinctly different flight behaviours, they are an ideal substrate to pinpoint potential neuronal adaptations to the different flight styles.In this article we describe the relationship between locomotion behaviour and motion vision on three different levels:1.We compare the different flight styles based on the categorisation of flight behaviour into prototypical movements.2.We measure the species specific dynamics of the optic flow under naturalistic flight conditions. We found the translational optic flow of both species to be very different.3.We describe possible adaptations of a homologue motion sensitive neuron. We stimulate this cell in blowflies (Calliphora and hoverflies (Eristalis with naturalistic optic flow generated by both species during free flight. The characterized hoverfly tangential cell responds faster to transient changes in the optic flow than its blowfly homologue. It is discussed whether and how the different dynamical response properties aid optic flow analysis.

Crystal structure of pira toxin-I: a calcium-independent, myotoxic phospholipase A2 - homologue from Bothrops pirajai venom

International Nuclear Information System (INIS)

Canduri, R.J.; Ward, R.J.; Azevedo Junior, G.W.F. de; Arni, R.K.; Soares, A.M.; Giglio, J.R.

1997-01-01

Full text. Phospho lipases A2 (PLA 2 ) are small enzymes that specifically hydrolysed the sn-2 ester bond of phospholipids, preferentially in lamellar or micellar aggregates at membrane surfaces. These enzymes are widely distributed in nature and have been extensively studied. Toxic proteins from venoms from Bothrops species include catalytically active PLA 2 s and calcium independent PLA 2L ys 49 homologues. The substitution of Asp49 by Lys greatly diminishes the ability of these PLA 2 to bind calcium, an ion that plays a critical role in the stabilization of the tetrahedral transition state intermediate in the catalytic mechanism. The Lys 49 PLA 2 homologues and therefore catalytically inactive yet maintain cytolytic and myotoxic activities and furthermore retain the ability to disrupt the integrity of both plasma membranes and model lipid bilayers by a poorly understood Ca 2+ independente mechanism. Lys49 PLA 2 homologues demonstrate a specific toxic activity against skeletal muscle, affecting only muscle fibers and leaving other tissue structure such as connective tissue, nerves and vessels essentially unharmed. In order to improve our understanding of the molecular basis of the myotoxic and Ca 2+ -independent membrane damaging activities, we have determined the crystal structure of Pr TX-I, a Lys49 variant from the venom of B. pirajai. The model presented has been determined at 2.8 angstrom resolution and refined to a crystallographic residual of 19.7% (R free =29.7%). (author)

Differential effects of voluntary wheel running and toy rotation on the mRNA expression of neurotrophic factors and FKBP5 in a post-traumatic stress disorder rat model with the shuttle-box task.

Science.gov (United States)

Tanichi, Masaaki; Toda, Hiroyuki; Shimizu, Kunio; Koga, Minori; Saito, Taku; Enomoto, Shingo; Boku, Shuken; Asai, Fumiho; Mitsui, Yumi; Nagamine, Masanori; Fujita, Masanori; Yoshino, Aihide

2018-06-18

Life-threatening experiences can result in the development of post-traumatic stress disorder. We have developed an animal model for post-traumatic stress disorder (PTSD) using a shuttle box in rats. In this paradigm, the rats were exposed to inescapable foot-shock stress (IS) in a shuttle box, and then an avoidance/escape task was performed in the same box 2 weeks after IS. A previous study using this paradigm revealed that environmental enrichment (EE) ameliorated avoidance/numbing-like behaviors, but not hyperarousal-like behaviors, and EE also elevated hippocampal brain-derived neurotrophic factor (BDNF) expression. However, the differential effects of EE components, i.e., running wheel (RW) or toy rotation, on PTSD-like behaviors has remained unclear. In this experiment, we demonstrated that RW, toy rotation, and EE (containing RW and toy rotation) ameliorated avoidance/numbing-like behaviors, induced learning of avoidance responses, and improved depressive-like behaviors in traumatized rats. The RW increased the hippocampal mRNA expression of neurotrophic factors, especially BDNF and glial-cell derived neurotrophic factor. Toy rotation influenced FK506 binding protein 5 mRNA expression, which is believed to be a regulator of the hypothalamic-pituitary-adrenal (HPA)-axis system, in the hippocampus and amygdala. This is the first report to elucidate the differential mechanistic effects of RW and toy rotation. The former appears to exert its effects via neurotrophic factors, while the latter exerts its effects via the HPA axis. Further studies will lead to a better understanding of the influence of environmental factors on PTSD. Copyright © 2018 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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.

[Effects of lipopolysaccharides extracted from Porphyromonas endodontalis on the expression of IL-1beta mRNA and IL-6 mRNA in osteoblasts].

Science.gov (United States)

Yang, Di; Li, Ren; Qiu, Li-Hong; Li, Chen

2009-04-01

To quantify the IL-1 beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides (LPS)extracted from Porphyromonas endodontalis(P.e) in osteoblasts, and to relate P.e-LPS to bone absorption pathogenesis in lesions of chronical apical periodontitis. MG63 was treated with different concentrations of P.e-LPS(0-50 microg/mL) for different hours(0-24h). The expression of IL-1 beta mRNA and IL-6 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR).Statistical analysis was performed using one- way ANOVA and Dunnett t test with SPSS11.0 software package. The level of IL-1 beta mRNA and IL-6 mRNA increased significantly after treatment with P.e-LPS at more than 5 microg/mL (P<0.01)and for more than 1 hour (P<0.01), which indicated that P.e-LPS induced osteoblasts to express IL-1 beta mRNA and IL-6 mRNA in dose and time dependent manners. P.e-LPS may promote bone resorption in lesions of chronical apical periodontitis by inducing IL-1 beta mRNA and IL-6 mRNA expression in osteoblasts.

Alpha subunit of glycoprotein hormones in the sera of acromegalic patients and its mRNA in the tumors.

Science.gov (United States)

Machiavelli, G A; Artese, R; Benencia, H; Bruno, O; Guerra, L; Basso, A; Burdman, J A

1999-04-01

Within a population of 16 pituitary adenomas we found high levels of glycoprotein alpha subunits in the sera of patients with somatotrophic tumors. This finding was correlated with the presence of mRNA alpha subunit in these tumors indicating the adenomas themselves as the origin of the circulating alpha-subunit. Synthesis of these two hormones, which are chemically very different, by the same tumor cells indicates a high degree of differentiation of these cells. We are unable at this time to conclusively correlate differentiation of these tumors aggressively.

CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

Science.gov (United States)

Busi, Florent; Cresteil, Thierry

2005-09-01

The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

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.

Science.gov (United States)

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.

Sirt6 regulates postnatal growth plate differentiation and proliferation via Ihh signaling.

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Piao, Jinying; Tsuji, Kunikazu; Ochi, Hiroki; Iwata, Munetaka; Koga, Daisuke; Okawa, Atsushi; Morita, Sadao; Takeda, Shu; Asou, Yoshinori

2013-10-23

Sirtuin 6 (Sirt6) is a mammalian homologue of NAD⁺-dependent histone deacetylase Sir2. Although Sirt6⁻/⁻ mice exhibit growth retardation, the role of Sirt6 in cartilage metabolism is unclear. The aim of this study was to investigate the Sirt6 signaling pathway in cartilage metabolism. Immunohistological evaluation of the tibial growth plate in Sirt6⁻/⁻ mice exhibited impaired proliferation and differentiation of chondrocytes, reduced expression of Indian hedgehog (Ihh), and a senescent phenotype. When Sirt6 was knocked down in chondrocytes in vitro, expression of Ihh and its downstream genes were reduced. Impaired differentiation by Sirt6 silencing was completely rescued by administration of a Hh signal agonist. When sirtuins were activated, chondrocyte differentiation was enhanced together with activation of Ihh signal, and these effects were abrogated by Sirt6 silencing. ChIP assay revealed the affinity of ATF4 to the Ihh promoter was markedly decreased by Sirt6 knockdown. These data indicate Sirt6 directly controls proliferation and differentiation of chondrocytes.

mRNA Fragments in In-Vitro Culture Media are Associated with Bovine Preimplantation Embryonic Development

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Jenna eKropp

2015-08-01

Full Text Available In vitro production (IVP systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerated conditioned media. Differential expression was confirmed by quantitative real-time PCR for

Exploratory Bioinformatics Study of lncRNAs in Alzheimer’s Disease mRNA Sequences with Application to Drug Development

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T. Holden

2013-01-01

Full Text Available Long noncoding RNA (lncRNA within mRNA sequences of Alzheimer’s disease genes, namely, APP, APOE, PSEN1, and PSEN2, has been analyzed using fractal dimension (FD computation and correlation analysis. We examined lncRNA by comparing mRNA FD to corresponding coding DNA sequences (CDSs FD. APP, APOE, and PSEN1 CDSs select slightly higher FDs compared to the mRNA, while PSEN2 CDSs FDs are lower. The correlation coefficient for these sequences is 0.969. A comparative study of differentially expressed MAPK signaling pathway lncRNAs in pancreatic cancer cells shows a correlation of 0.771. Selection of higher FD CDSs could indicate interaction of Alzheimer’s gene products APP, APOE, and PSEN1. Including hypocretin sequences (where all CDSs have higher fractal dimensions than mRNA in the APP, APOE, and PSEN1 sequence analyses improves correlation, but the inclusion of erythropoietin (where all CDSs have higher FD than mRNA would suppress correlation, suggesting that HCRT, a hypothalamus neurotransmitter related to the wake/sleep cycle, might be better when compared to EPO, a glycoprotein hormone, for targeting Alzheimer’s disease drug development. Fractal dimension and entropy correlation have provided supporting evidence, consistent with evolutionary studies, for using a zebrafish model together with a mouse model, in HCRT drug development.

Increased mRNA expression of a laminin-binding protein in human colon carcinoma: Complete sequence of a full-length cDNA encoding the protein

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Yow, Hsiukang; Wong, Jau Min; Chen, Hai Shiene; Lee, C.; Steele, G.D. Jr.; Chen, Lanbo

1988-01-01

Reliable markers to distinguish human colon carcinoma from normal colonic epithelium are needed particularly for poorly differentiated tumors where no useful marker is currently available. To search for markers the authors constructed cDNA libraries from human colon carcinoma cell lines and screened for clones that hybridize to a greater degree with mRNAs of colon carcinomas than with their normal counterparts. Here they report one such cDNA clone that hybridizes with a 1.2-kilobase (kb) mRNA, the level of which is ∼9-fold greater in colon carcinoma than in adjacent normal colonic epithelium. Blot hybridization of total RNA from a variety of human colon carcinoma cell lines shows that the level of this 1.2-kb mRNA in poorly differentiated colon carcinomas is as high as or higher than that in well-differentiated carcinomas. Molecular cloning and complete sequencing of cDNA corresponding to the full-length open reading frame of this 1.2-kb mRNA unexpectedly show it to contain all the partial cDNA sequence encoding 135 amino acid residues previously reported for a human laminin receptor. The deduced amino acid sequence suggests that this putative laminin-binding protein from human colon carcinomas consists of 295 amino acid residues with interesting features. There is an unusual C-terminal 70-amino acid segment, which is trypsin-resistant and highly negatively charged

ErbB3 mRNA leukocyte levels as a biomarker for major depressive disorder

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Milanesi Elena

2012-09-01

Full Text Available Abstract Background In recent years, the identification of peripheral biomarkers that are associated with psychiatric diseases, such as Major Depressive Disorder (MDD, has become relevant because these biomarkers may improve the efficiency of the differential diagnosis process and indicate targets for new antidepressant drugs. Two recent candidate genes, ErbB3 and Fgfr1, are growth factors whose mRNA levels have been found to be altered in the leukocytes of patients that are affected by bipolar disorder in a depressive state. On this basis, the aim of the study was to determine if ErbB3 and Fgfr1 mRNA levels could be a biomarkers of MDD. Methods We measured by Real Time PCR ErbB3 and Fgfr1 mRNA expression levels in leukocytes of MDD patients compared with controls. Successively, to assess whether ErbB3 mRNA levels were influenced by previous antidepressant treatment we stratified our patients sample in two cohorts, comparing drug-naive versus drug-free patients. Moreover, we evaluated the levels of the transcript in MDD patients after 12 weeks of antidepressant treatment, and in prefrontal cortex of rats stressed and treated with an antidepressant drug of the same class. Results These results showed that ErbB3 but not Fgfr1 mRNA levels were reduced in leukocytes of MDD patients compared to healthy subjects. Furthermore, ErbB3 levels were not affected by antidepressant treatment in either human or animal models Conclusions Our data suggest that ErbB3 might be considered as a biomarker for MDD and that its deficit may underlie the pathopsysiology of the disease and is not a consequence of treatment. Moreover the study supports the usefulness of leukocytes as a peripheral system for identifying biomarkers in psychiatric diseases.

The SPF27 homologue Num1 connects splicing and kinesin 1-dependent cytoplasmic trafficking in Ustilago maydis.

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Nikola Kellner

2014-01-01

Full Text Available The conserved NineTeen protein complex (NTC is an integral subunit of the spliceosome and required for intron removal during pre-mRNA splicing. The complex associates with the spliceosome and participates in the regulation of conformational changes of core spliceosomal components, stabilizing RNA-RNA- as well as RNA-protein interactions. In addition, the NTC is involved in cell cycle checkpoint control, response to DNA damage, as well as formation and export of mRNP-particles. We have identified the Num1 protein as the homologue of SPF27, one of NTC core components, in the basidiomycetous fungus Ustilago maydis. Num1 is required for polarized growth of the fungal hyphae, and, in line with the described NTC functions, the num1 mutation affects the cell cycle and cell division. The num1 deletion influences splicing in U. maydis on a global scale, as RNA-Seq analysis revealed increased intron retention rates. Surprisingly, we identified in a screen for Num1 interacting proteins not only NTC core components as Prp19 and Cef1, but several proteins with putative functions during vesicle-mediated transport processes. Among others, Num1 interacts with the motor protein Kin1 in the cytoplasm. Similar phenotypes with respect to filamentous and polar growth, vacuolar morphology, as well as the motility of early endosomes corroborate the genetic interaction between Num1 and Kin1. Our data implicate a previously unidentified connection between a component of the splicing machinery and cytoplasmic transport processes. As the num1 deletion also affects cytoplasmic mRNA transport, the protein may constitute a novel functional interconnection between the two disparate processes of splicing and trafficking.

Stem cell proliferation and differentiation and stochastic bistability in gene expression

International Nuclear Information System (INIS)

Zhdanov, V. P.

2007-01-01

The process of proliferation and differentiation of stem cells is inherently stochastic in the sense that the outcome of cell division is characterized by probabilities that depend on the intracellular properties, extracellular medium, and cell-cell communication. Despite four decades of intensive studies, the understanding of the physics behind this stochasticity is still limited, both in details and conceptually. Here, we suggest a simple scheme showing that the stochastic behavior of a single stem cell may be related to (i) the existence of a short stage of decision whether it will proliferate or differentiate and (ii) control of this stage by stochastic bistability in gene expression or, more specifically, by transcriptional 'bursts.' Our Monte Carlo simulations indicate that our proposed scheme may operate if the number of mRNA (or protein) molecules generated during the high-reactive periods of gene expression is below or about 50. The stochastic-burst window in the space of kinetic parameters is found to increase with decreasing the mRNA and/or regulatory-protein numbers and increasing the number of regulatory sites. For mRNA production with three regulatory sites, for example, the mRNA degradation rate constant may change in the range ±10%

Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway

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Shimamura, Mai; Kyotani, Akane [Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Azuma, Yumiko [Department of Neurology, Kyoto Prefectural University of Medicine, 465 Kajii-cho,Kamigyo-ku, Kyoto 602-8566 (Japan); Yoshida, Hideki; Binh Nguyen, Thanh [Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Mizuta, Ikuko; Yoshida, Tomokatsu; Mizuno, Toshiki [Department of Neurology, Kyoto Prefectural University of Medicine, 465 Kajii-cho,Kamigyo-ku, Kyoto 602-8566 (Japan); Nakagawa, Masanori [North Medical Center, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566 (Japan); Tokuda, Takahiko, E-mail: ttokuda@koto.kpu-m.ac.jp [Department of Neurology, Kyoto Prefectural University of Medicine, 465 Kajii-cho,Kamigyo-ku, Kyoto 602-8566 (Japan); Department of Molecular Pathobiology of Brain Diseases, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566 (Japan); Yamaguchi, Masamitsu, E-mail: myamaguc@kit.ac.jp [Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan)

2014-08-01

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila. - Highlights: • Knockdown of Cabeza induced rough eye phenotype. • Knockdown of Cabeza induced fusion of cone cells in pupal retinae. • Knockdown of Cabeza induced apoptosis in pupal retinae. • Mutation in EGFR pathway-related genes suppressed the rough eye phenotype. • Cabeza may negatively regulate the EGFR pathway.

The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach

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Tachibana Taro

2011-10-01

Full Text Available Abstract Background Cellular function is regulated by the balance of stringently regulated amounts of mRNA. Previous reports revealed that RNA polymerase II (RNAPII, which transcribes mRNA, can be classified into the pausing state and the active transcription state according to the phosphorylation state of RPB1, the catalytic subunit of RNAPII. However, genome-wide association between mRNA expression level and the phosphorylation state of RNAPII is unclear. While the functional importance of pausing genes is clear, such as in mouse Embryonic Stem cells for differentiation, understanding this association is critical for distinguishing pausing genes from active transcribing genes in expression profiling data, such as microarrays and RNAseq. Therefore, we examined the correlation between the phosphorylation of RNAPII and mRNA expression levels using a combined analysis by ChIPseq and RNAseq. Results We first performed a precise quantitative measurement of mRNA by performing an optimized calculation in RNAseq. We then visualized the recruitment of various phosphorylated RNAPIIs, such as Ser2P and Ser5P. A combined analysis using optimized RNAseq and ChIPseq for phosphorylated RNAPII revealed that mRNA levels correlate with the various phosphorylation states of RNAPII. Conclusions We demonstrated that the amount of mRNA is precisely reflected by the phased phosphorylation of Ser2 and Ser5. In particular, even the most "pausing" genes, for which only Ser5 is phosphorylated, were detectable at a certain level of mRNA. Our analysis indicated that the complexity of quantitative regulation of mRNA levels could be classified into three categories according to the phosphorylation state of RNAPII.

Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

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Alberto Miranda

2011-04-01

Full Text Available Cellular prion protein (PRNP is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs. Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB differentiation in mouse Prnp-null (KO and WT embryonic stem cell (ESC lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5 in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel and SPRN (Shadoo, whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

The Neurospora crassa UVS-3 epistasis group encodes homologues of the ATR/ATRIP checkpoint control system.

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Kazama, Yusuke; Ishii, Chizu; Schroeder, Alice L; Shimada, Hisao; Wakabayashi, Michiyoshi; Inoue, Hirokazu

2008-02-01

The mutagen sensitive uvs-3 and mus-9 mutants of Neurospora show mutagen and hydroxyurea sensitivity, mutator effects and duplication instability typical of recombination repair and DNA damage checkpoint defective mutants. To determine the nature of these genes we used cosmids from a genomic library to clone the uvs-3 gene by complementation for MMS sensitivity. Mutation induction by transposon insertion and RIP defined the coding sequence. RFLP analysis confirmed that this sequence maps in the area of uvs-3 at the left telomere of LG IV. Analysis of the cDNA showed that the UVS-3 protein contains an ORF of 969 amino acids with one intron. It is homologous to UvsD of Aspergillus nidulans, a member of the ATRIP family of checkpoint proteins. It retains the N' terminal coiled-coil motif followed by four basic amino acids typical of these proteins and shows the highest homology in this region. The uvsD cDNA partially complements the defects of the uvs-3 mutation. The uvs-3 mutant shows a higher level of micronuclei in conidia and failure to halt germination and nuclear division in the presence of hydroxyurea than wild type, suggesting checkpoint defects. ATRIP proteins bind tightly to ATR PI-3 kinase (phosphatidylinositol 3-kinase) proteins. Therefore, we searched the Neurospora genome sequence for homologues of the Aspergillus nidulans ATR, UvsB. A uvsB homologous sequence was present in the right arm of chromosome I where the mus-9 gene maps. A cosmid containing this genomic DNA complemented the mus-9 mutation. The putative MUS-9 protein is 2484 amino acids long with eight introns. Homology is especially high in the C-terminal 350 amino acids that correspond to the PI-3 kinase domain. In wild type a low level of constitutive mRNA is present for both genes. It is transiently induced upon UV exposure.

A La autoantigen homologue is required for the internal ribosome entry site mediated translation of giardiavirus.

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Srinivas Garlapati

2011-03-01

Full Text Available Translation of Giardiavirus (GLV mRNA is initiated at an internal ribosome entry site (IRES in the viral transcript. The IRES localizes to a downstream portion of 5' untranslated region (UTR and a part of the early downstream coding region of the transcript. Recent studies indicated that the IRES does not require a pre-initiation complex to initiate translation but may directly recruit the small ribosome subunit with the help of a number of trans-activating protein factors. A La autoantigen homologue in the viral host Giardia lamblia, GlLa, was proposed as one of the potential trans-activating factors based on its specific binding to GLV-IRES in vitro. In this study, we further elucidated the functional role of GlLa in GLV-IRES mediated translation in Giardia by knocking down GlLa with antisense morpholino oligo, which resulted in a reduction of GLV-IRES activity by 40%. An over-expression of GlLa in Giardia moderately stimulated GLV-IRES activity by 20%. A yeast inhibitory RNA (IRNA, known to bind mammalian and yeast La autoantigen and inhibit Poliovirus and Hepatitis C virus IRES activities in vitro and in vivo, was also found to bind to GlLa protein in vitro and inhibited GLV-IRES function in vivo. The C-terminal domain of La autoantigen interferes with the dimerization of La and inhibits its function. An over-expression of the C-terminal domain (200-348aa of GlLa in Giardia showed a dominant-negative effect on GLV-IRES activity, suggesting a potential inhibition of GlLa dimerization. HA tagged GlLa protein was detected mainly in the cytoplasm of Giardia, thus supporting a primary role of GlLa in translation initiation in Giardiavirus.

Highly efficient reprogramming to pluripotency and directed differentiation of human cells using synthetic modified mRNA

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Warren, Luigi; Manos, Philip D.; Ahfeldt, Tim; Loh, Yuin-Han; Li, Hu; Lau, Frank; Ebina, Wataru; Mandal, Pankaj; Smith, Zachary D.; Meissner, Alexander; Daley, George Q.; Brack, Andrew S.; Collins, James J.; Cowan, Chad; Schlaeger, Thorsten M.

2010-01-01

Clinical application of induced pluripotent stem (iPS) cells is limited by the low efficiency of iPS derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPS cells towards clinically useful cell types are lacking. Here we describe a simple, non-integrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate anti-viral re...

[Intersection point rule for the retention value with mobile phase composition and boiling point of the homologues and chlorobenzenes in soil leaching column chromatography].

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Xu, F; Liang, X; Lin, B; Su, F

1999-03-01

Based on the linear retention equation of the logarithm of the capacity factor (logk') vs. the methanol volume fraction (psi) of aqueous binary mobile phase in soil leaching column chromatography, the intersection point rule for the logk' of homologues and weak polar chlorobenzenes, with psi, as well as with boiling point, has been derived due to existence of the similar interactions among solutes of the same series, stationary phase (soil) and eluent (methanol-water). These rules were testified by experimental data of homologues (n-alkylbenzenes, methylbenzenes) and weak polar chlorobenzenes.

Mono-, di- and trimethylated homologues of isoprenoid tetraether lipid cores in archaea and environmental samples: mass spectrometric identification and significance.

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Knappy, Chris; Barillà, Daniela; Chong, James; Hodgson, Dominic; Morgan, Hugh; Suleman, Muhammad; Tan, Christine; Yao, Peng; Keely, Brendan

2015-12-01

Higher homologues of widely reported C(86) isoprenoid diglycerol tetraether lipid cores, containing 0-6 cyclopentyl rings, have been identified in (hyper)thermophilic archaea, representing up to 21% of total tetraether lipids in the cells. Liquid chromatography-tandem mass spectrometry confirms that the additional carbon atoms in the C(87-88) homologues are located in the etherified chains. Structures identified include dialkyl and monoalkyl ('H-shaped') tetraethers containing C(40-42) or C(81-82) hydrocarbons, respectively, many representing novel compounds. Gas chromatography-mass spectrometric analysis of hydrocarbons released from the lipid cores by ether cleavage suggests that the C(40) chains are biphytanes and the C(41) chains 13-methylbiphytanes. Multiple isomers, having different chain combinations, were recognised among the dialkyl lipids. Methylated tetraethers are produced by Methanothermobacter thermautotrophicus in varying proportions depending on growth conditions, suggesting that methylation may be an adaptive mechanism to regulate cellular function. The detection of methylated lipids in Pyrobaculum sp. AQ1.S2 and Sulfolobus acidocaldarius represents the first reported occurrences in Crenarchaeota. Soils and aquatic sediments from geographically distinct mesotemperate environments that were screened for homologues contained monomethylated tetraethers, with di- and trimethylated structures being detected occasionally. The structural diversity and range of occurrences of the C(87-89) tetraethers highlight their potential as complementary biomarkers for archaea in natural environments. Copyright © 2015 John Wiley & Sons, Ltd.

First report of a thioredoxin homologue in jellyfish: molecular cloning, expression and antioxidant activity of CcTrx1 from Cyanea capillata.

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Zengliang Ruan

Full Text Available Thioredoxins (Trx proteins are a family of small, highly-conserved and ubiquitous proteins that play significant roles in the resistance of oxidative damage. In this study, a homologue of Trx was identified from the cDNA library of tentacle of the jellyfish Cyanea capillata and named CcTrx1. The full-length cDNA of CcTrx1 was 479 bp with a 312 bp open reading frame encoding 104 amino acids. Bioinformatics analysis revealed that the putative CcTrx1 protein harbored the evolutionarily-conserved Trx active site 31CGPC34 and shared a high similarity with Trx1 proteins from other organisms analyzed, indicating that CcTrx1 is a new member of Trx1 sub-family. CcTrx1 mRNA was found to be constitutively expressed in tentacle, umbrella, oral arm and gonad, indicating a general role of CcTrx1 protein in various physiological processes. The recombinant CcTrx1 (rCcTrx1 protein was expressed in Escherichia coli BL21 (DE3, and then purified by affinity chromatography. The rCcTrx1 protein was demonstrated to possess the expected redox activity in enzymatic analysis and protection against oxidative damage of supercoiled DNA. These results indicate that CcTrx1 may function as an important antioxidant in C. capillata. To our knowledge, this is the first Trx protein characterized from jellyfish species.

CORRELATIONS BETWEEN HOMOLOGUE CONCENTRATIONS OF PCDD/FS AND TOXIC EQUIVALENCY VALUES IN LABORATORY-, PACKAGE BOILER-, AND FIELD-SCALE INCINERATORS

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The toxic equivalency (TEQ) values of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) are predicted with a model based on the homologue concentrations measured from a laboratory-scale reactor (124 data points), a package boiler (61 data points), and ...

Taurine Promotes the Cartilaginous Differentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Vitro.

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Yao, Xiuhua; Huang, Huiling; Li, Zhou; Liu, Xiaohua; Fan, Weijia; Wang, Xinping; Sun, Xuelian; Zhu, Jianmin; Zhou, Hongrui; Wei, Huaying

2017-08-01

Taurine has been reported to influence osteogenic differentiation, but the role of taurine on cartilaginous differentiation using human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) remains unclear. In this study, we investigated the effect of taurine (0, 1, 5 and 10 mM) on the proliferation and chondrogenesis of hUC-MSCs by analyzing cell proliferation, accumulation of glycosaminoglycans and expression of cartilage specific mRNA. The results show though taurine did not affected the proliferation of hUC-MSCs, 5 mM of taurine is sufficient to enhanced the accumulation of glycosaminoglycans and up-regulate cartilage specific mRNA expression, namely collagen type II, aggrecan and SOX9. Taurine also inhibits chondrocyte dedifferentiation by reducing expression of collagen type I mRNA. Taken together, our study reveals that taurine promotes and maintains the chondrogenesis of hUC-MSCs.

Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action

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Kihara, Tasuku; Ichikawa, Saki; Yonezawa, Takayuki; Lee, Ji-Won; Akihisa, Toshihiro; Woo, Je Tae; Michi, Yasuyuki; Amagasa, Teruo; Yamaguchi, Akira

2011-01-01

Research highlights: → Acerogenin A stimulated osteoblast differentiation in osteogenic cells. → Acerogenin A-induced osteoblast differentiation was inhibited by noggin. → Acerogenin A increased Bmp-2, Bmp-4 and Bmp-7 mRNA expression in MC3T3-E1 cells. → Acerogenin A is a candidate agent for stimulating bone formation. -- Abstract: We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3 days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.

Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine.

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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

Aplasia Ras homologue member Ⅰ overexpression inhibits tumor growth and induces apoptosis through inhibition of PI3K/Akt survival pathways in human osteosarcoma MG-63 cells in culture.

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Ye, Kaishan; Wang, Shuanke; Yang, Yong; Kang, Xuewen; Wang, Jing; Han, Hua

2015-09-01

Aplasia Ras homologue member Ⅰ (ARHI), an imprinted tumor-suppressor gene, is downregulated in various types of cancer. However, the expression, function and specific mechanisms of ARHI in human osteosarcoma (OS) cells remain unclear. The aim of the present study was to assess the effect of ARHI on OS cell proliferation and apoptosis and its associated mechanism. In the study, ARHI mRNA and protein levels were markedly downregulated in OS cells compared with the human osteoblast precursor cell line hFOB1.19. By generating stable transfectants, ARHI was overexpressed in OS cells that had low levels of ARHI. Overexpression of ARHI inhibited cell viability and proliferation and induced apoptosis. However, caspase‑3 activity was not changed by ARHI overexpression. In addition, phosphorylated Akt protein expression decreased in the ARHI overexpression group compared to that in the control vector group. The knockdown of ARHI also resulted in the promotion of cell proliferation and the attenuation of apoptosis in MG‑63 cells. Additionally, ARHI silencing increased the level of p‑Akt. The present results indicate that ARHI inhibits OS cell proliferation and may have a key role in the development of OS.

Differential Expression of Cysteine Dioxygenase 1 in Complex Karyotype Liposarcomas

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Mohammed Shaker

2014-01-01

Full Text Available Altered cysteine dioxygenase 1 (CDO1 gene expression has been observed in several cancers but has not yet been investigated in liposarcomas. The aim of this study was to evaluate CDO1 expression in a cohort of liposarcomas and to determine its association with clinicopathological features. Existing microarray data indicated variable CDO1 expression in liposarcoma subtypes. CDO1 mRNA from a larger cohort of liposarcomas was quantified by real time-PCR, and CDO1 protein expression was determined by immunohistochemistry (IHC in more than 300 tumor specimens. Well-differentiated liposarcomas (WDLSs had significantly higher CDO1 gene expression and protein levels than dedifferentiated liposarcomas (DDLSs ( P < 0.001. Location of the tumor was not predictive of the expression level of CDO1 mRNA in any histological subtype of liposarcoma. Recurrent tumors did not show any difference in CDO1 expression when compared to primary tumors. CDO1 expression was upregulated as human mesenchymal stem cells (hMSCs undergo differentiation into mature adipocytes. Our results suggest that CDO1 is a marker of liposarcoma progression and adipogenic differentiation.

Reference: 351 [Arabidopsis Phenome Database[Archive

Lifescience Database Archive (English)

Full Text Available similarly high levels of ABA. ABA levels decreased rapidly upon imbibition, although they fell further in ND than in D. Gene express...e family (CYP707A)] genes. Of these, only the AtCYP707A2 gene was differentially expressed between D and ND seeds, being express...ed to a much higher level in ND seeds. Similarly, a barley CYP707 homologue, (HvABA8'OH-1) was express...ins. Consistent with this, in situ hybridization studies showed HvABA8'OH-1 mRNA expression was stronger in ... plays a key role in dormancy release. Constitutive expression of a CYP707A gene in transgenic Arabidopsis r

Fip1 regulates mRNA alternative polyadenylation to promote stem cell self-renewal

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Lackford, Brad; Yao, Chengguo; Charles, Georgette M; Weng, Lingjie; Zheng, Xiaofeng; Choi, Eun-A; Xie, Xiaohui; Wan, Ji; Xing, Yi; Freudenberg, Johannes M; Yang, Pengyi; Jothi, Raja; Hu, Guang; Shi, Yongsheng

2014-01-01

mRNA alternative polyadenylation (APA) plays a critical role in post-transcriptional gene control and is highly regulated during development and disease. However, the regulatory mechanisms and functional consequences of APA remain poorly understood. Here, we show that an mRNA 3′ processing factor, Fip1, is essential for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Fip1 promotes stem cell maintenance, in part, by activating the ESC-specific APA profiles to ensure the optimal expression of a specific set of genes, including critical self-renewal factors. Fip1 expression and the Fip1-dependent APA program change during ESC differentiation and are restored to an ESC-like state during somatic reprogramming. Mechanistically, we provide evidence that the specificity of Fip1-mediated APA regulation depends on multiple factors, including Fip1-RNA interactions and the distance between APA sites. Together, our data highlight the role for post-transcriptional control in stem cell self-renewal, provide mechanistic insight on APA regulation in development, and establish an important function for APA in cell fate specification. PMID:24596251

Functions of Heterogeneous Nuclear Ribonucleoproteins in Stem Cell Potency and Differentiation

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Qishan Chen

2013-01-01

Full Text Available Stem cells possess huge importance in developmental biology, disease modelling, cell replacement therapy, and tissue engineering in regenerative medicine because they have the remarkable potential for self-renewal and to differentiate into almost all the cell types in the human body. Elucidation of molecular mechanisms regulating stem cell potency and differentiation is essential and critical for extensive application. Heterogeneous nuclear ribonucleoproteins (hnRNPs are modular proteins consisting of RNA-binding motifs and auxiliary domains characterized by extensive and divergent functions in nucleic acid metabolism. Multiple roles of hnRNPs in transcriptional and posttranscriptional regulation enable them to be effective gene expression regulators. More recent findings show that hnRNP proteins are crucial factors implicated in maintenance of stem cell self-renewal and pluripotency and cell differentiation. The hnRNPs interact with certain sequences in target gene promoter regions to initiate transcription. In addition, they recognize 3′UTR or 5′UTR of specific gene mRNA forming mRNP complex to regulate mRNA stability and translation. Both of these regulatory pathways lead to modulation of gene expression that is associated with stem cell proliferation, cell cycle control, pluripotency, and committed differentiation.

Purification of the spliced leader ribonucleoprotein particle from Leptomonas collosoma revealed the existence of an Sm protein in trypanosomes. Cloning the SmE homologue.

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Goncharov, I; Palfi, Z; Bindereif, A; Michaeli, S

1999-04-30

Trans-splicing in trypanosomes involves the addition of a common spliced leader (SL) sequence, which is derived from a small RNA, the SL RNA, to all mRNA precursors. The SL RNA is present in the cell in the form of a ribonucleoprotein, the SL RNP. Using conventional chromatography and affinity selection with 2'-O-methylated RNA oligonucleotides at high ionic strength, five proteins of 70, 16, 13, 12, and 8 kDa were co-selected with the SL RNA from Leptomonas collosoma, representing the SL RNP core particle. Under conditions of lower ionic strength, additional proteins of 28 and 20 kDa were revealed. On the basis of peptide sequences, the gene coding for a protein with a predicted molecular weight of 11.9 kDa was cloned and identified as homologue of the cis-spliceosomal SmE. The protein carries the Sm motifs 1 and 2 characteristic of Sm antigens that bind to all known cis-spliceosomal uridylic acid-rich small nuclear RNAs (U snRNAs), suggesting the existence of Sm proteins in trypanosomes. This finding is of special interest because trypanosome snRNPs are the only snRNPs examined to date that are not recognized by anti-Sm antibodies. Because of the early divergence of trypanosomes from the eukaryotic lineage, the trypanosome SmE protein represents one of the primordial Sm proteins in nature.

Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells.

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Kawasaki, Shunsuke; Fujita, Yoshihiko; Nagaike, Takashi; Tomita, Kozo; Saito, Hirohide

2017-07-07

Synthetic biology has great potential for future therapeutic applications including autonomous cell programming through the detection of protein signals and the production of desired outputs. Synthetic RNA devices are promising for this purpose. However, the number of available devices is limited due to the difficulty in the detection of endogenous proteins within a cell. Here, we show a strategy to construct synthetic mRNA devices that detect endogenous proteins in living cells, control translation and distinguish cell types. We engineered protein-binding aptamers that have increased stability in the secondary structures of their active conformation. The designed devices can efficiently respond to target proteins including human LIN28A and U1A proteins, while the original aptamers failed to do so. Moreover, mRNA delivery of an LIN28A-responsive device into human induced pluripotent stem cells (hiPSCs) revealed that we can distinguish living hiPSCs and differentiated cells by quantifying endogenous LIN28A protein expression level. Thus, our endogenous protein-driven RNA devices determine live-cell states and program mammalian cells based on intracellular protein information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Integrating microRNA and mRNA expression profiles in response to radiation-induced injury in rat lung

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Xie, Ling; Zhou, Jundong; Zhang, Shuyu; Chen, Qing; Lai, Rensheng; Ding, Weiqun; Song, ChuanJun; Meng, XingJun; Wu, Jinchang

2014-01-01

Exposure to radiation provokes cellular responses, which are likely regulated by gene expression networks. MicroRNAs are small non-coding RNAs, which regulate gene expression by promoting mRNA degradation or inhibiting protein translation. The expression patterns of both mRNA and miRNA during the radiation-induced lung injury (RILI) remain less characterized and the role of miRNAs in the regulation of this process has not been studied. The present study sought to evaluate miRNA and mRNA expression profiles in the rat lung after irradiation. Male Wistar rats were subjected to single dose irradiation with 20 Gy using 6 MV x-rays to the right lung. (A dose rate of 5 Gy/min was applied). Rats were sacrificed at 3, 12 and 26 weeks after irradiation, and morphological changes in the lung were examined by haematoxylin and eosin. The miRNA and mRNA expression profiles were evaluated by microarrays and followed by quantitative RT-PCR analysis. A cDNA microarray analysis found 2183 transcripts being up-regulated and 2917 transcripts down-regulated (P ≤ 0.05, ≥2.0 fold change) in the lung tissues after irradiation. Likewise, a miRNAs microarray analysis indicated 15 miRNA species being up-regulated and 8 down-regulated (P ≤ 0.05). Subsequent bioinformatics anal -yses of the differentially expressed mRNA and miRNAs revealed that alterations in mRNA expression following irradiation were negatively correlated with miRNAs expression. Our results provide evidence indicating that irradiation induces alterations of mRNA and miRNA expression in rat lung and that there is a negative correlation of mRNA and miRNA expression levels after irradiation. These findings significantly advance our understanding of the regulatory mechanisms underlying the pathophysiology of radiation-induced lung injury. In summary, RILI does not develop gradually in a linear process. In fact, different cell types interact via cytokines in a very complex network. Furthermore, this study suggests that

Combinatorial Control of mRNA Fates by RNA-Binding Proteins and Non-Coding RNAs

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Valentina Iadevaia

2015-09-01

Full Text Available Post-transcriptional control of gene expression is mediated by RNA-binding proteins (RBPs and small non-coding RNAs (e.g., microRNAs that bind to distinct elements in their mRNA targets. Here, we review recent examples describing the synergistic and/or antagonistic effects mediated by RBPs and miRNAs to determine the localisation, stability and translation of mRNAs in mammalian cells. From these studies, it is becoming increasingly apparent that dynamic rearrangements of RNA-protein complexes could have profound implications in human cancer, in synaptic plasticity, and in cellular differentiation.

Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPAR{gamma} activation

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Takahashi, Nobuyuki; Goto, Tsuyoshi; Taimatsu, Aki; Egawa, Kahori; Katoh, Sota; Kusudo, Tatsuya; Sakamoto, Tomoya; Ohyane, Chie; Lee, Joo-Young; Kim, Young-il; Uemura, Taku; Hirai, Shizuka [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan); Kawada, Teruo, E-mail: fat@kais.kyoto-u.ac.jp [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan)

2009-12-25

Insulin resistance is partly due to suppression of insulin-induced glucose uptake into adipocytes. The uptake is dependent on adipocyte differentiation, which is controlled at mRNA transcription level. The peroxisome proliferator-activated receptor (PPAR), a ligand-regulated nuclear receptor, is involved in the differentiation. Many food-derived compounds serve as ligands to activate or inactivate PPAR. In this study, we demonstrated that bixin and norbixin (annatto extracts) activate PPAR{gamma} by luciferase reporter assay using GAL4-PPAR chimera proteins. To examine the effects of bixin on adipocytes, 3T3-L1 adipocytes were treated with bixin or norbixin. The treatment induced mRNA expression of PPAR{gamma} target genes such as adipocyte-specific fatty acid-binding protein (aP2), lipoprotein lipase (LPL), and adiponectin in differentiated 3T3-L1 adipocytes and enhanced insulin-dependent glucose uptake. The observations indicate that bixin acts as an agonist of PPAR{gamma} and enhances insulin sensitivity in 3T3-L1 adipocytes, suggesting that bixin is a valuable food-derived compound as a PPAR ligand to regulate lipid metabolism and to ameliorate metabolic syndrome.

T-lymphocyte cytokine mRNA expression in cystic echinococcosis.

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Fauser, S; Kern, P

1997-04-01

In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response.

The effect of Dimethyl Sulfoxide on hepatogenic differentiation of Mesenchymal Stem Cells

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Effat Alizadeh

2014-05-01

The results demonstrate that DMSO speeds up hepatic differentiation of AT-MSCs characterized by rapid changes in morphology, higher expression of hepatic marker gene (AFP in both mRNA and protein level (P

A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement*

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Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

2016-01-01

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. PMID:26887951

A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement.

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Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

2016-04-15

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphorylation of eIF2α is required for mRNA translation inhibition and survival during moderate hypoxia

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Koritzinsky, Marianne; Rouschop, Kasper M.A.; Beucken, Twan van den; Magagnin, Michael G.; Savelkouls, Kim; Lambin, Philippe; Wouters, Bradly G.

