Benzoate-mediated changes on expression profile of soluble proteins in Serratia sp. DS001.

PubMed

Pandeeti, E V P; Chinnaboina, M R; Siddavattam, D

2009-05-01

To assess differences in protein expression profile associated with shift in carbon source from succinate to benzoate in Serratia sp. DS001 using a proteomics approach. A basic proteome map was generated for the soluble proteins extracted from Serratia sp. DS001 grown in succinate and benzoate. The differently and differentially expressed proteins were identified using ImageMaster 2D Platinum software (GE Healthcare). The identity of the proteins was determined by employing MS or MS/MS. Important enzymes such as Catechol 1,2 dioxygenase and transcriptional regulators that belong to the LysR superfamily were identified. Nearly 70 proteins were found to be differentially expressed when benzoate was used as carbon source. Based on the protein identity and degradation products generated from benzoate it is found that ortho pathway is operational in Serratia sp. DS001. Expression profile of the soluble proteins associated with shift in carbon source was mapped. The study also elucidates degradation pathway of benzoate in Serratia sp. DS001 by correlating the proteomics data with the catabolites of benzoate.

Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts.

PubMed

Imin, Nijat; De Jong, Femke; Mathesius, Ulrike; van Noorden, Giel; Saeed, Nasir A; Wang, Xin-Ding; Rose, Ray J; Rolfe, Barry G

2004-07-01

Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells. The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population. We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage. Over 3000 proteins could reproducibly be resolved over a pI range of 4-11. Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing. This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine. These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M. truncatula. The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.

Characterization of the Structural Gene Promoter of Aedes aegypti Densovirus

PubMed Central

Ward, Todd W.; Kimmick, Michael W.; Afanasiev, Boris N.; Carlson, Jonathan O.

2001-01-01

Aedes aegypti densonucleosis virus (AeDNV) has two promoters that have been shown to be active by reporter gene expression analysis (B. N. Afanasiev, Y. V. Koslov, J. O. Carlson, and B. J. Beaty, Exp. Parasitol. 79:322–339, 1994). Northern blot analysis of cells infected with AeDNV revealed two transcripts 1,200 and 3,500 nucleotides in length that are assumed to express the structural protein (VP) gene and nonstructural protein genes, respectively. Primer extension was used to map the transcriptional start site of the structural protein gene. Surprisingly, the structural protein gene transcript began at an initiator consensus sequence, CAGT, 60 nucleotides upstream from the map unit 61 TATAA sequence previously thought to define the promoter. Constructs with the β-galactosidase gene fused to the structural protein gene were used to determine elements necessary for promoter function. Deletion or mutation of the initiator sequence, CAGT, reduced protein expression by 93%, whereas mutation of the TATAA sequence at map unit 61 had little effect. An additional open reading frame was observed upstream of the structural protein gene that can express β-galactosidase at a low level (20% of that of VP fusions). Expression of the AeDNV structural protein gene was shown to be stimulated by the major nonstructural protein NS1 (Afanasiev et al., Exp. parasitol., 1994). To determine the sequences required for transactivation, expression of structural protein gene–β-galactosidase gene fusion constructs differing in AeDNV genome content was measured with and without NS1. The presence of NS1 led to an 8- to 10-fold increase in expression when either genomic end was present, compared to a 2-fold increase with a construct lacking the genomic ends. An even higher (37-fold) increase in expression occurred with both genomic ends present; however, this was in part due to template replication as shown by Southern blot analysis. These data indicate the location and importance of various elements necessary for efficient protein expression and transactivation from the structural protein gene promoter of AeDNV. PMID:11152505

Graded levels of FGF protein span the midbrain and can instruct graded induction and repression of neural mapping labels

PubMed Central

Chen, Yao; Mohammadi, Moosa; Flanagan, John G.

2009-01-01

Summary Graded guidance labels are widely used in neural map formation, but it is not well understood which potential strategy leads to their graded expression. In midbrain tectal map development, FGFs can induce an entire midbrain, but their protein distribution is unclear, nor is it known whether they may act instructively to produce graded gene expression. Using a receptor-alkaline phosphatase fusion probe, we find a long-range posterior>anterior FGF protein gradient spanning the midbrain. Heparan sulfate proteoglycan (HSPG) is required for this gradient. To test whether graded FGF concentrations can instruct graded gene expression, a quantitative tectal explant assay was developed. Engrailed-2 and ephrin-As, normally in posterior>anterior tectal gradients, showed graded upregulation. Moreover, EphAs, normally in anterior>posterior countergradients, showed coordinately graded downregulation. These results provide a mechanism to establish graded mapping labels, and more generally provide a developmental strategy to coordinately induce a structure and pattern its cell properties in gradients. PMID:19555646

mapDIA: Preprocessing and statistical analysis of quantitative proteomics data from data independent acquisition mass spectrometry.

PubMed

Teo, Guoshou; Kim, Sinae; Tsou, Chih-Chiang; Collins, Ben; Gingras, Anne-Claude; Nesvizhskii, Alexey I; Choi, Hyungwon

2015-11-03

Data independent acquisition (DIA) mass spectrometry is an emerging technique that offers more complete detection and quantification of peptides and proteins across multiple samples. DIA allows fragment-level quantification, which can be considered as repeated measurements of the abundance of the corresponding peptides and proteins in the downstream statistical analysis. However, few statistical approaches are available for aggregating these complex fragment-level data into peptide- or protein-level statistical summaries. In this work, we describe a software package, mapDIA, for statistical analysis of differential protein expression using DIA fragment-level intensities. The workflow consists of three major steps: intensity normalization, peptide/fragment selection, and statistical analysis. First, mapDIA offers normalization of fragment-level intensities by total intensity sums as well as a novel alternative normalization by local intensity sums in retention time space. Second, mapDIA removes outlier observations and selects peptides/fragments that preserve the major quantitative patterns across all samples for each protein. Last, using the selected fragments and peptides, mapDIA performs model-based statistical significance analysis of protein-level differential expression between specified groups of samples. Using a comprehensive set of simulation datasets, we show that mapDIA detects differentially expressed proteins with accurate control of the false discovery rates. We also describe the analysis procedure in detail using two recently published DIA datasets generated for 14-3-3β dynamic interaction network and prostate cancer glycoproteome. The software was written in C++ language and the source code is available for free through SourceForge website http://sourceforge.net/projects/mapdia/.This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

Fine mapping of canine parvovirus B cell epitopes.

PubMed

López de Turiso, J A; Cortés, E; Ranz, A; García, J; Sanz, A; Vela, C; Casal, J I

1991-10-01

In this report we describe the topological mapping of neutralizing domains of canine parvovirus (CPV). We obtained 11 CPV-specific monoclonal antibodies (MAbs), six of which are neutralizing. The reactivities were as determined by ELISA and Western blot (immunoblot) analysis. VP2, the most abundant protein of the CPV capsid, seemed to contain all the neutralization sites. Also, an almost full-length genomic clone of CPV was constructed in the bacterial plasmid pUC18 to enable expression of CPV proteins. All the neutralizing MAbs recognized recombinant VP2 when it was expressed as a free protein in Escherichia coli but not when expressed as a fusion protein with glutathione-S-transferase. When two large fragments containing about 85% and 67% of the C terminus of VP2 were expressed, no neutralization sites were detected. When fusion proteins containing the N terminus were expressed, two linear determinants were mapped, one between residues 1 to 10 of VP2, and the other between amino acids 11 and 23. The peptide 11 GQPAVRNERATGS 23, recognized by MAb 3C9, was synthesized chemically and checked for immunogenicity, not being able to induce neutralizing activity. Although the antibody response in rabbits to all the fusion proteins was uniformly high, the anti-CPV response was very variable. Protein from pCPVEx11, which contains a T cell epitope (peptide PKIFINLAKKKKAG) present in the VP1-specific region as well as the B cell epitopes, seemed to be the most effective in inducing virus neutralization.

Antigenicity of recombinant maltose binding protein-Mycobacterium avium subsp. paratuberculosis fusion proteins with and without factor Xa cleaving

USDA-ARS?s Scientific Manuscript database

Mycobacterium avium subsp paratuberculosis (MAP) causes Johne’s disease (JD) in ruminants. Proteomic studies have shown that MAP expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such prot...

Evaluation of prognostic and predictive value of microtubule associated protein tau in two independent cohorts

PubMed Central

2011-01-01

Introduction Microtubule associated proteins (MAPs) endogenously regulate microtubule stabilization and have been reported as prognostic and predictive markers for taxane response. The microtubule stabilizer, MAP-tau, has shown conflicting results. We quantitatively assessed MAP-tau expression in two independent breast cancer cohorts to determine prognostic and predictive value of this biomarker. Methods MAP-tau expression was evaluated in the retrospective Yale University breast cancer cohort (n = 651) using tissue microarrays and also in the TAX 307 cohort, a clinical trial randomized for TAC versus FAC chemotherapy (n = 140), using conventional whole tissue sections. Expression was measured using the AQUA method for quantitative immunofluorescence. Scores were correlated with clinicopathologic variables, survival, and response to therapy. Results Assessment of the Yale cohort using Cox univariate analysis indicated an improved overall survival (OS) in tumors with a positive correlation between high MAP-tau expression and overall survival (OS) (HR = 0.691, 95% CI = 0.489-0.974; P = 0.004). Kaplan Meier analysis showed 10-year survival for 65% of patients with high MAP-tau expression compared to 52% with low expression (P = .006). In TAX 307, high expression was associated with significantly longer median time to tumor progression (TTP) regardless of treatment arm (33.0 versus 23.4 months, P = 0.010) with mean TTP of 31.2 months. Response rates did not differ by MAP-tau expression (P = 0.518) or by treatment arm (P = 0.584). Conclusions Quantitative measurement of MAP-tau expression has prognostic value in both cohorts, with high expression associated with longer TTP and OS. Differences by treatment arm or response rate in low versus high MAP-tau groups were not observed, indicating that MAP-tau is not associated with response to taxanes and is not a useful predictive marker for taxane-based chemotherapy. PMID:21888627

MicroRNA 27a-3p Regulates Antimicrobial Responses of Murine Macrophages Infected by Mycobacterium avium subspecies paratuberculosis by Targeting Interleukin-10 and TGF-β-Activated Protein Kinase 1 Binding Protein 2

PubMed Central

Hussain, Tariq; Zhao, Deming; Shah, Syed Zahid Ali; Wang, Jie; Yue, Ruichao; Liao, Yi; Sabir, Naveed; Yang, Lifeng; Zhou, Xiangmei

2018-01-01

Mycobacterium avium subspecies paratuberculosis (MAP) persistently survive and replicate in mononuclear phagocytic cells by adopting various strategies to subvert host immune response. Interleukin-10 (IL-10) upregulation via inhibition of macrophage bactericidal activity is a critical step for MAP survival and pathogenesis within the host cell. Mitogen-activated protein kinase p38 signaling cascade plays a crucial role in the elevation of IL-10 and progression of MAP pathogenesis. The contribution of microRNAs (miRNAs) and their influence on the activation of macrophages during MAP pathogenesis are still unclear. In the current study, we found that miRNA-27a-3p (miR-27a) expression is downregulated during MAP infection both in vivo and in vitro. Moreover, miR-27a is also downregulated in toll-like receptor 2 (TLR2)-stimulated murine macrophages (RAW264.7 and bone marrow-derived macrophage). ELISA and real-time qRT-PCR results confirm that overexpression of miR-27a inhibited MAP-induced IL-10 production in macrophages and upregulated pro-inflammatory cytokines, while miR-27a inhibitor counteracted these effects. Luciferase reporter assay results revealed that IL-10 and TGF-β-activated protein kinase 1 binding protein 2 (TAB 2) are potential targets of miR-27a. In addition, we demonstrated that miR-27a negatively regulates TAB 2 expression and diminishes TAB 2-dependent p38/JNK phosphorylation, ultimately downregulating IL-10 expression in MAP-infected macrophages. Furthermore, overexpression of miR-27a significantly inhibited the intracellular survival of MAP in infected macrophages. Our data show that miR-27a augments antimicrobial activities of macrophages and inhibits the expression of IL-10, demonstrating that miR-27a regulates protective innate immune responses during MAP infection and can be exploited as a novel therapeutic target in the control of intracellular pathogens, including paratuberculosis. PMID:29375563

Heterogeneity mapping of protein expression in tumors using quantitative immunofluorescence.