2007-01-01

Abstracts: Background and purpose: Human tumors are characterized by temporal fluctuations in oxygen tension. The biological pathways that respond to the dynamic tumor microenvironment represent potential molecular targets for cancer therapy. Anoxic conditions result in eIF2α dependent inhibition of overall mRNA translation, differential gene expression, hypoxia tolerance and tumor growth. The signaling pathway which governs eIF2α phosphorylation has therefore emerged as a potential molecular target. In this study, we investigated the role of eIF2α in regulating mRNA translation and hypoxia tolerance during moderate hypoxia. Since other molecular pathways that regulate protein synthesis are frequently mutated in cancer, we also assessed mRNA translation in a panel of cell lines from different origins. Materials and methods: Immortalized human fibroblast, transformed mouse embryo fibroblasts (MEFs) and cells from six cancer cell lines were exposed to 0.2% or 0.0% oxygen. We assayed global mRNA translation efficiency by polysome analysis, as well as proliferation and clonogenic survival. The role of eIF2α was assessed in MEFs harboring a homozygous inactivating mutation (S51A) as well as in U373-MG cells overexpressing GADD34 (C-term) under a tetracycline-dependent promoter. The involvement of eIF4E regulation was investigated in HeLa cells stably expressing a short hairpin RNA (shRNA) targeting 4E-BP1. Results: All cells investigated inhibited mRNA translation severely in response to anoxia and modestly in response to hypoxia. Two independent genetic cell models demonstrated that inhibition of mRNA translation in response to moderate hypoxia was dependent on eIF2α phosphorylation. Disruption of eIF2α phosphorylation caused sensitivity to hypoxia and anoxia. Conclusions: Disruption of eIF2α phosphorylation is a potential target for hypoxia-directed molecular cancer therapy

Genetic analysis of the spindle checkpoint genes san-1, mdf-2, bub-3 and the CENP-F homologues hcp-1 and hcp-2 in Caenorhabditis elegans

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Moore Landon L

2008-02-01

Full Text Available Abstract Background The spindle checkpoint delays the onset of anaphase until all sister chromatids are aligned properly at the metaphase plate. To investigate the role san-1, the MAD3 homologue, has in Caenorhabditis elegans embryos we used RNA interference (RNAi to identify genes synthetic lethal with the viable san-1(ok1580 deletion mutant. Results The san-1(ok1580 animal has low penetrating phenotypes including an increased incidence of males, larvae arrest, slow growth, protruding vulva, and defects in vulva morphogenesis. We found that the viability of san-1(ok1580 embryos is significantly reduced when HCP-1 (CENP-F homologue, MDF-1 (MAD-1 homologue, MDF-2 (MAD-2 homologue or BUB-3 (predicted BUB-3 homologue are reduced by RNAi. Interestingly, the viability of san-1(ok1580 embryos is not significantly reduced when the paralog of HCP-1, HCP-2, is reduced. The phenotype of san-1(ok1580;hcp-1(RNAi embryos includes embryonic and larval lethality, abnormal organ development, and an increase in abnormal chromosome segregation (aberrant mitotic nuclei, anaphase bridging. Several of the san-1(ok1580;hcp-1(RNAi animals displayed abnormal kinetochore (detected by MPM-2 and microtubule structure. The survival of mdf-2(RNAi;hcp-1(RNAi embryos but not bub-3(RNAi;hcp-1(RNAi embryos was also compromised. Finally, we found that san-1(ok1580 and bub-3(RNAi, but not hcp-1(RNAi embryos, were sensitive to anoxia, suggesting that like SAN-1, BUB-3 has a functional role as a spindle checkpoint protein. Conclusion Together, these data suggest that in the C. elegans embryo, HCP-1 interacts with a subset of the spindle checkpoint pathway. Furthermore, the fact that san-1(ok1580;hcp-1(RNAi animals had a severe viability defect whereas in the san-1(ok1580;hcp-2(RNAi and san-1(ok1580;hcp-2(ok1757 animals the viability defect was not as severe suggesting that hcp-1 and hcp-2 are not completely redundant.

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

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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.

Identification and characterization of the ESAT-6 homologue of Mycobacterium leprae and T-cell cross-reactivity with Mycobacterium tuberculosis

NARCIS (Netherlands)

Geluk, Annemieke; van Meijgaarden, Krista E.; Franken, Kees L. M. C.; Subronto, Yanri W.; Wieles, Brigitte; Arend, Sandra M.; Sampaio, Elizabeth P.; de Boer, Tjitske; Faber, William R.; Naafs, Ben; Ottenhoff, Tom H. M.

2002-01-01

In this paper we describe identification and characterization of Mycobacterium leprae ESAT-6 (L-ESAT-6), the homologue of M. tuberculosis ESAT-6 (T-ESAT-6). T-ESAT-6 is expressed by all pathogenic strains belonging to the M. tuberculosis complex but is absent from virtually all other mycobacterial

4-Oxalocrotonate tautomerase, its homologue YwhB, and active vinylpyruvate hydratase : Synthesis and evaluation of 2-fluoro substrate analogues

NARCIS (Netherlands)

Johnson, William H; Wang, Susan C; Stanley, Thanuja M; Czerwinski, Robert M; Almrud, Jeffrey J; Poelarends, Gerrit J; Murzin, Alexey G; Whitman, Christian P

2004-01-01

A series of 2-fluoro-4-alkene and 2-fluoro-4-alkyne substrate analogues were synthesized and examined as potential inhibitors of three enzymes: 4-oxalocrotonate tautomerase (4-OT) and vinylpyruvate hydratase (VPH) from the catechol meta-fission pathway and a closely related 4-OT homologue found in

Transcriptome Sequencing, De Novo Assembly and Differential Gene Expression Analysis of the Early Development of Acipenser baeri.

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Wei Song

Full Text Available The molecular mechanisms that drive the development of the endangered fossil fish species Acipenser baeri are difficult to study due to the lack of genomic data. Recent advances in sequencing technologies and the reducing cost of sequencing offer exclusive opportunities for exploring important molecular mechanisms underlying specific biological processes. This manuscript describes the large scale sequencing and analyses of mRNA from Acipenser baeri collected at five development time points using the Illumina Hiseq2000 platform. The sequencing reads were de novo assembled and clustered into 278167 unigenes, of which 57346 (20.62% had 45837 known homologues proteins in Uniprot protein databases while 11509 proteins matched with at least one sequence of assembled unigenes. The remaining 79.38% of unigenes could stand for non-coding unigenes or unigenes specific to A. baeri. A number of 43062 unigenes were annotated into functional categories via Gene Ontology (GO annotation whereas 29526 unigenes were associated with 329 pathways by mapping to KEGG database. Subsequently, 3479 differentially expressed genes were scanned within developmental stages and clustered into 50 gene expression profiles. Genes preferentially expressed at each stage were also identified. Through GO and KEGG pathway enrichment analysis, relevant physiological variations during the early development of A. baeri could be better cognized. Accordingly, the present study gives insights into the transcriptome profile of the early development of A. baeri, and the information contained in this large scale transcriptome will provide substantial references for A. baeri developmental biology and promote its aquaculture research.

miRNA-720 controls stem cell phenotype, proliferation and differentiation of human dental pulp cells.

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Emilio Satoshi Hara

Full Text Available Dental pulp cells (DPCs are known to be enriched in stem/progenitor cells but not well characterized yet. Small non-coding microRNAs (miRNAs have been identified to control protein translation, mRNA stability and transcription, and have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In order to characterize dental pulp stem/progenitor cells and its mechanism of differentiation, we herein sorted stem-cell-enriched side population (SP cells from human DPCs and periodontal ligament cells (PDLCs, and performed a locked nucleic acid (LNA-based miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Concomitantly, mRNA levels of DNA methyltransferases (DNMTs, which are known to play crucial factors during stem cell differentiation, were also increased by miR-720 through unknown mechanism. Finally, miR-720 decreased DPC proliferation as determined by immunocytochemical analysis against ki-67, and promoted odontogenic differentiation as demonstrated by alizarin red staining, as well as alkaline phosphatase and osteopontin mRNA levels. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.

Effect of perfluorooctane sulfonate on pluripotency and differentiation factors in mouse embryoid bodies

International Nuclear Information System (INIS)

Xu, Bo; Ji, Xiaoli; Chen, Xiaojiao; Yao, Mengmeng; Han, Xiumei; Chen, Minjian; Tang, Wei; Xia, Yankai

2015-01-01

Perfluorooctane sulfonate (PFOS) poses potential risks to early development, but the molecular mechanisms how PFOS affects embryonic development are still unclear. Mouse embryoid bodies (mEBs) provide ideal models for testing safety or toxicity of chemicals in vitro. In this study, mEBs were exposed to PFOS up to 6 days and then their pluripotency and differentiation markers were evaluated. Our data showed that the mRNA and protein levels of pluripotency markers (Oct4, Sox2, Nanog) in mEBs were significantly increased following exposure to PFOS. Meanwhile, the expressions of miR-134, miR-145, miR-490-3p were decreased accordingly. PFOS reduced the mRNA levels of endodermal markers (Sox17, FOXA2), mesodermal markers (SMA, Brachyury) and ectodermal markers (Nestin, Fgf5) in mEBs. Meanwhile, PFOS increased the mRNA and protein levels of polycomb group (PcG) family members (Cbx4, Cbx7, Ezh2). Overall, our results showed that PFOS could increase the expression levels of pluripotency factors and decrease the differentiation markers

Dietary fatty acids regulate hepatic low density lipoprotein (LDL) transport by altering LDL receptor protein and mRNA levels.

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Horton, J D; Cuthbert, J A; Spady, D K

1993-01-01

The concentration of LDL in plasma is strongly influenced by the amount and the type of lipid in the diet. Recent studies in the hamster have shown that dietary fatty acids differentially affect circulating LDL levels primarily by altering receptor-dependent LDL uptake in the liver. To investigate the mechanistic basis of this effect, rates of receptor-dependent LDL transport in the liver were correlated with LDL receptor protein and mRNA levels in hamsters fed safflower oil or coconut oil and varying amounts of cholesterol. Hepatic LDL receptor activity was significantly lower in animals fed coconut oil than in animals fed safflower oil at all levels of cholesterol intake (26, 53, and 61% lower at cholesterol intakes of 0, 0.06, and 0.12%, respectively). These fatty acid-induced changes in hepatic LDL receptor activity were accompanied by parallel changes in hepatic LDL receptor protein and mRNA levels, suggesting that dietary fatty acids regulate the LDL receptor pathway largely at the mRNA level. Images PMID:8349814

BMP7 transfection induces in-vitro osteogenic differentiation of dental pulp mesenchymal stem cells

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Ka Po John Yau

2013-01-01

Full Text Available Objective: To assess whether in-vitro osteogenic differentiation of human dental pulp mesenchymal stem cells can be induced by transient transfection with the gene encoding human bone morphogenic protein 7 (BMP7. Materials and Methods: A mesenchymal stem cell population was isolated from the dental pulp of two extracted permanent premolars, expanded and characterized. The human BMP7 gene, as a recombinant pcDNA3.1/V5-His-TOPO-BMP7 plasmid, was transfected into the cells. Three negative controls were used: No plasmid, empty vector, and an unrelated vector encoding green fluorescent protein. After the interval of 24 and 48 h, mRNA levels of alkaline phosphatase and osteocalcin as markers of in-vitro osteogenic differentiation were measured by real-time polymerase chain reaction and standardized against β-actin mRNA levels. Results: The level of alkaline phosphatase mRNA was significantly higher for the BMP7 group than for all three negative controls 48 h after transfection (706.9 vs. 11.24 for untransfected cells, 78.05 for empty vector, and 73.10 for green fluorescent protein vector. The level of osteocalcin mRNA was significantly higher for the BMP7 group than for all three negative controls 24 h after transfection (1.0, however, decreased after another 24 h. Conclusions: In-vitro osteoblastic differentiation of human dental pulp mesenchymal stem cells, as indicated by expression of alkaline phosphatase and osteocalcin, can be induced by transient transfection with the BMP7 gene.

PPARg mRNA in the adult mouse hypothalamus: distribution and regulation in response to dietary challenges

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Yang eLiu

2015-09-01

Full Text Available Peroxisome proliferator-activated receptor gamma (PPARg is a ligand-activated transcription factor that was originally identified as a regulator of peroxisome proliferation and adipocyte differentiation. Emerging evidence suggests that functional PPARg signaling also occurs within the hypothalamus. However, the exact distribution and identities of PPARg-expressing hypothalamic cells remains under debate. The present study systematically mapped PPARg mRNA expression in the adult mouse brain using in situ hybridization histochemistry. PPARg mRNA was found to be expressed at high levels outside the hypothalamus including the neocortex, the olfactory bulb, the organ of the vasculosum of the lamina terminalis, and the subfornical organ. Within the hypothalamus, PPARg was present at moderate levels in the suprachiasmatic nucleus and the ependymal of the 3rd ventricle. In all examined feeding-related hypothalamic nuclei, PPARg was expressed at very low levels that were close to the limit of detection. Using qPCR techniques, we demonstrated that PPARg mRNA expression was upregulated in the suprachiasmatic nucleus in response to fasting. Double in situ hybridization further demonstrated that PPARg was primarily expressed in neurons. Collectively, our observations provide a comprehensive map of PPARg distribution and regulation in the intact adult mouse hypothalamus.

The use of polynuclear aromatic hydrocarbon (PAH) alkyl homologues in determining petroleum source identification and weathering

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Brown, J.S.; Boehm, P.D.; Sauer, T.C.; Wong, W.M.C.

1993-01-01

Techniques utilizing double ratio plots of selected polynuclear aromatic hydrocarbon (PAH) alkyl homologues were used to identify and distinguish crude oils and refined petroleum products from each other and to distinguish petroleum sources in complex pollutant regimes. Petroleum samples were fractionated by high performance liquid chromatography (HPLC) into saturated and aromatic (PAH) hydrocarbon fractions. The saturated hydrocarbon fractions were then analyzed by gas chromatography/flame ionization detection (GC/FID) to obtain a resolved/unresolved alkane fingerprint of each oil. The aromatic fractions of the oils were analyzed by gas chromatography/mass spectrometry (GC/MS) for PAH and selected alkyl homologues. Comparisons of the saturated hydrocarbon fingerprints indicated that some oils were indistinguishable based on the alkane fingerprint alone. Another double ratio plot of the alkyl chrysenes and alkyl dibenzothiophenes was effective in establishing the weathering of oil in environmental samples which were processed using the same analytical techniques, since the dibenzothiophenes are degraded more rapidly than the chrysenes. The application of selected ratios in oil spill source identification in complex environmental samples from Suisin Bay California and Boston Harbor are discussed. The use of ratios to measure the extent of weathering in oil spill samples from Prince William Sound and the Gulf of Alaska is examined

Identifying airway sensitizers: cytokine mRNA profiles induced by various anhydrides

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Plitnick, L.M.; Loveless, S.E.; Ladics, G.S.; Holsapple, M.P.; Smialowicz, R.J.; Woolhiser, M.R.; Anderson, P.K.; Smith, C.; Selgrade, M.J.K.

2003-01-01

Exposure to low molecular weight (LMW) chemicals in the workplace has been linked to a variety of respiratory effects. Within the LMW chemicals, one of the major classes involved in these effects are the acid anhydrides. The immunological basis of respiratory hypersensitivity involves CD4+ cells. By virtue of their induction of cytokines typical of CD4+ T-helper type 2 (Th2) cells--interleukin (IL)-4, 10, and 13--respiratory sensitizers may be identified and differentiated from contact sensitizers which induce Th1 cytokines (IL-2 and IFN-γ). Our previous work suggested that the ribonuclease protection assay (RPA) was useful in identifying the respiratory sensitizer, trimellitic anhydride (TMA), based on quantitative differences in Th2 cytokine mRNA as compared to the contact sensitizer dinitrochlorobenzene (DNCB). Therefore, the purpose of the studies described in this report was to expand the chemicals tested in the RPA. To this end, four acid anhydrides with known respiratory sensitization potential, TMA, maleic anhydride (MA), phthalic anhydride (PA) and hexahydrophthalic anhydride (HHPA), were tested. Although previously determined to induce immunologically equivalent responses in a local lymph node assay (LLNA), the initial dose chosen (2.5%) failed to induce Th2 cytokine mRNA expression. To determine if the lack of cytokine expression was related to dose, LLNAs were conducted at higher doses for each of the anhydrides. The highest doses evaluated (four- to six-fold higher than those used in the initial RPA) gave equivalent proliferative responses for the various anhydrides and were used for subsequent RPA testing. At these higher doses, significant increases in Th2 versus Th1 cytokine mRNA were observed for all anhydrides tested. These results suggest that the RPA has the potential to serve as a screen for the detection of LMW airway sensitizing chemicals. However, the basis for selecting immunologically equivalent doses may require some modification

The actin homologue MreB organizes the bacterial cell membrane.

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Strahl, Henrik; Bürmann, Frank; Hamoen, Leendert W

2014-03-07

The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate bacterial cell wall synthesis. We noticed that the MreB cytoskeleton influences fluorescent staining of the cytoplasmic membrane. Detailed analyses combining an array of mutants, using specific lipid staining techniques and spectroscopic methods, revealed that MreB filaments create specific membrane regions with increased fluidity (RIFs). Interference with these fluid lipid domains (RIFs) perturbs overall lipid homeostasis and affects membrane protein localization. The influence of MreB on membrane organization and fluidity may explain why the active movement of MreB stimulates membrane protein diffusion. These novel MreB activities add additional complexity to bacterial cell membrane organization and have implications for many membrane-associated processes.

BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells.

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Awe, Jason P; Crespo, Agustin Vega; Li, You; Kiledjian, Megerditch; Byrne, James A

2013-02-06

The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-κB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells. We tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4. We found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels. For the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics.

MiR-378 Plays an Important Role in the Differentiation of Bovine Preadipocytes.

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Liu, Si-Yuan; Zhang, Yang-Yang; Gao, Yan; Zhang, Lian-Jiang; Chen, Hong-Yan; Zhou, Qian; Chai, Meng-Long; Li, Qing-Ying; Jiang, Hao; Yuan, Bao; Dai, Li-Sheng; Zhang, Jia-Bao

2015-01-01

Adipocyte, the main cellular component of white adipose tissue, plays a vital role in energy balance in higher eukaryotes. In recent years, adipocytes have also been identified as a major endocrine organ involved in immunological responses, vascular diseases, and appetite regulation. In farm animals, fat content and categories are closely correlated with meat quality. MicroRNAs (miRNAs), a class of endogenous single-stranded non-coding RNA molecules, participate in the regulation of adipocyte differentiation and adipogenesis through regulating the transcription or translation of target mRNAs. MiR-378 plays an important role in a number of biological processes, including cell growth, cell differentiation, tumor cell survival and angiogenesis. In the present study, bioinformatics analysis and dual-luciferase reporter assay were used to identify and validate the target genes of miR-378. In vitro cell transfection, quantitative reverse transcription polymerase chain reaction (RT-qPCR), western blot analysis, Oil Red O staining, and triglyceride content measurement were conducted to analyze the effects of miR-378 on bovine preadipocyte differentiation. MiR-378 was induced during adipocyte differentiation. In the differentiated adipocytes overexpressing miR- 378, the volume of lipid droplets was enlarged, and the triglyceride content was increased. Moreover, the mRNA expression levels of the adipocyte differentiation marker genes, peroxisome proliferator-activated receptor gamma (PPARγ) and sterol regulatory element-binding protein (SREBP), were significantly elevated in the differentiated, mature adipocytes. In contrast, the mRNA expression level of preadipocyte factor 1 (Pref-1) was markedly reduced. E2F transcription factor 2 (E2F2) and Ras-related nuclear (RAN)-binding protein 10 (RANBP10) were the two target genes of miR-378. The mRNA expression levels of E2F2 and RANBP10 did not significantly change in bovine preadipocytes overexpressing miR-378. However, the

MiR-378 Plays an Important Role in the Differentiation of Bovine Preadipocytes

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Si-Yuan Liu

2015-07-01

Full Text Available Background: Adipocyte, the main cellular component of white adipose tissue, plays a vital role in energy balance in higher eukaryotes. In recent years, adipocytes have also been identified as a major endocrine organ involved in immunological responses, vascular diseases, and appetite regulation. In farm animals, fat content and categories are closely correlated with meat quality. MicroRNAs (miRNAs, a class of endogenous single-stranded non-coding RNA molecules, participate in the regulation of adipocyte differentiation and adipogenesis through regulating the transcription or translation of target mRNAs. MiR-378 plays an important role in a number of biological processes, including cell growth, cell differentiation, tumor cell survival and angiogenesis. Methods: In the present study, bioinformatics analysis and dual-luciferase reporter assay were used to identify and validate the target genes of miR-378. In vitro cell transfection, quantitative reverse transcription polymerase chain reaction (RT-qPCR, western blot analysis, Oil Red O staining, and triglyceride content measurement were conducted to analyze the effects of miR-378 on bovine preadipocyte differentiation. Results: MiR-378 was induced during adipocyte differentiation. In the differentiated adipocytes overexpressing miR-378, the volume of lipid droplets was enlarged, and the triglyceride content was increased. Moreover, the mRNA expression levels of the adipocyte differentiation marker genes, peroxisome proliferator-activated receptor gamma (PPARγ and sterol regulatory element-binding protein (SREBP, were significantly elevated in the differentiated, mature adipocytes. In contrast, the mRNA expression level of preadipocyte factor 1 (Pref-1 was markedly reduced. E2F transcription factor 2 (E2F2 and Ras-related nuclear (RAN-binding protein 10 (RANBP10 were the two target genes of miR-378. The mRNA expression levels of E2F2 and RANBP10 did not significantly change in bovine preadipocytes

Integrative testis transcriptome analysis reveals differentially expressed miRNAs and their mRNA targets during early puberty in Atlantic salmon.

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Skaftnesmo, K O; Edvardsen, R B; Furmanek, T; Crespo, D; Andersson, E; Kleppe, L; Taranger, G L; Bogerd, J; Schulz, R W; Wargelius, A

2017-10-18

Our understanding of the molecular mechanisms implementing pubertal maturation of the testis in vertebrates is incomplete. This topic is relevant in Atlantic salmon aquaculture, since precocious male puberty negatively impacts animal welfare and growth. We hypothesize that certain miRNAs modulate mRNAs relevant for the initiation of puberty. To explore which miRNAs regulate mRNAs during initiation of puberty in salmon, we performed an integrated transcriptome analysis (miRNA and mRNA-seq) of salmon testis at three stages of development: an immature, long-term quiescent stage, a prepubertal stage just before, and a pubertal stage just after the onset of single cell proliferation activity in the testis. Differentially expressed miRNAs clustered into 5 distinct expression profiles related to the immature, prepubertal and pubertal salmon testis. Potential mRNA targets of these miRNAs were predicted with miRmap and filtered for mRNAs displaying negatively correlated expression patterns. In summary, this analysis revealed miRNAs previously known to be regulated in immature vertebrate testis (miR-101, miR-137, miR-92b, miR-18a, miR-20a), but also miRNAs first reported here as regulated in the testis (miR-new289, miR-30c, miR-724, miR-26b, miR-new271, miR-217, miR-216a, miR-135a, miR-new194 and the novel predicted n268). By KEGG enrichment analysis, progesterone signaling and cell cycle pathway genes were found regulated by these differentially expressed miRNAs. During the transition into puberty we found differential expression of miRNAs previously associated (let7a/b/c), or newly associated (miR-15c, miR-2184, miR-145 and the novel predicted n7a and b) with this stage. KEGG enrichment analysis revealed that mRNAs of the Wnt, Hedgehog and Apelin signaling pathways were potential regulated targets during the transition into puberty. Likewise, several regulated miRNAs in the pubertal stage had earlier been associated (miR-20a, miR-25, miR-181a, miR-202, let7c/d/a, miR-125b

Differential expression of viral PAMP receptors mRNA in peripheral blood of patients with chronic hepatitis C infection

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Riñón Marta

2007-11-01

Full Text Available Abstract Background Pathogen-associated molecular patterns (PAMP receptors play a key role in the early host response to viruses. In this work, we determined mRNA levels of two members of the Toll-like Receptors family, (TLR3 and TLR7 and the helicase RIG-I, all of three recognizing viral RNA products, in peripheral blood of healthy donors and hepatitis C virus (HCV patients, to observe if their transcripts are altered in this disease. Methods IFN-α, TLR3, TLR7 and RIG-I levels in peripheral blood from healthy controls (n = 18 and chronic HCV patients (n = 18 were quantified by real-time polymerase chain reaction. Results Our results show that IFN-α, TLR3, TLR7 and RIG-I mRNA levels are significantly down-regulated in patients with chronic HCV infection when compared with healthy controls. We also found that the measured levels of TLR3 and TLR7, but not RIG-I, correlated significantly with those of IFN-α Conclusion Monitoring the expression of RNA-sensing receptors like TLR3, TLR7 and RIG-I during the different clinical stages of infection could bring a new source of data about the prognosis of disease.

Momordica charantia (bitter melon inhibits primary human adipocyte differentiation by modulating adipogenic genes

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Nerurkar Vivek R

2010-06-01

Full Text Available Abstract Background Escalating trends of obesity and associated type 2 diabetes (T2D has prompted an increase in the use of alternative and complementary functional foods. Momordica charantia or bitter melon (BM that is traditionally used to treat diabetes and complications has been demonstrated to alleviate hyperglycemia as well as reduce adiposity in rodents. However, its effects on human adipocytes remain unknown. The objective of our study was to investigate the effects of BM juice (BMJ on lipid accumulation and adipocyte differentiation transcription factors in primary human differentiating preadipocytes and adipocytes. Methods Commercially available cryopreserved primary human preadipocytes were treated with and without BMJ during and after differentiation. Cytotoxicity, lipid accumulation, and adipogenic genes mRNA expression was measured by commercial enzymatic assay kits and semi-quantitative RT-PCR (RT-PCR. Results Preadipocytes treated with varying concentrations of BMJ during differentiation demonstrated significant reduction in lipid content with a concomitant reduction in mRNA expression of adipocyte transcription factors such as, peroxisome proliferator-associated receptor γ (PPARγ and sterol regulatory element-binding protein 1c (SREBP-1c and adipocytokine, resistin. Similarly, adipocytes treated with BMJ for 48 h demonstrated reduced lipid content, perilipin mRNA expression, and increased lipolysis as measured by the release of glycerol. Conclusion Our data suggests that BMJ is a potent inhibitor of lipogenesis and stimulator of lipolysis activity in human adipocytes. BMJ may therefore prove to be an effective complementary or alternative therapy to reduce adipogenesis in humans.

Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in ras- and Rho-mediated signaling.

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Pazman, C; Mayes, C A; Fanto, M; Haynes, S R; Mlodzik, M

2000-04-01

The small GTPase Ras plays an important role in many cellular signaling processes. Ras activity is negatively regulated by GTPase activating proteins (GAPs). It has been proposed that RasGAP may also function as an effector of Ras activity. We have identified and characterized the Drosophila homologue of the RasGAP-binding protein G3BP encoded by rasputin (rin). rin mutants are viable and display defects in photoreceptor recruitment and ommatidial polarity in the eye. Mutations in rin/G3BP genetically interact with components of the Ras signaling pathway that function at the level of Ras and above, but not with Raf/MAPK pathway components. These interactions suggest that Rin is required as an effector in Ras signaling during eye development, supporting an effector role for RasGAP. The ommatidial polarity phenotypes of rin are similar to those of RhoA and the polarity genes, e.g. fz and dsh. Although rin/G3BP interacts genetically with RhoA, affecting both photoreceptor differentiation and polarity, it does not interact with the gain-of-function genotypes of fz and dsh. These data suggest that Rin is not a general component of polarity generation, but serves a function specific to Ras and RhoA signaling pathways.

The phocein homologue SmMOB3 is essential for vegetative cell fusion and sexual development in the filamentous ascomycete Sordaria macrospora.

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Bernhards, Yasmine; Pöggeler, Stefanie

2011-04-01

Members of the striatin family and their highly conserved interacting protein phocein/Mob3 are key components in the regulation of cell differentiation in multicellular eukaryotes. The striatin homologue PRO11 of the filamentous ascomycete Sordaria macrospora has a crucial role in fruiting body development. Here, we functionally characterized the phocein/Mob3 orthologue SmMOB3 of S. macrospora. We isolated the gene and showed that both, pro11 and Smmob3 are expressed during early and late developmental stages. Deletion of Smmob3 resulted in a sexually sterile strain, similar to the previously characterized pro11 mutant. Fusion assays revealed that ∆Smmob3 was unable to undergo self-fusion and fusion with the pro11 strain. The essential function of the SmMOB3 N-terminus containing the conserved mob domain was demonstrated by complementation analysis of the sterile S. macrospora ∆Smmob3 strain. Downregulation of either pro11 in ∆Smmob3, or Smmob3 in pro11 mutants by means of RNA interference (RNAi) resulted in synthetic sexual defects, demonstrating for the first time the importance of a putative PRO11/SmMOB3 complex in fruiting body development.

Induction of osteogenic markers in differentially treated cultures of embryonic stem cells

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Ommerborn Michelle A

2008-06-01

Full Text Available Abstract Background Facial trauma or tumor surgery in the head and face area often lead to massive destruction of the facial skeleton. Cell-based bone reconstruction therapies promise to offer new therapeutic opportunities for the repair of bone damaged by disease or injury. Currently, embryonic stem cells (ESCs are discussed to be a potential cell source for bone tissue engineering. The purpose of this study was to investigate various supplements in culture media with respect to the induction of osteogenic differentiation. Methods Murine ESCs were cultured in the presence of LIF (leukemia inhibitory factor, DAG (dexamethasone, ascorbic acid and β-glycerophosphate or bone morphogenetic protein-2 (BMP-2. Microscopical analyses were performed using von Kossa staining, and expression of osteogenic marker genes was determined by real time PCR. Results ESCs cultured with DAG showed by far the largest deposition of calcium phosphate-containing minerals. Starting at day 9 of culture, a strong increase in collagen I mRNA expression was detected in the DAG-treated cells. In BMP-2-treated ESCs the collagen I mRNA induction was less increased. Expression of osteocalcin, a highly specific marker for osteogentic differentiation, showed a double-peaked curve in DAG-treated cells. ESCs cultured in the presence of DAG showed a strong increase in osteocalcin mRNA at day 9 followed by a second peak starting at day 17. Conclusion Supplementation of ESC cell cultures with DAG is effective in inducing osteogenic differentiation and appears to be more potent than stimulation with BMP-2 alone. Thus, DAG treatment can be recommended for generating ESC populations with osteogenic differentiation that are intended for use in bone tissue engineering.

Mucin gene mRNA levels in broilers challenged with eimeria and/or Clostridium perfringens.

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Kitessa, Soressa M; Nattrass, Gregory S; Forder, Rebecca E A; McGrice, Hayley A; Wu, Shu-Biao; Hughes, Robert J

2014-09-01

The effects of Eimeria (EM) and Clostridium perfringens (CP) challenges on the mRNA levels of genes involved in mucin (Muc) synthesis (Muc2, Muc5ac, Muc13, and trefoil family factor-2 [TFF2]), inflammation (tumor necrosis factor alpha [TNF-alpha] and interleukin-18 [IL-18]), and metabolic processes (cluster of differentiation [CD]36) in the jejunum of broilers were investigated. Two parallel experiments involving 1) EM challenge and 2) EM and CP challenges were conducted. The first experiment was a 2 X 2 study with 12 birds per treatment (N = 48) involving fishmeal substitution (25%) in the diet (FM) and EM challenge. The treatments were: Control (FM-, EM-), Fishmeal (FM+, EM-), EM challenge (FM-, EM+), and fishmeal substitution and EM challenge (FM+, EM+). The second experiment was a 2 X 2 X 2 experiment with six birds per treatment (N = 48) involving fishmeal (FM-, FM+), Eimeria (EM-, EM+), and C perfringens (CP-, CP+). In both arms of the study, male broilers were given a starter diet for the whole period of 16 days, except those assigned to FM+, where 25% of the starter ration was replaced with fishmeal from days 8 to 14. EM inoculation was performed on day 9 and CP inoculation on days 14 and 15. The EM challenge birds were euthanatized for sampling on day 13; postmortem examination and sampling for the Eimeria plus C perfringens challenge arm of the study were on day 16. In the Eimeria challenge arm of the study, fishmeal supplementation significantly suppressed the mRNA levels of TNF-alpha, TFF2, and IL-18 pre-CP inoculation but simultaneously increased the levels of Muc13 and CD36 mRNAs. Birds challenged with Eimeria exhibited increased mRNA levels of Muc13, Muc5ac, TNF-alpha, and IL-18. In the Eimeria and C. perfringens challenge arm, birds exposed to EM challenge exhibited significantly lower mRNA levels of Muc2 and CD36. The mRNA levels of CD36 were also significantly suppressed by CP challenge. Our results showed that the transcription of mucin synthesis

Human pancreatic islet-derived extracellular vesicles modulate insulin expression in 3D-differentiating iPSC clusters.

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Diana Ribeiro

Full Text Available It has been suggested that extracellular vesicles (EVs can mediate crosstalk between hormones and metabolites within pancreatic tissue. However, the possible effect of pancreatic EVs on stem cell differentiation into pancreatic lineages remains unknown. Herein, human islet-derived EVs (h-Islet-EVs were isolated, characterized and subsequently added to human induced pluripotent stem cell (iPSC clusters during pancreatic differentiation. The h-islet-EVs had a mean size of 117±7 nm and showed positive expression of CD63 and CD81 EV markers as measured by ELISA. The presence of key pancreatic transcription factor mRNA, such as NGN3, MAFA and PDX1, and pancreatic hormone proteins such as C-peptide and glucagon, were confirmed in h-Islet-EVs. iPSC clusters were differentiated in suspension and at the end stages of the differentiation protocol, the mRNA expression of the main pancreatic transcription factors and pancreatic hormones was increased. H-Islet-EVs were supplemented to the iPSC clusters in the later stages of differentiation. It was observed that h-Islet-EVs were able to up-regulate the intracellular levels of C-peptide in iPSC clusters in a concentration-dependent manner. The effect of h-Islet-EVs on the differentiation of iPSC clusters cultured in 3D-collagen hydrogels was also assessed. Although increased mRNA expression for pancreatic markers was observed when culturing the iPSC clusters in 3D-collagen hydrogels, delivery of EVs did not affect the insulin or C-peptide intracellular content. Our results provide new information on the role of h-Islet-EVs in the regulation of insulin expression in differentiating iPSC clusters, and are highly relevant for pancreatic tissue engineering applications.

Developmental programming: Impact of fetal exposure to endocrine-disrupting chemicals on gonadotropin-releasing hormone and estrogen receptor mRNA in sheep hypothalamus

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Mahoney, Megan M.; Padmanabhan, Vasantha

2010-01-01

Bisphenol-A (BPA) and methoxychlor (MXC), two endocrine-disrupting chemicals (EDCs) with estrogenic and antiandrogenic effects, disrupt the reproductive system. BPA has profound effects on luteinizing hormone (LH) surge amplitude, and MXC has profound effects on on LH surge timing in sheep. The neural mechanisms involved in the differential disruption of the LH surge by these two EDCs remain to be elucidated. We tested the hypothesis that the differential effects of BPA and MXC on LH surge system involved changes in hypothalamic gonadotropin-releasing hormone (GnRH) and estrogen receptors (ESR), ESR1 and ESR2, mRNA expression. Pregnant sheep were given daily injections of cottonseed oil (controls), MXC, or BPA (5 mg/kg/day) from day 30 to 90 of gestation (term 147 d). Offspring from these animals were euthanized as adults, during the late follicular phase following synchronization of estrus with prostaglandin F 2α , just before the expected onset of preovulatory LH surge and changes in mRNA expression of hypothalamic GnRH, ESR1, and ESR2 quantified following in situ hybridization. GnRH mRNA expression was significantly lower in both groups of EDC-treated females compared to controls. ESR1 expression was increased in prenatal BPA- but not MXC-treated females in medial preoptic area relative to controls. In contrast, ESR2 expression was reduced in the medial preoptic area of both EDC-treated groups. Differences in expression of ESR1/ESR2 receptors may contribute to the differential effects of BPA and MXC on the LH surge system. These findings provide support that prenatal exposure to EDCs alters the neural developmental trajectory leading to long-term reproductive consequences in the adult female.

Developmental programming: impact of fetal exposure to endocrine-disrupting chemicals on gonadotropin-releasing hormone and estrogen receptor mRNA in sheep hypothalamus.

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Mahoney, Megan M; Padmanabhan, Vasantha

2010-09-01

Bisphenol-A (BPA) and methoxychlor (MXC), two endocrine-disrupting chemicals (EDCs) with estrogenic and antiandrogenic effects, disrupt the reproductive system. BPA has profound effects on luteinizing hormone (LH) surge amplitude, and MXC has profound effects on on LH surge timing in sheep. The neural mechanisms involved in the differential disruption of the LH surge by these two EDCs remain to be elucidated. We tested the hypothesis that the differential effects of BPA and MXC on LH surge system involved changes in hypothalamic gonadotropin-releasing hormone (GnRH) and estrogen receptors (ESR), ESR1 and ESR2, mRNA expression. Pregnant sheep were given daily injections of cottonseed oil (controls), MXC, or BPA (5mg/kg/day) from day 30 to 90 of gestation (term 147d). Offspring from these animals were euthanized as adults, during the late follicular phase following synchronization of estrus with prostaglandin F(2alpha), just before the expected onset of preovulatory LH surge and changes in mRNA expression of hypothalamic GnRH, ESR1, and ESR2 quantified following in situ hybridization. GnRH mRNA expression was significantly lower in both groups of EDC-treated females compared to controls. ESR1 expression was increased in prenatal BPA- but not MXC-treated females in medial preoptic area relative to controls. In contrast, ESR2 expression was reduced in the medial preoptic area of both EDC-treated groups. Differences in expression of ESR1/ESR2 receptors may contribute to the differential effects of BPA and MXC on the LH surge system. These findings provide support that prenatal exposure to EDCs alters the neural developmental trajectory leading to long-term reproductive consequences in the adult female. 2010 Elsevier Inc. All rights reserved.

[Impacts of the formula of Suoquanwan(SQW) on expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency].

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Cao, Hong-Ying; Wu, Qing-He; Huang, Ping; He, Jin-Yang

2009-06-01

To observe the impacts of the formula of Suoquanwan (SQW) on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency. The model rats were induced by adenine (250 mg/kg) for 4 weeks, then treated respectively with SQW or dDAVP. The expression of AQP-2 mRNA and AVPR-V2 mRNA in kidney of Yang-deficiency model by realtime fluorescence quantitative PCR method were investigated. In model rats, the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney decreased, dDAVP and SQW high dose could increased the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. The others had no influence on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. SQW can increase the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency.

Staphylococcus aureus RNAIII binds to two distant regions of coa mRNA to arrest translation and promote mRNA degradation.

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Clément Chevalier

2010-03-01

Full Text Available Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation.

Effects of Dendrobium officinale polysaccharide on adipogenic differentiation of rat bone marrow mesenchymal stem cells

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Yinjuan ZHAO

Full Text Available Abstract This study investigated the effect of Dendrobium officinale polysaccharide (DOP on the adipogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs. DOP was extracted fresh Dendrobium officinale. Rat BMSCs were prepared, and then were treated with 0 (control, 50, 100, 200, 400, 800 μg/mL DOP, respectively. The cell viability was determined by MTT assay. The adipogenic differentiation was quantitatively analyzed by oil red O staining assay. The mRNA expressions of adipogenic differentiation related gene peroxisome proliferator-activated receptor gamma (PPARG, lipoprotein lipase (LPL and fatty acid binding protein 4 (FABP4 were detected by RT-PCR. Results showed that, DOP with 0-800 μg/mL concentration had no significant toxicity to BMSCs. 200-800 μg/mL DOP could obviously inhibit the adipogenic differentiation of BMSCs. Compared with control group, the expression levels of PPARG, LPL and FABP4 mRNA 200, 400 and 800 μg/mL DOP groups were significantly decreased (P < 0.05 or P < 0.01. DOP can inhibit the adipogenic differentiation of BMSCs, which may be related with its down-regulation of PPARG, LPL and FABP4 expressions in BMSCs.

Structure of the gene encoding VGF, a nervous system-specific mRNA that is rapidly and selectively induced by nerve growth factor in PC12 cells.

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Salton, S R; Fischberg, D J; Dong, K W

1991-05-01

Nerve growth factor (NGF) plays a critical role in the development and survival of neurons in the peripheral nervous system. Following treatment with NGF but not epidermal growth factor, rat pheochromocytoma (PC12) cells undergo neural differentiation. We have cloned a nervous system-specific mRNA, NGF33.1, that is rapidly and relatively selectively induced by treatment of PC12 cells with NGF and basic fibroblast growth factor in comparison with epidermal growth factor. Analysis of the nucleic acid and predicted amino acid sequences of the NGF33.1 cDNA clone suggested that this clone corresponded to the NGF-inducible mRNA called VGF (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985; R. Possenti, J. D. Eldridge, B. M. Paterson, A. Grasso, and A. Levi, EMBO J. 8:2217-2223, 1989). We have used the NGF33.1 cDNA clone to isolate and characterize the VGF gene, and in this paper we report the complete sequence of the VGF gene, including 853 bases of 5' flank revealed TATAA and CCAAT elements, several GC boxes, and a consensus cyclic AMP response element-binding protein binding site. The VGF promoter contains sequences homologous to other NGF-inducible, neuronal promoters. We further show that VGF mRNA is induced in PC12 cells to a greater extent by depolarization and by phorbol-12-myristate-13-acetate treatment than by 8-bromo-cyclic AMP treatment. By Northern (RNA) and RNase protection analysis, VGF mRNA is detectable in embryonic and postnatal central and peripheral nervous tissues but not in a number of nonneural tissues. In the cascade of events which ultimately leads to the neural differentiation of NGF-treated PC12 cells, the VGF gene encodes the most rapidly and selectively regulated, nervous-system specific mRNA yet identified.

Differential conserted activity induced regulation of Nogo receptors (1-3, LOTUS and Nogo mRNA in mouse brain.