PubMed

Faratian, Dana; Christiansen, Jason; Gustavson, Mark; Jones, Christine; Scott, Christopher; Um, InHwa; Harrison, David J

2011-10-25

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor(1,2), and some of these sub-clones give rise to metastatic (and therefore lethal) disease. Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension(3), or on macrodissection(4). A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue(5), providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ. We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system(6). Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index(7). To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Up-regulation of microtubule-associated protein 2 accompanying the filial imprinting of domestic chicks (Gallus gallus domesticus).

PubMed

Yamaguchi, Shinji; Fujii-Taira, Ikuko; Murakami, Akio; Hirose, Naoki; Aoki, Naoya; Izawa, Ei-Ichi; Fujimoto, Yasuyuki; Takano, Tatsuya; Matsushima, Toshiya; Homma, Koichi J

2008-06-15

Using cDNA microarrays, we have identified elsewhere the genes of microtubule-associated proteins as a group up-regulated in newly hatched chick brains after filial imprinting training. Here we show by in situ hybridization that the mRNA for the microtubule-associated protein 2 (MAP2) gene was enriched in the mesopallium and the hippocampus in the trained chick brain. The regionally specific enrichments of MAP2 mRNA were not observed in the brain of dark-reared or light-exposed chick as controls, implying an association between the degree of expression and the strength of the learned preference. In agreement with the gene expression, MAP2 protein was accumulated in the mesopallium of the trained chick brain, but not in the brains of the controls. The accumulation of MAP2 was found in the cytosol of neurons and co-localized with beta-tubulin, suggesting a change in microtubule assembly. Our results suggest a postnatal reorganization of cytoskeleton following filial imprinting.

A high-throughput shotgun mutagenesis approach to mapping B-cell antibody epitopes.

PubMed

Davidson, Edgar; Doranz, Benjamin J

2014-09-01

Characterizing the binding sites of monoclonal antibodies (mAbs) on protein targets, their 'epitopes', can aid in the discovery and development of new therapeutics, diagnostics and vaccines. However, the speed of epitope mapping techniques has not kept pace with the increasingly large numbers of mAbs being isolated. Obtaining detailed epitope maps for functionally relevant antibodies can be challenging, particularly for conformational epitopes on structurally complex proteins. To enable rapid epitope mapping, we developed a high-throughput strategy, shotgun mutagenesis, that enables the identification of both linear and conformational epitopes in a fraction of the time required by conventional approaches. Shotgun mutagenesis epitope mapping is based on large-scale mutagenesis and rapid cellular testing of natively folded proteins. Hundreds of mutant plasmids are individually cloned, arrayed in 384-well microplates, expressed within human cells, and tested for mAb reactivity. Residues are identified as a component of a mAb epitope if their mutation (e.g. to alanine) does not support candidate mAb binding but does support that of other conformational mAbs or allows full protein function. Shotgun mutagenesis is particularly suited for studying structurally complex proteins because targets are expressed in their native form directly within human cells. Shotgun mutagenesis has been used to delineate hundreds of epitopes on a variety of proteins, including G protein-coupled receptor and viral envelope proteins. The epitopes mapped on dengue virus prM/E represent one of the largest collections of epitope information for any viral protein, and results are being used to design better vaccines and drugs. © 2014 John Wiley & Sons Ltd.

Mycobacterium paratuberculosis, Mycobacterium smegmatis, and lipopolysaccharide induce different transcriptional and post-transcriptional regulation of the IRG1 gene in murine macrophages.

PubMed

Basler, Tina; Jeckstadt, Sabine; Valentin-Weigand, Peter; Goethe, Ralph

2006-03-01

Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic enteritis in ruminants. In addition, MAP is presently the most favored pathogen linked to Crohn's disease. In this study, we were interested in dissecting the molecular mechanisms of macrophage activation or deactivation after infection with MAP. By subtractive hybridization of cDNAs, we identified the immune-responsive gene 1 (IRG1), which was expressed substantially higher in lipopolysaccharide (LPS)-stimulated than in MAP-infected murine macrophage cell lines. A nuclear run-on transcription assay revealed that the IRG1 gene was activated transcriptionally in LPS-stimulated and MAP-infected macrophages with higher expression in LPS-stimulated cells. Analysis of post-transcriptional regulation demonstrated that IRG1 mRNA stability was increased in LPS-stimulated but not in MAP-infected macrophages. Furthermore, IRG1 gene expression of macrophages infected with the nonpathogenic Mycobacterium smegmatis differed from those of LPS-stimulated and MAP-infected macrophages. At 2 h postinfection, M. smegmatis-induced IRG1 gene expression was as low as in MAP-infected, and 8 h postinfection, it increased nearly to the level in LPS-stimulated macrophages. Transient transfection experiments revealed similar IRG1 promoter activities in MAP- and M. smegmatis-infected cells. Northern analysis demonstrated increased IRG1 mRNA stability in M. smegmatis-infected macrophages. IRG1 mRNA stabilization was p38 mitogen-activated protein kinase-independent. Inhibition of protein synthesis revealed that constitutively expressed factors seemed to be responsible for IRG1 mRNA destabilization. Thus, our data demonstrate that transcriptional and post-transcriptional mechanisms are responsible for a differential IRG1 gene expression in murine macrophages treated with LPS, MAP, and M. smegmatis.

Porcine MAP3K5 analysis: molecular cloning, characterization, tissue expression pattern, and copy number variations associated with residual feed intake.

PubMed

Pu, L; Zhang, L C; Zhang, J S; Song, X; Wang, L G; Liang, J; Zhang, Y B; Liu, X; Yan, H; Zhang, T; Yue, J W; Li, N; Wu, Q Q; Wang, L X

2016-08-12

Mitogen-activated protein kinase kinase kinase 5 (MAP3K5) is essential for apoptosis, proliferation, differentiation, and immune responses, and is a candidate marker for residual feed intake (RFI) in pig. We cloned the full-length cDNA sequence of porcine MAP3K5 by rapid-amplification of cDNA ends. The 5451-bp gene contains a 5'-untranslated region (UTR) (718 bp), a coding region (3738 bp), and a 3'-UTR (995 bp), and encodes a peptide of 1245 amino acids, which shares 97, 99, 97, 93, 91, and 84% sequence identity with cattle, sheep, human, mouse, chicken, and zebrafish MAP3K5, respectively. The deduced MAP3K5 protein sequence contains two conserved domains: a DUF4071 domain and a protein kinase domain. Phylogenetic analysis showed that porcine MAP3K5 forms a separate branch to vicugna and camel MAP3K5. Tissue expression analysis using real-time quantitative polymerase chain reaction (qRT-PCR) revealed that MAP3K5 was expressed in the heart, liver, spleen, lung, kidney, muscle, fat, pancrea, ileum, and stomach tissues. Copy number variation was detected for porcine MAP3K5 and validated by qRT-PCR. Furthermore, a significant increase in average copy number was detected in the low RFI group when compared to the high RFI group in a Duroc pig population. These results provide useful information regarding the influence of MAP3K5 on RFI in pigs.

The TAM-family receptor Mer mediates production of HGF through the RhoA-dependent pathway in response to apoptotic cells.

PubMed

Park, Hyun-Jung; Baen, Ji-Yeon; Lee, Ye-Ji; Choi, Youn-Hee; Kang, Jihee Lee

2012-08-01

The TAM receptor protein tyrosine kinases Tyro3, Axl, and Mer play important roles in macrophage function. We investigated the roles of the TAM receptors in mediating the induction of hepatocyte growth factor (HGF) during the interaction of macrophages with apoptotic cells. Mer-specific neutralizing antibody, small interfering RNA (siRNA), and a recombinant Mer protein (Mer/Fc) inhibited HGF mRNA and protein expression, as well as activation of RhoA, Akt, and specific mitogen-activated protein (MAP) kinases in response to apoptotic cells. Inhibition of Axl or Tyro3 with specific antibodies, siRNA, or Fc-fusion proteins did not prevent apoptotic cell-induced HGF mRNA and protein expression and did not inhibit activation of the postreceptor signaling molecules RhoA and certain MAP kinases, including extracellular signal-regulated protein kinase and c-Jun NH(2)-terminal kinase. However, Axl- and Tyro3-specific blockers did inhibit the activation of Akt and p38 MAP kinase in response to apoptotic cells. In addition, none of the TAM receptors mediated the effects of apoptotic cells on transforming growth factor-β or epidermal growth factor mRNA expression. However, they were involved in the induction of vascular endothelial growth factor mRNA expression. Our data provide evidence that when macrophages interact with apoptotic cells, only Mer of the TAM-family receptors is responsible for mediating transcriptional HGF production through a RhoA-dependent pathway.

A Reference Proteomic Database of Lactobacillus plantarum CMCC-P0002

PubMed Central

Tian, Wanhong; Yu, Gang; Liu, Xiankai; Wang, Jie; Feng, Erling; Zhang, Xuemin; Chen, Bei; Zeng, Ming; Wang, Hengliang

2011-01-01

Lactobacillus plantarum is a widespread probiotic bacteria found in many fermented food products. In this study, the whole-cell proteins and secretory proteins of L. plantarum were separated by two-dimensional electrophoresis method. A total of 434 proteins were identified by tandem mass spectrometry, including a plasmid-encoded hypothetical protein pLP9000_05. The information of first 20 highest abundance proteins was listed for the further genetic manipulation of L. plantarum, such as construction of high-level expressions system. Furthermore, the first interaction map of L. plantarum was established by Blue-Native/SDS-PAGE technique. A heterodimeric complex composed of maltose phosphorylase Map3 and Map2, and two homodimeric complexes composed of Map3 and Map2 respectively, were identified at the same time, indicating the important roles of these proteins. These findings provided valuable information for the further proteomic researches of L. plantarum. PMID:21998671

A reference proteomic database of Lactobacillus plantarum CMCC-P0002.

PubMed

Zhu, Li; Hu, Wei; Liu, Datao; Tian, Wanhong; Yu, Gang; Liu, Xiankai; Wang, Jie; Feng, Erling; Zhang, Xuemin; Chen, Bei; Zeng, Ming; Wang, Hengliang

2011-01-01

Lactobacillus plantarum is a widespread probiotic bacteria found in many fermented food products. In this study, the whole-cell proteins and secretory proteins of L. plantarum were separated by two-dimensional electrophoresis method. A total of 434 proteins were identified by tandem mass spectrometry, including a plasmid-encoded hypothetical protein pLP9000_05. The information of first 20 highest abundance proteins was listed for the further genetic manipulation of L. plantarum, such as construction of high-level expressions system. Furthermore, the first interaction map of L. plantarum was established by Blue-Native/SDS-PAGE technique. A heterodimeric complex composed of maltose phosphorylase Map3 and Map2, and two homodimeric complexes composed of Map3 and Map2 respectively, were identified at the same time, indicating the important roles of these proteins. These findings provided valuable information for the further proteomic researches of L. plantarum.

Nitrosative/oxidative stress conditions regulate thioredoxin-interacting protein (TXNIP) expression and thioredoxin-1 (TRX-1) nuclear localization.

PubMed

Ogata, Fernando Toshio; Batista, Wagner Luiz; Sartori, Adriano; Gesteira, Tarsis Ferreira; Masutani, Hiroshi; Arai, Roberto Jun; Yodoi, Junji; Stern, Arnold; Monteiro, Hugo Pequeno

2013-01-01

Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras-ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H₂O₂, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases.

Angiotensin II regulation of neuromodulation: downstream signaling mechanism from activation of mitogen-activated protein kinase.

PubMed

Lu, D; Yang, H; Raizada, M K

1996-12-01

Angiotensin II (Ang II) stimulates expression of tyrosine hydroxylase and norepinephrine transporter genes in brain neurons; however, the signal-transduction mechanism is not clearly defined. This study was conducted to determine the involvement of the mitogen-activated protein (MAP) kinase signaling pathway in Ang II stimulation of these genes. MAP kinase was localized in the perinuclear region of the neuronal soma. Ang II caused activation of MAP kinase and its subsequent translocation from the cytoplasmic to nuclear compartment, both effects being mediated by AT1 receptor subtype. Ang II also stimulated SRE- and AP1-binding activities and fos gene expression and its translocation in a MAP kinase-dependent process. These observations are the first demonstration of a downstream signaling pathway involving MAP kinase in Ang II-mediated neuromodulation in noradrenergic neurons.

Microtubule-associated protein 1S-related autophagy inhibits apoptosis of intestinal epithelial cells via Wnt/β-catenin signaling in Crohn's disease.

PubMed

Bai, Wenxia; Bai, Jian'an; Li, Yanhai; Tian, Delong; Shi, Ruihua

2017-04-08

Many autophagy-related genes, to our knowledge, have been identified as Crohn's disease (CD) polymorphic sites by genomic wide studies. As a novel member of the microtubule-associated protein 1 (MAP1) family, MAP1S is a microtubule-binding proteins involved in autophagy. However, its expression and potential functions in CD have not been understood. For the first time, we discovered the up-regulated MAP1S and autophagy level (indicated by LC3-Ⅱ/LC3-Ⅰ) in inflamed epithelium among CD patients. Similarly, in TNBS-induced murine colitis model, MAP1S expression was obviously increased. Meanwhile, we found the co-location of MAP1S and active-caspase 3 which acted as "apoptotic executor" which might indicate the basis of their co-efficient. At the cellular level, MAP1S silencing inhibited starvation-induced over-expression of active-caspase 3 partially via Wnt/β-catenin signaling activation in HCT-116 cells. Finally, we demonstrated that IWP-2, an inhibitor of the Wnt/β-catenin signaling, reversed the down-regulation of active-caspase 3 induced by MAP1S siRNA in HCT-116 cells. Taken together, our results suggested that MAP1S were up-regulated among CD patients and MAP1S-related autophagy inhibits apoptosis of intestinal epithelial cells (IECs) through Wnt/β-catenin signaling pathway which might play a vital role in the protection of intestinal mucosal barrier and inhibition the progression of CD. Copyright © 2017 Elsevier Inc. All rights reserved.

Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries

DTIC Science & Technology

2012-07-01

Description HRAS Homo sapiens v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS), transcript 1 CDC25C Homo sapiens cell division cycle 25...homolog C (CDC25C), transcript variant 1 MYC Homo sapiens v-myc myeloctomatosis viral oncogene homolog (avian) (MYC) MAP3K7 Homo sapiens mitogen...activated protein kinase kinase kinase 7 (MAP3K7) MAP3K8 Homo sapiens mitogen-activated protein kinase kinase kinase 8 (MAP3K8) SF3B1 Homo sapiens splicing

Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries

DTIC Science & Technology

2013-01-01

Description HRAS Homo sapiens v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS), transcript 1 CDC25C Homo sapiens cell division cycle 25...homolog C (CDC25C), transcript variant 1 MYC Homo sapiens v-myc myeloctomatosis viral oncogene homolog (avian) (MYC) MAP3K7 Homo sapiens mitogen...activated protein kinase kinase kinase 7 (MAP3K7) MAP3K8 Homo sapiens mitogen-activated protein kinase kinase kinase 8 (MAP3K8) SF3B1 Homo sapiens

Intracellular Localization Map of Human Herpesvirus 8 Proteins▿

PubMed Central

Sander, Gaby; Konrad, Andreas; Thurau, Mathias; Wies, Effi; Leubert, Rene; Kremmer, Elisabeth; Dinkel, Holger; Schulz, Thomas; Neipel, Frank; Stürzl, Michael

2008-01-01

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma. We present a localization map of 85 HHV-8-encoded proteins in mammalian cells. Viral open reading frames were cloned with a Myc tag in expression plasmids, confirmed by full-length sequencing, and expressed in HeLa cells. Protein localizations were analyzed by immunofluorescence microscopy. Fifty-one percent of all proteins were localized in the cytoplasm, 22% were in the nucleus, and 27% were found in both compartments. Surprisingly, we detected viral FLIP (v-FLIP) in the nucleus and in the cytoplasm, whereas cellular FLIPs are generally localized exclusively in the cytoplasm. This suggested that v-FLIP may exert additional or alternative functions compared to cellular FLIPs. In addition, it has been shown recently that the K10 protein can bind to at least 15 different HHV-8 proteins. We noticed that K10 and only five of its 15 putative binding factors were localized in the nucleus when the proteins were expressed in HeLa cells individually. Interestingly, in coexpression experiments K10 colocalized with 87% (13 of 15) of its putative binding partners. Colocalization was induced by translocation of either K10 alone or both proteins. These results indicate active intracellular translocation processes in virus-infected cells. Specifically in this framework, the localization map may provide a useful reference to further elucidate the function of HHV-8-encoded genes in human diseases. PMID:18077714

Characterization of Mitogen-Activated Protein Kinase Expression in Nucleus Accumbens and Hippocampus of Rats Subjected to Food Selection in the Cafeteria Diet Protocol.

PubMed

Sarro-Ramírez, Andrea; Sánchez, Daniel; Tejeda-Padrón, Alma; Buenfil-Canto, Linda Vianey; Valladares-García, Jorge; Pacheco-Pantoja, Elda; Arias-Carrión, Oscar; Murillo-Rodríguez, Eric

2016-01-01

Obesity is a world-wide health problem that requires different experimental perspectives to understand the onset of this disease, including the neurobiological basis of food selection. From a molecular perspective, obesity has been related with activity of several endogenous molecules, including the mitogenactivated protein kinases (MAP-K). The aim of this study was to characterize MAP-K expression in hedonic and learning and memory brain-associated areas such as nucleus accumbens (AcbC) and hippocampus (HIPP) after food selection. We show that animals fed with cafeteria diet during 14 days displayed an increase in p38 MAP-K activity in AcbC if chose cheese. Conversely, a diminution was observed in animals that preferred chocolate in AcbC. Also, a decrease of p38 MAP-K phosphorylation was found in HIPP in rats that selected either cheese or chocolate. Our data demonstrate a putative role of MAP-K expression in food selection. These findings advance our understanding of neuromolecular basis engaged in obesity.

Macrophage differentiation increases expression of the ascorbate transporter (SVCT2)

PubMed Central

Qiao, Huan; May, James M.

2013-01-01

To determine whether macrophage differentiation involves increased uptake of vitamin C, or ascorbic acid, we assessed the expression and function of its transporter SVCT2 during phorbol ester-induced differentiation of human-derived THP-1 monocytes. Induction of THP-1 monocyte differentiation by phorbol 12-myristate 13-acetate (PMA) markedly increased SVCT2 mRNA, protein, and function. When ascorbate was present during PMA-induced differentiation, the increase in SVCT2 protein expression was inhibited, but differentiation was enhanced. PMA-induced SVCT2 protein expression was blocked by inhibitors of protein kinase C (PKC), with most of the affect due to the PKCβI and βII isoforms. Activation of MEK/ERK was sustained up to 48 h after PMA treatment, and the inhibitors completely blocked PMA-stimulated SVCT2 protein expression, indicating an exclusive role for the classical MAP kinase pathway. However, inhibitors of NF-κB activation, NADPH oxidase inhibitors, and several antioxidants also partially prevented SVCT2 induction, suggesting diverse distal routes for control of SVCT2 transcription. Both known promoters for the SVCT2 were involved in these effects. In conclusion, PMA-induced monocyte-macrophage differentiation is enhanced by ascorbate and associated with increased expression and function of the SVCT2 protein through a pathway involving sustained activation of PKCβI/II, MAP kinase, NADPH oxidase, and NF-κB. PMID:19232538

In silico design and expression of a novel fusion protein of HBHA and high antigenic region of FAP-P of Mycobacterium avium subsp. paratuberculosis in Pichia pastoris.

PubMed

Eraghi, Vida; Derakhshandeh, Abdollah; Hosseini, Arsalan; Motamedi-Boroojeni, Azar

2017-12-01

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in ruminants and there has been a shift in the public health approach to MAP and human diseases like Crohn's disease. The prevention of infection by MAP in ruminants is thought to deter the high impact of economic losses in the level of dairy industry and possible spreading of this pathogen in dairy products. The present study was done to investigate the construction and expression of the soluble form of a novel fusion protein, consisting of Heparin-binding hemagglutinin (HBHA) and high antigenic region of Fibronectin Attachment Protein-P (FAP-P), in order to introduce as a Th1 inducer subunit vaccine against MAP. HBHA is a mycobacterial adhesin and it has been demonstrated that a HBHA-specific IFN-γ response, in latent M. tuberculosis infection, depends on the methylation of the antigen. Further, FAP-P induces Th1 polarization. Because methylation of HBHA was not performed in E. coli , Pichia pastoris was chosen as the host. The desired fusion protein had a similar 3D structure to that of HBHA with its native form and post-translational methylation in C-terminal. Hence, the uptake of the purified fusion protein will be done by M cells because of HBHA, and cell-mediated immunity will be induced because of both antigens. Eventually, successful construction and expression of the newly-designed chimeric protein under the mentioned conditions is reported in this article.