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Tobias E Karlsson

Full Text Available Nogo Receptor 1 (NgR1 mRNA is downregulated in hippocampal and cortical regions by increased neuronal activity such as a kainic acid challenge or by exposing rats to running wheels. Plastic changes in cerebral cortex in response to loss of specific sensory inputs caused by spinal cord injury are also associated with downregulation of NgR1 mRNA. Here we investigate the possible regulation by neuronal activity of the homologous receptors NgR2 and NgR3 as well as the endogenous NgR1 antagonist LOTUS and the ligand Nogo. The investigated genes respond to kainic acid by gene-specific, concerted alterations of transcript levels, suggesting a role in the regulation of synaptic plasticity, Downregulation of NgR1, coupled to upregulation of the NgR1 antagonist LOTUS, paired with upregulation of NgR2 and 3 in the dentate gyrus suggest a temporary decrease of Nogo/OMgp sensitivity while CSPG and MAG sensitivity could remain. It is suggested that these activity-synchronized temporary alterations may serve to allow structural alterations at the level of local synaptic circuitry in gray matter, while maintaining white matter pathways and that subsequent upregulation of Nogo-A and NgR1 transcript levels signals the end of such a temporarily opened window of plasticity.

Effects of the deletion of the Escherichia coli frataxin homologue CyaY on the respiratory NADH:ubiquinone oxidoreductase

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Grauman Peter L

2007-07-01

Full Text Available Abstract Background Frataxin is discussed as involved in the biogenesis of iron-sulfur clusters. Recently it was discovered that a frataxin homologue is a structural component of the respiratory NADH:ubiquinone oxidoreductase (complex I in Thermus thermophilus. It was not clear whether frataxin is in general a component of complex I from bacteria. The Escherichia coli homologue of frataxin is coined CyaY. Results We report that complex I is completely assembled to a stable and active enzyme complex equipped with all known iron-sulfur clusters in a cyaY mutant of E. coli. However, the amount of complex I is reduced by one third compared to the parental strain. Western blot analysis and live cell imaging of CyaY engineered with a GFP demonstrated that CyaY is located in the cytoplasm and not attached to the membrane as to be expected if it were a component of complex I. Conclusion CyaY plays a non-essential role in the assembly of complex I in E. coli. It is not a structural component but may transiently interact with the complex.

Analysis of the 3’ untranslated regions of α-tubulin and S-crystallin mRNA and the identification of CPEB in dark- and light-adapted octopus retinas

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Kelly, Shannan; Yamamoto, Hideki

2008-01-01

Purpose We previously reported the differential expression and translation of mRNA and protein in dark- and light-adapted octopus retinas, which may result from cytoplasmic polyadenylation element (CPE)–dependent mRNA masking and unmasking. Here we investigate the presence of CPEs in α-tubulin and S-crystallin mRNA and report the identification of cytoplasmic polyadenylation element binding protein (CPEB) in light- and dark-adapted octopus retinas. Methods 3’-RACE and sequencing were used to isolate and analyze the 3’-UTRs of α-tubulin and S-crystallin mRNA. Total retinal protein isolated from light- and dark-adapted octopus retinas was subjected to western blot analysis followed by CPEB antibody detection, PEP-171 inhibition of CPEB, and dephosphorylation of CPEB. Results The following CPE-like sequence was detected in the 3’-UTR of isolated long S-crystallin mRNA variants: UUUAACA. No CPE or CPE-like sequences were detected in the 3’-UTRs of α-tubulin mRNA or of the short S-crystallin mRNA variants. Western blot analysis detected CPEB as two putative bands migrating between 60-80 kDa, while a third band migrated below 30 kDa in dark- and light-adapted retinas. Conclusions The detection of CPEB and the identification of the putative CPE-like sequences in the S-crystallin 3’-UTR suggest that CPEB may be involved in the activation of masked S-crystallin mRNA, but not in the regulation of α-tubulin mRNA, resulting in increased S-crystallin protein synthesis in dark-adapted octopus retinas. PMID:18682811

Evaluation of mRNA expression levels and electrophysiological function of neuron-like cells derived from canine bone marrow stromal cells.

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Nakano, Rei; Edamura, Kazuya; Sugiya, Hiroshi; Narita, Takanori; Okabayashi, Ken; Moritomo, Tadaaki; Teshima, Kenji; Asano, Kazushi; Nakayama, Tomohiro

2013-10-01

To investigate the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into functional, mature neurons. Bone marrow from 6 adult dogs. BMSCs were isolated from bone marrow and chemically induced to develop into neurons. The morphology of the BMSCs during neuronal induction was monitored, and immunocytochemical analyses for neuron markers were performed after the induction. Real-time PCR methods were used to evaluate the mRNA expression levels of markers for neural stem or progenitor cells, neurons, and ion channels, and western blotting was used to assess the expression of neuronal proteins before and after neuronal induction. The electrophysiological properties of the neuron-like cells induced from canine BMSCs were evaluated with fluorescent dye to monitor Ca(2)+ influx. Canine BMSCs developed a neuron-like morphology after neuronal induction. Immunocytochemical analysis revealed that these neuron-like cells were positive for neuron markers. After induction, the cells' mRNA expression levels of almost all neuron and ion channel markers increased, and the protein expression levels of nestin and neurofilament-L increased significantly. However, the neuron-like cells derived from canine BMSCs did not have the Ca(2)+ influx characteristic of spiking neurons. Although canine BMSCs had neuron-like morphological and biochemical properties after induction, they did not develop the electrophysiological characteristics of neurons. Thus, these results have suggested that canine BMSCs could have the capacity to differentiate into a neuronal lineage, but the differentiation protocol used may have been insufficient to induce development into functional neurons.

Changes in apoptotic microRNA and mRNA expression profiling in Caenorhabditis elegans during the Shenzhou-8 mission

International Nuclear Information System (INIS)

Gao Ying; Li Shuai; Xu Dan; Wang Junjun; Sun Yeqing

2015-01-01

Radiation and microgravity exposure have been proven to induce abnormal apoptosis in microRNA (miRNA) and mRNA expression, but whether space conditions, including radiation and microgravity, activate miRNAs to regulate the apoptosis is undetermined. For that purpose, we investigated miRNome and mRNA expression in the ced-1 Caenorhabditis elegans mutant vs the wild-type, both of which underwent spaceflight, spaceflight 1g-centrifuge control and ground control conditions during the Shenzhou-8 mission. Results showed that no morphological changes in the worms were detected, but differential miRNA expression increased from 43 (ground control condition) to 57 and 91 in spaceflight and spaceflight control conditions, respectively. Microgravity altered miRNA expression profiling by decreasing the number and significance of differentially expressed miRNA compared with 1 g incubation during spaceflight. Alterations in the miRNAs were involved in alterations in apoptosis, neurogenesis larval development, ATP metabolism and GTPase-mediated signal transduction. Among these, 17 altered miRNAs potentially involved in apoptosis were screened and showed obviously different expression signatures between space conditions. By integrated analysis of miRNA and mRNA, miR-797 and miR-81 may be involved in apoptosis by targeting the genes ced-10 and both drp-1 and hsp-1, respectively. Compared with ground condition, space conditions regulated apoptosis though a different manner on transcription, by altering expression of seven core apoptotic genes in spaceflight condition, and eight in spaceflight control condition. Results indicate that, miRNA of Caenorhabditis elegans probably regulates apoptotic gene expression in response to space environmental stress, and shows different behavior under microgravity condition compared with 1 g condition in the presence of space radiation. (author)

Effect of molecular weight and concentration of hyaluronan on cell proliferation and osteogenic differentiation in vitro

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Zhao, Ningbo, E-mail: curl-zhao@163.com; Wang, Xin, E-mail: 394041230@qq.com; Qin, Lei, E-mail: qinlei30@126.com; Guo, Zhengze, E-mail: zhzeguo@163.com; Li, Dehua, E-mail: lidehuafmmu@163.com

2015-09-25

Hyaluronan (HA), the simplest glycosaminoglycan and a major component of the extracellular matrix, exists in various tissues. It is involved in some critical biological procedures, including cellular signaling, cell adhesion and proliferation, and cell differentiation. The effect of molecular weight (MW) and concentration of HA on cell proliferation and differentiation was controversial. In this study, we investigated the effect of MW and concentration of HA on the proliferation and osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro. Results showed that high MW HA decreased the cell adhesion rate in a concentration-dependant manner. The cell adhesion rate was decreased by increasing MW of HA. Cell proliferation was significantly enhanced by low MW HA (P < 0.05). The factorial analysis indicated that MW and concentration had an interactive effect on the cell adhesion rate and cell proliferation (P < 0.05). High MW HA increased the mRNA expressions of ALP, RUNX-2 and OCN. The higher the MW was, the higher the mRNA expressions were. The factorial analysis indicated that MW and concentration had an interactive effect on ALP mRNA expression (P < 0.05). HA of higher MW and higher concentration promoted bone formation. These findings provide some useful information in understanding the mechanism underlying the effect of MW and concentration of HA on cell proliferation and differentiation. - Highlights: • Effect of hyaluronan on cell proliferation and differentiation is evaluated in vitro. • Hyaluronan of low molecular weight increases cell proliferation. • Hyaluronan of high molecular weight promotes cell osteogenic differentiation. • Molecular weight and concentration of hyaluronan show interactive effect.

Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells

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Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan

2016-01-01

Background We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. Objective We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. Methods To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Results Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. Conclusion These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation. PMID:27489424

Single-Crystal X-Ray Diffraction Studies of Homologues in the Series nBa(Nb,Zr)O 3+3 mNbO with n=2, 3, 4, 5 and m=1

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Nilsson, G.; Svensson, G.

2001-01-01

Single crystals of four homologues in the series nBa(Nb,Zr)O3+3mNbO, with n:m=2:1, 3:1, 4:1, and 5:1, were found in the reduced Ba-Nb-Zr-O system. Single-crystal X-ray diffraction data were collected for all the crystals. For all homologues the space group was found to be P4/mmm. The structures can be described as intergrowths of Ba(Nb,Zr)O3 perovskite and NbO slabs. The refined cell parameters and compositions of the 2:1, 3:1, and 4:1 homologues are a=4.1768(5) Å and c=12.269(2) Å for Ba2Nb4.5(1)Zr0.5(1)O9, a=4.1769(5) Å and c=16.493(3) Å for Ba3+δNb4.8(2)-δ Zr1.2(2)O12-δ (δ=0.098(4)), and a=4.1747(6) Å and c= 20.619(4) Å for Ba4+δNb5.1(4)-δZr1.9(4)O15-δ (δ=0.270(9)). The refined cell parameters of the 5:1 homologue are a=4.1727(3) Å and c=24.804(3) Å. Zr replaces Nb only in the NbO6 octahedra found in the perovskite slabs.

Differential Gene Expression of Longan Under Simulated Acid Rain Stress.

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Zheng, Shan; Pan, Tengfei; Ma, Cuilan; Qiu, Dongliang

2017-05-01

Differential gene expression profile was studied in Dimocarpus longan Lour. in response to treatments of simulated acid rain with pH 2.5, 3.5, and a control (pH 5.6) using differential display reverse transcription polymerase chain reaction (DDRT-PCR). Results showed that mRNA differential display conditions were optimized to find an expressed sequence tag (EST) related with acid rain stress. The potential encoding products had 80% similarity with a transcription initiation factor IIF of Gossypium raimondii and 81% similarity with a protein product of Theobroma cacao. This fragment is the transcription factor activated by second messenger substances in longan leaves after signal perception of acid rain.

The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans.

Science.gov (United States)

Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hua; Li, Hau; Florens, Laurence; Mak, Ho Yi

2012-04-15

The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.

Emerging role of LRRK2 in human neural progenitor cell cycle progression, survival and differentiation

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Meyer Anne K

2009-06-01

Full Text Available Abstract Despite a comprehensive mapping of the Parkinson's disease (PD-related mRNA and protein leucine-rich repeat kinase 2 (LRRK2 in the mammalian brain, its physiological function in healthy individuals remains enigmatic. Based on its structural features and kinase properties, LRRK2 may interact with other proteins involved in signalling pathways. Here, we show a widespread LRRK2 mRNA and/or protein expression in expanded or differentiated human mesencephalic neural progenitor cells (hmNPCs and in post-mortem substantia nigra PD patients. Using small interfering RNA duplexes targeting LRRK2 in hmNPCs following their differentiation into glia and neurons, we observed a reduced number of dopaminergic neurons due to apoptosis in LRRK2 knockdown samples. LRRK2-deficient hmNPCs exhibited elevated cell cycle- and cell death-related markers. In conclusion, a reduction of LRRK2 expression in hmNPCs severely impaired dopaminergic differentiation and/or survival of dopaminergic neurons most likely via preserving or reactivating the cell cycle.

Reversible effects of oxygen partial pressure on genes associated with placental angiogenesis and differentiation in primary-term cytotrophoblast cell culture.

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Debiève, F; Depoix, C; Gruson, D; Hubinont, C

2013-09-01

Timely regulated changes in oxygen partial pressure are important for placental formation. Disturbances could be responsible for pregnancy-related diseases like preeclampsia and intrauterine growth restriction. We aimed to (i) determine the effect of oxygen partial pressure on cytotrophoblast differentiation; (ii) measure mRNA expression and protein secretion from genes associated with placental angiogenesis; and (iii) determine the reversibility of these effects at different oxygen partial pressures. Term cytotrophoblasts were incubated at 21% and 2.5% O2 for 96 hr, or were switched between the two oxygen concentrations after 48 hr. Real-time PCR and enzyme-linked immunosorbent assays (ELISAs) were used to evaluate cell fusion and differentiation, measuring transcript levels for those genes involved in cell fusion and placental angiogenesis, including VEGF, PlGF, VEGFR1, sVEGFR1, sENG, INHA, and GCM1. Cytotrophoblasts underwent fusion and differentiation in 2.5% O2 . PlGF expression was inhibited while sVEGFR1 expression increased. VEGF and sENG mRNA expressions increased in 2.5% compared to 21% O2 , but no protein was detected in the cell supernatants. Finally, GCM1 mRNA expression increased during trophoblast differentiation at 21% O2 , but was inhibited at 2.5% O2 . These mRNA expression effects were reversed by returning the cells to 21% O2 . Thus, low-oxygen partial pressure does not inhibit term-cytotrophoblast cell fusion and differentiation in vitro. Lowering the oxygen partial pressure from 21% to 2.5% caused normal-term trophoblasts to reversibly modify their expression of genes associated with placental angiogenesis. This suggests that modifications observed in pregnancy diseases such as preeclampsia or growth retardation are probably due to an extrinsic effect on trophoblasts. Copyright © 2013 Wiley Periodicals, Inc.

WNT2B2 mRNA, up-regulated in primary gastric cancer, is a positive regulator of the WNT- beta-catenin-TCF signaling pathway.

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Katoh, M; Kirikoshi, H; Terasaki, H; Shiokawa, K

2001-12-21

Genetic alterations of WNT signaling molecules lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway. We have previously cloned and characterized WNT2B/WNT13 gene on human chromosome 1p13, which is homologous to proto-oncogene WNT2 on human chromosome 7q31. WNT2B1 and WNT2B2 mRNAs, generated from the WNT2B gene due to alternative splicing of the alternative promoter type, encode almost identical polypeptides with divergence in the N-terminal region. WNT2B2 mRNA rather than WNT2B1 mRNA is preferentially expressed in NT2 cells with the potential of neuronal differentiation. Here, we describe our investigations of expression of WNT2B mRNAs in various types of human primary cancer. Matched tumor/normal expression array analysis revealed that WNT2B mRNAs were significantly up-regulated in 2 of 8 cases of primary gastric cancer. WNT2B2 mRNA rather than WNT2B1 mRNA was found to be preferentially up-regulated in a case of primary gastric cancer (signet ring cell carcinoma). Function of WNT2B1 mRNA and that of WNT2B2 mRNA were investigated by using Xenopus axis duplication assay. Injection of synthetic WNT2B1 mRNA into the ventral marginal zone of fertilized Xenopus eggs at the 4-cell stage did not induce axis duplication. In contrast, ventral injection of synthetic WNT2B2 mRNA induced axis duplication in 90% of embryos (complete axis duplication, 24%). These results strongly suggest that WNT2B2 up-regulation in some cases of gastric cancer might lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway.

DNA–PKcs–SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation

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Xu, Yong; Fang, Shi-ji [The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000 (China); Zhu, Li-juan [Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and Institute of Neuroscience, Soochow University, Suzhou, Jiangsu 215021 (China); Zhu, Lun-qing, E-mail: xiaodongwangsz@163.com [The Center of Diagnosis and Treatment for Children’s Bone Diseases, The Children’s Hospital Affiliated to Soochow University, Suzhou, Jiangsu 215000 (China); Zhou, Xiao-zhong, E-mail: zhouxz@suda.edu.cn [The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000 (China)

2014-10-24

Highlights: • LDI increases ALP activity, promotes type I collagen (Col I)/Runx2 mRNA expression. • LDI induces DNA–PKcs activation, which is required for osteoblast differentiation. • Akt activation mediates LDI-induced ALP activity and Col I/Runx2 mRNA increase. • DNA–PKcs–SIN1 complexation mediates LDI-induced Akt Ser-473 phosphorylation. • DNA–PKcs–SIN1 complexation is important for osteoblast differentiation. - Abstract: Low-dose irradiation (LDI) induces osteoblast differentiation, however the underlying mechanisms are not fully understood. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA–PKcs)–Akt signaling in LDI-induced osteoblast differentiation. We confirmed that LDI promoted mouse calvarial osteoblast differentiation, which was detected by increased alkaline phosphatase (ALP) activity as well as mRNA expression of type I collagen (Col I) and runt-related transcription factor 2 (Runx2). In mouse osteoblasts, LDI (1 Gy) induced phosphorylation of DNA–PKcs and Akt (mainly at Ser-473). The kinase inhibitors against DNA–PKcs (NU-7026 and NU-7441) or Akt (LY294002, perifosine and MK-2206), as well as partial depletion of DNA–PKcs or Akt1 by targeted-shRNA, dramatically inhibited LDI-induced Akt activation and mouse osteoblast differentiation. Further, siRNA-knockdown of SIN1, a key component of mTOR complex 2 (mTORC2), also inhibited LDI-induced Akt Ser-473 phosphorylation as well as ALP activity increase and Col I/Runx2 expression in mouse osteoblasts. Co-immunoprecipitation (Co-IP) assay results demonstrated that LDI-induced DNA–PKcs–SIN1 complexation, which was inhibited by NU-7441 or SIN1 siRNA-knockdown in mouse osteoblasts. In summary, our data suggest that DNA–PKcs–SIN1 complexation-mediated Akt activation (Ser-473 phosphorylation) is required for mouse osteoblast differentiation.

mRNA processing in yeast

International Nuclear Information System (INIS)

Stevens, A.

1982-01-01

Investigations in this laboratory center on basic enzymatic reactions of RNA. Still undefined are reactions involved in the conversion of precursors of mRA (pre-mRNA) to mRNA in eukaryotes. The pre-mRNA is called heterogeneous nuclear RNA and is 2 to 6 times larger than mRNA. The conversion, called splicing, involves a removal of internal sequences called introns by endoribonuclease action followed by a rejoining of the 3'- and 5'-end fragments, called exons, by ligating activity. It has not been possible yet to study the enzymes involved in vitro. Also undefined are reactions involved in the turnover or discarding of certain of the pre-mRNA molecules. Yeast is a simple eukaryote and may be expected to have the same, but perhaps simpler, processing reactions as the higher eukaryotes. Two enzymes involved in the processing of pre-mRNA and mRNA in yeast are under investigation. Both enzymes have been partially purified from ribonucleoprotein particles of yeast. The first is a unique decapping enzyme which cleaves [ 3 H]m 7 Gppp [ 14 C]RNA-poly (A) of yeast, yielding [ 3 H]m 7 GDP and is suggested by the finding that the diphosphate product, m 7 GpppA(G), and UDP-glucose are not hydrolyzed. The second enzyme is an endoribonuclease which converts both the [ 3 H] and [ 14 C] labels of [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) from an oligo(dT)-cellulose bound form to an unbound, acid-insoluble form. Results show that the stimulation involves an interaction of the labeled RNA with the small nuclear RNA. The inhibition of the enzyme by ethidium bromide and its stimulation by small nuclear RNA suggest that it may be a processing ribonuclease, requiring specific double-stranded features in its substrate. The characterization of the unique decapping enzyme and endoribonuclease may help to understand reactions involved in the processing of pre-mRNA and mRNA in eukaryotes

mRNA transfection of mouse and human neural stem cell cultures

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McLenachan, Samuel; Zhang, D.; Palomo, A.B.; Edel, Michael John; Chen, F.K.

2013-01-01

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has ...

A novel link between Sus1 and the cytoplasmic mRNA decay machinery suggests a broad role in mRNA metabolism

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Llopis Ana

2010-03-01

Full Text Available Abstract Background Gene expression is achieved by the coordinated action of multiple factors to ensure a perfect synchrony from chromatin epigenetic regulation through to mRNA export. Sus1 is a conserved mRNA export/transcription factor and is a key player in coupling transcription initiation, elongation and mRNA export. In the nucleus, Sus1 is associated to the transcriptional co-activator SAGA and to the NPC associated complex termed TREX2/THSC. Through these associations, Sus1 mediates the nuclear dynamics of different gene loci and facilitate the export of the new transcripts. Results In this study, we have investigated whether the yeast Sus1 protein is linked to factors involved in mRNA degradation pathways. We provide evidence for genetic interactions between SUS1 and genes coding for components of P-bodies such as PAT1, LSM1, LSM6 and DHH1. We demonstrate that SUS1 deletion is synthetic lethal with 5'→3' decay machinery components LSM1 and PAT1 and has a strong genetic interaction with LSM6 and DHH1. Interestingly, Sus1 overexpression led to an accumulation of Sus1 in cytoplasmic granules, which can co-localise with components of P-bodies and stress granules. In addition, we have identified novel physical interactions between Sus1 and factors associated to P-bodies/stress granules. Finally, absence of LSM1 and PAT1 slightly promotes the Sus1-TREX2 association. Conclusions In this study, we found genetic and biochemical association between Sus1 and components responsible for cytoplasmic mRNA metabolism. Moreover, Sus1 accumulates in discrete cytoplasmic granules, which partially co-localise with P-bodies and stress granules under specific conditions. These interactions suggest a role for Sus1 in gene expression during cytoplasmic mRNA metabolism in addition to its nuclear function.

Aging-dependent DNA hypermethylation and gene expression of GSTM1 involved in T cell differentiation.

Science.gov (United States)

Yeh, Shu-Hui; Liu, Cheng-Ling; Chang, Ren-Chieh; Wu, Chih-Chiang; Lin, Chia-Hsueh; Yang, Kuender D

2017-07-25

This study investigated whether aging was associated with epigenetic changes of DNA hypermethylation on immune gene expression and lymphocyte differentiation. We screened CG sites of methylation in blood leukocytes from different age populations, picked up genes with age-related increase of CG methylation content more than 15%, and validated immune related genes with CG hypermethylation involved in lymphocyte differentiation in the aged population. We found that 12 genes (EXHX1、 IL-10、 TSP50、 GSTM1、SLC5A5、SPI1、F2R、LMO2、PTPN6、FGFR2、MMP9、MET) were associated with promoter or exon one DNA hypermethylation in the aged group. Two immune related genes, GSTM1 and LMO2, were chosen to validate its aging-related CG hypermethylation in different leukocytes. We are the first to validate that GSTM1_P266 and LMO2_E128 CG methylation contents in T lymphocytes but not polymorphonuclear cells (PMNs) or mononuclear cells (MNCs) were significantly increased in the aged population. The GSTM1 mRNA expression in T lymphocytes but not PMNs or MNCs was inversely associated with the GSTM1 CG hypermethylation levels in the aged population studied. Further studies showed that lower GSTM1 CG methylation content led to the higher GSTM1 mRNA expression in T cells and knockdown of GSTM1 mRNA expression decreased type 1 T helper cell (Th1) differentiation in Jurkat T cells and normal adult CD4 T cells. The GSTM1_P266 hypermethylation in the aged population associated with lower GSTM1 mRNA expression was involved in Th1 differentiation, highlighting that modulation of aging-associated GSTM1 methylation may be able to enhance T helper cell immunity in the elders.

L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy.

Science.gov (United States)

Mobley, Christopher Brooks; Fox, Carlton D; Ferguson, Brian S; Amin, Rajesh H; Dalbo, Vincent J; Baier, Shawn; Rathmacher, John A; Wilson, Jacob M; Roberts, Michael D

2014-01-01

The purpose of this study was to examine if L-leucine (Leu), β-hydroxy-β-methylbutyrate (HMB), or creatine monohydrate (Crea) prevented potential atrophic effects of myostatin (MSTN) on differentiated C2C12 myotubes. After four days of differentiation, myotubes were treated with MSTN (10 ng/ml) for two additional days and four treatment groups were studied: 1) 3x per day 10 mM Leu, 2) 3x per day 10 mM HMB, 3) 3x per day 10 mM Crea, 4) DM only. Myotubes treated with DM without MSTN were analyzed as the control condition (DM/CTL). Following treatment, cells were analyzed for total protein, DNA content, RNA content, muscle protein synthesis (MPS, SUnSET method), and fiber diameter. Separate batch treatments were analyzed for mRNA expression patterns of myostatin-related genes (Akirin-1/Mighty, Notch-1, Ski, MyoD) as well as atrogenes (MuRF-1, and MAFbx/Atrogin-1). MSTN decreased fiber diameter approximately 30% compared to DM/CTL myotubes (p HMB and Crea prevented MSTN-induced atrophy. MSTN did not decrease MPS levels compared to DM/CTL myotubes, but MSTN treatment decreased the mRNA expression of Akirin-1/Mighty by 27% (p HMB myotubes had similar Akirin-1/Mighty and MyoD mRNA levels compared to DM/CTL myotubes. Furthermore, MSTN + Crea myotubes exhibited a 36% (p HMB and Crea may reduce MSTN-induced muscle fiber atrophy by influencing Akirin-1/Mighty mRNA expression patterns. Future studies are needed to examine if Leu, HMB and Crea independently or synergistically affect Akirin-1/Mighty expression, and how Akirin-1/Mighty expression mechanistically relates to skeletal muscle hypertrophy in vivo.

PPARγ and MyoD are differentially regulated by myostatin in adipose-derived stem cells and muscle satellite cells

International Nuclear Information System (INIS)

Zhang, Feng; Deng, Bing; Wen, Jianghui; Chen, Kun; Liu, Wu; Ye, Shengqiang; Huang, Haijun; Jiang, Siwen; Xiong, Yuanzhu

2015-01-01

Myostatin (MSTN) is a secreted protein belonging to the transforming growth factor-β (TGF-β) family that is primarily expressed in skeletal muscle and also functions in adipocyte maturation. Studies have shown that MSTN can inhibit adipogenesis in muscle satellite cells (MSCs) but not in adipose-derived stem cells (ADSCs). However, the mechanism by which MSTN differently regulates adipogenesis in these two cell types remains unknown. Peroxisome proliferator-activated receptor-γ (PPARγ) and myogenic differentiation factor (MyoD) are two key transcription factors in fat and muscle cell development that influence adipogenesis. To investigate whether MSTN differentially regulates PPARγ and MyoD, we analyzed PPARγ and MyoD expression by assessing mRNA, protein and methylation levels in ADSCs and MSCs after treatment with 100 ng/mL MSTN for 0, 24, and 48 h. PPARγ mRNA levels were downregulated after 24 h and upregulated after 48 h of treatment in ADSCs, whereas in MSCs, PPARγ levels were downregulated at both time points. MyoD expression was significantly increased in ADSCs and decreased in MSCs. PPARγ and MyoD protein levels were upregulated in ADSCs and downregulated in MSCs. The CpG methylation levels of the PPARγ and MyoD promoters were decreased in ADSCs and increased in MSCs. Therefore, this study demonstrated that the different regulatory adipogenic roles of MSTN in ADSCs and MSCs act by differentially regulating PPARγ and MyoD expression. - Highlights: • PPARγ and MyoD mRNA and protein levels are upregulated by myostatin in ADSCs. • PPARγ and MyoD mRNA and protein levels are downregulated by myostatin in MSCs. • PPARγ exhibited different methylation levels in myostatin-treated ADSCs and MSCs. • MyoD exhibited different methylation levels in myostatin-treated ADSCs and MSCs. • PPARγ and MyoD are differentially regulated by myostatin in ADSCs and MSCs

PPARγ and MyoD are differentially regulated by myostatin in adipose-derived stem cells and muscle satellite cells

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Zhang, Feng [Key Laboratory of Swine Genetics and Breeding of the Agricultural Ministry and Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of the Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070 (China); Deng, Bing [Wuhan Institute of Animal Science and Veterinary Medicine, Wuhan Academy of Agricultural Science and Technology, Wuhan, Hubei, 430208 (China); Wen, Jianghui [Wu Han University of Technology, Wuhan 430074 (China); Chen, Kun [Key Laboratory of Swine Genetics and Breeding of the Agricultural Ministry and Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of the Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070 (China); Liu, Wu; Ye, Shengqiang; Huang, Haijun [Wuhan Institute of Animal Science and Veterinary Medicine, Wuhan Academy of Agricultural Science and Technology, Wuhan, Hubei, 430208 (China); Jiang, Siwen, E-mail: jiangsiwen@mail.hzau.edu.cn [Key Laboratory of Swine Genetics and Breeding of the Agricultural Ministry and Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of the Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070 (China); Xiong, Yuanzhu, E-mail: xiongyzhu@163.com [Key Laboratory of Swine Genetics and Breeding of the Agricultural Ministry and Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of the Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070 (China)

2015-03-06

Myostatin (MSTN) is a secreted protein belonging to the transforming growth factor-β (TGF-β) family that is primarily expressed in skeletal muscle and also functions in adipocyte maturation. Studies have shown that MSTN can inhibit adipogenesis in muscle satellite cells (MSCs) but not in adipose-derived stem cells (ADSCs). However, the mechanism by which MSTN differently regulates adipogenesis in these two cell types remains unknown. Peroxisome proliferator-activated receptor-γ (PPARγ) and myogenic differentiation factor (MyoD) are two key transcription factors in fat and muscle cell development that influence adipogenesis. To investigate whether MSTN differentially regulates PPARγ and MyoD, we analyzed PPARγ and MyoD expression by assessing mRNA, protein and methylation levels in ADSCs and MSCs after treatment with 100 ng/mL MSTN for 0, 24, and 48 h. PPARγ mRNA levels were downregulated after 24 h and upregulated after 48 h of treatment in ADSCs, whereas in MSCs, PPARγ levels were downregulated at both time points. MyoD expression was significantly increased in ADSCs and decreased in MSCs. PPARγ and MyoD protein levels were upregulated in ADSCs and downregulated in MSCs. The CpG methylation levels of the PPARγ and MyoD promoters were decreased in ADSCs and increased in MSCs. Therefore, this study demonstrated that the different regulatory adipogenic roles of MSTN in ADSCs and MSCs act by differentially regulating PPARγ and MyoD expression. - Highlights: • PPARγ and MyoD mRNA and protein levels are upregulated by myostatin in ADSCs. • PPARγ and MyoD mRNA and protein levels are downregulated by myostatin in MSCs. • PPARγ exhibited different methylation levels in myostatin-treated ADSCs and MSCs. • MyoD exhibited different methylation levels in myostatin-treated ADSCs and MSCs. • PPARγ and MyoD are differentially regulated by myostatin in ADSCs and MSCs.

Involvement of miRNAs in the differentiation of human glioblastoma multiforme stem-like cells.

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Beatriz Aldaz

Full Text Available Glioblastoma multiforme (GBM-initiating cells (GICs represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process.

Short-chain chlorinated paraffins in soil, paddy seeds (Oryza sativa) and snails (Ampullariidae) in an e-waste dismantling area in China: Homologue group pattern, spatial distribution and risk assessment.

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Yuan, Bo; Fu, Jianjie; Wang, Yawei; Jiang, Guibin

2017-01-01

Short-chain chlorinated paraffins (SCCPs) in multi-environmental matrices are studied in Taizhou, Zhejiang Province, China, which is a notorious e-waste dismantling area. The investigated matrices consist of paddy field soil, paddy seeds (Oryza sativa, separated into hulls and rice unpolished) and apple snails (Ampullariidae, inhabiting the paddy fields). The sampling area covered a 65-km radius around the contamination center. C 10 and C 11 are the two predominant homologue groups in the area, accounting for about 35.7% and 33.0% of total SCCPs, respectively. SCCPs in snails and hulls are generally higher than in soil samples (30.4-530 ng/g dw), and SCCPs in hulls are approximate five times higher than in corresponding rice samples (4.90-55.1 ng/g dw). Homologue pattern analysis indicates that paddy seeds (both hull and rice) tend to accumulate relatively high volatile SCCP homologues, especially the ones with shorter carbon chain length, while snails tend to accumulate relatively high lipophilic homologues, especially the ones with more substituted chlorines. SCCPs in both paddy seeds and snails are linearly related to those in the soil. The e-waste dismantling area, which covers a radius of approximate 20 km, shows higher pollution levels for SCCPs according to their spatial distribution in four matrices. The preliminary assessment indicates that SCCP levels in local soils pose no significant ecological risk for soil dwelling organisms, but higher risks from dietary exposure of SCCPs are suspected for people living in e-waste dismantling area. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Expression of ICAT and Wnt signaling-related proteins in the monocytic differentiation of HL-60 cells induced by a new steroidal drug NSC67657].

Science.gov (United States)

Wang, J S; Wang, W J; Wang, T; Zhang, Y

2016-04-01

To investigate the expression of mRNA and proteins of β-catenin, TCF-4 (ICAT) and Wnt signaling pathway-related genes in the monocytic differentiation of acute myeloid leukemia HL-60 cells induced by a new steroidal drug NSC67657. Wright's staining and α-NBE staining were used to observe the differentiation of HL-60 cells after 5 days of 10 μmol/L NSC67657 treatment. Flow cytometry (FCM) was used to detect the differentiation and cell cycles. The expressions of mRNA and proteins of ICAT and Wnt signaling pathway-related factors, including β-catenin, TCF-4, c-myc, cyclin D1 and TCF-1 before and after differentiation, were detected by RT-PCR and Western blot. Morphological observation showed that NSC67657 induced monocytic differentiation of HL-60 cells. At 5 days after 10 μmol/L NSC67657 treatment, the number of CD14(+) HL-60 cells was (94.37±2.84)%, significantly higher than the (1.31±0.09)% in control group (Pcells were of (18.76±0.98)%, significantly lower than that of (34.38±2.61) % in the control group (Pprotein, and down-regulated the expression of β-catenin mRNA and protin (Pprotein and nuclear protein in the HL-60 cells (P>0.05 for all). The target genes of Wnt signaling pathway, including c-myc, cyclinD1 and TCF-1 mRNA and proteins in the HL-60 cells were significantly down-regulated after NSC67657 treatment (Pcells, and down-regulates the expression of β-catenin and target genes of Wnt signaling pathway. These results indicate that Wnt signaling pathway may be directly or indirectly involved in the monocytic differentiation process of HL-60 cells.

Inhibition of mitogen-activated protein kinase kinase, DNA methyltransferase, and transforming growth factor-β promotes differentiation of human induced pluripotent stem cells into enterocytes.

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Kodama, Nao; Iwao, Takahiro; Kabeya, Tomoki; Horikawa, Takashi; Niwa, Takuro; Kondo, Yuki; Nakamura, Katsunori; Matsunaga, Tamihide

2016-06-01

We previously reported that small-molecule compounds were effective in generating pharmacokinetically functional enterocytes from human induced pluripotent stem (iPS) cells. In this study, to determine whether the compounds promote the differentiation of human iPS cells into enterocytes, we investigated the effects of a combination of mitogen-activated protein kinase kinase (MEK), DNA methyltransferase (DNMT), and transforming growth factor (TGF)-β inhibitors on intestinal differentiation. Human iPS cells cultured on feeder cells were differentiated into endodermal cells by activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, the cells were differentiated into enterocyte cells by epidermal growth factor and small-molecule compounds. After differentiation, mRNA expression levels and drug-metabolizing enzyme activities were measured. The mRNA expression levels of the enterocyte marker sucrase-isomaltase and the major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 were increased by a combination of MEK, DNMT, and TGF-β inhibitors. The mRNA expression of CYP3A4 was markedly induced by 1α,25-dihydroxyvitamin D3. Metabolic activities of CYP1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP3A4/5, UDP-glucuronosyltransferase, and sulfotransferase were also observed in the differentiated cells. In conclusion, MEK, DNMT, and TGF-β inhibitors can be used to promote the differentiation of human iPS cells into pharmacokinetically functional enterocytes. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study.

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García-Álvarez, Laura; Holden, Matthew T G; Lindsay, Heather; Webb, Cerian R; Brown, Derek F J; Curran, Martin D; Walpole, Enid; Brooks, Karen; Pickard, Derek J; Teale, Christopher; Parkhill, Julian; Bentley, Stephen D; Edwards, Giles F; Girvan, E Kirsty; Kearns, Angela M; Pichon, Bruno; Hill, Robert L R; Larsen, Anders Rhod; Skov, Robert L; Peacock, Sharon J; Maskell, Duncan J; Holmes, Mark A

2011-08-01

Animals can act as a reservoir and source for the emergence of novel meticillin-resistant Staphylococcus aureus (MRSA) clones in human beings. Here, we report the discovery of a strain of S aureus (LGA251) isolated from bulk milk that was phenotypically resistant to meticillin but tested negative for the mecA gene and a preliminary investigation of the extent to which such strains are present in bovine and human populations. Isolates of bovine MRSA were obtained from the Veterinary Laboratories Agency in the UK, and isolates of human MRSA were obtained from diagnostic or reference laboratories (two in the UK and one in Denmark). From these collections, we searched for mecA PCR-negative bovine and human S aureus isolates showing phenotypic meticillin resistance. We used whole-genome sequencing to establish the genetic basis for the observed antibiotic resistance. A divergent mecA homologue (mecA(LGA251)) was discovered in the LGA251 genome located in a novel staphylococcal cassette chromosome mec element, designated type-XI SCCmec. The mecA(LGA251) was 70% identical to S aureus mecA homologues and was initially detected in 15 S aureus isolates from dairy cattle in England. These isolates were from three different multilocus sequence type lineages (CC130, CC705, and ST425); spa type t843 (associated with CC130) was identified in 60% of bovine isolates. When human mecA-negative MRSA isolates were tested, the mecA(LGA251) homologue was identified in 12 of 16 isolates from Scotland, 15 of 26 from England, and 24 of 32 from Denmark. As in cows, t843 was the most common spa type detected in human beings. Although routine culture and antimicrobial susceptibility testing will identify S aureus isolates with this novel mecA homologue as meticillin resistant, present confirmatory methods will not identify them as MRSA. New diagnostic guidelines for the detection of MRSA should consider the inclusion of tests for mecA(LGA251). Department for Environment, Food and Rural Affairs

Quantitative analyses of postmortem heat shock protein mRNA profiles in the occipital lobes of human cerebral cortices: implications in cause of death.

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Chung, Ukhee; Seo, Joong-Seok; Kim, Yu-Hoon; Son, Gi Hoon; Hwang, Juck-Joon

2012-11-01

Quantitative RNA analyses of autopsy materials to diagnose the cause and mechanism of death are challenging tasks in the field of forensic molecular pathology. Alterations in mRNA profiles can be induced by cellular stress responses during supravital reactions as well as by lethal insults at the time of death. Here, we demonstrate that several gene transcripts encoding heat shock proteins (HSPs), a gene family primarily responsible for cellular stress responses, can be differentially expressed in the occipital region of postmortem human cerebral cortices with regard to the cause of death. HSPA2 mRNA levels were higher in subjects who died due to mechanical asphyxiation (ASP), compared with those who died by traumatic injury (TI). By contrast, HSPA7 and A13 gene transcripts were much higher in the TI group than in the ASP and sudden cardiac death (SCD) groups. More importantly, relative abundances between such HSP mRNA species exhibit a stronger correlation to, and thus provide more discriminative information on, the death process than does routine normalization to a housekeeping gene. Therefore, the present study proposes alterations in HSP mRNA composition in the occipital lobe as potential forensic biological markers, which may implicate the cause and process of death.

Consequences of metaphase II oocyte cryopreservation on mRNA content.

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Chamayou, S; Bonaventura, G; Alecci, C; Tibullo, D; Di Raimondo, F; Guglielmino, A; Barcellona, M L

2011-04-01

We studied the consequences of freezing/thawing processes on mRNA contents in MII oocytes after slow-freezing/rapid thawing (SF/RT) and vitrification/warming (V/W) protocols, and compared the results to fresh MII oocytes. We quantified the nuclear transcript mRNA responsible for the translation of proteins belonging either to trans-regulatory protein family or to functional structural proteins such as proteins involved in DNA structural organization (NAP1L1, TOP1, H1F0H1), chromosomal structure maintenance (SMC, SCC3, RAD21, SMC1A, SMC1B, STAG3, REC8), mitochondrial energetic pathways (ATP5GJ, SDHC), cell cycle regulation and processes (CLTA, MAPK6, CKS2) and staminal cell potency-development competence stage (DPPA3, OCT4, FOXJ2). Surplus MII oocytes were donated from patients in IVF cycles and divided in three groups of 15 oocytes. Group 1 was comprised of non-cryopreserved oocytes and Groups 2 and 3 underwent SF/RT and V/W procedures, respectively. There was an overall decrease of mRNA extracted from cryopreserved oocytes compared to control group. Only 39.4% of mRNA content were preserved after SF/RT while 63.3% of mRNA content were maintained after V/W. Oocyte cryopreservation is associated with molecular injury associated with the decrease of stored mRNA. However the V/W protocol is more conservative than SF/RT resulting in a level of mRNA sufficient to maintain biologic functions in the subsequent fertilized oocyte. Copyright © 2011 Elsevier Inc. All rights reserved.

Establishing in vitro Zinnia elegans cell suspension culture with high tracheary elements differentiation

NARCIS (Netherlands)

Twumasi, P.; Schel, J.H.N.; Ieperen, van W.; Woltering, E.J.; Emons, A.M.C.

2009-01-01

The Zinnia elegans mesophyll cell culture is a useful system for xylogenesis studies. The system is associated with highly synchronous tracheary element (TE) differentiation, making it more suitable for molecular studies requiring larger amounts of molecular isolates, such as mRNA and proteins and

Maternal nutrient restriction in mid-to-late gestation influences fetal mRNA expression in muscle tissues in beef cattle.