Mitogen-Activated Protein Kinase 14 Promotes AKI

PubMed Central

Husi, Holger; Gonzalez-Lafuente, Laura; Valiño-Rivas, Lara; Fresno, Manuel; Sanz, Ana Belen; Mullen, William; Albalat, Amaya; Mezzano, Sergio; Vlahou, Tonia; Mischak, Harald

2017-01-01

An improved understanding of pathogenic pathways in AKI may identify novel therapeutic approaches. Previously, we conducted unbiased liquid chromatography-tandem mass spectrometry–based protein expression profiling of the renal proteome in mice with acute folate nephropathy. Here, analysis of the dataset identified enrichment of pathways involving NFκB in the kidney cortex, and a targeted data mining approach identified components of the noncanonical NFκB pathway, including the upstream kinase mitogen-activated protein kinase kinase kinase 14 (MAP3K14), the NFκB DNA binding heterodimer RelB/NFκB2, and proteins involved in NFκB2 p100 ubiquitination and proteasomal processing to p52, as upregulated. Immunohistochemistry localized MAP3K14 expression to tubular cells in acute folate nephropathy and human AKI. In vivo, kidney expression levels of NFκB2 p100 and p52 increased rapidly after folic acid injection, as did DNA binding of RelB and NFκB2, detected in nuclei isolated from the kidneys. Compared with wild-type mice, MAP3K14 activity–deficient aly/aly (MAP3K14aly/aly) mice had less kidney dysfunction, inflammation, and apoptosis in acute folate nephropathy and less kidney dysfunction and a lower mortality rate in cisplatin-induced AKI. The exchange of bone marrow between wild-type and MAP3K14aly/aly mice did not affect the survival rate of either group after folic acid injection. In cultured tubular cells, MAP3K14 small interfering RNA targeting decreased inflammation and cell death. Additionally, cell culture and in vivo studies identified the chemokines MCP-1, RANTES, and CXCL10 as MAP3K14 targets in tubular cells. In conclusion, MAP3K14 promotes kidney injury through promotion of inflammation and cell death and is a promising novel therapeutic target. PMID:27620989

Adjustable under-expression of yeast mating pathway proteins in Saccharomyces cerevisiae using a programmed ribosomal frameshift.

PubMed

Choi, Min-Yeon; Park, Sang-Hyun

2016-06-01

Experimental research in molecular biology frequently relies on the promotion or suppression of gene expression, an important tool in the study of its functions. Although yeast is among the most studied model systems with the ease of maintenance and manipulation, current experimental methods are mostly limited to gene deletion, suppression or overexpression of genes. Therefore, the ability to reduce protein expressions and then observing the effects would promote a better understanding of the exact functions and their interactions. Reducing protein expression is mainly limited by the difficulties associated with controlling the reduction level, and in some cases, the initial endogenous abundance is too low. For the under-expression to be useful as an experimental tool, repeatability and stability of reduced expression is important. We found that cis-elements in programmed -1 ribosomal frameshifting (-1RFS) of beet western yellow virus (BWYV) could be utilized to reduced protein expression in Saccharomyces cerevisiae. The two main advantages of using -1RFS are adjustable reduction rates and ease of use. To demonstrate the utility of this under-expression system, examples of reduced protein abundance were shown using yeast mating pathway components. The abundance of MAP kinase Fus3 was reduced to approximately 28-75 % of the wild-type value. Other MAP kinase mating pathway components, including Ste5, Ste11, and Ste7, were also under-expressed to verify that the -1RFS system works with different proteins. Furthermore, reduced Fus3 abundance altered the overall signal transduction outcome of the mating pathway, demonstrating the potential for further studies of signal transduction adjustment via under-expression.

Relative gene expression of bile salt hydrolase and surface proteins in two putative indigenous Lactobacillus plantarum strains under in vitro gut conditions.

PubMed

Duary, Raj Kumar; Batish, Virender Kumar; Grover, Sunita

2012-03-01

Probiotic bacteria must overcome the toxicity of bile salts secreted in the gut and adhere to the epithelial cells to enable their better colonization with extended transit time. Expression of bile salt hydrolase and other proteins on the surface of probiotic bacteria can help in better survivability and optimal functionality in the gut. Two putative Lactobacillus plantarum isolates i.e., Lp9 and Lp91 along with standard strain CSCC5276 were used. A battery of six housekeeping genes viz. gapB, dnaG, gyrA, ldhD, rpoD and 16S rRNA were evaluated by using geNorm 3.4 excel based application for normalizing the expression of bile salt hydrolase (bsh), mucus-binding protein (mub), mucus adhesion promoting protein (mapA), and elongation factor thermo unstable (EF-Tu) in Lp9 and Lp91. The maximal level of relative bsh gene expression was recorded in Lp91 with 2.89 ± 0.14, 4.57 ± 0.37 and 6.38 ± 0.19 fold increase at 2% bile salt concentration after 1, 2 and 3 h, respectively. Similarly, mub and mapA genes were maximally expressed in Lp9 at the level of 20.07 ± 1.28 and 30.92 ± 1.51 fold, when MRS was supplemented with 0.05% mucin and 1% each of bile and pancreatin (pH 6.5). However, in case of EF-Tu, the maximal expression of 42.84 ± 5.64 fold was recorded in Lp91 in the presence of mucin alone (0.05%). Hence, the expression of bsh, mub, mapA and EF-Tu could be considered as prospective biomarkers for screening of novel probiotic lactobacillus strains for optimal functionality in the gut.

Protein interactome analysis of 12 mitogen-activated protein kinase kinase kinase in rice using a yeast two-hybrid system.

PubMed

Singh, Raksha; Lee, Jae-Eun; Dangol, Sarmina; Choi, Jihyun; Yoo, Ran Hee; Moon, Jae Sun; Shim, Jae-Kyung; Rakwal, Randeep; Agrawal, Ganesh Kumar; Jwa, Nam-Soo

2014-01-01

The mitogen-activated protein kinase (MAPK) cascade is composed at least of MAP3K (for MAPK kinase kinase), MAP2K, and MAPK family modules. These components together play a central role in mediating extracellular signals to the cell and vice versa by interacting with their partner proteins. However, the MAP3K-interacting proteins remain poorly investigated in plants. Here, we utilized a yeast two-hybrid system and bimolecular fluorescence complementation in the model crop rice (Oryza sativa) to map MAP3K-interacting proteins. We identified 12 novel nonredundant interacting protein pairs (IPPs) representing 11 nonredundant interactors using 12 rice MAP3Ks (available as full-length cDNA in the rice KOME (http://cdna01.dna.affrc.go.jp/cDNA/) at the time of experimental design and execution) as bait and a rice seedling cDNA library as prey. Of the 12 MAP3Ks, only six had interacting protein partners. The established MAP3K interactome consisted of two kinases, three proteases, two forkhead-associated domain-containing proteins, two expressed proteins, one E3 ligase, one regulatory protein, and one retrotransposon protein. Notably, no MAP3K showed physical interaction with either MAP2K or MAPK. Seven IPPs (58.3%) were confirmed in vivo by bimolecular fluorescence complementation. Subcellular localization of 14 interactors, together involved in nine IPPs (75%) further provide prerequisite for biological significance of the IPPs. Furthermore, GO of identified interactors predicted their involvement in diverse physiological responses, which were supported by a literature survey. These findings increase our knowledge of the MAP3K-interacting proteins, help in proposing a model of MAPK modules, provide a valuable resource for developing a complete map of the rice MAPK interactome, and allow discussion for translating the interactome knowledge to rice crop improvement against environmental factors. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nitrosative/Oxidative Stress Conditions Regulate Thioredoxin-Interacting Protein (TXNIP) Expression and Thioredoxin-1 (TRX-1) Nuclear Localization

PubMed Central

Ogata, Fernando Toshio; Batista, Wagner Luiz; Sartori, Adriano; Gesteira, Tarsis Ferreira; Masutani, Hiroshi; Arai, Roberto Jun; Yodoi, Junji; Stern, Arnold; Monteiro, Hugo Pequeno

2013-01-01

Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras - ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H2O2, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases. PMID:24376827

Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

PubMed Central

Roth Flach, Rachel J.; Skoura, Athanasia; Matevossian, Anouch; Danai, Laura V.; Zheng, Wei; Cortes, Christian; Bhattacharya, Samit K.; Aouadi, Myriam; Hagan, Nana; Yawe, Joseph C.; Vangala, Pranitha; Menendez, Lorena Garcia; Cooper, Marcus P.; Fitzgibbons, Timothy P.; Buckbinder, Leonard; Czech, Michael P.

2015-01-01

Signalling pathways that control endothelial cell (EC) permeability, leukocyte adhesion and inflammation are pivotal for atherosclerosis initiation and progression. Here we demonstrate that the Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), which has been implicated in inflammation, is abundantly expressed in ECs and in atherosclerotic plaques from mice and humans. On the basis of endothelial-specific MAP4K4 gene silencing and gene ablation experiments in Apoe−/− mice, we show that MAP4K4 in ECs markedly promotes Western diet-induced aortic macrophage accumulation and atherosclerotic plaque development. Treatment of Apoe−/− and Ldlr−/− mice with a selective small-molecule MAP4K4 inhibitor also markedly reduces atherosclerotic lesion area. MAP4K4 silencing in cultured ECs attenuates cell surface adhesion molecule expression while reducing nuclear localization and activity of NFκB, which is critical for promoting EC activation and atherosclerosis. Taken together, these results reveal that MAP4K4 is a key signalling node that promotes immune cell recruitment in atherosclerosis. PMID:26688060

Proteomic analysis to investigate color changes of chilled beef longissimus steaks held under carbon monoxide and high oxygen packaging.

PubMed

Yang, Xiaoyin; Wu, Shuang; Hopkins, David L; Liang, Rongrong; Zhu, Lixian; Zhang, Yimin; Luo, Xin

2018-08-01

This study investigated the proteome basis for color stability variations in beef steaks packaged under two modified atmosphere packaging (MAP) methods: HiOx-MAP (80% O 2 /20% CO 2 ) and CO-MAP (0.4% CO/30% CO 2 /69.6% N 2 ) during 15 days of storage. The color stability, pH, and sarcoplasmic proteome analysis of steaks were evaluated on days 0, 5, 10 and 15 of storage. Proteomic results revealed that the differential expression of the sarcoplasmic proteome during storage contributed to the variations in meat color stability between the two MAP methods. Compared with HiOx-MAP steaks, some glycolytic and energy metabolic enzymes important in NADH regeneration and antioxidant processes, antioxidant peroxiredoxins (thioredoxin-dependent peroxide reductase, peroxiredoxin-2, peroxiredoxin-6) and protein DJ-1 were more abundant in CO-MAP steaks. The over-expression of these proteins could induce CO-MAP steaks to maintain high levels of metmyoglobin reducing activity and oxygen consumption rate, resulting in CO-MAP steaks exhibiting better color stability than HiOx-MAP steaks during storage. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mapping-by-sequencing of Ligon-lintless-1 (Li 1 ) reveals a cluster of neighboring genes with correlated expression in developing fibers of Upland cotton (Gossypium hirsutum L.).

PubMed

Thyssen, Gregory N; Fang, David D; Turley, Rickie B; Florane, Christopher; Li, Ping; Naoumkina, Marina

2015-09-01

Mapping-by-sequencing and SNP marker analysis were used to fine map the Ligon-lintless-1 ( Li 1 ) short fiber mutation in tetraploid cotton to a 255-kb region that contains 16 annotated proteins. The Ligon-lintless-1 (Li 1 ) mutant of cotton (Gossypium hirsutum L.) has been studied as a model for cotton fiber development since its identification in 1929; however, the causative mutation has not been identified yet. Here we report the fine genetic mapping of the mutation to a 255-kb region that contains only 16 annotated genes in the reference Gossypium raimondii genome. We took advantage of the incompletely dominant dwarf vegetative phenotype to identify 100 mutants (Li 1 /Li 1 ) and 100 wild-type (li 1 /li 1 ) homozygotes from a mapping population of 2567 F2 plants, which we bulked and deep sequenced. Since only homozygotes were sequenced, we were able to use a high stringency in SNP calling to rapidly narrow down the region harboring the Li 1 locus, and designed subgenome-specific SNP markers to test the population. We characterized the expression of all sixteen genes in the region by RNA sequencing of elongating fibers and by RT-qPCR at seven time points spanning fiber development. One of the most highly expressed genes found in this interval in wild-type fiber cells is 40-fold under-expressed at the day of anthesis (DOA) in the mutant fiber cells.  This gene is a major facilitator superfamily protein, part of the large family of proteins that includes auxin and sugar transporters. Interestingly, nearly all genes in this region were most highly expressed at DOA and showed a high degree of co-expression. Further characterization is required to determine if transport of hormones or carbohydrates is involved in both the dwarf and lintless phenotypes of Li 1 plants.

Proteolysis of microtubule associated protein 2 and sensitivity of pancreatic tumours to docetaxel

PubMed Central

Veitia, R; David, S; Barbier, P; Vantard, M; Gounon, P; Bissery, M C; Fellous, A

2000-01-01

We have studied the state of microtubule associated protein 2 (MAP2) in the pancreatic ductal adenocarcinomas P03 and P02 (sensitive and refractory to docetaxel respectively) since they express the corresponding mRNA and MAP2-related peptides. Immunohistochemical localization showed that in tumour P03 the MAP2-related peptides are highly expressed and confined to the epithelial malignant cells while in P02 the intensity of the immunostaining is lower. However, anti α-tubulin staining followed a similar pattern suggesting that the net amount of macromolecular structures in the sensitive tumour is higher than in the refractory one. This may explain its higher sensitivity to docetaxel, because tubulin assembled into microtubules is the target of the drug. We found that protein extracts from both tumours differed in their proteolytic activity on rat brain MAP2. Since the proteolysis pattern obtained was similar to the one produced by Cathepsin D, we studied the effect of MAP2 proteolysed by this enzyme on microtubule formation in vitro. Proteolysis was found to increase the tendency of tubulin to assemble into macromolecular structures (microtubules and aggregates) in the presence of docetaxel. This suggests that in vivo proteolysis of MAP2 might increase microtubule alterations and potentiate the antitumour effect of docetaxel. © 2000 Cancer Research Campaign PMID:10945505

Human liver proteome project: plan, progress, and perspectives.

PubMed

He, Fuchu

2005-12-01

The Human Liver Proteome Project is the first initiative of the human proteome project for human organs/tissues and aims at writing a modern Prometheus myth. Its global scientific objectives are to reveal the "solar system" of the human liver proteome, expression profiles, modification profiles, a protein linkage (protein-protein interaction) map, and a proteome localization map, and to define an ORFeome, physiome, and pathome. Since it was first proposed in April 2002, the Human Liver Proteome Project has attracted more than 100 laboratories from all over the world. In the ensuing 3 years, we set up a management infrastructure, identified reference laboratories, confirmed standard operating procedures, initiated international research collaborations, and finally achieved the first set of expression profile data.

Hepatic expression of the GH/JAK/STAT/IGF pathway, acute-phase response signalling and complement system are affected in mouse offspring by prenatal and early postnatal exposure to maternal high-protein diet.

PubMed

Vanselow, Jens; Kucia, Marzena; Langhammer, Martina; Koczan, Dirk; Rehfeldt, Charlotte; Metges, Cornelia C

2011-12-01

Effects of pre- and early postnatal exposure to maternal high-protein diets are not well understood. Transcription profiling was performed in male mouse offspring exposed to maternal high-protein diet during pregnancy and/or lactation to identify affected hepatic molecular pathways. Dams were fed isoenergetic diets with control (20% w/w) or high protein levels (40%). The hepatic expression profiles were evaluated by differential microarray analysis 3 days (d3) and 3 weeks (d21) after birth. Offspring from three different high-protein dietary groups, HP (d3, high-protein diet during pregnancy), HPHP (d21, high-protein diet during pregnancy and lactation) and CHP (d21, control diet during pregnancy and high-protein diet during lactation), were compared with age-matched offspring from dams fed control diet. Offspring body and liver mass of all high-protein groups were decreased. Prenatal high-protein diet affected hepatic expression of genes mapping to the acute response/complement system and the GH/JAK/STAT/IGF signalling pathways. Maternal exposure to high-protein diet during lactation affected hepatic gene expression of the same pathways but additionally affected genes mapping to protein, fatty acid, hexose and pyruvate metabolism. (1) Genes of the acute response/complement system and GH/JAK/STAT/IGF pathways were down-regulated in offspring of dams exposed to high-protein diets during pregnancy and/or lactation. (2) Genes related to nutrient and energy metabolism, however, were only affected when high-protein diet was administered during lactation. (3) Modulation of the GH/JAK/STAT/IGF pathway might be responsible for reduced body and liver masses by maternal high-protein diet.

High-resolution Antibody Array Analysis of Childhood Acute Leukemia Cells*

PubMed Central

Kanderova, Veronika; Kuzilkova, Daniela; Stuchly, Jan; Vaskova, Martina; Brdicka, Tomas; Fiser, Karel; Hrusak, Ondrej; Lund-Johansen, Fridtjof

2016-01-01

Acute leukemia is a disease pathologically manifested at both genomic and proteomic levels. Molecular genetic technologies are currently widely used in clinical research. In contrast, sensitive and high-throughput proteomic techniques for performing protein analyses in patient samples are still lacking. Here, we used a technology based on size exclusion chromatography followed by immunoprecipitation of target proteins with an antibody bead array (Size Exclusion Chromatography-Microsphere-based Affinity Proteomics, SEC-MAP) to detect hundreds of proteins from a single sample. In addition, we developed semi-automatic bioinformatics tools to adapt this technology for high-content proteomic screening of pediatric acute leukemia patients. To confirm the utility of SEC-MAP in leukemia immunophenotyping, we tested 31 leukemia diagnostic markers in parallel by SEC-MAP and flow cytometry. We identified 28 antibodies suitable for both techniques. Eighteen of them provided excellent quantitative correlation between SEC-MAP and flow cytometry (p < 0.05). Next, SEC-MAP was applied to examine 57 diagnostic samples from patients with acute leukemia. In this assay, we used 632 different antibodies and detected 501 targets. Of those, 47 targets were differentially expressed between at least two of the three acute leukemia subgroups. The CD markers correlated with immunophenotypic categories as expected. From non-CD markers, we found DBN1, PAX5, or PTK2 overexpressed in B-cell precursor acute lymphoblastic leukemias, LAT, SH2D1A, or STAT5A overexpressed in T-cell acute lymphoblastic leukemias, and HCK, GLUD1, or SYK overexpressed in acute myeloid leukemias. In addition, OPAL1 overexpression corresponded to ETV6-RUNX1 chromosomal translocation. In summary, we demonstrated that SEC-MAP technology is a powerful tool for detecting hundreds of proteins in clinical samples obtained from pediatric acute leukemia patients. It provides information about protein size and reveals differences in protein expression between particular leukemia subgroups. Forty-seven of SEC-MAP identified targets were validated by other conventional method in this study. PMID:26785729

Loss of Mitogen-Activated Protein Kinase Kinase Kinase 4 (MAP3K4) Reveals a Requirement for MAPK Signalling in Mouse Sex Determination

PubMed Central

Bogani, Debora; Siggers, Pam; Brixey, Rachel; Warr, Nick; Beddow, Sarah; Edwards, Jessica; Williams, Debbie; Wilhelm, Dagmar; Koopman, Peter; Flavell, Richard A.; Chi, Hongbo; Ostrer, Harry; Wells, Sara; Cheeseman, Michael; Greenfield, Andy

2009-01-01

Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY) gonad, sex-determining region of the Y (SRY) protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK) signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg) mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4), a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas). These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and create a novel entry point into the molecular and cellular mechanisms underlying sex determination in mice and disorders of sexual development in humans. PMID:19753101

Amelioration of cognitive impairment and changes in microtubule-associated protein 2 after transient global cerebral ischemia are influenced by complex environment experience.

PubMed

Briones, Teresita L; Woods, Julie; Wadowska, Magdalena; Rogozinska, Magdalena

2006-04-03

In this study we examined whether expression of microtubule-associated protein 2 (MAP2) after transient global cerebral ischemia can be influenced by behavioral experience and if the changes are associated with functional improvement. Rats received either ischemia or sham surgery then assigned to: complex environment housing (EC) or social housing (SC) as controls for 14 days followed by water maze testing. Upregulation of MAP2 was seen in all ischemic animals with a significant overall increase evident in the EC housed rats. Behaviorally, all animals learned to perform the water maze task over time but the ischemia SC rats had the worst performance overall while all the EC housed animals demonstrated the best performance in general. Regression analysis showed that increase MAP2 expression was able to explain some of the variance in the behavioral parameters in the water maze suggesting that this cytoskeletal protein probably played a role in mediating enhanced functional outcomes.

MAP18 regulates the direction of pollen tube growth in Arabidopsis by modulating F-actin organization.