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Paradis, Francois; Wood, Katie M; Swanson, Kendall C; Miller, Stephen P; McBride, Brian W; Fitzsimmons, Carolyn

2017-08-18

Manipulating maternal nutrition during specific periods of gestation can result in re-programming of fetal and post-natal development. In this experiment we investigated how a feed restriction of 85% compared with 140% of total metabolizable energy requirements, fed to cows during mid-to-late gestation, influences phenotypic development of fetuses and mRNA expression of growth (Insulin-Like Growth Factor family and Insulin Receptor (INSR)), myogenic (Myogenic Differentiation 1 (MYOD1), Myogenin (MYOG), Myocyte Enhancer Factor 2A (MEF2A), Serum Response Factor (SRF)) and adipogenic (Peroxisome Proliferator Activated Receptor Gamma (PPARG)) genes in fetal longissimus dorsi (LD) and semitendinosus (ST) muscle. DNA methylation of imprinted genes, Insulin Like Growth Factor 2 (IGF2) and Insulin Like Growth Factor 2 Receptor (IGF2R), and micro RNA (miRNA) expression, were also examined as potential consequences of poor maternal nutrition, but also potential regulators of altered gene expression patterns. While the nutrient restriction impacted dam body weight, no differences were observed in phenotypic fetal measurements (weight, crown-rump length, or thorax circumference). Interestingly, LD and ST muscles responded differently to the differential pre-natal nutrient levels. While LD muscle of restricted fetal calves had greater mRNA abundances for Insulin Like Growth Factor 1 and its receptor (IGF1 and IGF1R), IGF2R, INSR, MYOD1, MYOG, and PPARG, no significant differences were observed for gene expression in ST muscle. Similarly, feed restriction had a greater impact on the methylation level of IGF2 Differentially Methylated Region 2 (DMR2) in LD muscle as compared to ST muscle between treatment groups. A negative correlation existed between IGF2 mRNA expression and IGF2 DMR2 methylation level in both LD and ST muscles. Differential expression of miRNAs 1 and 133a were also detected in LD muscle. Our data suggests that a nutrient restriction of 85% as compared to 140

In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

DEFF Research Database (Denmark)

Nielsen, M. E.; Rasmussen, I. A.; Kristensen, S. G.

2011-01-01

significantly with the expression of AMHRII, but did not correlate with any of the hormones in the follicular fluid. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the follicular......Human small antral follicles (diameter 3-9 mm) were obtained from ovaries surgically removed for fertility preservation. From the individual aspirated follicles, granulosa cells and the corresponding follicular fluid were isolated in 64 follicles, of which 55 were available for mRNA analysis (24...... and to the follicular fluid concentrations of AMH, inhibin-B, progesterone and estradiol. AR mRNA expression in granulosa cells and the follicular fluid content of androgens both showed a highly significant positive association with the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated...

Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

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Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A. (UNL)

2012-08-21

DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.

Differentially expressed microRNA in multiple sclerosis: A window into pathogenesis?

DEFF Research Database (Denmark)

Martin, Nellie Anne; Illés, Zsolt

2014-01-01

MicroRNA are small non-coding RNA that mediate mRNA translation repression or mRNA degradation, and thereby refine protein expression levels. More than 30–60% of all genes are regulated by microRNA. Exploring disease-related microRNA signatures is an emerging tool in biomarker discovery, and sile......MicroRNA are small non-coding RNA that mediate mRNA translation repression or mRNA degradation, and thereby refine protein expression levels. More than 30–60% of all genes are regulated by microRNA. Exploring disease-related microRNA signatures is an emerging tool in biomarker discovery......RNA related to multiple sclerosis has increased significantly in recent years. Differentially expressed microRNA have been identified in the whole blood, serum, plasma, cerebrospinal fluid, peripheral blood mononuclear cells, blood-derived cell subsets and brain lesions of patients with multiple sclerosis....... Most studies applied a non-candidate approach of screening by microarray and validation by quantitative polymerase chain reaction or next generation sequencing; others used a candidate-driven approach. Despite a relatively high number of multiple sclerosis-associated microRNA, just a few could...

Reduced Dnmt3a increases Gdf5 expression with suppressed satellite cell differentiation and impaired skeletal muscle regeneration.

Science.gov (United States)

Hatazawa, Yukino; Ono, Yusuke; Hirose, Yuma; Kanai, Sayaka; Fujii, Nobuharu L; Machida, Shuichi; Nishino, Ichizo; Shimizu, Takahiko; Okano, Masaki; Kamei, Yasutomi; Ogawa, Yoshihiro

2018-03-01

DNA methylation is an epigenetic mechanism regulating gene expression. In this study, we observed that DNA methyltransferase 3a (Dnmt3a) expression is decreased after muscle atrophy. We made skeletal muscle-specific Dnmt3a-knockout (Dnmt3a-KO) mice. The regeneration capacity after muscle injury was markedly decreased in Dnmt3a-KO mice. Diminished mRNA and protein expression of Dnmt3a were observed in skeletal muscles as well as in satellite cells, which are important for muscle regeneration, in Dnmt3a-KO mice. Dnmt3a-KO satellite cell showed smaller in size (length/area), suggesting suppressed myotube differentiation. Microarray analysis of satellite cells showed that expression of growth differentiation factor 5 (Gdf5) mRNA was markedly increased in Dnmt3a-KO mice. The DNA methylation level of the Gdf5 promoter was markedly decreased in Dnmt3a-KO satellite cells. In addition, DNA methylation inhibitor azacytidine treatment increased Gdf5 expression in wild-type satellite cells, suggesting Gdf5 expression is regulated by DNA methylation. Also, we observed increased inhibitor of differentiation (a target of Gdf5) mRNA expression in Dnmt3a-KO satellite cells. Thus, Dnmt3a appears to regulate satellite cell differentiation via DNA methylation. This mechanism may play a role in the decreased regeneration capacity during atrophy such as in aged sarcopenia.-Hatazawa, Y., Ono, Y., Hirose, Y., Kanai, S., Fujii, N. L., Machida, S., Nishino, I., Shimizu, T., Okano, M., Kamei, Y., Ogawa, Y. Reduced Dnmt3a increases Gdf5 expression with suppressed satellite cell differentiation and impaired skeletal muscle regeneration.

Dynamics of Chloroplast Translation during Chloroplast Differentiation in Maize.

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Prakitchai Chotewutmontri

2016-07-01

Full Text Available Chloroplast genomes in land plants contain approximately 100 genes, the majority of which reside in polycistronic transcription units derived from cyanobacterial operons. The expression of chloroplast genes is integrated into developmental programs underlying the differentiation of photosynthetic cells from non-photosynthetic progenitors. In C4 plants, the partitioning of photosynthesis between two cell types, bundle sheath and mesophyll, adds an additional layer of complexity. We used ribosome profiling and RNA-seq to generate a comprehensive description of chloroplast gene expression at four stages of chloroplast differentiation, as displayed along the maize seedling leaf blade. The rate of protein output of most genes increases early in development and declines once the photosynthetic apparatus is mature. The developmental dynamics of protein output fall into several patterns. Programmed changes in mRNA abundance make a strong contribution to the developmental shifts in protein output, but output is further adjusted by changes in translational efficiency. RNAs with prioritized translation early in development are largely involved in chloroplast gene expression, whereas those with prioritized translation in photosynthetic tissues are generally involved in photosynthesis. Differential gene expression in bundle sheath and mesophyll chloroplasts results primarily from differences in mRNA abundance, but differences in translational efficiency amplify mRNA-level effects in some instances. In most cases, rates of protein output approximate steady-state protein stoichiometries, implying a limited role for proteolysis in eliminating unassembled or damaged proteins under non-stress conditions. Tuned protein output results from gene-specific trade-offs between translational efficiency and mRNA abundance, both of which span a large dynamic range. Analysis of ribosome footprints at sites of RNA editing showed that the chloroplast translation machinery

Over-expression, purification and characterization of an Asc-1 homologue from Gloeobacter violaceus

DEFF Research Database (Denmark)

Wang, Xiaole; Hald, Helle; Ernst, Heidi Asschenfeldt

2010-01-01

The human alanine-serine-cysteine transporter 1 (Asc-1) belongs to the slc7a family of solute carrier transporters. Asc-1 mediates the uptake of D-serine in an exchanger-type fashion, coupling the process to the release of alanine and cysteine. Among the bacterial Asc-1 homologues, one transporter...... of auto-induction was crucial for obtaining high yields and purity of the transporter. The transporter was purified with yields in the range of 0.2-0.4 mg per L culture and eluted in a single peak from a size-exclusion column. The circular dichroism spectrum revealed a folded and apparently all...

Metabolic reprogramming during neuronal differentiation.

Science.gov (United States)

Agostini, M; Romeo, F; Inoue, S; Niklison-Chirou, M V; Elia, A J; Dinsdale, D; Morone, N; Knight, R A; Mak, T W; Melino, G

2016-09-01

Newly generated neurons pass through a series of well-defined developmental stages, which allow them to integrate into existing neuronal circuits. After exit from the cell cycle, postmitotic neurons undergo neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and synaptic maturation and plasticity. Lack of a global metabolic analysis during early cortical neuronal development led us to explore the role of cellular metabolism and mitochondrial biology during ex vivo differentiation of primary cortical neurons. Unexpectedly, we observed a huge increase in mitochondrial biogenesis. Changes in mitochondrial mass, morphology and function were correlated with the upregulation of the master regulators of mitochondrial biogenesis, TFAM and PGC-1α. Concomitant with mitochondrial biogenesis, we observed an increase in glucose metabolism during neuronal differentiation, which was linked to an increase in glucose uptake and enhanced GLUT3 mRNA expression and platelet isoform of phosphofructokinase 1 (PFKp) protein expression. In addition, glutamate-glutamine metabolism was also increased during the differentiation of cortical neurons. We identified PI3K-Akt-mTOR signalling as a critical regulator role of energy metabolism in neurons. Selective pharmacological inhibition of these metabolic pathways indicate existence of metabolic checkpoint that need to be satisfied in order to allow neuronal differentiation.

Identification of genes differentially expressed in ectomycorrhizal roots during the Pinus pinaster-Laccaria bicolor interaction.

Science.gov (United States)

Flores-Monterroso, Aranzazu; Canales, Javier; de la Torre, Fernando; Ávila, Concepción; Cánovas, Francisco M

2013-06-01

Ectomycorrhizal associations are of major ecological importance in temperate and boreal forests. The development of a functional ectomycorrhiza requires many genetic and biochemical changes. In this study, suppressive subtraction hybridization was used to identify differentially expressed genes in the roots of maritime pine (Pinus pinaster Aiton) inoculated with Laccaria bicolor, a mycorrhizal fungus. A total number of 200 unigenes were identified as being differentially regulated in maritime pine roots during the development of mycorrhiza. These unigenes were classified into 10 categories according to the function of their homologues in the GenBank database. Approximately, 40 % of the differentially expressed transcripts were genes that coded for unknown proteins in the databases or that had no homology to known genes. A group of these differentially expressed genes was selected to validate the results using quantitative real-time PCR. The transcript levels of the representative genes were compared between the non-inoculated and inoculated plants at 1, 5, 15 and 30 days after inoculation. The observed expression patterns indicate (1) changes in the composition of the wall cell, (2) tight regulation of defence genes during the development of mycorrhiza and (3) changes in carbon and nitrogen metabolism. Ammonium excess or deficiency dramatically affected the stability of ectomycorrhiza and altered gene expression in maritime pine roots.

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae

Directory of Open Access Journals (Sweden)

Susanne Huch

2016-10-01

Full Text Available The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization.

Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

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Beatriz M. A. Fontoura

2013-07-01

Full Text Available 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.

Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

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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

Leishmania donovani infection induces anemia in hamsters by differentially altering erythropoiesis in bone marrow and spleen.

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William P Lafuse

Full Text Available Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating in macrophages of the bone marrow, spleen, and liver. Severe anemia and leucopenia is associated with the disease. Although immune defense mechanisms against the parasite have been studied, we have a limited understanding of how L. donovani alters hematopoiesis. In this study, we used Syrian golden hamsters to investigate effects of L. donovani infection on erythropoiesis. Infection resulted in severe anemia and leucopenia by 8 weeks post-infection. Anemia was associated with increased levels of serum erythropoietin, which indicates the hamsters respond to the anemia by producing erythropoietin. We found that infection also increased numbers of BFU-E and CFU-E progenitor populations in the spleen and bone marrow and differentially altered erythroid gene expression in these organs. In the bone marrow, the mRNA expression of erythroid differentiation genes (α-globin, β-globin, ALAS2 were inhibited by 50%, but mRNA levels of erythroid receptor (c-kit, EpoR and transcription factors (GATA1, GATA2, FOG1 were not affected by the infection. This suggests that infection has a negative effect on differentiation of erythroblasts. In the spleen, erythroid gene expression was enhanced by infection, indicating that the anemia activates a stress erythropoiesis response in the spleen. Analysis of cytokine mRNA levels in spleen and bone marrow found that IFN-γ mRNA is highly increased by L. donovani infection. Expression of the IFN-γ inducible cytokine, TNF-related apoptosis-inducing ligand (TRAIL, was also up-regulated. Since TRAIL induces erythroblasts apoptosis, apoptosis of bone marrow erythroblasts from infected hamsters was examined by flow cytometry. Percentage of erythroblasts that were apoptotic was significantly increased by L. donovani infection. Together, our results suggest that L. donovani infection inhibits erythropoiesis in the bone marrow by

Integrated mRNA and microRNA transcriptome sequencing characterizes sequence variants and mRNA–microRNA regulatory network in nasopharyngeal carcinoma model systems

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Carol Ying-Ying Szeto

2014-01-01

Full Text Available Nasopharyngeal carcinoma (NPC is a prevalent malignancy in Southeast Asia among the Chinese population. Aberrant regulation of transcripts has been implicated in many types of cancers including NPC. Herein, we characterized mRNA and miRNA transcriptomes by RNA sequencing (RNASeq of NPC model systems. Matched total mRNA and small RNA of undifferentiated Epstein–Barr virus (EBV-positive NPC xenograft X666 and its derived cell line C666, well-differentiated NPC cell line HK1, and the immortalized nasopharyngeal epithelial cell line NP460 were sequenced by Solexa technology. We found 2812 genes and 149 miRNAs (human and EBV to be differentially expressed in NP460, HK1, C666 and X666 with RNASeq; 533 miRNA–mRNA target pairs were inversely regulated in the three NPC cell lines compared to NP460. Integrated mRNA/miRNA expression profiling and pathway analysis show extracellular matrix organization, Beta-1 integrin cell surface interactions, and the PI3K/AKT, EGFR, ErbB, and Wnt pathways were potentially deregulated in NPC. Real-time quantitative PCR was performed on selected mRNA/miRNAs in order to validate their expression. Transcript sequence variants such as short insertions and deletions (INDEL, single nucleotide variant (SNV, and isomiRs were characterized in the NPC model systems. A novel TP53 transcript variant was identified in NP460, HK1, and C666. Detection of three previously reported novel EBV-encoded BART miRNAs and their isomiRs were also observed. Meta-analysis of a model system to a clinical system aids the choice of different cell lines in NPC studies. This comprehensive characterization of mRNA and miRNA transcriptomes in NPC cell lines and the xenograft provides insights on miRNA regulation of mRNA and valuable resources on transcript variation and regulation in NPC, which are potentially useful for mechanistic and preclinical studies.

Effects of intermittent versus continuous parathyroid hormone administration on condylar chondrocyte proliferation and differentiation

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Liu, Qi; Wan, Qilong; Yang, Rongtao; Zhou, Haihua; Li, Zubing

2012-01-01

Highlights: ► Different PTH administration exerts different effects on condylar chondrocyte. ► Intermittent PTH administration suppresses condylar chondrocyte proliferation. ► Continuous PTH administration maintains condylar chondrocyte proliferating. ► Intermittent PTH administration enhances condylar chondrocyte differentiation. -- Abstract: Endochondral ossification is a complex process involving chondrogenesis and osteogenesis regulated by many hormones and growth factors. Parathyroid hormone (PTH), one of the key hormones regulating bone metabolism, promotes osteoblast differentiation and osteogenesis by intermittent administration, whereas continuous PTH administration inhibits bone formation. However, the effects of PTH on chondrocyte proliferation and differentiation are still unclear. In this study, intermittent PTH administration presented enhanced effects on condylar chondrocyte differentiation and bone formation, as demonstrated by increased mineral nodule formation and alkaline phosphatase (ALP) activity, up-regulated runt-related transcription factor 2 (RUNX2), ALP, collagen type X (COL10a1), collagen type I (COL1a1), osteocalcin (OCN), bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2) and osterix (OSX) mRNA and/or protein expression. On the contrary, continuous PTH administration promoted condylar chondrocyte proliferation and suppressed its differentiation, as demonstrated by up-regulated collagen type II (COL2a1) mRNA expression, reduced mineral nodule formation and down-regulated expression of the mRNAs and/or proteins mentioned above. Our data suggest that PTH can regulate condylar chondrocyte proliferation and differentiation, depending on the type of PTH administration. These results provide new insight into the effects of PTH on condylar chondrocytes and new evidence for using local PTH administration to cure mandibular asymmetry.

The TFIID components human TAF(II)140 and Drosophila BIP2 (TAF(II)155) are novel metazoan homologues of yeast TAF(II)47 containing a histone fold and a PHD finger.

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Gangloff, Y G; Pointud, J C; Thuault, S; Carré, L; Romier, C; Muratoglu, S; Brand, M; Tora, L; Couderc, J L; Davidson, I

2001-08-01

The RNA polymerase II transcription factor TFIID comprises the TATA binding protein (TBP) and a set of TBP-associated factors (TAF(II)s). TFIID has been extensively characterized for yeast, Drosophila, and humans, demonstrating a high degree of conservation of both the amino acid sequences of the constituent TAF(II)s and overall molecular organization. In recent years, it has been assumed that all the metazoan TAF(II)s have been identified, yet no metazoan homologues of yeast TAF(II)47 (yTAF(II)47) and yTAF(II)65 are known. Both of these yTAF(II)s contain a histone fold domain (HFD) which selectively heterodimerizes with that of yTAF(II)25. We have cloned a novel mouse protein, TAF(II)140, containing an HFD and a plant homeodomain (PHD) finger, which we demonstrated by immunoprecipitation to be a mammalian TFIID component. TAF(II)140 shows extensive sequence similarity to Drosophila BIP2 (dBIP2) (dTAF(II)155), which we also show to be a component of Drosophila TFIID. These proteins are metazoan homologues of yTAF(II)47 as their HFDs selectively heterodimerize with dTAF(II)24 and human TAF(II)30, metazoan homologues of yTAF(II)25. We further show that yTAF(II)65 shares two domains with the Drosophila Prodos protein, a recently described potential dTAF(II). These conserved domains are critical for yTAF(II)65 function in vivo. Our results therefore identify metazoan homologues of yTAF(II)47 and yTAF(II)65.

Analysis of dofA, a fruA-Dependent Developmental Gene, and Its Homologue, dofB, in Myxococcus xanthus

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Horiuchi, Takayuki; Akiyama, Takuya; Inouye, Sumiko; Komano, Teruya

2002-01-01

The developmentally regulated gene dofA, identified from pulse-labeling experiments by two-dimensional gel electrophoresis, and its homologue, dofB, were cloned and characterized in Myxococcus xanthus. Deletion of dofA and dofB did not affect the vegetative growth and development of M. xanthus. dofA was specifically expressed during development, while dofB expression was observed during vegetative growth and development. The dofA-lacZ fusion was introduced into a fruA mutant and A, B, C, D, a...

Regulation of T cell differentiation and function by EZH2

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THEODOROS KARANTANOS

2016-05-01

Full Text Available The enhancer of zeste homologue 2 (EZH2, one of the polycomb group (PcG proteins, is the catalytic subunit of Polycomb-repressive complex 2 (PRC2 and induces the trimethylation of the histone H3 lysine 27 (H3K27me3 promoting epigenetic gene silencing. EZH2 contains a SET domain promoting the methyltransferase activity while the three other protein components of PRC2, namely EED, SUZ12 and RpAp46/48 induce compaction of the chromatin permitting EZH2 enzymatic activity. Numerous studies highlight the role of this evolutionary conserved protein as a master regulator of differentiation in humans involved in the repression of the homeotic (Hox gene and the inactivation of X-chromosome. Through its effects in the epigenetic regulation of critical genes, EZH2 has been strongly linked to cell cycle progression, stem cell pluripotency and cancer biology. Most recently, EZH2 has been associated with hematopoietic stem cell proliferation and differentiation, thymopoiesis and lymphopoiesis. Several studies have evaluated the role of EZH2 in the regulation of T cell differentiation and plasticity as well as its implications in the development of autoimmune diseases and graft versus host disease (GvHD. In this review we will briefly summarize the current knowledge regarding the role of EZH2 in the regulation of T cell differentiation, effector function and homing in the tumor microenvironment and we will discuss possible therapeutic targeting of EZH2 in order to alter T cell immune functions.

Expression, purification, and characterization of a dentin phosphoprotein produced by Escherichia coli, and its odontoblastic differentiation effects on human dental pulp cells.

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Yun, Ye-Rang; Jeon, Eunyi; Lee, Sujin; Kang, Wonmo; Kim, Sang-Gi; Kim, Hae-Won; Suh, Chang Kook; Jang, Jun-Hyeog

2012-08-01

To investigate the functions of recombinant human dentin phosphoprotein (rhDPP), we examined cell adhesion, viability and the odontoblastic differentiation activity of human dental pulp cells (hDPCs). Firstly, rhDPP was constructed using pBAD-HisA plasmid in Escherichia coli. Cell adhesion and viability of hDPCs by rhDPP was examined using a crystal violet assay and a MTT assay, ALP, mineralization activity and odontoblastic differentiation-related mRNA levels of hDPCs were measured to elucidate the odontoblastic differentiation effect of rhDPP on hDPCs. Initially, rhDPP significantly and dose-dependently increased hDPCs adhesion versus the untreated control (p < 0.05). Cell viability was also significantly increased by rhDPP at 5 days (p < 0.001). Furthermore, the odontoblastic differentiation effect of rhDPP was verified by measuring ALP activity, mineralization activity and the mRNA levels of odontoblastic differentiation markers. Taken together, rhDPP is expected to play an important role on hDPCs, thereby suggesting its potential use for tooth repair and regeneration.

Localization of the Norrie disease gene mRNA by in situ hybridization.

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Hartzer, M K; Cheng, M; Liu, X; Shastry, B S

1999-07-15

Norrie disease is a rare X-linked recessive neurodevelopmental disorder. The affected males manifest congenital blindness, which is often associated with hearing loss, mental retardation and psychiatric problems. Genetic linkage studies have localized the gene to the short arm of the X-chromosome and the gene has been isolated recently. The encoded protein is a member of the superfamily of growth factors containing a cystine knot motif and may be involved in cell adhesion and neurodevelopment. Molecular genetic analysis revealed a large number of missense, nonsense, deletion, and splice-site mutations among Norrie patients. In order to further determine the role of the Norrie disease gene, we studied the distribution pattern of its mRNA in the retina and in brain by in situ hybridization. The results show abundant hybridization signals in outer nuclear, inner nuclear, and ganglion cell layers of the retina in all three species (mice, rabbit, and human) examined. There was no significant expression in the vitreous body, lens, and rod outer segment. High expression levels were also observed in the cerebellar granular layer, hippocampus, olfactory bulb, cortex, and epithelium of the rabbit brain. These data suggest that the Norrie disease gene could play a critical role in the differentiation or maintenance of the differentiated state of the retina.

Effect of resveratrol on proliferation and differentiation of embryonic cardiomyoblasts

International Nuclear Information System (INIS)

Leong, C.-W.; Wong, C.H.; Lao, S.-C.; Leong, Emilia Conceicao; Lao, Iok Fong; Law, Patrick Tik Wan; Fung, Kwok Pui; Tsang, Kam Sze; Waye, Mary Miu-Yee; Tsui, Stephen Kwok-Wing; Wang Yitao; Lee, Simon Ming-Yuen

2007-01-01

Resveratrol (trans-3,5,4'-trihydroxystilbene), a polyphenolic compound found largely in the skins of red grapes, has been used as a nutritional supplement or an investigational new drug for prevention of cardiovascular diseases. Previous reports showed that resveratrol had a protective effect against oxidative agent-induced cell injury. Our studies indicate that resveratrol plays a role in the differentiation of cardiomyoblasts. The cardiomyoblast cell line, H9c2, was exposed to 30-120 μM resveratrol for up to 5 days. Resveratrol inhibits cardiomyoblast proliferation without causing cells injury. Moreover, resveratrol treatment modulated the differentiation of morphological characteristics including elongation and cell fusion in cardiomyoblasts. Proliferation and differentiation of H9c2 cells were further revealed by measurement of the mRNA expression of a cell cycle marker (CDK2), a differentiation marker (myogenin), and a contractile apparatus protein (MLC-2). Gene expression analysis revealed that resveratrol promoted entry into cell cycle arrest but extended the myogenic differentiation progress. These results have implications for the role of resveratrol in modulating cell cycle control and differentiation in cardiomyoblasts

Use of isotope ratio mass spectrometry to differentiate between endogenous steroids and synthetic homologues in cattle: a review.

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Janssens, Geert; Courtheyn, Dirk; Mangelinckx, Sven; Prévost, Stéphanie; Bichon, Emmanuelle; Monteau, Fabrice; De Poorter, Geert; De Kimpe, Norbert; Le Bizec, Bruno

2013-04-15

Although substantial technical advances have been achieved during the past decades to extend and facilitate the analysis of growth promoters in cattle, the detection of abuse of synthetic analogs of naturally occurring hormones has remained a challenging issue. When it became clear that the exogenous origin of steroid hormones could be traced based on the (13)C/(12)C isotope ratio of the substances, GC/C/IRMS has been successfully implemented to this aim since the end of the past century. However, due to the costly character of the instrumental setup, the susceptibility of the equipment to errors and the complex and time consuming sample preparation, this method is up until now only applied by a limited number of laboratories. In this review, the general principles as well as the practical application of GC/C/IRMS to differentiate between endogenous steroids and exogenously synthesized homologous compounds in cattle will be discussed in detail, and will be placed next to other existing and to be developed methods based on isotope ratio mass spectrometry. Finally, the link will be made with the field of sports doping, where GC/C/IRMS has been established within the World Anti-Doping Agency (WADA) approved methods as the official technique to differentiate between exogenous and endogenous steroids over the past few years. Copyright © 2013 Elsevier B.V. All rights reserved.

Immediate-early gene response to repeated immobilization: Fos protein and arc mRNA levels appear to be less sensitive than c-fos mRNA to adaptation.

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Ons, Sheila; Rotllant, David; Marín-Blasco, Ignacio J; Armario, Antonio

2010-06-01

Stress exposure resulted in brain induction of immediate-early genes (IEGs), considered as markers of neuronal activation. Upon repeated exposure to the same stressor, reduction of IEG response (adaptation) has been often observed, but there are important discrepancies in literature that may be in part related to the particular IEG and methodology used. We studied the differential pattern of adaptation of the IEGs c-fos and arc (activity-regulated cytoskeleton-associated protein) after repeated exposure to a severe stressor: immobilization on wooden boards (IMO). Rats repeatedly exposed to IMO showed reduced c-fos mRNA levels in response to acute IMO in most brain areas studied: the medial prefrontal cortex (mPFC), lateral septum (LS), medial amygdala (MeA), paraventricular nucleus of the hypothalamus (PVN) and locus coeruleus. In contrast, the number of neurons showing Fos-like immunoreactivity was only reduced in the MeA and the various subregions of the PVN. IMO-induced increases in arc gene expression were restricted to telencephalic regions and reduced by repeated IMO only in the mPFC. Double-labelling in the LS of IMO-exposed rats revealed that arc was expressed in only one-third of Fos+ neurons, suggesting two populations of Fos+ neurons. These data suggest that c-fos mRNA levels are more affected by repeated IMO than corresponding protein, and that arc gene expression does not reflect adaptation in most brain regions, which may be related to its constitutive expression. Therefore, the choice of a particular IEG and the method of measurement are important for proper interpretation of the impact of chronic repeated stress on brain activation.

ATF3 inhibits adipocyte differentiation of 3T3-L1 cells

International Nuclear Information System (INIS)

Jang, Min Kyung; Kim, Cho Hee; Seong, Je Kyung; Jung, Myeong Ho

2012-01-01

Highlights: ► Overexpression of ATF3 inhibits adipocyte differentiation in 3T3-L1 cells. ► Overexpression of ATF3 represses C/EBPα expression. ► ATF3 directly binds to mouse C/EBPα promoter spanning from −1928 to −1907. ► ATF3 may play a role in hypoxia-mediated inhibition of adipocyte differentiation. -- Abstract: ATF3 is a stress-adaptive gene that regulates proliferation or apoptosis under stress conditions. However, the role of ATF3 is unknown in adipocyte cells. Therefore, in this study, we investigated the functional role of ATF3 in adipocytes. Both lentivirus-mediated overexpression of ATF3 and stably-overexpressed ATF3 inhibited adipocyte differentiation in 3T3-L1 cells, as revealed by decreased lipid staining with oil red staining and reduction in adipogenic genes. Thapsigargin treatment and overexpression of ATF3 decreased C/EBPα transcript and repressed the activity of the 3.6-kb mouse C/EBPα promoter, demonstrating that ATF3 downregulates C/EBPα expression. Transfection studies using mutant constructs containing 5′-deletions in the C/EBPα promoter revealed that a putative ATF/CRE element, GGATGTCA, is located between −1921 and −1914. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 directly binds to mouse C/EBPα promoter spanning from −1928 to −1907. Both chemical hypoxia-mimetics or physical hypoxia led to reduce the C/EBPα mRNA and repress the promoter activity of the C/EBPα gene, whereas increase ATF3 mRNA, suggesting that ATF3 may contribute to the inhibition of adipocyte differentiation in hypoxia through downregulation of C/EBPα expression. Collectively, these results demonstrate that ATF3 represses the C/EBPα gene, resulting in inhibition of adipocyte differentiation, and thus plays a role in hypoxia-mediated inhibition of adipocyte differentiation.

Low levels of PRB3 mRNA are associated with dopamine-agonist resistance and tumor recurrence in prolactinomas.

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Wang, Fei; Gao, Hua; Li, Chuzhong; Bai, Jiwei; Lu, Runchun; Cao, Lei; Wu, Yongtu; Hong, Lichuan; Wu, Yonggang; Lan, Xiaolei; Zhang, Yazhuo

2014-01-01

Prolactinomas, or prolactin-secreting adenomas, constitute the most common type of hyperfunctioning pituitary adenoma. Dopamine agonists are used as first-line medication for prolactinomas, but the tumors are resistant to the therapy in 5-18 % of patients. To explore potential mechanisms of resistance to bromocriptine (a dopamine agonist), we analyzed six responsive prolactinomas and six resistant prolactinomas by whole-exome sequencing. We identified ten genes with sequence variants that were differentially found in the two groups of tumors. The expression of these genes was then quantified by real-time reverse-transcription PCR (RT-qPCR) in the 12 prolactinomas and in six normal pituitary glands. The mRNA levels of one of the genes, PRB3, were about fourfold lower in resistant prolactinomas than in the responsive tumors (p = 0.02). Furthermore, low PRB3 expression was also associated with tumor recurrence. Our results suggest that low levels of PRB3 mRNA may have a role in dopamine-agonist resistance and tumor recurrence of prolactinomas.

Assessing mRNA nuclear export in mammalian cells by microinjection.

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Lee, Eliza S; Palazzo, Alexander F

2017-08-15

The nuclear export of mRNAs is an important yet little understood part of eukaryotic gene expression. One of the easiest methods for monitoring mRNA export in mammalian tissue culture cells is through the microinjection of DNA plasmids into the nucleus and monitoring the distribution of the transcribed product over time. Here we describe how to setup a microscope equipped with a micromanipulator used in cell microinjections, and we explain how to perform a nuclear mRNA export assay and obtain the nuclear export rate for any given mRNA. Copyright © 2017 Elsevier Inc. All rights reserved.

Quercetin promotes proliferation and differentiation of oligodendrocyte precursor cells after oxygen/glucose deprivation-induced injury.

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Wu, Xiuxiang; Qu, Xuebin; Zhang, Qiang; Dong, Fuxing; Yu, Hongli; Yan, Chen; Qi, Dashi; Wang, Meng; Liu, Xuan; Yao, Ruiqin

2014-04-01

The aim of this study was to investigate quercetin's (Qu) ability to promote proliferation and differentiation of oligodendrocyte precursor cells (OPCs) under oxygen/glucose deprivation (OGD)-induced injury in vitro. The results showed that after OGD, OPCs survival rate was significantly increased by Qu as measured by Cell Counting Kit-8. Furthermore, Qu treatment reduced apoptosis of OPCs surveyed by Hoechst 33258 nuclear staining. Qu at 9 and 27 μM promoted the proliferation of OPCs the most by Brdu and Olig2 immunocytochemical staining after OGD 3 days. Also, Qu treatment for 8 days after OGD, the differentiation of OPCs to oligodendrocyte was detected by immunofluorescence staining showing that O4, Olig2, and myelin basic protein (MBP) positive cells were significantly increased compared to control group. Additionally, the protein levels of Olig2 and MBP of OPCs were quantified using western blot and mRNA levels of Olig2 and Inhibitor of DNA binding 2 (Id2) were measured by RT-PCR. Western blot showed a significant increase in Olig2 and MBP expression levels compared with controls after OGD and Qu treatment with a linear does-response curve from 3 to 81 μM. After treatment with Qu compared to its control group, Olig2 mRNA level was significantly up-regulated, whereas Id2 mRNA level was down-regulated. In conclusion, Qu at 3-27 μM can promote the proliferation and differentiation of OPCs after OGD injury and may regulate the activity of Olig2 and Id2.

Recombination at DNA replication fork barriers is not universal and is differentially regulated by Swi1.

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Pryce, David W; Ramayah, Soshila; Jaendling, Alessa; McFarlane, Ramsay J

2009-03-24

DNA replication stress has been implicated in the etiology of genetic diseases, including cancers. It has been proposed that genomic sites that inhibit or slow DNA replication fork progression possess recombination hotspot activity and can form potential fragile sites. Here we used the fission yeast, Schizosaccharomyces pombe, to demonstrate that hotspot activity is not a universal feature of replication fork barriers (RFBs), and we propose that most sites within the genome that form RFBs do not have recombination hotspot activity under nonstressed conditions. We further demonstrate that Swi1, the TIMELESS homologue, differentially controls the recombination potential of RFBs, switching between being a suppressor and an activator of recombination in a site-specific fashion.

Single step production of Cas9 mRNA for zygote injection.

Science.gov (United States)

Redel, Bethany K; Beaton, Benjamin P; Spate, Lee D; Benne, Joshua A; Murphy, Stephanie L; O'Gorman, Chad W; Spate, Anna M; Prather, Randall S; Wells, Kevin D

2018-03-01

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

Single-cell mRNA transfection studies: delivery, kinetics and statistics by numbers.

Science.gov (United States)

Leonhardt, Carolin; Schwake, Gerlinde; Stögbauer, Tobias R; Rappl, Susanne; Kuhr, Jan-Timm; Ligon, Thomas S; Rädler, Joachim O

2014-05-01

In artificial gene delivery, messenger RNA (mRNA) is an attractive alternative to plasmid DNA (pDNA) since it does not require transfer into the cell nucleus. Here we show that, unlike for pDNA transfection, the delivery statistics and dynamics of mRNA-mediated expression are generic and predictable in terms of mathematical modeling. We measured the single-cell expression time-courses and levels of enhanced green fluorescent protein (eGFP) using time-lapse microscopy and flow cytometry (FC). The single-cell analysis provides direct access to the distribution of onset times, life times and expression rates of mRNA and eGFP. We introduce a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby the dose-response relation. Our results establish a statistical framework for mRNA transfection and as such should advance the development of RNA carriers and small interfering/micro RNA-based drugs. This team of authors established a statistical framework for mRNA transfection by using a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby their dose-response relation. This study establishes a nice connection between theory and experimental planning and will aid the cellular delivery of mRNA molecules. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Nonsense mutations in the human β-globin gene affect mRNA metabolism

International Nuclear Information System (INIS)

Baserga, S.J.; Benz, E.J. Jr.

1988-01-01

A number of premature translation termination mutations (nonsense mutations) have been described in the human α- and β-globin genes. Studies on mRNA isolated from patients with β 0 -thalassemia have shown that for both the β-17 and the β-39 mutations less than normal levels of β-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human β-globin mRNA). In vitro studies using the cloned β-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. The authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human β-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation

Differentiation of human multipotent dermal fibroblasts into islet-like cell clusters

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Liu Wei

2010-06-01

Full Text Available Abstract Background We have previously obtained a clonal population of cells from human foreskin that is able to differentiate into mesodermal, ectodermal and endodermal progenies. It is of great interest to know whether these cells could be further differentiated into functional insulin-producing cells. Results Sixty-one single-cell-derived dermal fibroblast clones were established from human foreskin by limiting dilution culture. Of these, two clones could be differentiated into neuron-, adipocyte- or hepatocyte-like cells under certain culture conditions. In addition, those two clones were able to differentiate into islet-like clusters under pancreatic induction. Insulin, glucagon and somatostatin were detectable at the mRNA and protein levels after induction. Moreover, the islet-like clusters could release insulin in response to glucose in vitro. Conclusions This is the first study to demonstrate that dermal fibroblasts can differentiate into insulin-producing cells without genetic manipulation. This may offer a safer cell source for future stem cell-based therapies.

Effects of 2-deoxy-D-glucose and quercetin on the expression of osteonectin and osteopontin during the differentiation of irradiated MC3T3-E1 osteoblastic cells

International Nuclear Information System (INIS)

Yu, Su Kyung; Koh, Kwang Joon; Kim, Kyoung A

2008-01-01

To characterize the effects of 2-deoxy-D-glucose (2-DG) and quercetin (QCT) on gene expression of osteonectin (ON) and osteopontin (OP) in irradiated MC3T3-E1 cells. When MC3T3-E1 osteoblastic cells had reached 70-80% confluence, cultures were transferred to a differentiating medium supplemented with 5 mM 2-DG or 10 μM QCT and then irradiated with 2, 4, 6, and 8 Gy. At various times after irradiation, the cells were analyzed for the expression of bone mineralization genes such as ON and OP. The mRNA expression of both ON and OP was increased according to the culture time in the differentiation medium, and the increase of the genes peaked at 14 days after the differentiation induction. In the case of OP, the increase of mRNA expression was maintained to 28 days after the differentiation, while the mRNA level of ON was reduced to the basal level at the same time. Irradiation adding 2-DG showed a significant peak value in the expression pattern of ON at 4 Gy 7 days after irradiation. Irradiation adding QCT increased the mRNA expression of ON and OP in a dose-dependant manner, but irradiation adding 2-DG did not show any differences between the control and experiments 14 days after irradiation. Irradiation adding QCT increased significantly the expression patterns of ON 21 days after irradiation. The results showed that QCT acted as a radiosensitizer in the gene expression of ON and OP during differentiation of the late stage of irradiated MC3T3-E1 osteoblastic cells in vitro.

Secreted Frizzled related protein-4 (sFRP4) promotes epidermal differentiation and apoptosis

International Nuclear Information System (INIS)

Maganga, Richard; Giles, Natalie; Adcroft, Katharine; Unni, Ambili; Keeney, Diane; Wood, Fiona; Fear, Mark; Dharmarajan, Arunasalam

2008-01-01

The skin provides vital protection from infection and dehydration. Maintenance of the skin is through a constant program of proliferation, differentiation and apoptosis of epidermal cells, whereby proliferating cells in the basal layer differentiating to form the keratinized, anucleated stratum corneum. The WNT signalling pathway is known to be important in the skin. WNT signalling has been shown to be important both in epidermal development and in the maintenance and cycling of hair follicles and epidermal stem cells. However, the precise role for this pathway in epidermal differentiation remains unknown. We investigated the role of the WNT signalling inhibitor sFRP4 in epidermal differentiation. sFRP4 is expressed in both normal skin and keratinocytes in culture. Expression of sFRP4 mRNA and protein increases with keratinocyte differentiation and apoptosis, whilst exposure of keratinocytes to exogenous sFRP4 promotes apoptosis and expression of the terminal differentiation marker Involucrin. These data suggest sFRP4 promotes epidermal differentiation.

Osteoblast Differentiation and Bone Formation Gene Expression in Strontium-inducing Bone Marrow Mesenchymal Stem Cell

OpenAIRE

SILA-ASNA, MONNIPHA; BUNYARATVEJ, AHNOND; Maeda, Sakan; Kitaguchi, Hiromichi; BUNYARATAVEJ, NARONG

2007-01-01

Osteoblastic differentiation from human mesenchymal stem cell (hMSCs) is animportant step of bone formation. We studied the in vitro induction of hMSCs byusing strontium ranelate, a natural trace amount in water, food and human skeleton.The mRNA synthesis of various osteoblast specific genes was assessed by means ofreverse transcription polymerase chain reaction (RT-PCR). In the hMSCs culture,strontium ranelate could enhance the induction of hMSCs to differentiate intoosteoblasts. Cbfa1 gene ...

Clinical values of AFP, GPC3 mRNA in peripheral blood for prediction of hepatocellular carcinoma recurrence following OLT: AFP, GPC3 mRNA for prediction of HCC.

Science.gov (United States)

Wang, Yuliang; Shen, Zhongyang; Zhu, Zhijun; Han, Ruifa; Huai, Mingsheng

2011-03-01

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Annually, about 200,000 patients died of HCC in China. Liver transplantation (LT) holds great theoretical appeal in treating HCC. However, the high recurrence rate after transplantation is the most important limiting factor for long-term survival. To assess the value of alpha-fetoprotein (AFP) messenger RNA (mRNA), Glypican-3 (GPC3) mRNA-expressing cells in the peripheral blood (PB) for prediction of HCC recurrence following orthotopic liver transplantation (OLT). 29 patients with HCC who underwent OLT with a minimum clinical follow-up of 12 months were included in this retrospective study. We detected AFP mRNA, GPC3 mRNA-expressing cells in the PB by TaqMan real-time reverse transcriptase-polymerase chain reaction (RT-PCR), pre-, intra- and post-operatively. The early recurrence of patients was evaluated. 8 (28%), 15 (52%), and 9 (31%) patients had AFP mRNA detected pre-, intra-, and post-operatively, respectively. With 12 months of follow-up, HCC recurred in 7 (24%) patients. Univariate analysis revealed that positive pre- and post-operative AFP mRNA, TNM stage as well as vascular invasion were significant predictors for the HCC recurrence. Multivariate analysis revealed that being positive for AFP mRNA pre-operatively remained a significant risk factor for HCC recurrence after OLT. GPC3 mRNA was expressed in all PB samples. There was no significant difference in the expression levels of GPC3 mRNA between the HCC and control groups. There were no significant differences in GPC3 mRNA expression values between those patients with and without tumor recurrence. The pre-operative detection of circulating AFP mRNA-expressing cells could be a useful predictor for HCC recurrence following OLT. GPC3 mRNA-expressing cells in PB seem to have no diagnostic value.