PubMed

Zhu, Lei; Zhang, Yan; Kang, Erfang; Xu, Qiangyi; Wang, Miaoying; Rui, Yue; Liu, Baoquan; Yuan, Ming; Fu, Ying

2013-03-01

For fertilization to occur in plants, the pollen tube must be guided to enter the ovule via the micropyle. Previous reports have implicated actin filaments, actin binding proteins, and the tip-focused calcium gradient as key contributors to polar growth of pollen tubes; however, the regulation of directional pollen tube growth is largely unknown. We reported previously that Arabidopsis thaliana MICROTUBULE-ASSOCIATED PROTEIN18 (MAP18) contributes to directional cell growth and cortical microtubule organization. The preferential expression of MAP18 in pollen and in pollen tubes suggests that MAP18 also may function in pollen tube growth. In this study, we demonstrate that MAP18 functions in pollen tubes by influencing actin organization, rather than microtubule assembly. In vitro biochemical results indicate that MAP18 exhibits Ca(2+)-dependent filamentous (F)-actin-severing activity. Abnormal expression of MAP18 in map18 and MAP18 OX plants was associated with disorganization of the actin cytoskeleton in the tube apex, resulting in aberrant pollen tube growth patterns and morphologies, inaccurate micropyle targeting, and fewer fertilization events. Experiments with MAP18 mutants created by site-directed mutagenesis suggest that F-actin-severing activity is essential to the effects of MAP18 on pollen tube growth direction. Our study demonstrates that in Arabidopsis, MAP18 guides the direction of pollen tube growth by modulating actin filaments.

A glycosylated recombinant subunit candidate vaccine consisting of Ehrlichia ruminantium major antigenic protein1 induces specific humoral and Th1 type cell responses in sheep.

PubMed

Faburay, Bonto; McGill, Jodi; Jongejan, Frans

2017-01-01

Heartwater, or cowdriosis, is a tick-borne disease of domestic and wild ruminants that is endemic in the Caribbean and sub-Saharan Africa. The disease is caused by an intracellular pathogen, Ehrlichia ruminantium and may be fatal within days of the onset of clinical signs with mortality rates of up to 90% in susceptible hosts. Due to the presence of competent tick vectors in North America, there is substantial risk of introduction of heartwater with potentially devastating consequences to the domestic livestock industry. There is currently no reliable or safe vaccine for use globally. To develop a protective DIVA (differentiate infected from vaccinated animals) subunit vaccine for heartwater, we targeted the E. ruminantium immunodominant major antigenic protein1 (MAP1) with the hypothesis that MAP1 is a glycosylated protein and glycans contained in the antigenic protein are important epitope determinants. Using a eukaryotic recombinant baculovirus expression system, we expressed and characterized, for the first time, a glycoform profile of MAP1 of two Caribbean E. ruminantium isolates, Antigua and Gardel. We have shown that the 37-38 kDa protein corresponded to a glycosylated form of the MAP1 protein, whereas the 31-32 kDa molecular weight band represented the non-glycosylated form of the protein frequently reported in scientific literature. Three groups of sheep (n = 3-6) were vaccinated with increasing doses of a bivalent (Antigua and Gardel MAP1) rMAP1 vaccine cocktail formulation with montanide ISA25 as an adjuvant. The glycosylated recombinant subunit vaccine induced E. ruminantium-specific humoral and Th1 type T cell responses, which are critical for controlling intracellular pathogens, including E. ruminantium, in infected hosts. These results provide an important basis for development of a subunit vaccine as a novel strategy to protect susceptible livestock against heartwater in non-endemic and endemic areas.

A glycosylated recombinant subunit candidate vaccine consisting of Ehrlichia ruminantium major antigenic protein1 induces specific humoral and Th1 type cell responses in sheep

PubMed Central

McGill, Jodi; Jongejan, Frans

2017-01-01

Heartwater, or cowdriosis, is a tick-borne disease of domestic and wild ruminants that is endemic in the Caribbean and sub-Saharan Africa. The disease is caused by an intracellular pathogen, Ehrlichia ruminantium and may be fatal within days of the onset of clinical signs with mortality rates of up to 90% in susceptible hosts. Due to the presence of competent tick vectors in North America, there is substantial risk of introduction of heartwater with potentially devastating consequences to the domestic livestock industry. There is currently no reliable or safe vaccine for use globally. To develop a protective DIVA (differentiate infected from vaccinated animals) subunit vaccine for heartwater, we targeted the E. ruminantium immunodominant major antigenic protein1 (MAP1) with the hypothesis that MAP1 is a glycosylated protein and glycans contained in the antigenic protein are important epitope determinants. Using a eukaryotic recombinant baculovirus expression system, we expressed and characterized, for the first time, a glycoform profile of MAP1 of two Caribbean E. ruminantium isolates, Antigua and Gardel. We have shown that the 37–38 kDa protein corresponded to a glycosylated form of the MAP1 protein, whereas the 31–32 kDa molecular weight band represented the non-glycosylated form of the protein frequently reported in scientific literature. Three groups of sheep (n = 3–6) were vaccinated with increasing doses of a bivalent (Antigua and Gardel MAP1) rMAP1 vaccine cocktail formulation with montanide ISA25 as an adjuvant. The glycosylated recombinant subunit vaccine induced E. ruminantium-specific humoral and Th1 type T cell responses, which are critical for controlling intracellular pathogens, including E. ruminantium, in infected hosts. These results provide an important basis for development of a subunit vaccine as a novel strategy to protect susceptible livestock against heartwater in non-endemic and endemic areas. PMID:28957443

Oxidative-stress-related proteome changes in Helicobacter pylori-infected human gastric mucosa.

PubMed Central

Baek, Hye Yeon; Lim, Joo Weon; Kim, Hyeyoung; Kim, Jung Mogg; Kim, Joo Sung; Jung, Hyun Chae; Kim, Kyung Hwan

2004-01-01

Helicobacter pylori infection leads to gastroduodenal inflammation, peptic ulceration and gastric carcinoma. Proteomic analysis of the human gastric mucosa from the patients with erosive gastritis, peptic ulcer or gastric cancer, which were either infected or not with H. pylori, was used to determine the differentially expressed proteins by H. pylori in the human gastric mucosa in order to investigate the pathogenic mechanism of H. pylori -induced gastric diseases. Prior to the experiment, the expression of the main 18 proteins were identified in the gastric mucosa and used for a proteome map of the human gastric mucosa. Using two-dimensional electrophoresis of the protein isolated from the H. pylori -infected tissues, Coomassie Brilliant Blue staining and computerized analysis of the stained gel, the expression of eight proteins were altered in the H. pylori -infected tissues compared with the non-infected tissues. MS analysis (matrix-assisted laser desorption/ionization-time of flight MS) of the tryptic fragment and a data search allowed the the identification of the four increased proteins (78 kDa glucose-regulated protein precursor, endoplasmin precursor, aldehyde dehydrogenase 2 and L-lactate dehydrogenase B chain) and the four decreased proteins (intracellular chloride channel protein 1, glutathione S-transferase, heat-shock protein 60 and cytokeratin 8) caused by H. pylori infection in the gastric mucosa. These proteins are related to cell proliferation, carcinogenesis, cytoskeletal function and cellular defence mechanism. The common feature is that these proteins are related to oxidative-stress-mediated cell damage. In conclusion, the established gastric mucosal proteome map might be useful for detecting the disease-related protein changes. The H. pylori -induced alterations in protein expression demonstrate the involvement of oxidative stress in the pathogenesis of H. pylori -induced gastric diseases, including inflammation, ulceration and carcinogenesis. PMID:14711373

Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases

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

Arai, Roberto J.; Ogata, Fernando T.; Batista, Wagner L.

2008-12-01

Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects ofmore » GSNO on decreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases.« less

A New MAP Kinase Protein Involved in Estradiol-Stimulated Reproduction of the Helminth Parasite Taenia crassiceps

PubMed Central

Escobedo, Galileo; Soldevila, Gloria; Ortega-Pierres, Guadalupe; Chávez-Ríos, Jesús Ramsés; Nava, Karen; Fonseca-Liñán, Rocío; López-Griego, Lorena; Hallal-Calleros, Claudia; Ostoa-Saloma, Pedro; Morales-Montor, Jorge

2010-01-01

MAP kinases (MAPK) are involved in the regulation of cellular processes such as reproduction and growth. In parasites, the role of MAPK has been scarcely studied. Here, we describe the participation of an ERK-like protein in estrogen-dependent reproduction of the helminth parasite Taenia crassiceps. Our results show that 17β-estradiol induces a concentration-dependent increase in the bud number of in vitro cultured cysticerci. If parasites are also incubated in presence of an ERK-inhibitor, the stimulatory effect of estrogen is blocked. The expression of ERK-like mRNA and its corresponding protein was detected in the parasite. The ERK-like protein was over-expressed by all treatments. Nevertheless, a strong induction of phosphorylation of this protein was observed only in response to 17β-estradiol. Cross-contamination by host cells was discarded by flow cytometry analysis. Parasite cells expressing the ERK-like protein were exclusively located at the subtegument tissue by confocal microscopy. Finally, the ERK-like protein was separated by bidimensional electrophoresis and then sequenced, showing the conserved TEY activation motif, typical of all known ERK 1/2 proteins. Our results show that an ERK-like protein is involved in the molecular signalling during the interaction between the host and T. crassiceps, and may be considered as target for anti-helminth drugs design. PMID:20145710

Enhanced basal activation of mitogen-activated protein kinases in adipocytes from type 2 diabetes: potential role of p38 in the downregulation of GLUT4 expression.

PubMed

Carlson, Christian J; Koterski, Sandra; Sciotti, Richard J; Poccard, German Braillard; Rondinone, Cristina M

2003-03-01

Serine and threonine kinases may contribute to insulin resistance and the development of type 2 diabetes. To test the potential for members of the mitogen-activated protein (MAP) kinase family to contribute to type 2 diabetes, we examined basal and insulin-stimulated Erk 1/2, JNK, and p38 phosphorylation in adipocytes isolated from healthy and type 2 diabetic individuals. Maximal insulin stimulation increased the phosphorylation of Erk 1/2 and JNK in healthy control subjects but not type 2 diabetic patients. Insulin stimulation did not increase p38 phosphorylation in either healthy control subjects or type 2 diabetic patients. In type 2 diabetic adipocytes, the basal phosphorylation status of these MAP kinases was significantly elevated and was associated with decreased IRS-1 and GLUT4 in these fat cells. To determine whether MAP kinases were involved in the downregulation of IRS-1 and GLUT4 protein levels, selective inhibitors were used to inhibit these MAP kinases in 3T3-L1 adipocytes treated chronically with insulin. Inhibition of Erk 1/2, JNK, or p38 had no effect on insulin-stimulated reduction of IRS-1 protein levels. However, inhibition of the p38 pathway prevented the insulin-stimulated decrease in GLUT4 protein levels. In summary, type 2 diabetes is associated with an increased basal activation of the MAP kinase family. Furthermore, upregulation of the p38 pathway might contribute to the loss of GLUT4 expression observed in adipose tissue from type 2 diabetic patients.

The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs.