Primary induction of vitellogenin mRNA in the rooster by 17beta-estradiol.

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Burns, A T; Deeley, R G; Gordon, J I; Udell, D S; Mullinix, K P; Goldberger, R F

1978-01-01

We have studied the kinetics of vitellogenin mRNA accumulation in rooster liver after a primary injection of 17beta-estradiol. The levels of vitellogenin mRNA have been determined both by hybridization of total cellular RNA to vitellogenin cDNA and by translation of vitellogenin mRNA in a wheat germ cell-free system. The results obtained by both methods of analysis are in good agreement and indicate that vitellogenin mRNA is present in the liver of normal roosters at a level of 0-5 molecules per liver cell and increases in amount during the 3 days following injection of estrogen, reaching a level of almost 6000 molecules per cell at the peak of the response. The level of vitellogenin mRNA declined exponentially during the next 14 days with a half-life of 29 hr, reaching a level of less than 10 molecules per cell at 17 days after injection of the hormone. The levels of vitellogenin mRNA after stimulation with estrogen have been correlated with the in vivo rate of synthesis of the vitellogenin polypeptide. The results indicate that the rate of vitellogenin synthesis is closely correlated with the level of vitellogenin mRNA. On the basis of these findings, we conclude that vitellogenin mRNA does not exist in the liver in an untranslated form after withdrawal from estrogen. PMID:273910

Effects of intermittent versus continuous parathyroid hormone administration on condylar chondrocyte proliferation and differentiation

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Liu, Qi; Wan, Qilong; Yang, Rongtao; Zhou, Haihua [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); Li, Zubing, E-mail: lizubing0827@163.com [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China)

2012-07-20

Highlights: Black-Right-Pointing-Pointer Different PTH administration exerts different effects on condylar chondrocyte. Black-Right-Pointing-Pointer Intermittent PTH administration suppresses condylar chondrocyte proliferation. Black-Right-Pointing-Pointer Continuous PTH administration maintains condylar chondrocyte proliferating. Black-Right-Pointing-Pointer Intermittent PTH administration enhances condylar chondrocyte differentiation. -- Abstract: Endochondral ossification is a complex process involving chondrogenesis and osteogenesis regulated by many hormones and growth factors. Parathyroid hormone (PTH), one of the key hormones regulating bone metabolism, promotes osteoblast differentiation and osteogenesis by intermittent administration, whereas continuous PTH administration inhibits bone formation. However, the effects of PTH on chondrocyte proliferation and differentiation are still unclear. In this study, intermittent PTH administration presented enhanced effects on condylar chondrocyte differentiation and bone formation, as demonstrated by increased mineral nodule formation and alkaline phosphatase (ALP) activity, up-regulated runt-related transcription factor 2 (RUNX2), ALP, collagen type X (COL10a1), collagen type I (COL1a1), osteocalcin (OCN), bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2) and osterix (OSX) mRNA and/or protein expression. On the contrary, continuous PTH administration promoted condylar chondrocyte proliferation and suppressed its differentiation, as demonstrated by up-regulated collagen type II (COL2a1) mRNA expression, reduced mineral nodule formation and down-regulated expression of the mRNAs and/or proteins mentioned above. Our data suggest that PTH can regulate condylar chondrocyte proliferation and differentiation, depending on the type of PTH administration. These results provide new insight into the effects of PTH on condylar chondrocytes and new evidence for using local PTH administration to cure mandibular

Global mRNA expression analysis in myosin II deficient strains of Saccharomyces cerevisiae reveals an impairment of cell integrity functions

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Rivera-Molina Félix E

2008-01-01

Full Text Available Abstract Background The Saccharomyces cerevisiae MYO1 gene encodes the myosin II heavy chain (Myo1p, a protein required for normal cytokinesis in budding yeast. Myo1p deficiency in yeast (myo1Δ causes a cell separation defect characterized by the formation of attached cells, yet it also causes abnormal budding patterns, formation of enlarged and elongated cells, increased osmotic sensitivity, delocalized chitin deposition, increased chitin synthesis, and hypersensitivity to the chitin synthase III inhibitor Nikkomycin Z. To determine how differential expression of genes is related to these diverse cell wall phenotypes, we analyzed the global mRNA expression profile of myo1Δ strains. Results Global mRNA expression profiles of myo1Δ strains and their corresponding wild type controls were obtained by hybridization to yeast oligonucleotide microarrays. Results for selected genes were confirmed by real time RT-PCR. A total of 547 differentially expressed genes (p ≤ 0.01 were identified with 263 up regulated and 284 down regulated genes in the myo1Δ strains. Gene set enrichment analysis revealed the significant over-representation of genes in the protein biosynthesis and stress response categories. The SLT2/MPK1 gene was up regulated in the microarray, and a myo1Δslt2Δ double mutant was non-viable. Overexpression of ribosomal protein genes RPL30 and RPS31 suppressed the hypersensitivity to Nikkomycin Z and increased the levels of phosphorylated Slt2p in myo1Δ strains. Increased levels of phosphorylated Slt2p were also observed in wild type strains under these conditions. Conclusion Following this analysis of global mRNA expression in yeast myo1Δ strains, we conclude that 547 genes were differentially regulated in myo1Δ strains and that the stress response and protein biosynthesis gene categories were coordinately regulated in this mutant. The SLT2/MPK1 gene was confirmed to be essential for myo1Δ strain viability, supporting that the up

NGF induces adult stem Leydig cells to proliferate and differentiate during Leydig cell regeneration.

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Zhang, Lei; Wang, Huaxi; Yang, Yan; Liu, Hui; Zhang, Qihao; Xiang, Qi; Ge, Renshan; Su, Zhijian; Huang, Yadong

2013-06-28

Nerve growth factor (NGF) has been reported to be involved in male reproductive physiology. However, few reports have described the activity of NGF during Leydig cell development. The objective of the present study was to examine the role of NGF during stem-Leydig-cell (SLC) regeneration. We investigated the effects of NGF on Leydig-cell (LC) regeneration by measuring mRNA levels in the adult rat testis after ethane dimethanesulfonate (EDS) treatment. Furthermore, we used the established organ culture model of rat seminiferous tubules to examine the regulation of NGF during SLC proliferation and differentiation using EdU staining, real-time PCR and western blotting. Progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs) were also used to investigate the effects of NGF on LCs at different developmental stages. NGF mRNA levels changed significantly during Leydig-cell regeneration in vivo. In vitro, NGF significantly promoted the proliferation of stem Leydig cells and also induced steroidogenic enzyme gene expression and 3β-HSD protein expression. The data from PLCs and ILCs showed that NGF could increase Cyclin D1 and Hsd 17b3 mRNA levels in PLCs and Cyclin D1 mRNA levels in ILCs. These results indicate that NGF may play an important role during LC regeneration by regulating the proliferation and differentiation of LCs at different developmental stages, from SLCs to PLCs and from PLCs to ILCs. The discovery of this effect of NGF on Leydig cells will provide useful information for developing new potential therapies for PADAM (Partial Androgen Deficiency in the Aging Male). Copyright © 2013 Elsevier Inc. All rights reserved.

A new dawn for plant mitochondrial NAD(P)H dehydrogenases

DEFF Research Database (Denmark)

Møller, I.M.

2002-01-01

The expression of complex I and two homologues of bacterial and yeast NADH dehydrogenases, NDA and NDB, have been studied in potato leaf mitochondria. The mRNA level of NDA is completely light dependent and shows a diurnal rhythm with a sharp maximum just after dawn. NDA protein quantity and inte...

Increased uncoupling protein-2 mRNA abundance and glucocorticoid action in adipose tissue in the sheep fetus during late gestation is dependent on plasma cortisol and triiodothyronine

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Gnanalingham, MG; Mostyn, A; Forhead, AJ; Fowden, AL; Symonds, ME; Stephenson, T

2005-01-01

The endocrine regulation of uncoupling protein-2 (UCP2), an inner mitochondrial protein, in fetal adipose tissue remains unclear. The present study aimed to determine if fetal plasma cortisol and triiodothyronine (T3) influenced the mRNA abundance of UCP2, glucocorticoid receptor (GR) and 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and 2 (11βHSD2) in fetal adipose tissue in the sheep during late gestation. Perirenal–abdominal adipose tissue was sampled from ovine fetuses to which either cortisol (2–3 mg kg−1 day−1) or saline was infused for 5 days up to 127–130 days gestation, or near term fetuses (i.e. 142–145 days gestation) that were either adrenalectomised (AX) or remained intact. Fetal plasma cortisol and T3 concentrations were higher in the cortisol infused animals and lower in AX fetuses compared with their corresponding control group, and increased with gestational age. UCP2 and GR mRNA abundance were significantly lower in AX fetuses compared with age-matched controls, and increased with gestational age and by cortisol infusion. Glucocorticoid action in fetal adipose tissue was augmented by AX and suppressed by cortisol infusion, the latter also preventing the gestational increase in 11βHSD1 mRNA and decrease in 11βHSD2 mRNA. When all treatment groups were combined, both fetal plasma cortisol and T3 concentrations were positively correlated with UCP2, GR and 11βHSD2 mRNA abundance, but negatively correlated with 11βHSD1 mRNA abundance. In conclusion, plasma cortisol and T3 are both required for the late gestation rise in UCP2 mRNA and differentially regulate glucocorticoid action in fetal adipose tissue in the sheep during late gestation. PMID:15961419

High ALK mRNA expression has a negative prognostic significance in rhabdomyosarcoma

Science.gov (United States)

Bonvini, P; Zin, A; Alaggio, R; Pawel, B; Bisogno, G; Rosolen, A

2013-01-01

Background: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in cancer, but its clinical and functional importance remain controversial. Mutation or amplification of ALK, as well as its expression levels assessed by conventional immunohistochemistry methods, has been linked to prognosis in cancer, although with potential bias because of the semi-quantitative approaches. Herein, we measured ALK mRNA expression in rhabdomyosarcoma (RMS) and determined its clinical impact on patients' stratification and outcome. Methods: Specimens were obtained from RMS patients and cell lines, and ALK expression was analysed by quantitative RT–PCR, western blotting, IHC, and copy number analysis. Results: High ALK mRNA expression was detected in the vast majority of PAX3/7-FOXO1-positive tumours, whereas PAX3/7-FOXO1-negative RMS displayed considerably lower amounts of both mRNA and protein. Notably, ALK mRNA distinguished unfavourable PAX3/7-FOXO1-positive tumours from PAX3/7-FOXO1-negative RMS (Ptumour size (PALK mRNA levels were of prognostic relevance by Cox univariate regression analysis and correlated with increased risk of relapse (P=0.001) and survival (P=0.01), whereas by multivariate analysis elevated ALK mRNA expression resulted a negative prognostic marker when clinical stage was not included. Conclusion: Quantitative assessment of ALK mRNA expression helps to improve risk stratification of RMS patients and identifies tumours with adverse biological characteristics and aggressive behaviour. PMID:24149177

Prelamin A is involved in early steps of muscle differentiation

International Nuclear Information System (INIS)

Capanni, Cristina; Del Coco, Rosalba; Squarzoni, Stefano; Columbaro, Marta; Mattioli, Elisabetta; Camozzi, Daria; Rocchi, Anna; Scotlandi, Katia; Maraldi, Nadir; Foisner, Roland; Lattanzi, Giovanna

2008-01-01

Lamin A is a nuclear lamina constituent implicated in a number of human disorders including Emery-Dreifuss muscular dystrophy. Since increasing evidence suggests a role of the lamin A precursor in nuclear functions, we investigated the processing of prelamin A during differentiation of C2C12 mouse myoblasts. We show that both protein levels and cellular localization of prelamin A are modulated during myoblast activation. Similar changes of lamin A-binding proteins emerin and LAP2α were observed. Furthermore, prelamin A was found in a complex with LAP2α in differentiating myoblasts. Prelamin A accumulation in cycling myoblasts by expressing unprocessable mutants affected LAP2α and PCNA amount and increased caveolin 3 mRNA and protein levels, while accumulation of prelamin A in differentiated muscle cells following treatment with a farnesyl transferase inhibitor appeared to inhibit caveolin 3 expression. Our data provide evidence for a critical role of the lamin A precursor in the early steps of muscle cell differentiation

Differentiation of PC12 Cells Results in Enhanced VIP Expression and Prolonged Rhythmic Expression of Clock Genes

DEFF Research Database (Denmark)

Pretzmann, C.P.; Fahrenkrug, J.; Georg, B.

2008-01-01

To examine for circadian rhythmicity, the messenger RNA (mRNA) amount of the clock genes Per1 and Per2 was measured in undifferentiated and nerve-growth-factor-differentiated PC12 cells harvested every fourth hour. Serum shock was needed to induce circadian oscillations, which in undifferentiated...... PC12 cultures lasted only one 24-h period, while in differentiated cultures, the rhythms continued for at least 3 days. Thus, neuronal differentiation provided PC12 cells the ability to maintain rhythmicity for an extended period. Both vasoactive intestinal polypeptide (VIP) and its receptor VPAC(2...

Over-expression of the miRNA cluster at chromosome 14q32 in the alcoholic brain correlates with suppression of predicted target mRNA required for oligodendrocyte proliferation.

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Manzardo, A M; Gunewardena, S; Butler, M G

2013-09-10

We examined miRNA expression from RNA isolated from the frontal cortex (Broadman area 9) of 9 alcoholics (6 males, 3 females, mean age 48 years) and 9 matched controls using both the Affymetrix GeneChip miRNA 2.0 and Human Exon 1.0 ST Arrays to further characterize genetic influences in alcoholism and the effects of alcohol consumption on predicted target mRNA expression. A total of 12 human miRNAs were significantly up-regulated in alcohol dependent subjects (fold change≥1.5, false discovery rate (FDR)≤0.3; p<0.05) compared with controls including a cluster of 4 miRNAs (e.g., miR-377, miR-379) from the maternally expressed 14q32 chromosome region. The status of the up-regulated miRNAs was supported using the high-throughput method of exon microarrays showing decreased predicted mRNA gene target expression as anticipated from the same RNA aliquot. Predicted mRNA targets were involved in cellular adhesion (e.g., THBS2), tissue differentiation (e.g., CHN2), neuronal migration (e.g., NDE1), myelination (e.g., UGT8, CNP) and oligodendrocyte proliferation (e.g., ENPP2, SEMA4D1). Our data support an association of alcoholism with up-regulation of a cluster of miRNAs located in the genomic imprinted domain on chromosome 14q32 with their predicted gene targets involved with oligodendrocyte growth, differentiation and signaling. Copyright © 2013 Elsevier B.V. All rights reserved.

Protein Structure and the Sequential Structure of mRNA

DEFF Research Database (Denmark)

Brunak, Søren; Engelbrecht, Jacob

1996-01-01

entries in the Brookhaven Protein Data Bank produced 719 protein chains with matching mRNA sequence, amino acid sequence, and secondary structure assignment, By neural network analysis, we found strong signals in mRNA sequence regions surrounding helices and sheets, These signals do not originate from......A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed, We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting...... protein, The degeneracy of the genetic code allows for a biased selection of codons which may control the translational rate of the ribosome, and may thus in vivo have a catalyzing effect on the folding of the polypeptide chain, A complete search for GenBank nucleotide sequences coding for structural...

The differentiation status of primary gonadal germ cell tumors correlates inversely with telomerase activity and the expression level of the gene encoding the catalytic subunit of telomerase

International Nuclear Information System (INIS)

Schrader, Mark; Burger, Angelika M; Müller, Markus; Krause, Hans; Straub, Bernd; Schostak, Martin; Schulze, Wolfgang; Lauke, Heidrun; Miller, Kurt

2002-01-01

The activity of the ribonucleoprotein enzyme telomerase is detectable in germ, stem and tumor cells. One major component of telomerase is human telomerase reverse transcriptase (hTERT), which encodes the catalytic subunit of telomerase. Here we investigate the correlation of telomerase activity and hTERT gene expression and the differentiation status of primary testicular germ cell tumors (TGCT). Telomerase activity (TA) was detected by a quantitative telomerase PCR ELISA, and hTERT mRNA expression was quantified by online RT-PCR in 42 primary testicular germ cell tumors. The control group consisted of benign testicular biopsies from infertile patients. High levels of telomerase activity and hTERT expression were detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content. In contrast, differentiated teratomas and testicular control tissue without germ cells (Sertoli-cell-only syndrome) showed no telomerase activity and only minimal hTERT expression. These findings demonstrate an inverse relationship between the level of telomerase activity and hTERT mRNA expression and the differentiation state of germ cell tumors. Quantification of telomerase activity and hTERT mRNA expression enables a new molecular-diagnostic subclassification of germ cell tumors that describes their proliferation potential and differentiation status

The differentiation status of primary gonadal germ cell tumors correlates inversely with telomerase activity and the expression level of the gene encoding the catalytic subunit of telomerase

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Schulze Wolfgang

2002-11-01

Full Text Available Abstract Background The activity of the ribonucleoprotein enzyme telomerase is detectable in germ, stem and tumor cells. One major component of telomerase is human telomerase reverse transcriptase (hTERT, which encodes the catalytic subunit of telomerase. Here we investigate the correlation of telomerase activity and hTERT gene expression and the differentiation status of primary testicular germ cell tumors (TGCT. Methods Telomerase activity (TA was detected by a quantitative telomerase PCR ELISA, and hTERT mRNA expression was quantified by online RT-PCR in 42 primary testicular germ cell tumors. The control group consisted of benign testicular biopsies from infertile patients. Results High levels of telomerase activity and hTERT expression were detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content. In contrast, differentiated teratomas and testicular control tissue without germ cells (Sertoli-cell-only syndrome showed no telomerase activity and only minimal hTERT expression. Conclusions These findings demonstrate an inverse relationship between the level of telomerase activity and hTERT mRNA expression and the differentiation state of germ cell tumors. Quantification of telomerase activity and hTERT mRNA expression enables a new molecular-diagnostic subclassification of germ cell tumors that describes their proliferation potential and differentiation status.

Kinetics of lipid-nanoparticle-mediated intracellular mRNA delivery and function

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Zhdanov, Vladimir P.

2017-10-01

mRNA delivery into cells forms the basis for one of the new and promising ways to treat various diseases. Among suitable carriers, lipid nanoparticles (LNPs) with a size of about 100 nm are now often employed. Despite high current interest in this area, the understanding of the basic details of LNP-mediated mRNA delivery and function is limited. To clarify the kinetics of mRNA release from LNPs, the author uses three generic models implying (i) exponential, (ii) diffusion-controlled, and (iii) detachment-controlled kinetic regimes, respectively. Despite the distinct differences in these kinetics, the associated transient kinetics of mRNA translation to the corresponding protein and its degradation are shown to be not too sensitive to the details of the mRNA delivery by LNPs (or other nanocarriers). In addition, the author illustrates how this protein may temporarily influence the expression of one gene or a few equivalent genes. The analysis includes positive or negative regulation of the gene transcription via the attachment of the protein without or with positive or negative feedback in the gene expression. Stable, bistable, and oscillatory schemes have been scrutinized in this context.

Effect of conjugated linoleic acids on the activity and mRNA expression of 5- and 15-lipoxygenases in human macrophages.

Science.gov (United States)

Stachowska, Ewa; Dziedziejko, Violetta; Safranow, Krzysztof; Jakubowska, Katarzyna; Olszewska, Maria; Machaliñski, Bogusław; Chlubek, Dariusz

2007-06-27

Lipoxygenases are a family of non-heme enzyme dioxygenases. The role of lipoxygenases is synthesis of hydroperoxides of fatty acids, which perform signaling functions in the body. Studies on conjugated linoleic acids (CLAs) as fatty acids with a potential anti-atherosclerotic function have recently been initiated. The aim of the study was to test the effect of CLAs and linoleic acid on 5- and 15-lipoxygenase (5-LO, 15-LO-1) enzyme activity, their mRNA expression, and concentration in the cells. It was also desired to determine whether the CLAs are substrates for the enzymes. For the experiments monocytic cell line (THP-1) and monocytes obtained from human venous blood were used. Monocytes were differentiated to macrophages: THP-1 (CD14+) by PMA administration (100 nM for 24 h) and monocytes from blood (CD14+) by 7-day cultivation with the autologous serum (10%). After differentiation, macrophages were cultured with 30 microM CLAs or linoleic acid for 48 h. The 15- and 5-lipoxygenase products were measured by HPLC method. mRNA expression and protein content were analyzed by real-time PCR and Western blot analysis. The in vitro studies proved that both CLA isomers are not substrates for 15-LO-1; in ex vivo studies hydroxydecadienoic acid (HODE) concentration was significantly reduced (p = 0.019). The trans-10,cis-12 CLA isomer reduced HODE concentration by 28% (p = 0.046) and the cis-9,trans-11 CLA isomer by 35% (p = 0.028). In macrophages obtained from THP-1 fatty acids did not change significantly mRNA expression of the majority of the investigated genes. CLAs did not change the content of 5-LO and 15-LO-1 proteins in macrophages obtained from peripheral blood. Linoleic acid induced 15-LO-1 expression (2.6 times, p < 0.05). CLAs may perform the function of an inhibitor of lipoxygenase 15-LO-1 activity in macrophages.

Synthesis of novel vitamin K derivatives with alkylated phenyl groups introduced at the ω-terminal side chain and evaluation of their neural differentiation activities.

Science.gov (United States)

Sakane, Rie; Kimura, Kimito; Hirota, Yoshihisa; Ishizawa, Michiyasu; Takagi, Yuta; Wada, Akimori; Kuwahara, Shigefumi; Makishima, Makoto; Suhara, Yoshitomo

2017-11-01

Vitamin K is an essential cofactor of γ-glutamylcarboxylase as related to blood coagulation and bone formation. Menaquinone-4, one of the vitamin K homologues, is biosynthesized in the body and has various biological activities such as being a ligand for steroid and xenobiotic receptors, protection of neuronal cells from oxidative stress, and so on. From this background, we focused on the role of menaquinone in the differentiation activity of progenitor cells into neuronal cells and we synthesized novel vitamin K derivatives with modification of the ω-terminal side chain. We report here new vitamin K analogues, which introduced an alkylated phenyl group at the ω-terminal side chain. These compounds exhibited potent differentiation activity as compared to control. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Regulation of mRNA Translation Is a Novel Mechanism for Phthalate Toxicity.

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Jun Ling

Full Text Available Phthalates are a group of plasticizers that are widely used in many consumer products and medical devices, thus generating a huge burden to human health. Phthalates have been known to cause a number of developmental and reproductive disorders functioning as endocrine modulators. They are also involved in carcinogenesis with mechanisms less understood. To further understand the molecular mechanisms of phthalate toxicity, in this study we reported a new effect of phthalates on mRNA translation/protein synthesis, a key regulatory step of gene expression. Butyl benzyl phthalate (BBP was found to directly inhibit mRNA translation in vitro but showed a complicated pattern of affecting mRNA translation in cells. In human kidney embryonic cell (HEK-293T, BBP increased cap-dependent mRNA translation at lower concentrations but showed inhibitory effect at higher concentrations. Cap-independent translation was not affected. On the other hand, mono (2-ethylhexyl phthalate (MEHP as a major metabolite of another important phthalate di (2-ethylhexyl phthalate (DEHP inhibited both can-dependent and -independent mRNA translation in vivo. In contrast, BBP and MEHP exhibited an overall promoting effect on mRNA translation in cancer cells. Mechanistic studies identified that the level and phosphorylation of eIF4E-BP (eIF4E binding protein and the amount of eIF4GI in eIF4F complex were altered in accordance with the effect of BBP on translation. BBP was also identified to directly bind to eIF4E, providing a further mechanism underlying the regulation of mRNA by phthalate. At the cellular level BBP inhibited normal cell growth but slightly promoted cancer cells (HT29 growth. Overall, this study provides the first evidence that phthalates can directly regulate mRNA translation as a novel mechanism to mediate their biological toxicities.

Sexual phenotype differences in zic2 mRNA abundance in the preoptic area of a protogynous teleost, Thalassoma bifasciatum.

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Katherine McCaffrey

Full Text Available The highly conserved members of the zic family of zinc-finger transcription factors are primarily known for their roles in embryonic signaling pathways and regulation of cellular proliferation and differentiation. This study describes sexual phenotype differences in abundances of zic2 mRNA in the preoptic area of the hypothalamus, a region strongly implicated in sexual behavior and function, in an adult teleost, Thalassoma bifasciatum. The bluehead wrasse (Thalassoma bifasciatum is a valuable model for studying neuroendocrine processes because it displays two discrete male phenotypes, initial phase (IP males and territorial, terminal phase (TP males, and undergoes socially-controlled protogynous sex change. Previously generated microarray-based comparisons suggested that zic2 was upregulated in the brains of terminal phase males relative to initial phase males. To further explore this difference, we cloned a 727 bp sequence for neural zic2 from field-collected animals. Riboprobe-based in situ hybridization was employed to localize zic2 signal in adult bluehead brains and assess the relative abundance of brain zic2 mRNA across sexual phenotypes. We found zic2 mRNA expression was extremely abundant in the granular cells of the cerebellum and widespread in other brain regions including in the thalamus, hypothalamus, habenula, torus semicircularis, torus longitudinalis, medial longitudinal fascicle and telencephalic areas. Quantitative autoradiography and phosphorimaging showed zic2 mRNA hybridization signal in the preoptic area of the hypothalamus was significantly higher in terminal phase males relative to both initial phase males and females, and silver grain analysis confirmed this relationship between phenotypes. No significant difference in abundance was found in zic2 signal across phenotypes in the habenula, a brain region not implicated in the control of sexual behavior, or cerebellum.

The trypanosome transcriptome is remodelled during differentiation but displays limited responsiveness within life stages

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Sergeenko Tatiana

2008-06-01

Full Text Available Abstract Background Trypanosomatids utilise polycistronic transcription for production of the vast majority of protein-coding mRNAs, which operates in the absence of gene-specific promoters. Resolution of nascent transcripts by polyadenylation and trans-splicing, together with specific rates of mRNA turnover, serve to generate steady state transcript levels that can differ in abundance across several orders of magnitude and can be developmentally regulated. We used a targeted oligonucleotide microarray, representing the strongly developmentally-regulated T. brucei membrane trafficking system and ~10% of the Trypanosoma brucei genome, to investigate both between-stage, or differentiation-dependent, transcriptome changes and within-stage flexibility in response to various challenges. Results 6% of the gene cohort are developmentally regulated, including several small GTPases, SNAREs, vesicle coat factors and protein kinases both consistent with and extending previous data. Therefore substantial differentiation-dependent remodeling of the trypanosome transcriptome is associated with membrane transport. Both the microarray and qRT-PCR were then used to analyse transcriptome changes resulting from specific gene over-expression, knockdown, altered culture conditions and chemical stress. Firstly, manipulation of Rab5 expression results in co-ordinate changes to clathrin protein expression levels and endocytotic activity, but no detectable changes to steady-state mRNA levels, which indicates that the effect is mediated post-transcriptionally. Secondly, knockdown of clathrin or the variant surface glycoprotein failed to perturb transcription. Thirdly, exposure to dithiothreitol or tunicamycin revealed no evidence for a classical unfolded protein response, mediated in higher eukaryotes by transcriptional changes. Finally, altered serum levels invoked little transcriptome alteration beyond changes to expression of ESAG6/7, the transferrin receptor

Tissue transglutaminase (TG2 activity regulates osteoblast differentiation and mineralization in the SAOS-2 cell line

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Xiaoxue Yin

2012-08-01

Full Text Available Tissue transglutaminase (type II, TG2 has long been postulated to directly promote skeletal matrix calcification and play an important role in ossification. However, limited information is available on the expression, function and modulating mechanism of TG2 during osteoblast differentiation and mineralization. To address these issues, we cultured the well-established human osteosarcoma cell line SAOS-2 with osteo-inductive conditioned medium and set up three time points (culture days 4, 7, and 14 to represent different stages of SAOS-2 differentiation. Osteoblast markers, mineralization, as well as TG2 expression and activity, were then assayed in each stage. Furthermore, we inhibited TG activity with cystamine and then checked SAOS-2 differentiation and mineralization in each stage. The results showed that during the progression of osteoblast differentiation SAOS-2 cells presented significantly high levels of osteocalcin (OC mRNA, bone morphogenetic protein-2 (BMP-2 and collagen I, significantly high alkaline phosphatase (ALP activity, and the increased formation of calcified matrix. With the same tendency, TG2 expression and activity were up-regulated. Furthermore, inhibition of TG activity resulted in a significant decrease of OC, collagen I, and BMP-2 mRNA and of ALP activity and mineralization. This study demonstrated that TG2 is involved in osteoblast differentiation and may play a role in the initiation and regulation of the mineralization processes. Moreover, the modulating effects of TG2 on osteoblasts may be related to BMP-2.

Differentially-Expressed Pseudogenes in HIV-1 Infection

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Aditi Gupta

2015-09-01

Full Text Available Not all pseudogenes are transcriptionally silent as previously thought. Pseudogene transcripts, although not translated, contribute to the non-coding RNA pool of the cell that regulates the expression of other genes. Pseudogene transcripts can also directly compete with the parent gene transcripts for mRNA stability and other cell factors, modulating their expression levels. Tissue-specific and cancer-specific differential expression of these “functional” pseudogenes has been reported. To ascertain potential pseudogene:gene interactions in HIV-1 infection, we analyzed transcriptomes from infected and uninfected T-cells and found that 21 pseudogenes are differentially expressed in HIV-1 infection. This is interesting because parent genes of one-third of these differentially-expressed pseudogenes are implicated in HIV-1 life cycle, and parent genes of half of these pseudogenes are involved in different viral infections. Our bioinformatics analysis identifies candidate pseudogene:gene interactions that may be of significance in HIV-1 infection. Experimental validation of these interactions would establish that retroviruses exploit this newly-discovered layer of host gene expression regulation for their own benefit.

Differentially-Expressed Pseudogenes in HIV-1 Infection.

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Gupta, Aditi; Brown, C Titus; Zheng, Yong-Hui; Adami, Christoph

2015-09-29

Not all pseudogenes are transcriptionally silent as previously thought. Pseudogene transcripts, although not translated, contribute to the non-coding RNA pool of the cell that regulates the expression of other genes. Pseudogene transcripts can also directly compete with the parent gene transcripts for mRNA stability and other cell factors, modulating their expression levels. Tissue-specific and cancer-specific differential expression of these "functional" pseudogenes has been reported. To ascertain potential pseudogene:gene interactions in HIV-1 infection, we analyzed transcriptomes from infected and uninfected T-cells and found that 21 pseudogenes are differentially expressed in HIV-1 infection. This is interesting because parent genes of one-third of these differentially-expressed pseudogenes are implicated in HIV-1 life cycle, and parent genes of half of these pseudogenes are involved in different viral infections. Our bioinformatics analysis identifies candidate pseudogene:gene interactions that may be of significance in HIV-1 infection. Experimental validation of these interactions would establish that retroviruses exploit this newly-discovered layer of host gene expression regulation for their own benefit.

Theobromine inhibits differentiation of 3T3-L1 cells during the early stage of adipogenesis via AMPK and MAPK signaling pathways.

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Jang, Yeon Jeong; Koo, Hyun Jung; Sohn, Eun-Hwa; Kang, Se Chan; Rhee, Dong-Kwon; Pyo, Suhkneung

2015-07-01

Obesity is characterized by hypertrophy and/or by the differentiation or adipogenesis of pre-existing adipocytes. In this study, we investigated the inhibitory effects of theobromine, a type of alkaloid in cocoa, on adipocyte differentiation of 3T3-L1 preadipocytes and its mechanisms of action. Theobromine inhibited the accumulation of lipid droplets, the expression of PPARγ and C/EBPα, and the mRNA expression of aP2 and leptin. The inhibition of adipogenic differentiation by theobromine occurred primarily in the early stages of differentiation. In addition, theobromine arrested the cell cycle at the G0/G1 phase and regulated the expressions of CDK2, p27, and p21. Theobromine treatment increased AMPK phosphorylation and knockdown of AMPKα1/α2 prevented the ability of theobromine to inhibit PPARγ expression in the differentiating 3T3-L1 cells. Theobromine reduced the phosphorylation of ERK and JNK. Moreover, the secretion and the mRNA level of TNF-α and IL-6 were inhibited by theobromine treatment. These data suggest that theobromine inhibits adipocyte differentiation during the early stages of adipogenesis by regulating the expression of PPARγ and C/EBPα through the AMPK and ERK/JNK signaling pathways in 3T3-L1 preadipocytes.

Role of Alternative Polyadenylation during Adipogenic Differentiation: An In Silico Approach

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Spangenberg, Lucía; Correa, Alejandro; Dallagiovanna, Bruno; Naya, Hugo

2013-01-01

Post-transcriptional regulation of stem cell differentiation is far from being completely understood. Changes in protein levels are not fully correlated with corresponding changes in mRNAs; the observed differences might be partially explained by post-transcriptional regulation mechanisms, such as alternative polyadenylation. This would involve changes in protein binding, transcript usage, miRNAs and other non-coding RNAs. In the present work we analyzed the distribution of alternative transcripts during adipogenic differentiation and the potential role of miRNAs in post-transcriptional regulation. Our in silico analysis suggests a modest, consistent, bias in 3′UTR lengths during differentiation enabling a fine-tuned transcript regulation via small non-coding RNAs. Including these effects in the analyses partially accounts for the observed discrepancies in relative abundance of protein and mRNA. PMID:24143171

Simultaneous isolation of mRNA and native protein from minute samples of cells

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Petersen, Tonny Studsgaard; Andersen, Claus Yding

2014-01-01

Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native...... in their native state for traditional protein assays. We validated the procedure using neonatal rat ovaries and small numbers of human granulosa cells, demonstrating the extraction of mRNA suitable for gene expression analysis with simultaneous isolation of native proteins suitable for downstream characterization...... proteins from samples containing small numbers of cells. Our approach utilizes oligodeoxythymidylate [oligo(dT)25]-coated paramagnetic beads in an optimized reaction buffer to isolate mRNA comparable in quantity and quality to mRNA isolated with existing methods, while maintaining the proteins...

Characterisation of the Whole Blood mRNA Transcriptome in Holstein-Friesian and Jersey Calves in Response to Gradual Weaning.

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D Johnston

Full Text Available Weaning of dairy calves is an early life husbandry management practice which involves the changeover from a liquid to a solid feed based diet. The objectives of the study were to use RNA-seq technology to examine the effect of (i breed and (ii gradual weaning, on the whole blood mRNA transcriptome of artificially reared Holstein-Friesian and Jersey calves. The calves were gradually weaned over 14 days (day (d -13 to d 0 and mRNA transcription was examined one day before gradual weaning was initiated (d -14, one day after weaning (d 1, and 8 days after weaning (d 8. On d -14, 550 genes were differentially expressed between Holstein-Friesian and Jersey calves, while there were 490 differentially expressed genes (DEG identified on d 1, and 411 DEG detected eight days after weaning (P 0.05. The pathways, gene ontology terms, and biological functions consistently over-represented among the DEG between Holstein-Friesian and Jersey were associated with the immune response and immune cell signalling, specifically chemotaxis. Decreased transcription of several cytokines, chemokines, immunoglobulin-like genes, phagocytosis-promoting receptors and g-protein coupled receptors suggests decreased monocyte, natural killer cell, and T lymphocyte, chemotaxis and activation in Jersey compared to Holstein-Friesian calves. Knowledge of breed-specific immune responses could facilitate health management practices better tailored towards specific disease sensitivities of Holstein-Friesian and Jersey calves. Gradual weaning did not compromise the welfare of artificially-reared dairy calves, evidenced by the lack of alterations in the expression of any genes in response to gradual weaning.

Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription

International Nuclear Information System (INIS)

Dreyfuss, G.; Adam, S.A.; Choi, Y.D.

1984-01-01

Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared

Downregulation of adenomatous polyposis coli by microRNA-663 promotes odontogenic differentiation through activation of Wnt/beta-catenin signaling

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Kim, Jae-Sung; Park, Min-Gyeong; Lee, Seul Ah; Park, Sun-Young; Kim, Heung-Joong; Yu, Sun-Kyoung; Kim, Chun Sung; Kim, Su-Gwan; Oh, Ji-Su; You, Jae-Seek; Kim, Jin-Soo; Seo, Yo-Seob [Oral Biology Research Institute, School of Dentistry, Chosun University, Gwangju 501-759 (Korea, Republic of); Chun, Hong Sung [Department of Biomedical Science, Chosun University, Gwangju 501-759 (Korea, Republic of); Park, Joo-Cheol [Department of Oral Histology-Developmental Biology, School of Dentistry and Dental Research Institute, BK 21, Seoul National University, Seoul 110-749 (Korea, Republic of); Kim, Do Kyung, E-mail: kdk@chosun.ac.kr [Oral Biology Research Institute, School of Dentistry, Chosun University, Gwangju 501-759 (Korea, Republic of)

2014-04-18

Highlights: • miR-663 is significantly up-regulated during MDPC-23 odontoblastic cell differentiation. • miR-663 accelerates mineralization in MDPC-23 odontoblastic cells without cell proliferation. • miR-663 promotes odontoblastic cell differentiation by targeting APC and activating Wnt/β-catenin signaling in MDPC-23 cells. - Abstract: MicroRNAs (miRNAs) regulate cell differentiation by inhibiting mRNA translation or by inducing its degradation. However, the role of miRNAs in odontogenic differentiation is largely unknown. In this present study, we observed that the expression of miR-663 increased significantly during differentiation of MDPC-23 cells to odontoblasts. Furthermore, up-regulation of miR-663 expression promoted odontogenic differentiation and accelerated mineralization without proliferation in MDPC-23 cells. In addition, target gene prediction for miR-663 revealed that the mRNA of the adenomatous polyposis coli (APC) gene, which is associated with the Wnt/β-catenin signaling pathway, has a miR-663 binding site in its 3′-untranslated region (3′UTR). Furthermore, APC expressional was suppressed significantly by miR-663, and this down-regulation of APC expression triggered activation of Wnt/β-catenin signaling through accumulation of β-catenin in the nucleus. Taken together, these findings suggest that miR-663 promotes differentiation of MDPC-23 cells to odontoblasts by targeting APC-mediated activation of Wnt/β-catenin signaling. Therefore, miR-663 can be considered a critical regulator of odontoblast differentiation and can be utilized for developing miRNA-based therapeutic agents.

Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

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Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M.

1990-01-01

The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures

A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

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Toki, Yasumichi; Sasaki, Katsunori; Tanaka, Hiroki; Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro; Torimoto, Yoshihiro; Ohtake, Takaaki; Kohgo, Yutaka

2016-01-01

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.

A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

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Toki, Yasumichi [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp [Department of Gastrointestinal Immunology and Regenerative Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Tanaka, Hiroki [Department of Legal Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Torimoto, Yoshihiro [Oncology Center, Asahikawa Medical University Hospital, Hokkaido 078-8510 (Japan); Ohtake, Takaaki; Kohgo, Yutaka [Department of Gastroenterology, International University of Health and Welfare Hospital, Tochigi 329-2763 (Japan)

2016-08-05

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.

Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells

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Yamauchi Mika

2007-11-01

Full Text Available Abstract Background Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. Results Adiponectin receptor type 1 (AdipoR1 mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR, in the cells. AdipoR1 small interfering RNA (siRNA transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01–0.5 mM in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01–1.0 μg/ml also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively. Conclusion Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions.

Creatine kinase and alpha-actin mRNA levels decrease in diabetic rat hearts

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Popovich, B.; Barrieux, A.; Dillmann, W.H.

1987-01-01

Diabetic cardiomyopathy is associated with cardiac atrophy and isoenzyme redistribution. To determine if tissue specific changes occur in mRNAs coding for α-actin and creatine kinase (CK), they performed RNA blot analysis. Total ventricular RNA from control (C) and 4 wk old diabetic (D) rats were hybridized with 32 P cDNA probes for α-actin and CK. A tissue independent cDNA probe, CHOA was also used. Signal intensity was quantified by photodensitometry. D CK mRNA was 47 +/- 16% lower in D vs C. Insulin increases CK mRNA by 20% at 1.5 hs, and completely reverses the deficit after 4 wks. D α-actin mRNA is 66 +/- 18% lower in D vs C. Insulin normalized α-actin mRNA by 5 hs. CHOA mRNA is unchanged in D vs C, but D + insulin CHOA mRNA is 30 +/- 2% lower than C. In rats with diabetic cardiomyopathy, muscle specific CK and α-actin mRNAs are decreased. Insulin treatment reverses these changes

Programmed mitophagy is essential for the glycolytic switch during cell differentiation.

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Esteban-Martínez, Lorena; Sierra-Filardi, Elena; McGreal, Rebecca S; Salazar-Roa, María; Mariño, Guillermo; Seco, Esther; Durand, Sylvère; Enot, David; Graña, Osvaldo; Malumbres, Marcos; Cvekl, Ales; Cuervo, Ana María; Kroemer, Guido; Boya, Patricia

2017-06-14

Retinal ganglion cells (RGCs) are the sole projecting neurons of the retina and their axons form the optic nerve. Here, we show that embryogenesis-associated mouse RGC differentiation depends on mitophagy, the programmed autophagic clearance of mitochondria. The elimination of mitochondria during RGC differentiation was coupled to a metabolic shift with increased lactate production and elevated expression of glycolytic enzymes at the mRNA level. Pharmacological and genetic inhibition of either mitophagy or glycolysis consistently inhibited RGC differentiation. Local hypoxia triggered expression of the mitophagy regulator BCL2/adenovirus E1B 19-kDa-interacting protein 3-like (BNIP3L, best known as NIX) at peak RGC differentiation. Retinas from NIX-deficient mice displayed increased mitochondrial mass, reduced expression of glycolytic enzymes and decreased neuronal differentiation. Similarly, we provide evidence that NIX-dependent mitophagy contributes to mitochondrial elimination during macrophage polarization towards the proinflammatory and more glycolytic M1 phenotype, but not to M2 macrophage differentiation, which primarily relies on oxidative phosphorylation. In summary, developmentally controlled mitophagy promotes a metabolic switch towards glycolysis, which in turn contributes to cellular differentiation in several distinct developmental contexts. © 2017 The Authors.

Lactoferrin promote primary rat osteoblast proliferation and differentiation via up-regulation of insulin-like growth factor-1 expression.

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Hou, Jian-ming; Wu, Man; Lin, Qing-ming; Lin, Fan; Xue, Ying; Lan, Xu-hua; Chen, En-yu; Wang, Mei-li; Yang, Hai-yan; Wang, Feng-xiong

2014-08-01

The aim of this study was to explore the effect of lactoferrin (LF) in primary fetal rat osteoblasts proliferation and differentiation and investigate the underlying molecular mechanisms. Primary rat osteoblasts were obtained from the calvarias of neonatal rats. Osteoblasts were treated with LF (0.1-1000 μg/mL), or OSI-906 [a selective inhibitor of insulin-like growth factor 1 (IGF-1) receptor and insulin receptor]. The IGF-1 was then knocked down by small hairpin RNA (shRNA) technology and then was treated with recombinant human IGF-1 or LF. Cell proliferation and differentiation were measured by MTT assay and alkaline phosphatase (ALP) assay, respectively. The expression of IGF-1 and IGF binding protein 2 (IGFBP2) mRNA were analyzed using real-time PCR. LF promotes the proliferation and differentiation of osteoblasts in a certain range (1-100 μg/mL) in time- and dose-dependent manner. The mRNA level of IGF-1 was significantly increased, while the expression of IGFBP2 was suppressed by LF treatment. Knockdown of IGF-1 by shRNA in primary rat osteoblast dramatically decreased the abilities of proliferation and differentiation of osteoblasts and blocked the proliferation and differentiation effect of LF in osteoblasts. OSI906 (5 μM) blocked the mitogenic and differentiation of LF in osteoblasts. Proliferation and differentiation of primary rat osteoblasts in response to LF are mediated in part by stimulating of IGF-1 gene expression and alterations in the gene expression of IGFBP2.

The urokinase receptor and its structural homologue C4.4A in human cancer

DEFF Research Database (Denmark)

Jacobsen, B; Ploug, M

2008-01-01

The urokinase-type plasminogen activator receptor (uPAR) and its structural homologue C4.4A are multidomain members of the Ly6/uPAR/alpha-neurotoxin protein domain family. Both are glycosylphosphatidylinositol-anchored membrane glycoproteins encoded by neighbouring genes located on chromosome 19q13...... that high protein expression in tumour cells of non-small cell pulmonary adenocarcinomas is associated with a particularly severe disease progression. This review will evaluate structural-functional and disease-related aspects of uPAR and C4.4A with a view to possible pharmacological targeting strategies...... in the human genome. The structural relationship between the two proteins is, however, not reflected at the functional level. Whereas uPAR has a well-established role in regulating and focalizing uPA-mediated plasminogen activation to the surface of those cells expressing the receptor, the biological function...

Neurocognitive and neuroinflammatory correlates of PDYN and OPRK1 mRNA expression in the anterior cingulate in postmortem brain of HIV-infected subjects.

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Yuferov, Vadim; Butelman, Eduardo R; Ho, Ann; Morgello, Susan; Kreek, Mary Jeanne

2014-01-09

Chronic inflammation may contribute to neuropsychological impairments in individuals with HIV, and modulation of this inflammatory response by opiate receptor ligands is important in light of the prevalence of drug use in HIV populations. Exogenous MOR and KOR agonists have differential effects on central nervous system (CNS) immunity and, while some data suggest KOR agonists are immunosuppressive, the KOR agonist dynorphin has been shown to stimulate human monocyte chemotaxis. In this study, we examined mRNA levels of endogenous opioid receptors OPRK1 and OPRM1, prodynorphin (PDYN), macrophage scavenger receptor CD163, and microglia/macrophage marker CD68 in the caudate and anterior cingulate of postmortem brains from HIV-positive and HIV-negative subjects. Brain tissues of HIV-infected (n = 24) and control subjects (n = 15) were obtained from the Manhattan HIV Brain Bank. Quantification of the gene mRNA was performed using SYBR Green RT-PCR. CD68 and CD163 were increased in HIV-positive (HIV+) compared to HIV-negative (HIV-) individuals in both brain regions. There were higher OPRK1 (P <0.005), and lower PDYN mRNA (P <0.005) levels in the anterior cingulate of HIV+ compared to HIV- subjects. This difference between the clinical groups was not found in the caudate. There was no difference in the levels of OPRM1 mRNA between HIV+ and HIV- subjects. Using linear regression analysis, we examined the relationship of OPRK1 and PDYN mRNA levels in the HIV+ subjects with seven cognitive domain T scores of a neuropsychological test battery. Within the HIV+ subjects, there was a positive correlation between anterior cingulate PDYN mRNA levels and better T-scores in the motor domain. Within the HIV+ subjects there were also positive correlations of both OPRK1 and PDYN mRNA levels with the anti-inflammatory marker CD163, but not with proinflammatory CD68 levels. In this setting, decreased PDYN mRNA may reflect a homeostatic mechanism to reduce monocyte

Intestinal cellular localization of PCNA protein and CYP1A mRNA in Atlantic salmon Salmo salar L. exposed to a model toxicant

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Olsvik Pål A

2009-03-01

Full Text Available Abstract Background The aim of the study was to examine the intestinal cellular localization of proliferating cell nuclear antigen (PCNA and cytochrome P450 A1 (CYP1A expression in Atlantic salmon Salmo salar L. exposed to a model toxicant. The stress response was induced by intraperitoneal injection of four salmon with a single dose (50 mg/kg of the CYP1A inducer β-naphthoflavone (BNF and intestinal tissue (mid and distal intestine; MI and DI was sampled seven days later. Samples for histology and gene transcription analysis were collected from four exposed fish and four control fish. PCNA was assessed by immunohistochemistry, CYP1A mRNA was studied by in situ hybridization (ISH and finally the transcription of five genes was quantified by real-time quantitative RT-PCR (real-time RT-PCR; two detoxifying genes (CYP1A and glutathione S-transferase; GST, a stress marker gene (heat shock protein 70; HSP70, PCNA and a gene marker of apoptosis (caspase 6A. Results PCNA protein and CYP1A mRNA were successfully localized in the intestinal cells (MI of both experimental groups. At the cellular level, BNF significantly lowered intestinal cell proliferation and increased the CYP1A mRNA levels compared to the control group. The real-time RT-PCR data, which showed an increased mRNA expression both in the MI and DI of 139- and 62-fold, respectively, confirmed the increased cellular CYP1A mRNA levels detected using ISH. HSP70 expression was also up-regulated in the exposed fish. The other examined genes did not show any differential regulation in the experimental fish group. Conclusion This study showed that CYP1A mRNA had a specific intestinal cellular transcription pattern in Atlantic salmon exposed to BNF. At the cellular level CYP1A mRNA expression was always observed at or around the cell nucleus close to the basolateral cell membrane and at the tissue level CYP1A mRNA expression was most frequently observed in the basal and apex area of the intestinal

The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells.

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Suriguga; Li, Xiao-Fei; Li, Yang; Yu, Chun-Hong; Li, Yi-Ran; Yi, Zong-Chun

2013-12-15

Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation. © 2013.

Detection of melatonin receptor mRNA in human muscle

International Nuclear Information System (INIS)

Li Lei

2004-01-01

To verify the expression of melatonin receptor mRNA in human, muscle, muscle beside vertebrae was collected to obtain total RNA and the mRNA of melatonin receptor was detected by RT-PCR method. The electrophoretic results of RT-PCR products by mt 1 and MT 2 primer were all positive and the sequence is corresponding with human melatonin receptor cDNA. It suggests that melatonin may act on the muscle beside vertebrae directly and regulate its growth and development. (authors)

Identification and characterisation of the angiotensin converting enzyme-3 (ACE3 gene: a novel mammalian homologue of ACE

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Phelan Anne

2007-06-01

Full Text Available Abstract Background Mammalian angiotensin converting enzyme (ACE plays a key role in blood pressure regulation. Although multiple ACE-like proteins exist in non-mammalian organisms, to date only one other ACE homologue, ACE2, has been identified in mammals. Results Here we report the identification and characterisation of the gene encoding a third homologue of ACE, termed ACE3, in several mammalian genomes. The ACE3 gene is located on the same chromosome downstream of the ACE gene. Multiple sequence alignment and molecular modelling have been employed to characterise the predicted ACE3 protein. In mouse, rat, cow and dog, the predicted protein has mutations in some of the critical residues involved in catalysis, including the catalytic Glu in the HEXXH zinc binding motif which is Gln, and ESTs or reverse-transcription PCR indicate that the gene is expressed. In humans, the predicted ACE3 protein has an intact HEXXH motif, but there are other deletions and insertions in the gene and no ESTs have been identified. Conclusion In the genomes of several mammalian species there is a gene that encodes a novel, single domain ACE-like protein, ACE3. In mouse, rat, cow and dog ACE3, the catalytic Glu is replaced by Gln in the putative zinc binding motif, indicating that in these species ACE3 would lack catalytic activity as a zinc metalloprotease. In humans, no evidence was found that the ACE3 gene is expressed and the presence of deletions and insertions in the sequence indicate that ACE3 is a pseudogene.

Rifampin modulation of xeno- and endobiotic conjugating enzyme mRNA expression and associated microRNAs in human hepatocytes.

Science.gov (United States)

Gufford, Brandon T; Robarge, Jason D; Eadon, Michael T; Gao, Hongyu; Lin, Hai; Liu, Yunlong; Desta, Zeruesenay; Skaar, Todd C

2018-04-01

Rifampin is a pleiotropic inducer of multiple drug metabolizing enzymes and transporters. This work utilized a global approach to evaluate rifampin effects on conjugating enzyme gene expression with relevance to human xeno- and endo-biotic metabolism. Primary human hepatocytes from 7 subjects were treated with rifampin (10 μmol/L, 24 hours). Standard methods for RNA-seq library construction, EZBead preparation, and NextGen sequencing were used to measure UDP-glucuronosyl transferase UGT, sulfonyltransferase SULT, N acetyltransferase NAT, and glutathione-S-transferase GST mRNA expression compared to vehicle control (0.01% MeOH). Rifampin-induced (>1.25-fold) mRNA expression of 13 clinically important phase II drug metabolizing genes and repressed (>1.25-fold) the expression of 3 genes ( P  accounting for simultaneous induction of both CYP3A4 and UGT1A4 predicted a ~10-fold decrease in parent midazolam exposure with only a ~2-fold decrease in midazolam N-glucuronide metabolite exposure. These data reveal differential effects of rifampin on the human conjugating enzyme transcriptome and potential associations with miRNAs that form the basis for future mechanistic studies to elucidate the interplay of conjugating enzyme regulatory elements.

Embryonal carcinoma cell induction of miRNA and mRNA changes in co-cultured prostate stromal fibromuscular cells

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VÊNCIO, ENEIDA F.; PASCAL, LAURA E.; PAGE, LAURA S.; DENYER, GARETH; WANG, AMY J.; RUOHOLA-BAKER, HANNELE; ZHANG, SHILE; WANG, KAI; GALAS, DAVID J.; LIU, ALVIN Y.

2014-01-01

The prostate stromal mesenchyme controls organ-specific development. In cancer, the stromal compartment shows altered gene expression compared to non-cancer. The lineage relationship between cancer-associated stromal cells and normal tissue stromal cells is not known. Nor is the cause underlying the expression difference. Previously, the embryonal carcinoma (EC) cell line, NCCIT, was used by us to study the stromal induction property. In the current study, stromal cells from non-cancer (NP) and cancer (CP) were isolated from tissue specimens and co-cultured with NCCIT cells in a trans-well format to preclude heterotypic cell contact. After 3 days, the stromal cells were analyzed by gene arrays for microRNA (miRNA) and mRNA expression. In co-culture, NCCIT cells were found to alter the miRNA and mRNA expression of NP stromal cells to one like that of CP stromal cells. In contrast, NCCIT had no significant effect on the gene expression of CP stromal cells. We conclude that the gene expression changes in stromal cells can be induced by diffusible factors synthesized by EC cells, and suggest that cancer-associated stromal cells represent a more primitive or less differentiated stromal cell type. PMID:20945389

Phospholipase C δ-type consists of three isozymes: bovine PLCδ2 is a homologue of human/mouse PLCδ4

International Nuclear Information System (INIS)

Irino, Yasuhiro; Cho, Hiroyuki; Nakamura, Yoshikazu; Nakahara, Masamichi; Furutani, Masahiro; Suh, Pann-Ghill; Takenawa, Tadaomi; Fukami, Kiyoko

2004-01-01

To date, 12 phospholipase C (PLC) isozymes have been identified in mammals, and they are divided into five classes, β-, γ-, δ-, ε-, and ζ-type. PLCδ-type is reported to be composed of four isozymes, PLCδ1-δ4. Here we report that a screening for mouse PLCδ2 from a BAC library with primers that amplify a specific region of bovine PLCδ2 resulted in isolation of one clone containing the mouse PLCδ4 gene. Furthermore, a database search revealed that there is only one gene corresponding to PLCδ2 and PLCδ4 in the mouse and human genomes, indicating that bovine PLCδ2 is a homologue of human and mouse PLCδ4. However, PLCδ2 Western blot analysis with a widely used commercial anti-PLCδ2 antibody showed an expression pattern distinct from that of PLCδ4 in wild-type mice. In addition, an 80-kDa band, which was recognized by antibody against PLCδ2, was smaller than an 85-kDa band detected by anti-PLCδ4 antibody, and the 80-kDa band was detectable in lysates of brain, testis, and spleen from PLCδ4-deficient mice. We also found that immunoprecipitates from brain lysates with this PLCδ2 antibody contained no PLC activity. From these data, we conclude that bovine PLCδ2 is a homologue of human and mouse PLCδ4, and that three isozymes (δ1, δ3, and δ4) exist in the PLCδ family

Inhibition of odontogenic differentiation of human dental pulp cells by dental resin monomers.

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Kwon, Ji Hyun; Park, Hee Chul; Zhu, Tingting; Yang, Hyeong-Cheol

2015-01-01

Dental resin monomers that are leached from the resin matrix due to incomplete polymerization can affect the viability and various functions of oral tissues and cells. In this study, the effects of triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) on odontogenic differentiation of human dental pulp cells (HDPCs) were examined. To mimic clinical situations, dental pulp cells were treated with resin monomers for 24 h prior to the analysis of alkaline phosphatase (ALP) activity and mRNA expression of genes related to pulp cell differentiation. To elucidate the underlying signaling pathways, regulation of mitogen-activated protein (MAP) kinases by resin monomers was also investigated. The ALP activity of HDPCs was reduced by TEGDMA and HEMA at noncytotoxic concentrations. The mRNA expression of dentin sialophosphoprotein (DSPP), osteocalcin (OCN), and osteopontin (OPN) was also downregulated by resin monomers. However, DSPP expression was not affected by hydrogen peroxide (H2O2). Among the MAP kinases examined, ERK activation (ERK phosphorylation) was not affected by either resin monomers or H2O2, whereas JNK was phosphorylated by TEGDMA and HEMA. Phospho-p38 was upregulated by HEMA, while TEGDMA and H2O2 suppressed p38 phosphorylation. Exposure to TEGDMA and HEMA for a limited period suppresses differentiation of HDPCs via different signaling pathways.

Homologue Structure of the SLAC1 Anion Channel for Closing Stomata in Leaves

Energy Technology Data Exchange (ETDEWEB)

Y Chen; L Hu; M Punta; R Bruni; B Hillerich; B Kloss; B Rost; J Love; S Siegelbaum; W Hendrickson

2011-12-31

The plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating the exchange of water vapour and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. Here we determine the crystal structure of a bacterial homologue (Haemophilus influenzae) of SLAC1 at 1.20 {angstrom} resolution, and use structure-inspired mutagenesis to analyse the conductance properties of SLAC1 channels. SLAC1 is a symmetrical trimer composed from quasi-symmetrical subunits, each having ten transmembrane helices arranged from helical hairpin pairs to form a central five-helix transmembrane pore that is gated by an extremely conserved phenylalanine residue. Conformational features indicate a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics indicate that selectivity among different anions is largely a function of the energetic cost of ion dehydration.

Recombinant myostatin reduces highly expressed microRNAs in differentiating C2C12 cells

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Zachary A. Graham

2017-03-01

Full Text Available Myostatin is small glycopeptide that is produced and secreted by skeletal muscle. It is a potent negative regulator of muscle growth that has been associated with conditions of frailty. In C2C12 cells, myostatin limits cell differentiation. Myostatin acts through activin receptor IIB, activin receptor-like kinase (ALK and Smad transcription factors. microRNAs (miRNA are short, 22 base pair nucleotides that bind to the 3′ UTR of target mRNA to repress translation or reduce mRNA stability. In the present study, expression in differentiating C2C12 cells of the myomiRs miR-1 and 133a were down-regulated following treatment with 1 µg of recombinant myostatin at 1 d post-induction of differentiation while all myomiRs (miR-1, 133a/b and 206 were upregulated by SB431542, a potent ALK4/5/7 inhibitor which reduces Smad2 signaling, at 1 d and all, with the exception of miR-206, were upregulated by SB431542 at 3 d. The expression of the muscle-enriched miR-486 was greater following treatment with SB431542 but not altered by myostatin. Other highly expressed miRNAs in skeletal muscle, miR-23a/b and 145, were altered only at 1 d post-induction of differentiation. miR-27b responded differently to treatments at 1 d, where it was upregulated, as compared to 3 d, where it was downregulated. Neither myostatin nor SB431542 altered cell size or cell morphology. The data indicate that myostatin represses myomiR expression in differentiating C2C12 cells and that inhibition of Smad signaling with SB431542 can result in large changes in highly expressed miRNAs in differentiating myoblasts.

Three-Dimensional Mapping of mRNA Export through the Nuclear Pore Complex

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Steven J. Schnell

2014-11-01

Full Text Available The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE. Plenty of nuclear pore complexes (NPCs embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms.

Evidence for a Complex Class of Nonadenylated mRNA in Drosophila

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Zimmerman, J. Lynn; Fouts, David L.; Manning, Jerry E.

1980-01-01

The amount, by mass, of poly(A+) mRNA present in the polyribosomes of third-instar larvae of Drosophila melanogaster, and the relative contribution of the poly(A+) mRNA to the sequence complexity of total polysomal RNA, has been determined. Selective removal of poly(A+) mRNA from total polysomal RNA by use of either oligo-dT-cellulose, or poly(U)-sepharose affinity chromatography, revealed that only 0.15% of the mass of the polysomal RNA was present as poly(A+) mRNA. The present study shows that this RNA hybridized at saturation with 3.3% of the single-copy DNA in the Drosophila genome. After correction for asymmetric transcription and reactability of the DNA, 7.4% of the single-copy DNA in the Drosophila genome is represented in larval poly(A+) mRNA. This corresponds to 6.73 x 106 nucleotides of mRNA coding sequences, or approximately 5,384 diverse RNA sequences of average size 1,250 nucleotides. However, total polysomal RNA hybridizes at saturation to 10.9% of the single-copy DNA sequences. After correcting this value for asymmetric transcription and tracer DNA reactability, 24% of the single-copy DNA in Drosophila is represented in total polysomal RNA. This corresponds to 2.18 x 107 nucleotides of RNA coding sequences or 17,440 diverse RNA molecules of size 1,250 nucleotides. This value is 3.2 times greater than that observed for poly(A+) mRNA, and indicates that ≃69% of the polysomal RNA sequence complexity is contributed by nonadenylated RNA. Furthermore, if the number of different structural genes represented in total polysomal RNA is ≃1.7 x 104, then the number of genes expressed in third-instar larvae exceeds the number of chromomeres in Drosophila by about a factor of three. This numerology indicates that the number of chromomeres observed in polytene chromosomes does not reflect the number of structural gene sequences in the Drosophila genome. PMID:6777246

Unique variability of tocopherol composition in various seed oils recovered from by-products of apple industry: rapid and simple determination of all four homologues (α, β, γ and δ) by RP-HPLC/FLD.

Science.gov (United States)

Górnaś, Paweł

2015-04-01

The tocochromanol profile was studied in seed oils recovered from by-products of fruit industry, five dessert and seven crab apple varieties grown in Eastern Europe (Latvia). The seed oils obtained from dessert apples were characterized by higher contents of tocopherols (191.05-379.08 mg/100g oil) when compared to seed oils recovered from crab apples (130.55-202.54 mg/100g oil). The predominant homologues of tocopherol in all the studied samples were α and β over γ and δ. However, seed oils recovered from the apple cultivars 'Antej' and 'Beforest' had a unique profile of four tocopherol homologues (α:β:γ:δ) 91.41:80.55:72.46:79.03 and 114.55:112.84:78.69:73.00 mg/100g oil, respectively. A single dilution of seed oils in 2-propanol facilitated the direct use samples in the DPPH assay as well as injection into the RP-HPLC system containing a PFP (pentafluorophenyl) column, which resulted in a rapid separation of all four tocopherol homologues with excellent repeatability and reproducibility. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Systematic Analysis on mRNA and MicroRNA Expression in Runting and Stunting Chickens

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Xu, Haiping; Xu, Zhenqiang; Ma, Jinge; Li, Bixiao; Lin, Shudai; Nie, Qinghua; Luo, Qingbin; Zhang, Xiquan

2015-01-01

Runting and stunting syndrome (RSS), which is characterized by lower body weight, widely occurs in broilers. Some RSS chickens simply exhibit slow growth without pathological changes. An increasing number of studies indicate that broiler strains differ in susceptibility to infectious diseases, most likely due to their genetic differences. The objective of this study was to detect the differentially expressed miRNAs and mRNAs in RSS and normal chickens. By integrating miRNA with mRNA expression profiling, potential molecular mechanisms involved in RSS could be further explored. Twenty-two known miRNAs and 1,159 genes were differentially expressed in RSS chickens compared with normal chickens (P chicken liver albeit with reduced abundance. Dual-luciferase reporter assay indicated that gga-miR-30b/c directly target CARS through binding to its 3′UTR. The miR-30b/c: CARS regulation mainly occurred in liver. In thigh muscle and the hypothalamus, miR-30b/c are expressed at higher levels in RSS chickens compared with normal chickens from 2 to 6 w of age, and notably significant differences are observed at 4 w of age. PMID:26010155

A glimpse at mRNA dynamics reveals cellular domains and rapid trafficking through granules

NARCIS (Netherlands)

Gemert, Alice Myriam Christi van

2011-01-01

mRNA transport and targeting are essential to gene expression regulation. Specific mRNA sequences can bind several proteins and together form RiboNucleoProtein particles (RNP). The various proteins within the RNP determine mRNA fate: translation, transport or decay. RNP composition varies with

NLS-RARα promotes proliferation and inhibits differentiation in HL-60 cells.

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Hu, Xiu-Xiu; Zhong, Liang; Zhang, Xi; Gao, Yuan-Mei; Liu, Bei-Zhong

2014-01-01

A unique mRNA produced in leukemic cells from a t(15;17) acute promyelocytic leukemia (APL) patient encodes a fusion protein between the retinoic acid receptor α (RARα) and a myeloid gene product called PML. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RARα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. The resultant NLS-RARα fusion protein mainly localizes to, and functions within, the cell nucleus. It is speculated that NLS-RARα may act in different ways from the wild-type RARα, but its biological characteristics have not been reported. This study takes two approaches. Firstly, the NLS-RARα was silenced with pNLS-RARα-shRNA. The mRNA and protein expression of NLS-RARα were detected by RT-PCR and Western blot respectively. Cell proliferation in vitro was assessed by MTT assay. Flow cytometry (FCM) was used to detect the differentiation of cells. Secondly, the NLS-RARα was over-expressed by preparation of recombinant adenovirus HL-60/pAd-NLS-RARα. The assays of mRNA and protein expression of NLS-RARα, and cell proliferation, were as above. By contrast, cell differentiation was stimulated by all trans retinoic acid (ATRA) (2.5µmol/L) at 24h after virus infection of pAd-NLS-RARα, and then detected by CD11b labeling two days later. The transcription and translation of C-MYC was detected in HL-60/pAd-NLS-RARα cells which treated by ATRA. Our results showed that compared to the control groups, the expression of NLS-RARα was significantly reduced in the HL-60/pNLS-RARα-shRNA cells, and increased dramatically in the HL-60/pAd-NLS-RARα cells. The proliferation was remarkably inhibited in the HL-60/pNLS-RARα-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-NLS-RARα cells. FCM outcome revealed the differentiation increased in HL-60/pNLS-RARα-shRNA cells, and decreased in the HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. The

Human apolipoprotein B (apoB) mRNA: Identification of two distinct apoB mRNAs, an mRNA with the apoB-100 sequence and an apoB mRNA containing a premature in-frame translational stop codon, in both liver and intestine

International Nuclear Information System (INIS)

Higuchi, K.; Hospattankar, A.V.; Law, S.W.; Meglin, N.; Cortright, J.; Brewer, H.B. Jr.

1988-01-01

Human apolipoprotein B (apoB) is present in plasma as two separate isoproteins, designated apoB-100 (512 kDa) and apoB-48 (250 kDa). ApoB is encoded by a single gene on chromosome 2, and a single nuclear mRNA is edited and processed into two separate apoB mRNAs. A 14.1-kilobase apoB mRNA codes for apoB-100, and the second mRNA, which codes for apoB-48, contains a premature stop codon generated by a single base substitution of cytosine to uracil at nucleotide 6,538, which converts the translated CAA codon coding for the amino acid glutamine at residue 2,153 in apoB-100 to a premature in-frame stop codon (UAA). Two 30-base synthetic oligonucleotides, designated apoB-Stop and apoB-Gln, were synthesized containing the complementary sequence to the stop codon (UAA) and glutamine codon (CAA), respectively. The combined results from these studies establish that both human intestine and liver contain the two distinct apoB mRNAs, an mRNA that codes for apoB-100 and an apoB mRNA that contains the premature stop codon, which codes for apoB-48. The premature in-frame stop codon is not tissue specific and is present in both human liver and intestine

Co-ordinate expression of activin A and its type I receptor mRNAs during phorbol ester-induced differentiation of human K562 erythroleukemia cells.

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Hildén, K; Tuuri, T; Erämaa, M; Ritvos, O

1999-07-20

Activins were originally isolated based on their ability to stimulate follicle-stimulating hormone secretion but later they have been shown to regulate a number of different cellular functions such as nerve cell survival, mesoderm induction during early embryogenesis as well as hematopoiesis. We studied the regulation of activin A, a homodimer of betaA-subunits, mRNA and protein in K562 erythroleukemia cells, which are known to be induced toward the erythroid lineage in response to activin or TGF-beta or toward the megakaryocytic lineage by the phorbol ester protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). Here we show by Northern blot analysis as well as by Western and ligand blotting that TPA strongly promotes activin betaA-subunit mRNA and activin A protein expression in K562 cells in time- and concentration dependent manner. In contrast, neither activin A nor TGF-beta induced betaA-subunit mRNA expression during erythroid differentiation in K562 cells. Interestingly, whereas activin type II receptors are not regulated during K562 cell differentiation (Hilden et al. (1994) Blood 83, 2163-2170), we now show that the activin type I and IB receptor mRNAs are clearly induced by TPA but not by activin or TGF-beta. We also show that the inducing effect of TPA on expression of activin betaA-subunit mRNA is potentiated by the protein kinase A activator 8-bromo-cAMP. We conclude that activin A and its type I receptors appear to be co-ordinately up-regulated during megakaryocytic differentiation of K562 cells.

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

Science.gov (United States)

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

Complexity on Acute Myeloid Leukemia mRNA Transcript Variant

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Carlo Cattani

2011-01-01

Full Text Available This paper deals with the sequence analysis of acute myeloid leukemia mRNA. Six transcript variants of mlf1 mRNA, with more than 2000 bps, are analyzed by focusing on the autocorrelation of each distribution. Through the correlation matrix, some patches and similarities are singled out and commented, with respect to similar distributions. The comparison of Kolmogorov fractal dimension will be also given in order to classify the six variants. The existence of a fractal shape, patterns, and symmetries are discussed as well.

Genetic and physical mapping of homologues of the virus resistance gene Rx1 and the cyst nematode resistance gene Gpa2 in potato.

Science.gov (United States)

Bakker, E; Butterbach, P; Rouppe van der Voort, J; van der Vossen, E; van Vliet, J; Bakker, J; Goverse, A

2003-05-01

Nine resistance gene homologues (RGHs) were identified in two diploid potato clones (SH and RH), with a specific primer pair based on conserved motifs in the LRR domain of the potato cyst nematode resistance gene Gpa2 and the potato virus X resistance gene Rx1. A modified AFLP method was used to facilitate the genetic mapping of the RGHs in the four haplotypes under investigation. All nine RGHs appeared to be located in the Gpa2/ Rx1 cluster on chromosome XII. Construction of a physical map using bacterial artificial chromosome (BAC) clones for both the Solanum tuberosum ssp. tuberosum and the S. tuberosum ssp. andigena haplotype of SH showed that the RGHs are located within a stretch of less than 200 kb. Sequence analysis of the RGHs revealed that they are highly similar (93 to 95%) to Gpa2 and Rx1. The sequence identities among all RGHs range from 85 to 100%. Two pairs of RGHs are identical, or nearly so (100 and 99.9%), with each member located in a different genotype. Southern-blot analysis on genomic DNA revealed no evidence for additional homologues outside the Gpa2/ Rx1 cluster on chromosome XII.

Use of isotope ratio mass spectrometry to differentiate between endogenous steroids and synthetic homologues in cattle: A review

International Nuclear Information System (INIS)

Janssens, Geert; Courtheyn, Dirk; Mangelinckx, Sven; Prévost, Stéphanie; Bichon, Emmanuelle; Monteau, Fabrice; De Poorter, Geert; De Kimpe, Norbert; Le Bizec, Bruno

2013-01-01

Graphical abstract: Scheme: Representation of the observed isotope ratios and the relation to exogenous and endogenous natural steroids. AS stands for “amount of steroid”. Highlights: ► The difference between endogenous and exogenous steroids is thoroughly laid out. ► Factors influencing the carbon ratio and the use of Δ 13 C-values are explained. ► Implementation of GC/C/IRMS to detect steroid abuse in cattle is reviewed. ► Alternative methods and upcoming techniques are discussed. ► The differences and similarities with sports doping control are highlighted. -- Abstract: Although substantial technical advances have been achieved during the past decades to extend and facilitate the analysis of growth promoters in cattle, the detection of abuse of synthetic analogs of naturally occurring hormones has remained a challenging issue. When it became clear that the exogenous origin of steroid hormones could be traced based on the 13 C/ 12 C isotope ratio of the substances, GC/C/IRMS has been successfully implemented to this aim since the end of the past century. However, due to the costly character of the instrumental setup, the susceptibility of the equipment to errors and the complex and time consuming sample preparation, this method is up until now only applied by a limited number of laboratories. In this review, the general principles as well as the practical application of GC/C/IRMS to differentiate between endogenous steroids and exogenously synthesized homologous compounds in cattle will be discussed in detail, and will be placed next to other existing and to be developed methods based on isotope ratio mass spectrometry. Finally, the link will be made with the field of sports doping, where GC/C/IRMS has been established within the World Anti-Doping Agency (WADA) approved methods as the official technique to differentiate between exogenous and endogenous steroids over the past few years

Extracellular tumor-related mRNA in plasma of lymphoma patients and survival implications.

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Vanesa Garcia

Full Text Available BACKGROUND: We studied anomalous extracellular mRNAs in plasma from patients with diffuse large B-cell lymphoma (DLBCL and their survival implications. mRNAs studied have been reported in the literature as markers of poor (BCL2, CCND2, MYC and favorable outcome (LMO2, BCL6, FN1 in tumors. These markers were also analyzed in lymphoma tissues to test possible associations with their presence in plasma. METHODOLOGY/PRINCIPAL FINDINGS: mRNA from 42 plasma samples and 12 tumors from patients with DLBCL was analyzed by real-time PCR. Samples post-treatment were studied. The immunohistochemistry of BCL2 and BCL6 was defined. Presence of circulating tumor cells was determined by analyzing the clonality of the immunoglobulin heavy-chain genes by PCR. In DLBCL, MYC mRNA was associated with short overall survival. mRNA targets with unfavorable outcome in tumors were associated with characteristics indicative of poor prognosis, with partial treatment response and with short progression-free survival in patients with complete response. In patients with low IPI score, unfavorable mRNA targets were related to shorter overall survival, partial response, high LDH levels and death. mRNA disappeared in post-treatment samples of patients with complete response, and persisted in those with partial response or death. No associations were found between circulating tumor cells and plasma mRNA. Absence of BCL6 protein in tumors was associated with presence of unfavorable plasma mRNA. CONCLUSIONS/SIGNIFICANCE: Through a non-invasive procedure, tumor-derived mRNAs can be obtained in plasma. mRNA detected in plasma did not proceed from circulating tumor cells. In our study, unfavorable targets in plasma were associated with poor prognosis in B-cell lymphomas, mainly MYC mRNA. Moreover, the unfavorable targets in plasma could help us to classify patients with poor outcome within the good prognosis group according to IPI.

HemaExplorer: a database of mRNA expression profiles in normal and malignant haematopoiesis

DEFF Research Database (Denmark)

Bagger, Frederik Otzen; Rapin, Nicolas; Theilgaard-Mönch, Kim

2013-01-01

lead to full integrity of the data in the database. The HemaExplorer has comprehensive visualization interface that can make it useful as a daily tool for biologists and cancer researchers to assess the expression patterns of genes encountered in research or literature. HemaExplorer is relevant for all......The HemaExplorer (http://servers.binf.ku.dk/hemaexplorer) is a curated database of processed mRNA Gene expression profiles (GEPs) that provides an easy display of gene expression in haematopoietic cells. HemaExplorer contains GEPs derived from mouse/human haematopoietic stem and progenitor cells...... as well as from more differentiated cell types. Moreover, data from distinct subtypes of human acute myeloid leukemia is included in the database allowing researchers to directly compare gene expression of leukemic cells with those of their closest normal counterpart. Normalization and batch correction...

Impairment of FOS mRNA stabilization following translation arrest in granulocytes from myelodysplastic syndrome patients.

Science.gov (United States)

Feng, Xiaomin; Shikama, Yayoi; Shichishima, Tsutomu; Noji, Hideyoshi; Ikeda, Kazuhiko; Ogawa, Kazuei; Kimura, Hideo; Takeishi, Yasuchika; Kimura, Junko

2013-01-01

Although quantitative and qualitative granulocyte defects have been described in myelodysplastic syndromes (MDS), the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. We recently found that FOS mRNA elevation under translation-inhibiting stimuli was significantly smaller in granulocytes from MDS patients than in healthy individuals. The aim of this study is to clarify the cause of the impaired FOS induction in MDS. We first examined the mechanisms of FOS mRNA elevation using granulocytes from healthy donors cultured with the translation inhibitor emetine. Emetine increased both transcription and mRNA stability of FOS. p38 MAPK inhibition abolished the emetine-induced increase of FOS transcription but did not affect FOS mRNA stabilization. The binding of an AU-rich element (ARE)-binding protein HuR to FOS mRNA containing an ARE in 3'UTR was increased by emetine, and the knockdown of HuR reduced the FOS mRNA stabilizing effect of emetine. We next compared the emetine-induced transcription and mRNA stabilization of FOS between MDS patients and healthy controls. Increased rates of FOS transcription by emetine were similar in MDS and controls. In the absence of emetine, FOS mRNA decayed to nearly 17% of initial levels in 45 min in both groups. In the presence of emetine, however, 76.7±19.8% of FOS mRNA remained after 45 min in healthy controls, versus 37.9±25.5% in MDS (Pknowledge, this is the first report demonstrating attenuation of stress-induced FOS mRNA stabilization in MDS granulocytes.

Fibroblast growth factor-21 and omentin-1 hepatic mRNA expression and serum levels in morbidly obese women with non-alcoholic fatty liver disease.

Science.gov (United States)

Waluga, M; Kukla, M; Zorniak, M; Kajor, M; Liszka, L; Dyaczynski, M; Kowalski, G; Zadlo, D; Waluga, E; Olczyk, P; Buldak, R J; Berdowska, A; Hartleb, M

2017-06-01

Fibroblast growth factor-21 (FGF21) and omentin-1 have been recognized as potent antidiabetic agents with potential hepatoprotective activity. The aim of this study was to evaluate hepatic FGF21 and omentin-1 mRNA expression as well as their serum levels as predictive markers of liver injury and insulin resistance in morbidly obese women with non-alcoholic fatty liver disease (NAFLD). This study included 56 severely obese women who underwent intraoperative wedge liver biopsy during the bariatric surgery. Hepatic FGF21 and omentin-1 mRNA were assessed by quantitative real-time PCR, while their serum concentrations were measured with commercially available enzyme-linked immunosorbent assays. The FGF21 serum level was significantly higher in patients with a greater extent of steatosis (grade 2 and 3) compared to those without or with mild steatosis (grade 0 and 1) (P = 0.049). Receiver Operating Characteristic analysis, however, showed poor discriminant power for the FGF21 serum levels in differentiating between more and less extensive steatosis with an AUC = 0.666. There was a tendency towards higher levels of hepatic FGF21 mRNA in patients with lobular inflammation and fibrosis and towards lower levels in the case of hepatocyte ballooning and steatosis. There was a positive mutual correlation between hepatic FGF21 and omentin-1 mRNA levels (r = 0.78; P hepatic omentin-1 mRNA levels showed a tendency to be lower in patients with advanced steatosis and hepatocyte ballooning. In conclusion, our study, which focused on hepatic FGF21 and omentin-1 mRNA expression, confirmed marked expression of both molecules in the liver of morbidly obese patients with NAFLD. More extensive steatosis was associated with evident changes in the serum FGF21 concentration in morbidly obese women with NAFLD, but the difference did not reach statistical significance. The vast amount of fat, both visceral and subcutaneous, in severely obese patients may be the additional source and influence

mRNA and microRNA transcriptomics analyses in a murine model of dystrophin loss and therapeutic restoration

Directory of Open Access Journals (Sweden)

Thomas C. Roberts

2016-03-01

Full Text Available Duchenne muscular dystrophy (DMD is a pediatric, X-linked, progressive muscle-wasting disorder caused by loss of function mutations affecting the gene encoding the dystrophin protein. While the primary genetic insult in DMD is well described, many details of the molecular and cellular pathologies that follow dystrophin loss are incompletely understood. To investigate gene expression in dystrophic muscle we have applied mRNA and microRNA (miRNA microarray technology to the mdx mouse model of DMD. This study was designed to generate a complete description of gene expression changes associated with dystrophic pathology and the response to an experimental therapy which restores dystrophin protein function. These datasets have enabled (1 the determination of gene expression changes associated with dystrophic pathology, (2 identification of differentially expressed genes that are restored towards wild-type levels after therapeutic dystrophin rescue, (3 investigation of the correlation between mRNA and protein expression (determined by parallel mass spectrometry proteomics analysis, and (4 prediction of pathology associated miRNA-target interactions. Here we describe in detail how the data were generated including the basic analysis as contained in the manuscript published in Human Molecular Genetics with PMID 26385637. The data have been deposited in the Gene Expression Omnibus (GEO with the accession number GSE64420.

The flavonoid fisetin promotes osteoblasts differentiation through Runx2 transcriptional activity.

Science.gov (United States)

Léotoing, Laurent; Davicco, Marie-Jeanne; Lebecque, Patrice; Wittrant, Yohann; Coxam, Véronique

2014-06-01

Flavonoids represent a group of polyphenolic compounds commonly found in daily nutrition with proven health benefits. Among this group, the flavonol fisetin has been previously shown to protect bone by repressing osteoclast differentiation. In the present study, we investigated the role of fisetin in regulating osteoblasts physiology. In vivo mice treated with LPSs exhibited osteoporosis features associated with a dramatic repression of osteoblast marker expression. In this model, inhibition of osteocalcin and type I collagen alpha 1 transcription was partially countered by a daily consumption of fisetin. Interestingly, in vitro, fisetin promoted both osteoblast alkaline phosphatase activity and mineralization process. To decipher how fisetin may exert its positive effect on osteoblastogenesis, we analyzed its ability to control the runt-related transcription factor 2 (Runx2), a key organizer in developing and maturing osteoblasts. While fisetin did not impact Runx2 mRNA and protein levels, it upregulated its transcriptional activity. Actually, fisetin stimulated the luciferase activity of a reporter plasmid driven by the osteocalcin gene promoter that contains Runx2 binding sites and promoted the mRNA expression of osteocalcin and type I collagen alpha 1 targets. Bone sparing properties of fisetin also rely on its positive influence on osteoblast differentiation and activity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

Energy Technology Data Exchange (ETDEWEB)

Han, Yanfu [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Chai, Jiake, E-mail: cjk304@126.com [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Sun, Tianjun; Li, Dongjie; Tao, Ran [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China)

2011-10-07

Highlights: {yields} Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. {yields} Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. {yields} We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. {yields} Collagen type I and collagen type III mRNA level was higher in differentiated cells. {yields} UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. In this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue

Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

International Nuclear Information System (INIS)

Han, Yanfu; Chai, Jiake; Sun, Tianjun; Li, Dongjie; Tao, Ran

2011-01-01

Highlights: → Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. → Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. → We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. → Collagen type I and collagen type III mRNA level was higher in differentiated cells. → UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. In this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue-engineered dermis.

Characterization of a major late herpes simplex virus type 1 mRNA.