PubMed Central

Nebreda, A R; Hunt, T

1993-01-01

During studies of the activation and inactivation of the cyclin B-p34cdc2 protein kinase (MPF) in cell-free extracts of Xenopus oocytes and eggs, we found that a bacterially expressed fusion protein between the Escherichia coli maltose-binding protein and the Xenopus c-mos protein kinase (malE-mos) activated a 42 kDa MAP kinase. The activation of MAP kinase on addition of malE-mos was consistent, whereas the activation of MPF was variable and failed to occur in some oocyte extracts in which cyclin A or okadaic acid activated both MPF and MAP kinase. In cases when MPF activation was transient, MAP kinase activity declined after MPF activity was lost, and MAP kinase, but not MPF, could be maintained at a high level by the presence of malE-mos. When intact oocytes were treated with progesterone, however, the activation of MPF and MAP kinase occurred simultaneously, in contrast to the behaviour of extracts. These observations suggest that one role of c-mos may be to maintain high MAP kinase activity in meiosis. They also imply that the activation of MPF and MAP kinase in vivo are synchronous events that normally rely on an agent that has still to be identified. Images PMID:8387916

Sequence, molecular properties, and chromosomal mapping of mouse lumican

NASA Technical Reports Server (NTRS)

Funderburgh, J. L.; Funderburgh, M. L.; Hevelone, N. D.; Stech, M. E.; Justice, M. J.; Liu, C. Y.; Kao, W. W.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

1995-01-01

PURPOSE. Lumican is a major proteoglycan of vertebrate cornea. This study characterizes mouse lumican, its molecular form, cDNA sequence, and chromosomal localization. METHODS. Lumican sequence was determined from cDNA clones selected from a mouse corneal cDNA expression library using a bovine lumican cDNA probe. Tissue expression and size of lumican mRNA were determined using Northern hybridization. Glycosidase digestion followed by Western blot analysis provided characterization of molecular properties of purified mouse corneal lumican. Chromosomal mapping of the lumican gene (Lcn) used Southern hybridization of a panel of genomic DNAs from an interspecific murine backcross. RESULTS. Mouse lumican is a 338-amino acid protein with high-sequence identity to bovine and chicken lumican proteins. The N-terminus of the lumican protein contains consensus sequences for tyrosine sulfation. A 1.9-kb lumican mRNA is present in cornea and several other tissues. Antibody against bovine lumican reacted with recombinant mouse lumican expressed in Escherichia coli and also detected high molecular weight proteoglycans in extracts of mouse cornea. Keratanase digestion of corneal proteoglycans released lumican protein, demonstrating the presence of sulfated keratan sulfate chains on mouse corneal lumican in vivo. The lumican gene (Lcn) was mapped to the distal region of mouse chromosome 10. The Lcn map site is in the region of a previously identified developmental mutant, eye blebs, affecting corneal morphology. CONCLUSIONS. This study demonstrates sulfated keratan sulfate proteoglycan in mouse cornea and describes the tools (antibodies and cDNA) necessary to investigate the functional role of this important corneal molecule using naturally occurring and induced mutants of the murine lumican gene.

CsMAP34, a teleost MAP with dual role: A promoter of MASP-assisted complement activation and a regulator of immune cell activity.

PubMed

Li, Mo-Fei; Li, Jun; Sun, Li

2016-12-23

In teleost fish, the immune functions of mannan-binding lectin (MBL) associated protein (MAP) and MBL associated serine protease (MASP) are scarcely investigated. In the present study, we examined the biological properties both MAP (CsMAP34) and MASP (CsMASP1) molecules from tongue sole (Cynoglossus semilaevis). We found that CsMAP34 and CsMASP1 expressions occurred in nine different tissues and were upregulated by bacterial challenge. CsMAP34 protein was detected in blood, especially during bacterial infection. Recombinant CsMAP34 (rCsMAP34) bound C. semilaevis MBL (rCsBML) when the latter was activated by bacteria, while recombinant CsMASP1 (rCsMASP1) bound activated rCsBML only in the presence of rCsMAP34. rCsMAP34 stimulated the hemolytic and bactericidal activities of serum complement, whereas anti-CsMAP34 antibody blocked complement activities. Knockdown of CsMASP1 in C. semilaevis resulted in significant inhibition of complement activities. Furthermore, rCsMAP34 interacted directly with peripheral blood leukocytes (PBL) and enhanced the respiratory burst, acid phosphatase activity, chemotactic activity, and gene expression of PBL. These results indicate for the first time that a teleost MAP acts one hand as a regulator that promotes the lectin pathway of complement activation via its ability to recruit MBL to MASP, and other hand as a modulator of immune cell activity.

Overexpression of peptide deformylase in breast, colon, and lung cancers.

PubMed

Randhawa, Harsharan; Chikara, Shireen; Gehring, Drew; Yildirim, Tuba; Menon, Jyotsana; Reindl, Katie M

2013-07-01

Human mitochondrial peptide deformylase (PDF) has been proposed as a novel cancer therapeutic target. However, very little is known about its expression and regulation in human tissues. The purpose of this study was to characterize the expression pattern of PDF in cancerous tissues and to identify mechanisms that regulate its expression. The mRNA expression levels of PDF and methionine aminopeptidase 1D (MAP1D), an enzyme involved in a related pathway with PDF, were determined using tissue panels containing cDNA from patients with various types of cancer (breast, colon, kidney, liver, lung, ovarian, prostate, or thyroid) and human cell lines. Protein levels of PDF were also determined in 2 colon cancer patients via western blotting. Colon cancer cells were treated with inhibitors of ERK, Akt, and mTOR signaling pathways and the resulting effects on PDF and MAP1D mRNA levels were determined by qPCR for colon and lung cancer cell lines. Finally, the effects of a PDF inhibitor, actinonin, on the proliferation of breast, colon, and prostate cell lines were determined using the CyQUANT assay. PDF and MAP1D mRNA levels were elevated in cancer cell lines compared to non-cancer lines. PDF mRNA levels were significantly increased in breast, colon, and lung cancer samples while MAP1D mRNA levels were increased in just colon cancers. The expression of PDF and MAP1D varied with stage in these cancers. Further, PDF protein expression was elevated in colon cancer tissue samples. Inhibition of the MEK/ERK, but not PI3K or mTOR, pathway reduced the expression of PDF and MAP1D in both colon and lung cancer cell lines. Further, inhibition of PDF with actinonin resulted in greater reduction of breast, colon, and prostate cancer cell proliferation than non-cancer cell lines. This is the first report showing that PDF is over-expressed in breast, colon, and lung cancers, and the first evidence that the MEK/ERK pathway plays a role in regulating the expression of PDF and MAP1D. The over-expression of PDF in several cancers and the inhibition of cancer cell growth by a PDF inhibitor suggest this enzyme may act as an oncogene to promote cancer cell proliferation.

Overexpression of peptide deformylase in breast, colon, and lung cancers

PubMed Central

2013-01-01

Background Human mitochondrial peptide deformylase (PDF) has been proposed as a novel cancer therapeutic target. However, very little is known about its expression and regulation in human tissues. The purpose of this study was to characterize the expression pattern of PDF in cancerous tissues and to identify mechanisms that regulate its expression. Methods The mRNA expression levels of PDF and methionine aminopeptidase 1D (MAP1D), an enzyme involved in a related pathway with PDF, were determined using tissue panels containing cDNA from patients with various types of cancer (breast, colon, kidney, liver, lung, ovarian, prostate, or thyroid) and human cell lines. Protein levels of PDF were also determined in 2 colon cancer patients via western blotting. Colon cancer cells were treated with inhibitors of ERK, Akt, and mTOR signaling pathways and the resulting effects on PDF and MAP1D mRNA levels were determined by qPCR for colon and lung cancer cell lines. Finally, the effects of a PDF inhibitor, actinonin, on the proliferation of breast, colon, and prostate cell lines were determined using the CyQUANT assay. Results PDF and MAP1D mRNA levels were elevated in cancer cell lines compared to non-cancer lines. PDF mRNA levels were significantly increased in breast, colon, and lung cancer samples while MAP1D mRNA levels were increased in just colon cancers. The expression of PDF and MAP1D varied with stage in these cancers. Further, PDF protein expression was elevated in colon cancer tissue samples. Inhibition of the MEK/ERK, but not PI3K or mTOR, pathway reduced the expression of PDF and MAP1D in both colon and lung cancer cell lines. Further, inhibition of PDF with actinonin resulted in greater reduction of breast, colon, and prostate cancer cell proliferation than non-cancer cell lines. Conclusions This is the first report showing that PDF is over-expressed in breast, colon, and lung cancers, and the first evidence that the MEK/ERK pathway plays a role in regulating the expression of PDF and MAP1D. The over-expression of PDF in several cancers and the inhibition of cancer cell growth by a PDF inhibitor suggest this enzyme may act as an oncogene to promote cancer cell proliferation. PMID:23815882

miR-188 promotes senescence of lineage-negative bone marrow cells by targeting MAP3K3 expression.

PubMed

Zheng, Yue; Liu, Hua; Kong, Ye

2017-08-01

Lineage-negative bone marrow cells (lin-BMCs) have reparative potential for overcoming endothelial dysfunction and reducing cardiovascular risk. Here, we found that miR-188 is upregulated and mitogen-activated protein kinase kinase kinase 3 (MAP3K3) is downregulated in aged lin-BMCs, whereas their expression is reversed in young lin-BMCs. We identified and confirmed MAP3K3 as a direct target of miR-188. MiR-188 overexpression or MAP3K3 silencing in young lin-BMCs increases p16 and p21 expression, enhances cell senescence, and decreases the ability for cell proliferation, migration, and tube formation. Conversely, miR-188 suppression in aged lin-BMCs yields the opposite results. We further found that MAP3K3 is involved in miR-188-induced promotion of lin-BMC senescence. All data reveal that miR-188 induces lin-BMC senescence by targeting MAP3K3 expression, thus, providing new theoretical basis for the prevention and treatment of cardiovascular diseases. © 2017 Federation of European Biochemical Societies.

The eukaryotic cofactor for the human immunodeficiency virus type 1 (HIV-1) Rev protein, elF-5A, maps to chromosome 17p12-p13: Three elF-5A pseudogenes map to 10q23.3, 17q25, and 19q13.2

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

Steinkasserer, A.; Koettnitz, K.; Hauber, J.

1995-02-10

The eukaryotic initiation factor 5A (eIF-5A) has been identified as an essential cofactor for the HIV-1 transactivator protein Rev. Rev plays a key role in the complex regulation of HIV-1 gene expression and thereby in the generation of infectious virus particles. Expression of eIF-5A is vital for Rev function, and inhibition of this interaction leads to a block of the viral replication cycle. In humans, four different eIF-5A genes have been identified. One codes for the eIF-5A protein and the other three are pseudogenes. Using a panel of somatic rodent-human cell hybrids in combination with fluorescence in situ hybridization analysis,more » we show that the four genes map to three different chromosomes. The coding eIF-5A gene (EIF5A) maps to 17p12-p13, and the three pseudogenes EIF5AP1, EIF5AP2, and EIF5AP3 map to 10q23.3, 17q25, and 19q13.2, respectively. This is the first localization report for a eukaryotic cofactor for a regulatory HIV-1 protein. 16 refs., 1 fig.« less

Acrylamide inhibits cellular differentiation of human neuroblastoma and glioblastoma cells.