Science.gov (United States)

Costa, R H; Devi, B G; Anderson, K P; Gaylord, B H; Wagner, E K

1981-05-01

A major, late 6-kilobase (6-kb) mRNa mapping in the large unique region of herpes simplex virus type 1 (HSV-1) was characterized by using two recombinant DNA clones, one containing EcoRI fragment G (0.190 to 0.30 map units) in lambda. WES.B (L. Enquist, M. Madden, P. Schiop-Stansly, and G. Vandl Woude, Science 203:541-544, 1979) and one containing HindIII fragment J (0.181 to 0.259 map units) in pBR322. This 6-kb mRNA had its 3' end to the left of 0.231 on the prototypical arrangement of the HSV-1 genome and was transcribed from right to left. It was bounded on both sides by regions containing a large number of distinct mRNA species, and its 3' end was partially colinear with a 1.5-kb mRNA which encoded a 35,000-dalton polypeptide. The 6-kb mRNA encoded a 155,000-dalton polypeptide which was shown to be the only one of this size detectable by hybrid-arrested translation encoded by late polyadenylated polyribosomal RNA. The S1 nuclease mapping experiments indicated that there were no introns in the coding sequence for this mRNA and that its 3' end mapped approximately 800 nucleotides to the left of the BglII site at 0.231, whereas its 5' end extended very close to the BamHI site at 0.266.

Fibroblast growth factor-2 stimulates adipogenic differentiation of human adipose-derived stem cells

International Nuclear Information System (INIS)

Kakudo, Natsuko; Shimotsuma, Ayuko; Kusumoto, Kenji

2007-01-01

Adipose-derived stem cells (ASCs) have demonstrated a capacity for differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and factors. It is acknowledged that fibroblast growth factor-2 (FGF-2) promotes chondrogenic and inhibits osteogenic differentiation of ASCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate at the cellular and molecular levels the effect of FGF-2 on adipogenic differentiation of ASCs, as induced by an adipogenic hormonal cocktail consisting of 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and indomethacin. FGF-2 significantly enhances the adipogenic differentiation of human ASCs. Furthermore, in cultures receiving FGF-2 before adipogenic induction, mRNA expression of peroxisome proliferator-activated receptor γ2 (PPARγ2), a key transcription factor in adipogenesis, was upregulated. The results of FGF-2 supplementation suggest the potential applications of FGF-2 and ASCs in adipose tissue regeneration

Coordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

KAUST Repository

Arae, Toshihiro

2017-04-18

Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.

2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation.

Science.gov (United States)

Choi, Junhong; Indrisiunaite, Gabriele; DeMirci, Hasan; Ieong, Ka-Weng; Wang, Jinfan; Petrov, Alexey; Prabhakar, Arjun; Rechavi, Gideon; Dominissini, Dan; He, Chuan; Ehrenberg, Måns; Puglisi, Joseph D

2018-03-01

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2'-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2'-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

Quantitative PCR--new diagnostic tool for quantifying specific mRNA and DNA molecules

DEFF Research Database (Denmark)

Schlemmer, B O; Sorensen, B S; Overgaard, J

2004-01-01

of a subset of ligands from the EGF system is increased in bladder cancer. Furthermore, measurement of the mRNA concentration gives important information such as the expression of these ligands correlated to the survival of the patients. In addition to the alterations at the mRNA level, changes also can occur...... at the DNA level in the EGF system. Thus, it has been demonstrated that the number of genes coding for the human epidermal growth factor receptor 2 (HER2) is increased in a number of breast tumors. It is now possible to treat breast cancer patients with a humanized antibody reacting with HER2...... of mRNA or DNA in biological samples. In this study quantitative PCR was used to investigate the role of the EGF (epidermal growth factor) system in cancer both for measurements of mRNA concentrations and for measurements of the number of copies of specific genes. It is shown that the mRNA expression...

Applying the breaks on gene expression - mRNA deadenylation by Pop2p

DEFF Research Database (Denmark)

Andersen, Kasper Røjkjær; Jonstrup, Anette Thyssen; Van, Lan Bich

When driving a car, control of the brakes is just as important as control of the accelerator pedal. Likewise, in gene expression, regulation of mRNA degradation is as important as regulation of its synthesis (Mühlemann, 2005). The rate-determining step of mRNA decay in eukaryotes seems to be the ......When driving a car, control of the brakes is just as important as control of the accelerator pedal. Likewise, in gene expression, regulation of mRNA degradation is as important as regulation of its synthesis (Mühlemann, 2005). The rate-determining step of mRNA decay in eukaryotes seems...... to be the shortening of the poly(A) tail (deadenylation), as this step is slower than the subsequent decapping and degradation of the mRNA body. The Mega-Dalton Ccr4-Not complex contains two exonucleases, Ccr4p and Pop2p, responsible for this process. It is not known at present why two conserved nucleases are needed...

Differential Expression of Osteo-Modulatory Molecules in Periodontal Ligament Stem Cells in Response to Modified Titanium Surfaces

Directory of Open Access Journals (Sweden)

So Yeon Kim

2014-01-01

Full Text Available This study assessed differential gene expression of signaling molecules involved in osteogenic differentiation of periodontal ligament stem cells (PDLSCs subjected to different titanium (Ti surface types. PDLSCs were cultured on tissue culture polystyrene (TCPS, and four types of Ti discs (PT, SLA, hydrophilic PT (pmodPT, and hydrophilic SLA (modSLA with no osteoinductive factor and then osteogenic activity, including alkaline phosphatase (ALP activity, mRNA expression of runt-related gene 2, osterix, FOSB, FRA1, and protein levels of osteopontin and collagen type IA, were examined. The highest osteogenic activity appeared in PDLSCs cultured on SLA, compared with the TCPS and other Ti surfaces. The role of surface properties in affecting signaling molecules to modulate PDLSC behavior was determined by examining the regulation of Wnt pathways. mRNA expression of the canonical Wnt signaling molecules, Wnt3a and β-catenin, was higher on SLA and modSLA than on smooth surfaces, but gene expression of the calcium-dependent Wnt signaling molecules Wnt5a, calmodulin, and NFATc1 was increased significantly on PT and pmodPT. Moreover, integrin α2/β1, sonic hedgehog, and Notch signaling molecules were affected differently by each surface modification. In conclusion, surface roughness and hydrophilicity can affect differential Wnt pathways and signaling molecules, targeting the osteogenic differentiation of PDLSCs.

HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels

DEFF Research Database (Denmark)

Hviid, Thomas Vauvert F; Hylenius, Sine; Rørbye, Christina

2003-01-01

between mother and fetus in several ways. Finally, the expression of membrane-bound HLA-G and soluble HLA-G has been proposed to influence the outcome of pregnancy, and an aberrant HLA-G expression in pre-eclamptic placentas and spontaneous abortions has been reported. Here, an association between certain...... HLA-G polymorphisms and the mRNA levels of the different alternatively spliced HLA-G isoforms in first trimester trophoblast cell populations is reported. Several alternatively spliced HLA-G mRNA isoforms, including a 14-bp polymorphism in the 3'UTR end (exon 8) of the HLA-G gene, are expressed...

Effect of in vitro estrogenic pesticides on human oestrogen receptor α and β mRNA levels

DEFF Research Database (Denmark)

Theander Grünfeld, Heidi; Bonefeld-Jørgensen, Eva Cecilie

2004-01-01

of the ERα mRNA level, but only significantly for prochloraz, dieldrin, and tolchlofos-methyl. Alone no pesticides affected the ERβ mRNA level significantly, but chlorpyrifos increased the mRNA level weakly. Co-exposure with E2 elicited a significant increased ERβ mRNA level by prochloraz, fenarimol...

An investigation of nutrient-dependent mRNA translation in Drosophila larvae

Directory of Open Access Journals (Sweden)

Sabarish Nagarajan

2014-10-01

Full Text Available The larval period of the Drosophila life cycle is characterized by immense growth. In nutrient rich conditions, larvae increase in mass approximately two hundred-fold in five days. However, upon nutrient deprivation, growth is arrested. The prevailing view is that dietary amino acids drive this larval growth by activating the conserved insulin/PI3 kinase and Target of rapamycin (TOR pathways and promoting anabolic metabolism. One key anabolic process is protein synthesis. However, few studies have attempted to measure mRNA translation during larval development or examine the signaling requirements for nutrient-dependent regulation. Our work addresses this issue. Using polysome analyses, we observed that starvation rapidly (within thirty minutes decreased larval mRNA translation, with a maximal decrease at 6–18 hours. By analyzing individual genes, we observed that nutrient-deprivation led to a general reduction in mRNA translation, regardless of any starvation-mediated changes (increase or decrease in total transcript levels. Although sugars and amino acids are key regulators of translation in animal cells and are the major macronutrients in the larval diet, we found that they alone were not sufficient to maintain mRNA translation in larvae. The insulin/PI3 kinase and TOR pathways are widely proposed as the main link between nutrients and mRNA translation in animal cells. However, we found that genetic activation of PI3K and TOR signaling, or regulation of two effectors – 4EBP and S6K – could not prevent the starvation-mediated translation inhibition. Similarly, we showed that the nutrient stress-activated eIF2α kinases, GCN2 and PERK, were not required for starvation-induced inhibition of translation in larvae. These findings indicate that nutrient control of mRNA translation in larvae is more complex than simply amino acid activation of insulin and TOR signaling.

Localization of insulin receptor mRNA in rat brain by in situ hybridization

International Nuclear Information System (INIS)

Marks, J.L.; Porte, D. Jr.; Stahl, W.L.; Baskin, D.G.

1990-01-01

Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three 35 S-oligonucleotide probes and contact film autoradiography. Specificity was confirmed by showing that (a) excess unlabeled probe abolished the signal, (b) an oligonucleotide probe for rat neuropeptide Y mRNA showed a different distribution of hybridization signal, and (c) the distribution of insulin receptor binding was consistent with the distribution of insulin receptor mRNA. Insulin receptor mRNA was most abundant in the granule cell layers of the olfactory bulb, cerebellum and dentate gyrus, in the pyramidal cell body layers of the pyriform cortex and hippocampus, in the choroid plexus and in the arcuate nucleus of the hypothalamus

Anatomy of rat semaphorin III/collapsin-1 mRNA expression and relationship to developing nerve tracts during neuroembryogenesis

NARCIS (Netherlands)

Giger, Roman J; Wolfer, D P; De Wit, G M; Verhaagen, J

1996-01-01

Semaphorin III/collapsin-1 (semaIII/coll-1) is a chemorepellent that exhibits a repulsive effect on growth cones of dorsal root ganglion neurons. To identify structures that express semaIII/coll-1 in developing mammals, we cloned the rat homologue and performed in situ hybridization on embryonic,

Interleukin-21 mRNA expression during virus infections

DEFF Research Database (Denmark)

Holm, Christian; Nyvold, Charlotte Guldborg; Paludan, Søren Riis

2006-01-01

and activational effects of IL-21 on different leukocytes come into play in vivo in an immune response has so far not been fully investigated. We show here for the first time in vivo, that IL-21 mRNA is produced in the spleen when mice are challenged with herpes simplex virus type 2 (HSV-2) or lymphocytic...... choriomeningitis virus (LCMV). We show in HSV-2 challenged mice that this production takes place in CD4+ T cell fractions and is absent in CD4+ T cell-depleted fractions. We also show that the peak of IL-21 mRNA production in both the HSV-2 and LCMV-challenged mice coincides with the onset of the adaptive immune...

Parvovirus B19 Replication and Expression in Differentiating Erythroid Progenitor Cells.

Directory of Open Access Journals (Sweden)

Gloria Bua

Full Text Available The pathogenic Parvovirus B19 (B19V is characterized by a strict adaptation to erythroid progenitor cells (EPCs, a heterogeneous population of differentiating cells with diverse phenotypic and functional properties. In our work, we studied the dynamics of B19V infection in EPCs in dependence on the cell differentiation stage, in terms of distribution of infected cells, synthesis of viral nucleic acids and production of infectious virus. EPCs at early differentiation stage led to an abortive infection, without viral genome replication and a very low transcriptional activity. EPCs at later stages were permissive, with highest levels of viral replicative activity at day 9 (+3.0 Log from 2 to 48 hpi and lower levels at day 18 (+1.5 Log from 2 to 48 hpi. B19V DNA increment was in accordance with the percentage of cells positive to flow-FISH assay (41.4% at day 9, 1.1% at day 18. Quantitation of total RNA indicated a close association of genome replication and transcription with viral RNA accumulation within infected cells related to viral DNA increase during the course of infection. Analysis of the different classes of mRNAs revealed two distinct pattern of genome expression profile with a fine regulation in the frequency utilization of RNA processing signals: an early phase, when cleavage at the proximal site leading to a higher relative production of mRNA for NS protein, and a late phase, when cleavage at the distal site was more frequent leading to higher relative abundance of mRNA for VP and 11 kDA proteins. Infectious virus was released from cells at day 6-15, but not at day 18. Our results, providing a detailed description of B19V replication and expression profile in differentiating EPCs, highlight the very tight adaptation of B19V to a specific cellular target defined both by its erythroid lineage and its differentiation stage.

Parvovirus B19 Replication and Expression in Differentiating Erythroid Progenitor Cells

Science.gov (United States)

Bua, Gloria; Manaresi, Elisabetta; Bonvicini, Francesca; Gallinella, Giorgio

2016-01-01

The pathogenic Parvovirus B19 (B19V) is characterized by a strict adaptation to erythroid progenitor cells (EPCs), a heterogeneous population of differentiating cells with diverse phenotypic and functional properties. In our work, we studied the dynamics of B19V infection in EPCs in dependence on the cell differentiation stage, in terms of distribution of infected cells, synthesis of viral nucleic acids and production of infectious virus. EPCs at early differentiation stage led to an abortive infection, without viral genome replication and a very low transcriptional activity. EPCs at later stages were permissive, with highest levels of viral replicative activity at day 9 (+3.0 Log from 2 to 48 hpi) and lower levels at day 18 (+1.5 Log from 2 to 48 hpi). B19V DNA increment was in accordance with the percentage of cells positive to flow-FISH assay (41.4% at day 9, 1.1% at day 18). Quantitation of total RNA indicated a close association of genome replication and transcription with viral RNA accumulation within infected cells related to viral DNA increase during the course of infection. Analysis of the different classes of mRNAs revealed two distinct pattern of genome expression profile with a fine regulation in the frequency utilization of RNA processing signals: an early phase, when cleavage at the proximal site leading to a higher relative production of mRNA for NS protein, and a late phase, when cleavage at the distal site was more frequent leading to higher relative abundance of mRNA for VP and 11 kDA proteins. Infectious virus was released from cells at day 6–15, but not at day 18. Our results, providing a detailed description of B19V replication and expression profile in differentiating EPCs, highlight the very tight adaptation of B19V to a specific cellular target defined both by its erythroid lineage and its differentiation stage. PMID:26845771

The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells

Energy Technology Data Exchange (ETDEWEB)

Suriguga,; Li, Xiao-Fei; Li, Yang; Yu, Chun-Hong; Li, Yi-Ran; Yi, Zong-Chun, E-mail: yizc@buaa.edu.cn

2013-12-15

Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation. - Highlights: • Catechol enhanced hemin-induced hemoglobin accumulation. • COMT-catalyzed methylation acted as detoxication of catechol. • COMT involved in catechol-enhanced erythroid differentiation.

The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells

International Nuclear Information System (INIS)

Suriguga,; Li, Xiao-Fei; Li, Yang; Yu, Chun-Hong; Li, Yi-Ran; Yi, Zong-Chun

2013-01-01

Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation. - Highlights: • Catechol enhanced hemin-induced hemoglobin accumulation. • COMT-catalyzed methylation acted as detoxication of catechol. • COMT involved in catechol-enhanced erythroid differentiation

S100A16 promotes differentiation and contributes to a less aggressive tumor phenotype in oral squamous cell carcinoma

International Nuclear Information System (INIS)

Sapkota, Dipak; Bruland, Ove; Parajuli, Himalaya; Osman, Tarig A.; Teh, Muy-Teck; Johannessen, Anne C.; Costea, Daniela Elena

2015-01-01

Altered expression of S100A16 has been reported in human cancers, but its biological role in tumorigenesis is not fully understood. This study aimed to investigate the clinical significance and functional role of S100A16 in oral squamous cell carcinoma (OSCC) suppression. S100A16 mRNA and/or protein levels were examined by quantitative RT-PCR and immunohistochemistry in whole- and laser microdissected-specimens of normal human oral mucosa (NHOM, n = 65), oral dysplastic lesions (ODL, n = 21), OSCCs (n = 132) and positive cervical nodes (n = 17). S100A16 protein expression in OSCC was examined for correlations with clinicopathological variables and patient survival. S100A16 was over-expressed and knocked-down in OSCC-derived (CaLH3 and H357) cells by employing retroviral constructs to investigate its effects on cell proliferation, sphere formation and three dimensional (3D)-organotypic invasive abilities in vitro and tumorigenesis in a mouse xenograft model. Both S100A16 mRNA and protein levels were found to be progressively down-regulated from NHOM to ODL and OSCC. Low S100A16 protein levels in OSCC significantly correlated with reduced 10-year overall survival and poor tumor differentiation. Analysis of two external OSCC microarray datasets showed a positive correlation between the mRNA expression levels of S100A16 and keratinocyte differentiation markers. CaLH3 and H357 cell fractions enriched for differentiated cells either by lack of adherence to collagen IV or FACS sorting for low p75NTR expression expressed significantly higher S100A16 mRNA levels than the subpopulations enriched for less differentiated cells. Corroborating these findings, retroviral mediated S100A16 over-expression and knock-down in CaLH3 and H357 cells led to respective up- and down-regulation of differentiation markers. In vitro functional studies showed significant reduction in cell proliferation, sphere formation and 3D-invasive abilities of CaLH3 and H357 cells upon S100A16 over

Analysis of E2F factors during epidermal differentiation.

Science.gov (United States)

Chang, Wing Y; Dagnino, Lina

2005-01-01

The multigene E2F family of transcription factors is central in the control of cell cycle progression. The expression and activity of E2F proteins is tightly regulated transcriptionally and posttranslationally as a function of the proliferation and differentiation status of the cell. In this chapter, we review protocols designed to determine E2F mRNA abundance in tissues by in situ hybridization techniques. The ability to culture primary epidermal keratinocytes and maintain them as either undifferentiated or terminally differentiated cells allows the biochemical and molecular characterization of changes in E2F expression and activity. Thus, we also discuss in detail methods to analyze E2F protein abundance by immunoblot and their ability to bind DNA in cultured cells using electrophoretic mobility shift assays.

Combinations of ERK and p38 MAPK inhibitors ablate tumor necrosis factor-alpha (TNF-alpha ) mRNA induction. Evidence for selective destabilization of TNF-alpha transcripts.

Science.gov (United States)

Rutault, K; Hazzalin, C A; Mahadevan, L C

2001-03-02

Tumor necrosis factor-alpha (TNF-alpha) is a potent proinflammatory cytokine whose synthesis and secretion are implicated in diverse pathologies. Hence, inhibition of TNF-alpha transcription or translation and neutralization of its protein product represent major pharmaceutical strategies to control inflammation. We have studied the role of ERK and p38 mitogen-activated protein (MAP) kinase in controlling TNF-alpha mRNA levels in differentiated THP-1 cells and in freshly purified human monocytes. We show here that it is possible to produce virtually complete inhibition of lipopolysaccharide-stimulated TNF-alpha mRNA accumulation by using a combination of ERK and p38 MAP kinase inhibitors. Furthermore, substantial inhibition is achievable using combinations of 1 microm of each inhibitor, whereas inhibitors used individually are incapable of producing complete inhibition even at high concentrations. Finally, addressing mechanisms involved, we show that inhibition of p38 MAP kinase selectively destabilizes TNF-alpha transcripts but does not affect degradation of c-jun transcripts. These results impinge on the controversy in the literature surrounding the mode of action of MAP kinase inhibitors on TNF-alpha mRNA and suggest the use of combinations of MAP kinase inhibitors as an effective anti-inflammatory strategy.

Region specific regulation of glutamic acid decarboxylase mRNA expression by dopamine neurons in rat brain.

Science.gov (United States)

Lindefors, N; Brene, S; Herrera-Marschitz, M; Persson, H

1989-01-01

In situ hybridization histochemistry and RNA blots were used to study the expression of glutamic acid decarboxylase (GAD) mRNA in rats with or without a unilateral lesion of midbrain dopamine neurons. Two populations of GAD mRNA positive neurons were found in the intact caudate-putamen, substantia nigra and fronto-parietal cortex. In caudate-putamen, only one out of ten of the GAD mRNA positive neurons expressed high levels, while in substantia nigra every second of the positive neurons expressed high levels of GAD mRNA. Relatively few, but intensively labelled neurons were found in the intact fronto-parietal cerebral cortex. In addition, one out of six of the GAD mRNA positive neurons in the fronto-parietal cortex showed a low labeling. On the ipsilateral side, the forebrain dopamine deafferentation induced an increase in the number of neurons expressing high levels of GAD mRNA in caudate-putamen, and a decrease in fronto-parietal cortex. A smaller decrease was also seen in substantia nigra. However, the total number of GAD mRNA positive neurons were not significantly changed in any of these brain regions. The changes in the levels of GAD mRNA after the dopamine lesion were confirmed by RNA blot analysis. Hence, midbrain dopamine neurons appear to control neuronal expression of GAD mRNA by a tonic down-regulation in a fraction of GAD mRNA positive neurons in caudate-putamen, and a tonic up-regulation in a fraction of GAD mRNA positive neurons in fronto-parietal cortex and substantia nigra.

Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons

NARCIS (Netherlands)

Mochida, Sumiko; Poulain, Bernard; Eisel, Ulrich; Binz, Thomas; Kurazono, Hisao; Niemann, Heiner; Tauc, Ladislav; Bullock, Theodore H.

1990-01-01

Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters). No evidence for expression of injected exogenous mRNA, however, has been reported in

Natural selection and algorithmic design of mRNA.

Science.gov (United States)

Cohen, Barry; Skiena, Steven

2003-01-01

Messenger RNA (mRNA) sequences serve as templates for proteins according to the triplet code, in which each of the 4(3) = 64 different codons (sequences of three consecutive nucleotide bases) in RNA either terminate transcription or map to one of the 20 different amino acids (or residues) which build up proteins. Because there are more codons than residues, there is inherent redundancy in the coding. Certain residues (e.g., tryptophan) have only a single corresponding codon, while other residues (e.g., arginine) have as many as six corresponding codons. This freedom implies that the number of possible RNA sequences coding for a given protein grows exponentially in the length of the protein. Thus nature has wide latitude to select among mRNA sequences which are informationally equivalent, but structurally and energetically divergent. In this paper, we explore how nature takes advantage of this freedom and how to algorithmically design structures more energetically favorable than have been built through natural selection. In particular: (1) Natural Selection--we perform the first large-scale computational experiment comparing the stability of mRNA sequences from a variety of organisms to random synonymous sequences which respect the codon preferences of the organism. This experiment was conducted on over 27,000 sequences from 34 microbial species with 36 genomic structures. We provide evidence that in all genomic structures highly stable sequences are disproportionately abundant, and in 19 of 36 cases highly unstable sequences are disproportionately abundant. This suggests that the stability of mRNA sequences is subject to natural selection. (2) Artificial Selection--motivated by these biological results, we examine the algorithmic problem of designing the most stable and unstable mRNA sequences which code for a target protein. We give a polynomial-time dynamic programming solution to the most stable sequence problem (MSSP), which is asymptotically no more complex

Lower FOXO3 mRNA expression in granulosa cells is involved in unexplained infertility.

Science.gov (United States)

Yamamoto, Hikaru; Yamashita, Yoshiki; Saito, Natsuho; Hayashi, Atsushi; Hayashi, Masami; Terai, Yoshito; Ohmichi, Masahide

2017-06-01

The aim of this study was to investigate whether FOXO1 and FOXO3 mRNA expression in granulosa cells is the cause of unexplained infertility. Thirty-one patients aged infertility and 18 with male partner infertility as a control group) whose serum anti-Müllerian hormone level was >0.5 ng/μL were enrolled in the study. All patients underwent oocyte retrieval under a short protocol from June 2012 to October 2013. Real-time PCR was carried out using mRNA extracted from granulosa cells retrieved from mature follicles. We compared FOXO1 and FOXO3 mRNA expression ratios in granulosa cells between the unexplained infertility group and the male infertility group. The relation between FOXO1 and FOXO3 mRNA expression ratios in granulosa cells and assisted reproduction technology clinical outcome was also examined. FOXO3 mRNA expression ratio was significantly lower in the unexplained infertility group than in the male infertility group. Moreover, FOXO3 mRNA expression ratio showed a positive correlation with both the number of retrieved oocytes and serum anti-Müllerian hormone level. A positive correlation was also identified between FOXO1 mRNA expression and total dose of hMG. As well, the number of retrieved oocytes in the unexplained infertility group was statistically lower than that in the male infertility group. A lower FOXO3 mRNA expression in granulosa cells leads to poor oocyte development in patients with unexplained infertility undergoing controlled ovarian stimulation for in vitro fertilization-embryo transfer. © 2017 Japan Society of Obstetrics and Gynecology.

Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export.

Science.gov (United States)

Larsen, Sean; Bui, Steven; Perez, Veronica; Mohammad, Adeba; Medina-Ramirez, Hilario; Newcomb, Laura L

2014-08-28

Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1. Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3N2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA. Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export. These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we

Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines

Directory of Open Access Journals (Sweden)

Barredo Julio C

2002-03-01

Full Text Available Abstract Background We describe an alternative method to determine mRNA half-life (t1/2 based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability. Results Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGreen® fluorescent dye with the VersaFluor Fluorometer System. One μg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-actin gene (231 bp. To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR® Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h. Conclusions We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate.

1,25 dihydroxyvitamin D3 (1,25) regulation of c-myc mRNA in HL-60 leukemia cells

International Nuclear Information System (INIS)

Simpson, R.U.; Bresnick, E.H.; Begley, D.A.

1986-01-01

Recently, 1,25 was shown to induce differentiation and decrease c-myc levels in HL-60 cells. The authors have confirmed these observations by RNA dot blot analysis. Cells treated with 50 nM 1,25 for 4, 24 and 48 hr showed c-myc mRNA levels of 26, 17 and 15% of control respectively. β-Actin mRNA levels were not altered. To ascertain whether 1, 25 regulated c-myc transcriptionally, an HL-60 nuclear RNA runoff assay was developed. Assay of total nuclei transcriptional activity revealed that 50% of RNA elongation was α-amanitin (0.8 μg/ml) sensitive and was linear with nuclei concentration (0.1-1 x 10 7 nuclei). 1,25 (50 nM) treated (45-96 hr) cells had decreased (approx.40%) total transcription rate relative to control. Decreased total RNA synthesis occurred concomitant with NBT reducing activity. 32 P-RNA runoff transcripts from HL-60 nuclei were hybridized to excess (5 μg DNA was excess) Pst I linearized c-myc and β-actin clones (in pBR322) immobilized on nitrocellulose filter. 32 P-RNA input from 2 x 10 6 to 2 x 10 7 cpm yielded linear hybridization signal. Analysis of blot dot intensity revealed no difference in transcription of c-myc in nuclei from 1,25 dosed or control cells. (myc/actin ratios: 1,25 (50 nM, 72 hr) =1.1 +/- 0.3 and control (72 hr) = 1.0, N=3 or 2 or 3 dots ea). These preliminary data suggest 1,25 does not affect c-myc transcription in HL-60 nuclei and may regulate c-myc mRNA post-transcriptionally

Post-embryonic nerve-associated precursors to adult pigment cells: genetic requirements and dynamics of morphogenesis and differentiation.

Directory of Open Access Journals (Sweden)

Erine H Budi

2011-05-01

Full Text Available The pigment cells of vertebrates serve a variety of functions and generate a stunning variety of patterns. These cells are also implicated in human pathologies including melanoma. Whereas the events of pigment cell development have been studied extensively in the embryo, much less is known about morphogenesis and differentiation of these cells during post-embryonic stages. Previous studies of zebrafish revealed genetically distinct populations of embryonic and adult melanophores, the ectotherm homologue of amniote melanocytes. Here, we use molecular markers, vital labeling, time-lapse imaging, mutational analyses, and transgenesis to identify peripheral nerves as a niche for precursors to adult melanophores that subsequently migrate to the skin to form the adult pigment pattern. We further identify genetic requirements for establishing, maintaining, and recruiting precursors to the adult melanophore lineage and demonstrate novel compensatory behaviors during pattern regulation in mutant backgrounds. Finally, we show that distinct populations of latent precursors having differential regenerative capabilities persist into the adult. These findings provide a foundation for future studies of post-embryonic pigment cell precursors in development, evolution, and neoplasia.

Involvement of CRF2 signaling in enterocyte differentiation.

Science.gov (United States)

Ducarouge, Benjamin; Pelissier-Rota, Marjolaine; Powell, Rebecca; Buisson, Alain; Bonaz, Bruno; Jacquier-Sarlin, Muriel

2017-07-28

To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation. For this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method. CRF2 protein is preferentially expressed in undifferentiated epithelial cells from

Selective translation of the measles virus nucleocapsid mRNA by La protein

Directory of Open Access Journals (Sweden)

Yoshihisa eInoue

2011-08-01

Full Text Available Measles, caused by measles virus (MeV infection, is the leading cause of death in children because of secondary infections attributable to MeV-induced immune suppression. Recently, we have shown that wild-type MeVs induce the suppression of protein synthesis in host cells (referred to as "shutoff" and that viral mRNAs are preferentially translated under shutoff conditions in infected cells. To determine the mechanism behind the preferential translation of viral mRNA, we focused on the 5 untranslated region (UTR of nucleocapsid (N mRNA. The La/SSB autoantigen (La was found to specifically bind to an N-5UTR probe. Recombinant La enhanced the translation of luciferase mRNA containing the N-5UTR (N-fLuc, and RNA interference of La suppressed N-fLuc translation. Furthermore, recombinant MeV lacking the La-binding motif in the N-5UTR displayed delayed viral protein synthesis and growth kinetics at an early phase of infection. These results suggest that La induced predominant translation of N mRNA via binding to its 5UTR under shutoff conditions. This is the first report on a cellular factor that specifically regulates paramyxovirus mRNA translation.

Excision of a viral reprogramming cassette by delivery of synthetic Cre mRNA

Science.gov (United States)

Loh, Yuin-Han; Yang, Jimmy Chen; De Los Angeles, Alejandro; Guo, Chunguang; Cherry, Anne; Rossi, Derrick J.; Park, In-Hyun; Daley, George Q.

2012-01-01

The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy, in vitro modeling of human disease, and drug screening. To date, most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively, transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This report describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector, which includes the coding sequences of human OCT4, SOX2, KLF4, and cMYC linked with picornaviral 2A plasmids. Moreover, after reprogramming has been achieved, this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells. PMID:22605648

Role of extrathyroidal TSHR expression in adipocyte differentiation and its association with obesity

Directory of Open Access Journals (Sweden)

Lu Sumei

2012-01-01

Full Text Available Abstract Background Obesity is known to be associated with higher risks of cardiovascular disease, metabolic syndrome, and diabetes mellitus. Thyroid-stimulating hormone (TSHR is the receptor for thyroid-stimulating hormone (TSH, or thyrotropin, the key regulator of thyroid functions. The expression of TSHR, once considered to be limited to thyrocytes, has been so far detected in many extrathyroidal tissues including liver and fat. Previous studies have shown that TSHR expression is upregulated when preadipocytes differentiate into mature adipocytes, suggestive of a possible role of TSHR in adipogenesis. However, it remains unclear whether TSHR expression in adipocytes is implicated in the pathogenesis of obesity. Methods In the present study, TSHR expression in adipose tissues from both mice and human was analyzed, and its association with obesity was evaluated. Results We here showed that TSHR expression was increased at both mRNA and protein levels when 3T3-L1 preadipocytes were induced to differentiate. Knockdown of TSHR blocked the adipocyte differentiation of 3T3-L1 preadipocytes as evaluated by Oil-red-O staining for lipid accumulation and by RT-PCR analyses of PPAR-γ and ALBP mRNA expression. We generated obesity mice (C57/BL6 by high-fat diet feeding and found that the TSHR protein expression in visceral adipose tissues from obesity mice was significantly higher in comparison with the non-obesity control mice (P Conclusion Taken together, these results suggested that TSHR is an important regulator of adipocyte differentiation. Dysregulated expression of TSHR in adipose tissues is associated with obesity, which may involve a mechanism of excess adipogenesis.

Use of isotope ratio mass spectrometry to differentiate between endogenous steroids and synthetic homologues in cattle: A review

Energy Technology Data Exchange (ETDEWEB)

Janssens, Geert, E-mail: Geert.janssens@favv.be [Federal Agency for the Safety of the Food Chain, Directorate General Laboratories, Kruidtuinlaan 55, 1000 Brussels (Belgium); Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent (Belgium); Courtheyn, Dirk [Federal Agency for the Safety of the Food Chain, Directorate General Laboratories, Kruidtuinlaan 55, 1000 Brussels (Belgium); Mangelinckx, Sven [Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent (Belgium); Prévost, Stéphanie; Bichon, Emmanuelle; Monteau, Fabrice [LUNAM Université, Oniris, Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA), F-44307 Nantes (France); De Poorter, Geert [Federal Agency for the Safety of the Food Chain, Directorate General Laboratories, Kruidtuinlaan 55, 1000 Brussels (Belgium); De Kimpe, Norbert [Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent (Belgium); Le Bizec, Bruno [LUNAM Université, Oniris, Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA), F-44307 Nantes (France)

2013-04-15

Graphical abstract: Scheme: Representation of the observed isotope ratios and the relation to exogenous and endogenous natural steroids. AS stands for “amount of steroid”. Highlights: ► The difference between endogenous and exogenous steroids is thoroughly laid out. ► Factors influencing the carbon ratio and the use of Δ{sup 13}C-values are explained. ► Implementation of GC/C/IRMS to detect steroid abuse in cattle is reviewed. ► Alternative methods and upcoming techniques are discussed. ► The differences and similarities with sports doping control are highlighted. -- Abstract: Although substantial technical advances have been achieved during the past decades to extend and facilitate the analysis of growth promoters in cattle, the detection of abuse of synthetic analogs of naturally occurring hormones has remained a challenging issue. When it became clear that the exogenous origin of steroid hormones could be traced based on the {sup 13}C/{sup 12}C isotope ratio of the substances, GC/C/IRMS has been successfully implemented to this aim since the end of the past century. However, due to the costly character of the instrumental setup, the susceptibility of the equipment to errors and the complex and time consuming sample preparation, this method is up until now only applied by a limited number of laboratories. In this review, the general principles as well as the practical application of GC/C/IRMS to differentiate between endogenous steroids and exogenously synthesized homologous compounds in cattle will be discussed in detail, and will be placed next to other existing and to be developed methods based on isotope ratio mass spectrometry. Finally, the link will be made with the field of sports doping, where GC/C/IRMS has been established within the World Anti-Doping Agency (WADA) approved methods as the official technique to differentiate between exogenous and endogenous steroids over the past few years.

Differential expression of the human thymosin-β4 gene in lymphocytes, macrophages, and granulocytes

International Nuclear Information System (INIS)

Gondo, H.; Kudo, J.; White, J.W.; Barr, C.; Selvanayagam, P.; Saunders, G.F.

1987-01-01

A cDNA clone encoding human thymosin-β 4 was isolated from a cDNA library prepared from peripheral blood leukocytes of a patient with acute lymphocytic leukemia. This clone contained the entire coding sequence of 43 amino acid residues of thymosin-β 4 and had an initiation codon and two termination codons. The amino acid and nucleotide sequences in the coding region were well conserved between rat and human. No signal peptide was found in the deduced protein sequence. Human thymosin-β 4 mRNA, approximately 830 nucleotides in length, was about 30 nucleotides larger than rat thymosin-β 4 mRNA. Expression of the human thymosin-β 4 gene in various primary myeloid and lymphoid malignant cells and in a few human hemopoietic cell lines was studied. Northern blot analyses of different neoplastic B lymphocytes revealed that steady state levels of thymosin-β 4 mRNA varied as a function of differentiation stage. Thymosin-β 4 mRNA levels were decreased in myeloma cells as are class II human leukocyte antigen, Fc receptor, and complement receptor, suggesting a relationship between thymosin-β 4 and the immune response. Treatment of THP-1 cells, a human monocytic cell line, with recombinant human interferon-γ reduced the levels of thymosin-β 4 mRNA. The pattern of thymosin-β 4 gene expression suggests that it may play a fundamental role in the host defense mechanism

RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

KAUST Repository

Köster, Tino

2017-04-13

RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

KAUST Repository

Kö ster, Tino; Marondedze, Claudius; Meyer, Katja; Staiger, Dorothee

2017-01-01

RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

Postage for the messenger: Designating routes for Nuclear mRNA Export

Science.gov (United States)

Natalizio, Barbara J.; Wente, Susan R.

2013-01-01

Transcription of messenger(m) RNA occurs in the nucleus, making the translocation of mRNA across the nuclear envelope (NE) boundary a critical determinant of proper gene expression and cell survival. A major mRNA export route occurs via the NXF1-dependent pathway through the nuclear pore complexes (NPCs) embedded in the NE. However, recent findings have discovered new evidence supporting the existence of multiple mechanisms for crossing the NE, including both NPC-mediated and NE budding-mediated pathways. An analysis of the trans-acting factors and cis components that define these pathways reveals shared elements as well as mechanistic differences. We review here the current understanding of the mechanisms that characterize each pathway and highlight the determinants that influence mRNA transport fate. PMID:23583578

Integrated analysis of miRNA and mRNA expression in childhood medulloblastoma compared with neural stem cells.

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Laura A Genovesi

Full Text Available Medulloblastoma (MB is the most common malignant brain tumor in children and a leading cause of cancer-related mortality and morbidity. Several molecular sub-types of MB have been identified, suggesting they may arise from distinct cells of origin. Data from animal models indicate that some MB sub-types arise from multipotent cerebellar neural stem cells (NSCs. Hence, microRNA (miRNA expression profiles of primary MB samples were compared to CD133+ NSCs, aiming to identify deregulated miRNAs involved in MB pathogenesis. Expression profiling of 662 miRNAs in primary MB specimens, MB cell lines, and human CD133+ NSCs and CD133- neural progenitor cells was performed by qRT-PCR. Clustering analysis identified two distinct sub-types of MB primary specimens, reminiscent of sub-types obtained from their mRNA profiles. 21 significantly up-regulated and 12 significantly down-regulated miRNAs were identified in MB primary specimens relative to CD133+ NSCs (p<0.01. The majority of up-regulated miRNAs mapped to chromosomal regions 14q32 and 17q. Integration of the predicted targets of deregulated miRNAs with mRNA expression data from the same specimens revealed enrichment of pathways regulating neuronal migration, nervous system development and cell proliferation. Transient over-expression of a down-regulated miRNA, miR-935, resulted in significant down-regulation of three of the seven predicted miR-935 target genes at the mRNA level in a MB cell line, confirming the validity of this approach. This study represents the first integrated analysis of MB miRNA and mRNA expression profiles and is the first to compare MB miRNA expression profiles to those of CD133+ NSCs. We identified several differentially expressed miRNAs that potentially target networks of genes and signaling pathways that may be involved in the transformation of normal NSCs to brain tumor stem cells. Based on this integrative approach, our data provide an important platform for future

Possible promotion of neuronal differentiation in fetal rat brain neural progenitor cells after sustained exposure to static magnetism.

Science.gov (United States)

Nakamichi, Noritaka; Ishioka, Yukichi; Hirai, Takao; Ozawa, Shusuke; Tachibana, Masaki; Nakamura, Nobuhiro; Takarada, Takeshi; Yoneda, Yukio

2009-08-15

We have previously shown significant potentiation of Ca(2+) influx mediated by N-methyl-D-aspartate receptors, along with decreased microtubules-associated protein-2 (MAP2) expression, in hippocampal neurons cultured under static magnetism without cell death. In this study, we investigated the effects of static magnetism on the functionality of neural progenitor cells endowed to proliferate for self-replication and differentiate into neuronal, astroglial, and oligodendroglial lineages. Neural progenitor cells were isolated from embryonic rat neocortex and hippocampus, followed by culture under static magnetism at 100 mT and subsequent determination of the number of cells immunoreactive for a marker protein of particular progeny lineages. Static magnetism not only significantly decreased proliferation of neural progenitor cells without affecting cell viability, but also promoted differentiation into cells immunoreactive for MAP2 with a concomitant decrease in that for an astroglial marker, irrespective of the presence of differentiation inducers. In neural progenitors cultured under static magnetism, a significant increase was seen in mRNA expression of several activator-type proneural genes, such as Mash1, Math1, and Math3, together with decreased mRNA expression of the repressor type Hes5. These results suggest that sustained static magnetism could suppress proliferation for self-renewal and facilitate differentiation into neurons through promoted expression of activator-type proneural genes by progenitor cells in fetal rat brain.

HOXB7 mRNA is overexpressed in pancreatic ductal adenocarcinomas and its knockdown induces cell cycle arrest and apoptosis

International Nuclear Information System (INIS)

Chile, Thais; Bacchella, Telésforo; Giorgi, Ricardo Rodrigues; Fortes, Maria Angela Henriques Zanella; Corrêa-Giannella, Maria Lúcia Cardillo; Brentani, Helena Paula; Maria, Durvanei Augusto; Puga, Renato David; Paula, Vanessa de Jesus R de; Kubrusly, Marcia Saldanha; Novak, Estela Maria

2013-01-01

Human homeobox genes encode nuclear proteins that act as transcription factors involved in the control of differentiation and proliferation. Currently, the role of these genes in development and tumor progression has been extensively studied. Recently, increased expression of HOXB7 homeobox gene (HOXB7) in pancreatic ductal adenocarcinomas (PDAC) was shown to correlate with an invasive phenotype, lymph node metastasis and worse survival outcomes, but no influence on cell proliferation or viability was detected. In the present study, the effects arising from the knockdown of HOXB7 in PDAC cell lines was investigated. Real time quantitative PCR (qRT-PCR) (Taqman) was employed to assess HOXB7 mRNA expression in 29 PDAC, 6 metastatic tissues, 24 peritumoral tissues and two PDAC cell lines. siRNA was used to knockdown HOXB7 mRNA in the cell lines and its consequences on apoptosis rate and cell proliferation were measured by flow cytometry and MTT assay respectively. Overexpression of HOXB7 mRNA was observed in the tumoral tissues and in the cell lines MIA PaCa-2 and Capan-1. HOXB7 knockdown elicited (1) an increase in the expression of the pro-apoptotic proteins BAX and BAD in both cell lines; (2) a decrease in the expression of the anti-apoptotic protein BCL-2 and in cyclin D1 and an increase in the number of apoptotic cells in the MIA PaCa-2 cell line; (3) accumulation of cell in sub-G1 phase in both cell lines; (4) the modulation of several biological processes, especially in MIA PaCa-2, such as proteasomal ubiquitin-dependent catabolic process and cell cycle. The present study confirms the overexpression of HOXB7 mRNA expression in PDAC and demonstrates that decreasing its protein level by siRNA could significantly increase apoptosis and modulate several biological processes. HOXB7 might be a promising target for future therapies

Differentially displayed expressed sequence tags in Melipona scutellaris (Hymenoptera, Apidae, Meliponini) development.

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Santana, Flávia A; Nunes, Francis M F; Vieira, Carlos U; Machado, Maria Alice M S; Kerr, Warwick E; Silva, Wilson A; Bonetti, Ana Maria

2006-03-01

We have compared gene expression, using the Differential Display Reverse Transcriptase-Polymerase Chain Reaction (DDRT-PCR) technique, by means of mRNA profile in Melipona scutellaris during ontogenetic postembryonic development, in adult worker, and in both Natural and Juvenile Hormone III-induced adult queen. Six, out of the nine ESTs described here, presented differentially expressed in the phases L1 or L2, or even in both of them, suggesting that key mechanisms to the development of Melipona scutellaris are regulated in these stages. The combination HT11G-AP05 revealed in L1 and L2 a product which matches to thioredoxin reductase protein domain in the Clostridium sporogenes, an important protein during cellular oxidoreduction processes. This study represents the first molecular evidence of differential gene expression profiles toward a description of the genetic developmental traits in the genus Melipona.

Differential patterns of serum concentration and adipose tissue expression of chemerin in obesity: adipose depot specificity and gender dimorphism.

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Alfadda, Assim A; Sallam, Reem M; Chishti, Muhammad Azhar; Moustafa, Amr S; Fatma, Sumbul; Alomaim, Waleed S; Al-Naami, Mohammed Y; Bassas, Abdulelah F; Chrousos, George P; Jo, Hyunsun

2012-06-01

Chemerin, a recognized chemoattractant, is expressed in adipose tissue and plays a role in adipocytes differentiation and metabolism. Gender- and adipose tissue-specific differences in human chemerin expression have not been well characterized. Therefore, these differences were assessed in the present study. The body mass index (BMI) and the circulating levels of chemerin and other inflammatory, adiposity and insulin resistance markers were assessed in female and male adults of varying degree of obesity. Chemerin mRNA expression was also measured in paired subcutaneous and visceral adipose tissue samples obtained from a subset of the study subjects. Serum chemerin concentrations correlated positively with BMI and serum leptin levels and negatively with high density lipoprotein (HDL)-cholesterol levels. No correlation was found between serum chemerin concentrations and fasting glucose, total cholesterol, low density lipoprotein (LDL)-cholesterol, triglycerides, insulin, C-reactive protein or adiponectin. Similarly, no relation was observed with the homeostasis model assessment for insulin resistance (HOMA-IR) values. Gender- and adipose tissue-specific differences were observed in chemerin mRNA expression levels, with expression significantly higher in women than men and in subcutaneous than visceral adipose tissue. Interestingly, we found a significant negative correlation between circulating chemerin levels and chemerin mRNA expression in subcutaneous fat. Among the subjects studied, circulating chemerin levels were associated with obesity markers but not with markers of insulin resistance. At the tissue level, fat depot-specific differential regulation of chemerin mRNA expression might contribute to the distinctive roles of subcutaneous vs. visceral adipose tissue in human obesity.

Stereoselectivity of the demethylation of nicotine piperidine homologues by Nicotiana plumbaginifolia cell suspension cultures.

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Bartholomeusz, Trixie Ann; Molinié, Roland; Roscher, Albrecht; Felpin, François-Xavier; Gillet, Françoise; Lebreton, Jacques; Mesnard, François; Robins, Richard J

2005-08-01

The metabolism of (R,S)-N-methylanabasine and (R,S)-N-methylanatabine has been studied in a cell suspension culture of Nicotiana plumbaginifolia. Both substrates are effectively demethylated, anabasine or anatabine, respectively, accumulating in the medium. Similarly, there is strong stereoselectivity for the (R)-isomers of both substrates. The kinetics of metabolism of (R,S)-N-methylanabasine differ significantly from those of nicotine in that no further degradation of the initial demethylation product occurs. (R,S)-N-Methylanatabine, however, shows kinetics closer to those of nicotine, with loss of alkaloid from the system. Further more, (R,S)-N-methylanabasine does not diminish (S)-nicotine demethylation, indicating a lack of competition. However, the metabolism of (S)-nicotine is affected by the presence of (R,S)-N-methylanabasine. Hence, the demethylation of the piperidine homologues of nicotine is seen to be similar but not identical to that of the pyridine analogues. The implications of these different metabolic profiles in relation to the demethylation activity are discussed.

The potential role of IGF-I receptor mRNA in rats with diabetic retinopathy

Institute of Scientific and Technical Information of China (English)

匡洪宇; 邹伟; 刘丹; 史榕荇; 程丽华; 殷慧清; 刘晓民

2003-01-01

Objective To evaluate the potential role of insulin-like growth factor-1 receptor mRNA(IGF-IR mRNA) in the onset and development of retinopathy in diabetic rats.Methods A diabetic model was duplicated in Wistar rats. The early changes in the retina were examined using light and transmission electron microscopy. Expression of IGF-IR mRNA was analyzed using in situ hybridization.Results Weak expression of IGF-IR mRNA(5%) was found in retinas of normal rats, but was significantly increased (15% and 18%) in the retinas of diabetic rats after 3 and 6 months of diabetes (P＜0.01). In situ hybridization and morphological study demonstrated that there was a positive correlation between IGF-IR mRNA expression and retinal changes at various stages.Conclusion Increased IGF-IR mRNA might play an important role in the onset and development of diabetic retinopathy.

Ensembles generated from crystal structures of single distant homologues solve challenging molecular-replacement cases in AMPLE.

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Rigden, Daniel J; Thomas, Jens M H; Simkovic, Felix; Simpkin, Adam; Winn, Martyn D; Mayans, Olga; Keegan, Ronan M

2018-03-01

Molecular replacement (MR) is the predominant route to solution of the phase problem in macromolecular crystallography. Although routine in many cases, it becomes more effortful and often impossible when the available experimental structures typically used as search models are only distantly homologous to the target. Nevertheless, with current powerful MR software, relatively small core structures shared between the target and known structure, of 20-40% of the overall structure for example, can succeed as search models where they can be isolated. Manual sculpting of such small structural cores is rarely attempted and is dependent on the crystallographer's expertise and understanding of the protein family in question. Automated search-model editing has previously been performed on the basis of sequence alignment, in order to eliminate, for example, side chains or loops that are not present in the target, or on the basis of structural features (e.g. solvent accessibility) or crystallographic parameters (e.g. B factors). Here, based on recent work demonstrating a correlation between evolutionary conservation and protein rigidity/packing, novel automated ways to derive edited search models from a given distant homologue over a range of sizes are presented. A variety of structure-based metrics, many readily obtained from online webservers, can be fed to the MR pipeline AMPLE to produce search models that succeed with a set of test cases where expertly manually edited comparators, further processed in diverse ways with MrBUMP, fail. Further significant performance gains result when the structure-based distance geometry method CONCOORD is used to generate ensembles from the distant homologue. To our knowledge, this is the first such approach whereby a single structure is meaningfully transformed into an ensemble for the purposes of MR. Additional cases further demonstrate the advantages of the approach. CONCOORD is freely available and computationally inexpensive, so

Naturally occurring BRCA2 alternative mRNA splicing events in clinically relevant samples

DEFF Research Database (Denmark)

Fackenthal, James D; Yoshimatsu, Toshio; Zhang, Bifeng

2016-01-01

patterns and thereby disrupt gene function. mRNA analyses are therefore among the tests used to interpret the clinical significance of some genetic variants. However, these could be confounded by the appearance of naturally occurring alternative transcripts unrelated to germline sequence variation...... to characterise the spectrum of naturally occurring BRCA2 mRNA alternate-splicing events. METHODS: mRNA was prepared from several blood and breast tissue-derived cells and cell lines by contributing ENIGMA laboratories. cDNA representing BRCA2 alternate splice sites was amplified and visualised using capillary...... or agarose gel electrophoresis, followed by sequencing. RESULTS: We demonstrate the existence of 24 different BRCA2 mRNA alternate-splicing events in lymphoblastoid cell lines and both breast cancer and non-cancerous breast cell lines. CONCLUSIONS: These naturally occurring alternate-splicing events...

Crystallization and preliminary diffraction analysis of a DsbA homologue from Wolbachia pipientis

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Kurz, M. [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, St Lucia, QLD 4072 (Australia); Iturbe-Ormaetxe, I. [School of Integrative Biology, The University of Queensland, St Lucia, QLD 4072 (Australia); Jarrott, R. [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, St Lucia, QLD 4072 (Australia); O’Neill, S. L. [School of Integrative Biology, The University of Queensland, St Lucia, QLD 4072 (Australia); Byriel, K. A.; Martin, J. L., E-mail: j.martin@imb.uq.edu.au; Heras, B., E-mail: j.martin@imb.uq.edu.au [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, St Lucia, QLD 4072 (Australia)

2008-02-01

The first crystallization of a W. pipientis protein, α-DsbA1, was achieved using hanging-drop and sitting-drop vapour diffusion. α-DsbA1 is one of two DsbA homologues encoded by the Gram-negative α-proteobacterium Wolbachia pipientis, an endosymbiont that can behave as a reproductive parasite in insects and as a mutualist in medically important filarial nematodes. The α-DsbA1 protein is thought to be important for the folding and secretion of Wolbachia proteins involved in the induction of reproductive distortions. Crystals of native and SeMet α-DsbA1 were grown by vapour diffusion and belong to the monoclinic space group C2, with unit-cell parameters a = 71.4, b = 49.5, c = 69.3 Å, β = 107.0° and one molecule in the asymmetric unit (44% solvent content). X-ray data were recorded from native crystals to a resolution of 2.01 Å using a copper anode and data from SeMet α-DsbA1 crystals were recorded to 2.45 Å resolution using a chromium anode.

FSHD myoblasts fail to downregulate intermediate filament protein vimentin during myogenic differentiation.

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Lipinski M.

2011-10-01

Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is an autosomal dominant hereditary neuromuscular disorder. The clinical features of FSHD include weakness of the facial and shoulder girdle muscles followed by wasting of skeletal muscles of the pelvic girdle and lower extremities. Although FSHD myoblasts grown in vitro can be induced to differentiate into myotubes by serum starvation, the resulting FSHD myotubes have been shown previously to be morphologically abnormal. Aim. In order to find the cause of morphological anomalies of FSHD myotubes we compared in vitro myogenic differentiation of normal and FSHD myoblasts at the protein level. Methods. We induced myogenic differentiation of normal and FSHD myoblasts by serum starvation. We then compared protein extracts from proliferating myoblasts and differentiated myotubes using SDS-PAGE followed by mass spectrometry identification of differentially expressed proteins. Results. We demonstrated that the expression of vimentin was elevated at the protein and mRNA levels in FSHD myotubes as compared to normal myotubes. Conclusions. We demonstrate for the first time that in contrast to normal myoblasts, FSHD myoblasts fail to downregulate vimentin after induction of in vitro myogenic differentiation. We suggest that vimentin could be an easily detectable marker of FSHD myotubes

FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia.

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Nasilowska-Adamska, Barbara; Solarska, Iwona; Paluszewska, Monika; Malinowska, Iwona; Jedrzejczak, Wieslaw W; Warzocha, Krzysztof

2014-04-01

Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are aberrations associated with leukemia which indicate unsatisfactory prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined. We have analyzed the expression of MDR-1, multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) messenger RNA (mRNA) in relation to the mutational status of FLT3-ITD and MLL-PTD in 185 acute myeloid leukemia (AML) adult patients. The real-time quantitative polymerase chain reaction method was performed to assess the expression of the MDR-1, MRP-1, BCRP, and LRP mRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. Significantly higher expressions of MDR-1 mRNA were found in patients who did not harbor FLT3-ITD (0.20 vs. 0.05; p = 0.0001) and MRP-1 mRNA in patients with this mutation (0.96 vs. 0.70; p = 0.002) and of BCRP mRNA in patients with MLL-PTD (0.61 vs. 0.38; p = 0.03). In univariate analysis, the high expression of MDR-1 mRNA (≥0.1317) negatively influenced the outcome of induction therapy (p = 0.05), whereas the high expression of BCRP mRNA (≥1.1487) was associated with a high relapse rate (RR) (p = 0.013). We found that the high expression of MDR-1 (≥0.1317), MRP-1 (≥0.8409), and BCRP mRNA (≥1.1487) significantly influenced disease-free survival (DFS; p = 0.059, 0.032, and 0.009, respectively) and overall survival (0.048, 0.014, and 0.059, respectively). Moreover, a high expression of BCRP mRNA (≥1.1487) proved to be an independent prognostic factor for RR (p = 0.01) and DFS (p = 0.002) in multivariate analysis. The significant correlation between the expression of MDR-1, MRP-1, and BCRP mRNA and FLT3-ITD or MLL-PTD in AML patients requires further investigation.

Differential Gene Expression in Ovaries of Qira Black Sheep and Hetian Sheep Using RNA-Seq Technique

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Jia, Bin; Zhang, Yong Sheng; Wang, Xu Hai; Zeng, Xian Cun

2015-01-01

The Qira black sheep and the Hetian sheep are two local breeds in the Northwest of China, which are characterized by high-fecundity and low-fecundity breed respectively. The elucidation of mRNA expression profiles in the ovaries among different sheep breeds representing fecundity extremes will helpful for identification and utilization of major prolificacy genes in sheep. In the present study, we performed RNA-seq technology to compare the difference in ovarian mRNA expression profiles between Qira black sheep and Hetian sheep. From the Qira black sheep and the Hetian sheep libraries, we obtained a total of 11,747,582 and 11,879,968 sequencing reads, respectively. After aligning to the reference sequences, the two libraries included 16,763 and 16,814 genes respectively. A total of 1,252 genes were significantly differentially expressed at Hetian sheep compared with Qira black sheep. Eight differentially expressed genes were randomly selected for validation by real-time RT-PCR. This study provides a basic data for future research of the sheep reproduction. PMID:25790350

Differential gene expression in ovaries of Qira black sheep and Hetian sheep using RNA-Seq technique.

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Han Ying Chen

Full Text Available The Qira black sheep and the Hetian sheep are two local breeds in the Northwest of China, which are characterized by high-fecundity and low-fecundity breed respectively. The elucidation of mRNA expression profiles in the ovaries among different sheep breeds representing fecundity extremes will helpful for identification and utilization of major prolificacy genes in sheep. In the present study, we performed RNA-seq technology to compare the difference in ovarian mRNA expression profiles between Qira black sheep and Hetian sheep. From the Qira black sheep and the Hetian sheep libraries, we obtained a total of 11,747,582 and 11,879,968 sequencing reads, respectively. After aligning to the reference sequences, the two libraries included 16,763 and 16,814 genes respectively. A total of 1,252 genes were significantly differentially expressed at Hetian sheep compared with Qira black sheep. Eight differentially expressed genes were randomly selected for validation by real-time RT-PCR. This study provides a basic data for future research of the sheep reproduction.

Prognostic significance of CDH13 hypermethylation and mRNA in NSCLC

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Xue R

2014-10-01

Full Text Available Ruilin Xue,1 Cuili Yang,1 Fang Zhao,2 Dejia Li1 1Global Health Institute, School of Public Health, 2Zhongnan Hospital, Wuhan University, Wuhan, People's Republic of ChinaAbstract: Aberrant methylation of CpG dinucleotides is a commonly observed epigenetic modification in human cancer. Thus, detection of aberrant gene promoter methylation as a tool for diagnosis of tumors or as a prognostic marker has been widely described for many types of cancers, including nonsmall cell lung cancer (NSCLC. Emerging evidence indicates that CDH13 is a candidate tumor suppressor in several types of human tumors, including NSCLC. However, the correlation between CDH13 hypermethylation and clinicopathological characteristics of NSCLC remains unclear. In the current study, we conducted a systematic review and meta-analysis to quantitatively evaluate the effects of CDH13 hypermethylation on the incidence of NSCLC and clinicopathological characteristics. Final analysis of 803 NSCLC patients from eleven eligible studies was performed. CDH13 hypermethylation was observed to be significantly higher in NSCLC than in normal lung tissue, with the pooled odds ratio (OR from seven studies including 448 NSCLC and 345 normal lung tissue (OR, 7.85; 95% confidence interval, 5.12–12.03; P<0.00001. CDH13 hypermethylation was also associated with pathological types. The pooled OR was obtained from four studies, including 111 squamous cell carcinoma and 106 adenocarcinoma (OR, 0.35; 95% confidence interval, 0.19–0.66; P=0.001, which indicated that CDH13 hypermethylation plays a more important role in the pathogenesis of adenocarcinoma. NSCLC with CDH13 hypermethylation was found more frequently in poorly differentiated NSCLC patients. NSCLC patients with CDH13 hypermethylation had a lower survival rate than those without CDH13 hypermethylation. In addition, CDH13 mRNA high expression was found to correlate with better overall survival for all NSCLC patients followed for 20 years

Rift Valley fever virus NSS gene expression correlates with a defect in nuclear mRNA export.

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Copeland, Anna Maria; Van Deusen, Nicole M; Schmaljohn, Connie S

2015-12-01

We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NSS gene, but not the N, GN or NSM genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NSS, confirming that expression of NSS is likely responsible for this phenomenon. Published by Elsevier Inc.

Enrichment of Female Germline Stem Cells from Mouse Ovaries Using the Differential Adhesion Method

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Meng Wu

2018-04-01

Full Text Available Background/Aims: The isolation and establishment of female germline stem cells (FGSCs is controversial because of questions regarding the reliability and stability of the isolation method using antibody targeting mouse vasa homologue (MVH, and the molecular mechanism of FGSCs self-renewal remains unclear. Thus, there needs to be a simple and reliable method for sorting FGSCs to study them. Methods: We applied the differential adhesion method to enrich FGSCs (DA-FGSCs from mouse ovaries. Through four rounds of purification and 7-9 subsequent passages, DA-FGSC lines were established. In addition, we assessed the role of the phosphoinositide-3 kinase (PI3K-AKT pathway in regulating FGSC self-renewal. Results: The obtained DA-FGSCs spontaneously differentiated into oocyte-like cells in vitro and formed functional eggs in vivo that were fertilized and produced healthy offspring. AKT was rapidly phosphorylated when the proliferation rate of FGSCs increased after 10 passages, and the addition of a chemical PI3K inhibitor prevented FGSCs self-renewal. Furthermore, over-expression of AKT-induced proliferation and differentiation of FGSCs, c-Myc, Oct-4 and Gdf-9 levels were increased. Conclusions: The differential adhesion method provides a more feasible approach and is an easier procedure to establish FGSC lines than traditional methods. The AKT pathway plays an important role in regulation of the proliferation and maintenance of FGSCs. These findings could help promote stem cell studies and provide a better understanding of causes of ovarian infertility, thereby providing potential treatments for infertility.

Enrichment of Female Germline Stem Cells from Mouse Ovaries Using the Differential Adhesion Method.

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Wu, Meng; Xiong, Jiaqiang; Ma, Lingwei; Lu, Zhiyong; Qin, Xian; Luo, Aiyue; Zhang, Jinjin; Xie, Huan; Shen, Wei; Wang, Shixuan

2018-04-28

The isolation and establishment of female germline stem cells (FGSCs) is controversial because of questions regarding the reliability and stability of the isolation method using antibody targeting mouse vasa homologue (MVH), and the molecular mechanism of FGSCs self-renewal remains unclear. Thus, there needs to be a simple and reliable method for sorting FGSCs to study them. We applied the differential adhesion method to enrich FGSCs (DA-FGSCs) from mouse ovaries. Through four rounds of purification and 7-9 subsequent passages, DA-FGSC lines were established. In addition, we assessed the role of the phosphoinositide-3 kinase (PI3K)-AKT pathway in regulating FGSC self-renewal. The obtained DA-FGSCs spontaneously differentiated into oocyte-like cells in vitro and formed functional eggs in vivo that were fertilized and produced healthy offspring. AKT was rapidly phosphorylated when the proliferation rate of FGSCs increased after 10 passages, and the addition of a chemical PI3K inhibitor prevented FGSCs self-renewal. Furthermore, over-expression of AKT-induced proliferation and differentiation of FGSCs, c-Myc, Oct-4 and Gdf-9 levels were increased. The differential adhesion method provides a more feasible approach and is an easier procedure to establish FGSC lines than traditional methods. The AKT pathway plays an important role in regulation of the proliferation and maintenance of FGSCs. These findings could help promote stem cell studies and provide a better understanding of causes of ovarian infertility, thereby providing potential treatments for infertility. © 2018 The Author(s). Published by S. Karger AG, Basel.

Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein Augments mRNA Translation.

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Jeeva, Subbiah; Cheng, Erdong; Ganaie, Safder S; Mir, Mohammad A

2017-08-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus of the Bunyaviridae family, causing severe illness with high mortality rates in humans. Here, we demonstrate that CCHFV nucleocapsid protein (CCHFV-NP) augments mRNA translation. CCHFV-NP binds to the viral mRNA 5' untranslated region (UTR) with high affinity. It facilitates the translation of reporter mRNA both in vivo and in vitro with the assistance of the viral mRNA 5' UTR. CCHFV-NP equally favors the translation of both capped and uncapped mRNAs, demonstrating the independence of this translation strategy on the 5' cap. Unlike the canonical host translation machinery, inhibition of eIF4F complex, an amalgam of three initiation factors, eIF4A, eIF4G, and eIF4E, by the chemical inhibitor 4E1RCat did not impact the CCHFV-NP-mediated translation mechanism. However, the proteolytic degradation of eIF4G alone by the human rhinovirus 2A protease abrogated this translation strategy. Our results demonstrate that eIF4F complex formation is not required but eIF4G plays a critical role in this translation mechanism. Our results suggest that CCHFV has adopted a unique translation mechanism to facilitate the translation of viral mRNAs in the host cell cytoplasm where cellular transcripts are competing for the same translation apparatus. IMPORTANCE Crimean-Congo hemorrhagic fever, a highly contagious viral disease endemic to more than 30 countries, has limited treatment options. Our results demonstrate that NP favors the translation of a reporter mRNA harboring the viral mRNA 5' UTR. It is highly likely that CCHFV uses an NP-mediated translation strategy for the rapid synthesis of viral proteins during the course of infection. Shutdown of this translation mechanism might selectively impact viral protein synthesis, suggesting that an NP-mediated translation strategy is a target for therapeutic intervention against this viral disease. Copyright © 2017 American Society for Microbiology.

The mRNA expression of XRCC repair genes in mice after γ-ray radiation

International Nuclear Information System (INIS)

Wang Qin; Yue Jingyin; Li Jin; Mu Chuanjie; Fan Feiyue

2006-01-01

Objective: To investigate the role of XRCC repair genes in radioresistance of IRM-2 inbred mice. Methods: Northern hybridization was used to measure mRNA expression of XRCC1 and XRCC5 genes in IRM-2 inbred mice. ICR/JCL and 615 after exposure to different doses of γ-ray radiation at different postirradiation time. Results: The levels of XRCC1 and XRCC5 mRNA expression in control IRM-2 mice were higher significantly than those in their control parental mice (P<0.01 and P<0.05). The mRNA expression of XRCC genes in ICR/JCL and 615 mice all increased to some extent after exposure 1, 2 and 4 Gy radiation. But the levels were significantly higher at 2h postirradiation (P<0.05) . The levels of XRCC mRNA expression in IRM-2 mice did not increase significnatly compared with the control mice after exposure 1 and 2 Gy radiation. But the levels of XRCC1 and XRCC5 mRNA expression increased markedly at 4Gy 1h postirradiation (P<0.05 and P<0.01). Conclusion: The basal levels of XRCC1 and XRCC5 mRNA expression in IRM-2 mice were high. The high level of XRCC5 mRNA expression was involved in the repair of DNA double strand breaks induced by higher dose radiation, which perhaps was one of radioresistance causes of IRM-2 mice. (authors)

All-in-one detector of circulating mRNA based on a smartphone

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Cmiel, Vratislav; Gumulec, Jaromir; Svoboda, Ondrej; Raudenska, Martina; Hudcova, Kristyna; Sekora, Jiri; Balogh, Jaroslav; Masarik, Michal; Provaznik, Ivo

2016-03-01

Metallothionein is significantly elevated in various tumors, notably in prostate cancer on both mRNA and protein level. We demonstrated a strong predictive potential of free circulating metallothionein 2A isoform mRNA for patients with this cancer. Circulating mRNA detection relies on expensive equipment and requires high level of expertise. In this work we developed compact "all-in-one" laboratory system which replace microvolume spectrophotometer, thermocycler and realtime PCR machines. We managed to design and construct a microprocessor controlled heating/cooling chamber that ensures required temperature gradient. The chamber includes implemented optical system to enable fluorescence excitation and fluorescence analysis using a smart-phone.

Relative workload determines exercise-induced increases in PGC-1alpha mRNA

DEFF Research Database (Denmark)

Nordsborg, Nikolai Baastrup; Lundby, Carsten; Leick, Lotte

2010-01-01

INTRODUCTION:: The hypothesis that brief intermittent exercise induced increases in human skeletal muscle metabolic mRNA is dependent on relative workload was investigated. METHODS:: Trained (n=10) and untrained (n=8) subjects performed exhaustive intermittent cycling exercise (4x4 min @ 85% of VO2...... peak, interspersed by 3 min). Trained subjects also performed the intermittent exercise at the same absolute workload as untrained, corresponding to 70% of VO2 peak (n=6). RESULTS:: Exercise at 85% of VO2 peak elevated (P... and untrained, respectively. PGC-1alpha mRNA expression was increased (Pelevated (3.1+/-0.7 mM) and PGC-1alpha mRNA content was less (P

Expression of galectin-9 mRNA in obese children with polymorphism of the lactase gene

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A.E. Abaturov

2018-02-01

Full Text Available Background. The aim of the study is to investigate the association of expression of galectin-9 (Gal-9 mRNA and lactose malabsorption in obese children with polymorphism (SNP of the lactase gene (LCT and to study the efficacy of lactase deficiency therapy using exogenous lactase preparations. Materials and methods. Seventy obese children (BMI > 95th percentile and 16 children without obesity aged 6–18 years were examined. There was studied SNP LCT (material for investigation venous blood by real-time PCR, expression of Gal-9 mRNA (study material buccal epithelium by real-time PCR with reverse transcription, malabsorption of lactose by hydrogen breath test (HBT. Among obese children, 38 children with genotype C/C 13910 presented the first observation group, 32 children with phenotype identical genotypes C/T 13910 and T/T 13910, p > 0.05, presented the second group. Children from the first observation group also determined the level of expression of Gal-9 mRNA and lactose malabsorption after using exogenous lactase preparations. Results. The genotype C/C 13910 was determined in 38 (54.3 %, genotype C/T 13910 in 22 (31.4 % and genotype T/T in 10 (14.3 % patients. Malabsorption of lactose in children with genotype C/C 13910 averaged 32.7 ± 10.4 pmm, in children with genotypes C/T 13910 — 26.3 ± 4.9 pmm (p > 0.05 and with genotype T/T 13910 and was absent in children without obesity (p < 0.05. The average level of expression of Gal-9 mRNA in children with genotype C/C 13910 was 564.3 ± 32.8 RU DmRNA Gal-9/mRNA actin, in children with genotypes C/T and T/T 13910 — 61.04 ± 15.30 RU DmRNA Gal-9/mRNA actin, p < 0.01. It is of great importance that the children with genotype C/C 13910 and lactose malabsorption (n = 20 had the lowest average level of expression of Gal-9 mRNA (42.47 ± 13.30 RU DmRNA Gal-9/mRNA actin whereas the children with genotype C/C 13910 and without lactose malabsorption (n =18 had the largest level (1086

Photodynamic antisense regulation of mRNA having a point mutation with psoralen-conjugated oligonucleotide.

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Higuchi, Maiko; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira

2008-01-01

Nucleic acid-based drugs, such as antisense oligonucleotide, ribozyme, and small interfering RNA, are specific compounds that inhibit gene expression at the post-transcriptional level. To develop more effective nucleic acid-based drugs, we focused on photo-reactive antisense oligonucleotides. We have optimized the structure of psoralen-conjugated oligonucleotide to improve their sequence selectivity and photo-crosslinking efficiency. Previously, we reported that photo reactive oligonucleotides containing 2'-O-psoralenyl-methoxyethyl adenosine (2'-Ps-eom) showed drastic photo-reactivity with a strictly sequence specific manner in vitro. In this report, we evaluated the binding ability toward intracellular target mRNA. The 2'-Ps-eom selectively photo-cross-linked to the target mRNA extracted from cells. The 2'-Ps-eom also cross-linked to target mRNA in cells. Furthermore, 2'-Ps-eom did not cross-link to mRNA having a mismatch base. These results suggest that 2'-Ps-eom is a powerful antisense molecule to inhibit the expression of mRNA having a point mutation.

ZMS regulation of M2 muscarinic receptor mRNA stability requires protein factor

International Nuclear Information System (INIS)

Zhang Yongfang; Xia Zongqin; Hu Ya'er

2010-01-01

Aim The aim of this work is to study the elevation mechanism of ZMS on muscarinic M2 receptor mRNA expression. Methods Actinomycin D was added to cultured CHOm2 cells to stop the de novo synthesis of M2 receptor mRNA and samples were taken at various times to determine the time course of mRNA of M2 receptor with real-time quantitative RT-PCR. Half-life of M2 receptor mRNA and the effect of ZMS on the half-life was obtained from the slope of the exponential curves. Cycloheximide was added at 4 h prior to and 24 h after the addition of ZMS to examine the effect of de novo protein synthesis on the action of ZMS. Results The half-life of m2 mRNA was prolonged by ZMS treatment without cycloheximide (4.75±0.54 h and 2.13 h±0.23 h for ZMS and vehicle treated groups, respectively, P<0.05). When cycloheximide was added to the culture medium 4h prior to the addition of ZMS, the effect of ZMS in prolonging the half-life of m2 mRNA disappeared (3.06 h±0.23 h and 3.00 h±l.20 h for cells with and without ZMS, respectively). However, when the ZMS was added to the medium 24h prior to the addition of cycloheximide, the action of ZMS was not abolished by cycloheximide (half-life was 5.43 h±1.13 h and 2.46 h±0.09 h for cells with and without ZMS, respectively). Conclusion These data suggest that de novo protein synthesis was required for the increase in M2 mRNA stability induced by ZMS. (authors)

UCP2 mRNA expression is dependent on glucose metabolism in pancreatic islets

International Nuclear Information System (INIS)

Dalgaard, Louise T.

2012-01-01

Highlights: ► UCP2 mRNA levels are decreased in islets of Langerhans from glucokinase deficient mice. ► UCP2 mRNA up-regulation by glucose is dependent on glucokinase. ► Absence of UCP2 increases GSIS of glucokinase heterozygous pancreatic islets. ► This may protect glucokinase deficient mice from hyperglycemic damages. -- Abstract: Uncoupling Protein 2 (UCP2) is expressed in the pancreatic β-cell, where it partially uncouples the mitochondrial proton gradient, decreasing both ATP-production and glucose-stimulated insulin secretion (GSIS). Increased glucose levels up-regulate UCP2 mRNA and protein levels, but the mechanism for UCP2 up-regulation in response to increased glucose is unknown. The aim was to examine the effects of glucokinase (GK) deficiency on UCP2 mRNA levels and to characterize the interaction between UCP2 and GK with regard to glucose-stimulated insulin secretion in pancreatic islets. UCP2 mRNA expression was reduced in GK+/− islets and GK heterozygosity prevented glucose-induced up-regulation of islet UCP2 mRNA. In contrast to UCP2 protein function UCP2 mRNA regulation was not dependent on superoxide generation, but rather on products of glucose metabolism, because MnTBAP, a superoxide dismutase mimetic, did not prevent the glucose-induced up-regulation of UCP2. Glucose-stimulated insulin secretion was increased in UCP2−/− and GK+/− islets compared with GK+/− islets and UCP2 deficiency improved glucose tolerance of GK+/− mice. Accordingly, UCP2 deficiency increased ATP-levels of GK+/− mice. Thus, the compensatory down-regulation of UCP2 is involved in preserving the insulin secretory capacity of GK mutant mice and might also be implicated in limiting disease progression in MODY2 patients.

Protein functional features are reflected in the patterns of mRNA translation speed.

Science.gov (United States)

López, Daniel; Pazos, Florencio

2015-07-09

The degeneracy of the genetic code makes it possible for the same amino acid string to be coded by different messenger RNA (mRNA) sequences. These "synonymous mRNAs" may differ largely in a number of aspects related to their overall translational efficiency, such as secondary structure content and availability of the encoded transfer RNAs (tRNAs). Consequently, they may render different yields of the translated polypeptides. These mRNA features related to translation efficiency are also playing a role locally, resulting in a non-uniform translation speed along the mRNA, which has been previously related to some protein structural features and also used to explain some dramatic effects of "silent" single-nucleotide-polymorphisms (SNPs). In this work we perform the first large scale analysis of the relationship between three experimental proxies of mRNA local translation efficiency and the local features of the corresponding encoded proteins. We found that a number of protein functional and structural features are reflected in the patterns of ribosome occupancy, secondary structure and tRNA availability along the mRNA. One or more of these proxies of translation speed have distinctive patterns around the mRNA regions coding for certain protein local features. In some cases the three patterns follow a similar trend. We also show specific examples where these patterns of translation speed point to the protein's important structural and functional features. This support the idea that the genome not only codes the protein functional features as sequences of amino acids, but also as subtle patterns of mRNA properties which, probably through local effects on the translation speed, have some consequence on the final polypeptide. These results open the possibility of predicting a protein's functional regions based on a single genomic sequence, and have implications for heterologous protein expression and fine-tuning protein function.

The ribosome structure controls and directs mRNA entry, translocation and exit dynamics

International Nuclear Information System (INIS)

Kurkcuoglu, Ozge; Doruker, Pemra; Jernigan, Robert L; Sen, Taner Z; Kloczkowski, Andrzej

2008-01-01

The protein-synthesizing ribosome undergoes large motions to effect the translocation of tRNAs and mRNA; here, the domain motions of this system are explored with a coarse-grained elastic network model using normal mode analysis. Crystal structures are used to construct various model systems of the 70S complex with/without tRNA, elongation factor Tu and the ribosomal proteins. Computed motions reveal the well-known ratchet-like rotational motion of the large subunits, as well as the head rotation of the small subunit and the high flexibility of the L1 and L7/L12 stalks, even in the absence of ribosomal proteins. This result indicates that these experimentally observed motions during translocation are inherently controlled by the ribosomal shape and only partially dependent upon GTP hydrolysis. Normal mode analysis further reveals the mobility of A- and P-tRNAs to increase in the absence of the E-tRNA. In addition, the dynamics of the E-tRNA is affected by the absence of the ribosomal protein L1. The mRNA in the entrance tunnel interacts directly with helicase proteins S3 and S4, which constrain the mRNA in a clamp-like fashion, as well as with protein S5, which likely orients the mRNA to ensure correct translation. The ribosomal proteins S7, S11 and S18 may also be involved in assuring translation fidelity by constraining the mRNA at the exit site of the channel. The mRNA also interacts with the 16S 3' end forming the Shine–Dalgarno complex at the initiation step; the 3' end may act as a 'hook' to reel in the mRNA to facilitate its exit

Regulation of the growth hormone (GH) receptor and GH-binding protein mRNA

Energy Technology Data Exchange (ETDEWEB)

Kaji, Hidesuke; Ohashi, Shin-Ichirou; Abe, Hiromi; Chihara, Kazuo [Kobe Univ. School of Medicine, Kobe (Japan)

1994-12-31

In fasting rats, a transient increase in growth hormone-binding protein (GHBP) mRNA levels was observed after 1 day, in muscle, heart, and liver, but not in fat tissues. The liver GH receptor (GHR) mRNA level was significantly increased after 1 day (but not after 5 days) of bovine GH (bGH) treatment in fed rats. Both the liver GHR mRNA level and the net increment of plasma IGF-I markedly decreased after 5 days of bGH administration in fasting rats. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in a different way and that the expression of GHBP mRNA is regulated differently between tissues, at least in rats. The results also suggest that refractoriness to GH in a sustained fasting state might be beneficial in preventing anabolic effects of GH. In humans, GHR mRNA in lymphocytes, from subjects with either GH-deficiency or acromegaly, could be detected by the reverse transcription-polymerase chain reaction method. In one patient with partial GH insensitivity, a heterozygous missense mutation (P561T) was identified in the cytoplasmic domain of GHR. 15 refs., 4 figs.

Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

International Nuclear Information System (INIS)

Lazarus, Kyren A.; Zhao, Zhe; Knower, Kevin C.; To, Sarah Q.; Chand, Ashwini L.; Clyne, Colin D.

2013-01-01

Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E 2 ), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E 2 , showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E 2 treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer

Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

Energy Technology Data Exchange (ETDEWEB)

Lazarus, Kyren A. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122 (Australia); Zhao, Zhe; Knower, Kevin C. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); To, Sarah Q. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia); Chand, Ashwini L. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Clyne, Colin D., E-mail: Colin.clyne@princehenrys.org [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia)

2013-08-30

Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.

Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes.

Science.gov (United States)

Pardi, Norbert; Tuyishime, Steven; Muramatsu, Hiromi; Kariko, Katalin; Mui, Barbara L; Tam, Ying K; Madden, Thomas D; Hope, Michael J; Weissman, Drew

2015-11-10

In recent years, in vitro transcribed messenger RNA (mRNA) has emerged as a potential therapeutic platform. To fulfill its promise, effective delivery of mRNA to specific cell types and tissues needs to be achieved. Lipid nanoparticles (LNPs) are efficient carriers for short-interfering RNAs and have entered clinical trials. However, little is known about the potential of LNPs to deliver mRNA. Here, we generated mRNA-LNPs by incorporating HPLC purified, 1-methylpseudouridine-containing mRNA comprising codon-optimized firefly luciferase into stable LNPs. Mice were injected with 0.005-0.250mg/kg doses of mRNA-LNPs by 6 different routes and high levels of protein translation could be measured using in vivo imaging. Subcutaneous, intramuscular and intradermal injection of the LNP-encapsulated mRNA translated locally at the site of injection for up to 10days. For several days, high levels of protein production could be achieved in the lung from the intratracheal administration of mRNA. Intravenous and intraperitoneal and to a lesser extent intramuscular and intratracheal deliveries led to trafficking of mRNA-LNPs systemically resulting in active translation of the mRNA in the liver for 1-4 days. Our results demonstrate that LNPs are appropriate carriers for mRNA in vivo and have the potential to become valuable tools for delivering mRNA encoding therapeutic proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

Dwell-Time Distribution, Long Pausing and Arrest of Single-Ribosome Translation through the mRNA Duplex.

Science.gov (United States)

Xie, Ping

2015-10-09

Proteins in the cell are synthesized by a ribosome translating the genetic information encoded on the single-stranded messenger RNA (mRNA). It has been shown that the ribosome can also translate through the duplex region of the mRNA by unwinding the duplex. Here, based on our proposed model of the ribosome translation through the mRNA duplex we study theoretically the distribution of dwell times of the ribosome translation through the mRNA duplex under the effect of a pulling force externally applied to the ends of the mRNA to unzip the duplex. We provide quantitative explanations of the available single molecule experimental data on the distribution of dwell times with both short and long durations, on rescuing of the long paused ribosomes by raising the pulling force to unzip the duplex, on translational arrests induced by the mRNA duplex and Shine-Dalgarno(SD)-like sequence in the mRNA. The functional consequences of the pauses or arrests caused by the mRNA duplex and the SD sequence are discussed and compared with those obtained from other types of pausing, such as those induced by "hungry" codons or interactions of specific sequences in the nascent chain with the ribosomal exit tunnel.

Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

Science.gov (United States)

Thess, Andreas; Grund, Stefanie; Mui, Barbara L; Hope, Michael J; Baumhof, Patrick; Fotin-Mleczek, Mariola; Schlake, Thomas

2015-01-01

Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies. PMID:26050989

Integrator complex plays an essential role in adipose differentiation

International Nuclear Information System (INIS)

Otani, Yuichiro; Nakatsu, Yusuke; Sakoda, Hideyuki; Fukushima, Toshiaki; Fujishiro, Midori; Kushiyama, Akifumi; Okubo, Hirofumi; Tsuchiya, Yoshihiro; Ohno, Haruya; Takahashi, Shin-Ichiro; Nishimura, Fusanori; Kamata, Hideaki; Katagiri, Hideki; Asano, Tomoichiro

2013-01-01

Highlights: •IntS6 and IntS11 are subunits of the Integrator complex. •Expression levels of IntS6 and IntS11 were very low in 3T3-L1 fibroblast. •IntS6 and IntS11 were upregulated during adipose differentiation. •Suppression of IntS6 or IntS11 expression inhibited adipose differentiation. -- Abstract: The dynamic process of adipose differentiation involves stepwise expressions of transcription factors and proteins specific to the mature fat cell phenotype. In this study, it was revealed that expression levels of IntS6 and IntS11, subunits of the Integrator complex, were increased in 3T3-L1 cells in the period when the cells reached confluence and differentiated into adipocytes, while being reduced to basal levels after the completion of differentiation. Suppression of IntS6 or IntS11 expression using siRNAs in 3T3-L1 preadipocytes markedly inhibited differentiation into mature adipocytes, based on morphological findings as well as mRNA analysis of adipocyte-specific genes such as Glut4, perilipin and Fabp4. Although Pparγ2 protein expression was suppressed in IntS6 or IntS11-siRNA treated cells, adenoviral forced expression of Pparγ2 failed to restore the capacity for differentiation into mature adipocytes. Taken together, these findings demonstrate that increased expression of Integrator complex subunits is an indispensable event in adipose differentiation. Although further study is necessary to elucidate the underlying mechanism, the processing of U1, U2 small nuclear RNAs may be involved in cell differentiation steps