PubMed

Chen, Jong-Hang; Chou, Chin-Cheng

2015-08-01

This study explores human neuroblastoma (SH-SY5Y) and human glioblastoma (U-1240 MG) cellular differentiation changes under exposure to acrylamide (ACR). Differentiation of SH-SY5Y and U-1240 MG cells were induced by retinoic acid (RA) and butyric acid (BA), respectively. Morphological observations and MTT assay showed that the induced cellular differentiation and cell proliferation were inhibited by ACR in a time- and dose-dependent manner. ACR co-treatment with RA attenuated SH-SY5Y expressions of neurofilament protein-L (NF-L), microtubule-associated protein 1b (MAP1b; 1.2 to 0.7, p < 0.001), MAP2c (2.2 to 0.8, p < 0.05), and Janus kinase1 (JAK1; 1.9 to 0.6, p < 0.001), while ACR co-treatment with BA attenuated U-1240 MG expressions of glial fibrillary acidic protein (GFAP), MAP1b (1.2 to 0.6, p < 0.001), MAP2c (1.5 to 0.7, p < 0.01), and JAK1 (2.1 to 0.5, p < 0.001), respectively. ACR also decreased the phosphorylation of extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) in U-1240 MG cells, while caffeine reversed this suppression of ERK and JNK phosphorylation caused by ACR treatment. These results showed that RA-induced neurogenesis of SH-SY5Y and BA-induced astrogliogenesis of U-1240 MG cells were attenuated by ACR and were associated with down-regulation of MAPs expression and JAK-STAT signaling. Copyright © 2015 Elsevier Ltd. All rights reserved.

Phosphorylated and Nonphosphorylated PfMAP2 Are Localized in the Nucleus, Dependent on the Stage of Plasmodium falciparum Asexual Maturation

PubMed Central

Dahalan, Farah Aida; Sidek, Hasidah Mohd; Murtey, Mogana Das; Embi, Mohammed Noor; Ibrahim, Jamaiah; Fei Tieng, Lim; Zakaria, Nurul Aiezzah

2016-01-01

Plasmodium falciparum mitogen-activated protein (MAP) kinases, a family of enzymes central to signal transduction processes including inflammatory responses, are a promising target for antimalarial drug development. Our study shows for the first time that the P. falciparum specific MAP kinase 2 (PfMAP2) is colocalized in the nucleus of all of the asexual erythrocytic stages of P. falciparum and is particularly elevated in its phosphorylated form. It was also discovered that PfMAP2 is expressed in its highest quantity during the early trophozoite (ring form) stage and significantly reduced in the mature trophozoite and schizont stages. Although the phosphorylated form of the kinase is always more prevalent, its ratio relative to the nonphosphorylated form remained constant irrespective of the parasites' developmental stage. We have also shown that the TSH motif specifically renders PfMAP2 genetically divergent from the other plasmodial MAP kinase activation sites using Neighbour Joining analysis. Furthermore, TSH motif-specific designed antibody is crucial in determining the location of the expression of the PfMAP2 protein. However, by using immunoelectron microscopy, PPfMAP2 were detected ubiquitously in the parasitized erythrocytes. In summary, PfMAP2 may play a far more important role than previously thought and is a worthy candidate for research as an antimalarial. PMID:27525262

Cloning and characterization of mouse extracellular-signal-regulated protein kinase 3 as a unique gene product of 100 kDa.

PubMed

Turgeon, B; Saba-El-Leil, M K; Meloche, S

2000-02-15

MAP (mitogen-activated protein) kinases are a family of serine/threonine kinases that have a pivotal role in signal transduction. Here we report the cloning and characterization of a mouse homologue of extracellular-signal-regulated protein kinase (ERK)3. The mouse Erk3 cDNA encodes a predicted protein of 720 residues, which displays 94% identity with human ERK3. Transcription and translation of this cDNA in vitro generates a 100 kDa protein similar to the human gene product ERK3. Immunoblot analysis with an antibody raised against a unique sequence of ERK3 also recognizes a 100 kDa protein in mouse tissues. A single transcript of Erk3 was detected in every adult mouse tissue examined, with the highest expression being found in the brain. Interestingly, expression of Erk3 mRNA is acutely regulated during mouse development, with a peak of expression observed at embryonic day 11. The mouse Erk3 gene was mapped to a single locus on central mouse chromosome 9, adjacent to the dilute mutation locus and in a region syntenic to human chromosome 15q21. Finally, we provide several lines of evidence to support the existence of a unique Erk3 gene product of 100 kDa in mammalian cells.

Diagnostic utility and limitations of glutamine synthetase and serum amyloid-associated protein immunohistochemistry in the distinction of focal nodular hyperplasia and inflammatory hepatocellular adenoma.

PubMed

Joseph, Nancy M; Ferrell, Linda D; Jain, Dhanpat; Torbenson, Michael S; Wu, Tsung-Teh; Yeh, Matthew M; Kakar, Sanjay

2014-01-01

Inflammatory hepatocellular adenoma can show overlapping histological features with focal nodular hyperplasia, including inflammation, fibrous stroma, and ductular reaction. Expression of serum amyloid-associated protein in inflammatory hepatocellular adenoma and map-like pattern of glutamine synthetase in focal nodular hyperplasia can be helpful in this distinction, but the pitfalls and limitations of these markers have not been established. Morphology and immunohistochemistry were analyzed in 54 inflammatory hepatocellular adenomas, 40 focal nodular hyperplasia, and 3 indeterminate lesions. Morphological analysis demonstrated that nodularity, fibrous stroma, dystrophic blood vessels, and ductular reaction were more common in focal nodular hyperplasia, while telangiectasia, hemorrhage, and steatosis were more common in inflammatory hepatocellular adenoma, but there was frequent overlap of morphological features. The majority of inflammatory hepatocellular adenomas demonstrated perivascular and/or patchy glutamine synthetase staining (73.6%), while the remaining cases had diffuse (7.5%), negative (3.8%), or patchy pattern of staining (15%) that showed subtle differences from the classic map-like staining pattern and was designated as pseudo map-like staining. Positive staining for serum amyloid-associated protein was seen in the majority of inflammatory hepatocellular adenomas (92.6%) and in the minority of focal nodular hyperplasia (17.5%). The glutamine synthetase staining pattern was map-like in 90% of focal nodular hyperplasia cases, with the remaining 10% of cases showing pseudo map-like staining. Three cases were labeled as indeterminate and showed focal nodular hyperplasia-like morphology but lacked map-like glutamine synthetase staining pattern; these cases demonstrated a patchy pseudo map-like glutamine synthetase pattern along with the expression of serum amyloid-associated protein. Our results highlight the diagnostic errors that can be caused by variant patterns of staining with glutamine synthetase and serum amyloid-associated protein in inflammatory hepatocellular adenoma and focal nodular hyperplasia.

The Proteome of Shigella flexneri 2a 2457T Grown at 30 and 37 °C*

PubMed Central

Zhu, Li; Zhao, Ge; Stein, Robert; Zheng, Xuexue; Hu, Wei; Shang, Na; Bu, Xin; Liu, Xiankai; Wang, Jie; Feng, Erling; Wang, Bin; Zhang, Xuemin; Ye, Qinong; Huang, Peitang; Zeng, Ming; Wang, Hengliang

2010-01-01

To upgrade the proteome reference map of Shigella flexneri 2a 2457T, the protein expression profiles of log phase and stationary phase cells grown at 30 and 37 °C were thoroughly analyzed using multiple overlapping narrow pH range (between pH 4.0 and 11.0) two-dimensional gel electrophoresis. A total of 723 spots representing 574 protein entries were identified by MALDI-TOF/TOF MS, including the majority of known key virulence factors. 64 hypothetical proteins and six misannotated proteins were also experimentally identified. A comparison between the four proteome maps showed that most of the virulence-related proteins were up-regulated at 37 °C, and the differences were more notable in stationary phase cells, suggesting that the expressions of these virulence factors were not only controlled by temperature but also controlled by the nutrients available in the environment. The expression patterns of some virulence-related genes under the four different conditions suggested that they might also be regulated at the post-transcriptional level. A further significant finding was that the expression of the protein ArgT was dramatically up-regulated at 30 °C. The results of semiquantitative RT-PCR analysis showed that expression of argT was not regulated at the transcriptional level. Therefore, we carried out a series of experiments to uncover the mechanism regulating ArgT levels and found that the differential expression of ArgT was due to its degradation by a periplasmic protease, HtrA, whose activity, but not its synthesis, was affected by temperature. The cleavage site in ArgT was between position 160 (Val) and position 161 (Ala). These results may provide useful insights for understanding the physiology and pathogenesis of S. flexneri. PMID:20164057

The proteome of Shigella flexneri 2a 2457T grown at 30 and 37 degrees C.

PubMed

Zhu, Li; Zhao, Ge; Stein, Robert; Zheng, Xuexue; Hu, Wei; Shang, Na; Bu, Xin; Liu, Xiankai; Wang, Jie; Feng, Erling; Wang, Bin; Zhang, Xuemin; Ye, Qinong; Huang, Peitang; Zeng, Ming; Wang, Hengliang

2010-06-01

To upgrade the proteome reference map of Shigella flexneri 2a 2457T, the protein expression profiles of log phase and stationary phase cells grown at 30 and 37 degrees C were thoroughly analyzed using multiple overlapping narrow pH range (between pH 4.0 and 11.0) two-dimensional gel electrophoresis. A total of 723 spots representing 574 protein entries were identified by MALDI-TOF/TOF MS, including the majority of known key virulence factors. 64 hypothetical proteins and six misannotated proteins were also experimentally identified. A comparison between the four proteome maps showed that most of the virulence-related proteins were up-regulated at 37 degrees C, and the differences were more notable in stationary phase cells, suggesting that the expressions of these virulence factors were not only controlled by temperature but also controlled by the nutrients available in the environment. The expression patterns of some virulence-related genes under the four different conditions suggested that they might also be regulated at the post-transcriptional level. A further significant finding was that the expression of the protein ArgT was dramatically up-regulated at 30 degrees C. The results of semiquantitative RT-PCR analysis showed that expression of argT was not regulated at the transcriptional level. Therefore, we carried out a series of experiments to uncover the mechanism regulating ArgT levels and found that the differential expression of ArgT was due to its degradation by a periplasmic protease, HtrA, whose activity, but not its synthesis, was affected by temperature. The cleavage site in ArgT was between position 160 (Val) and position 161 (Ala). These results may provide useful insights for understanding the physiology and pathogenesis of S. flexneri.

Mapping of the antigenic determinants of the Leishmania infantum gp63 protein recognized by antibodies elicited during canine visceral leishmaniasis.

PubMed

Morales, G; Carrillo, G; Requena, J M; Guzman, F; Gomez, L C; Patarroyo, M E; Alonso, C

1997-06-01

The gp63 gene encoding the major surface antigen of Leishmania infantum has been cloned and sequenced. In spite of the overall sequence homology with the gp63 genes from other Leishmania species, particularly with the constitutively expressed Leishmania chagasi Gp63 gene, the carboxy-terminal ends of these genes are clearly divergent (62% homology). To study the prevalence of anti-gp63 antibodies in the sera from dogs with visceral leishmaniasis, a recombinant L. infantum gp63 protein was expressed in Escherichia coli. It was found that 100% of the sera from these dogs recognized the recombinant gp63 protein, suggesting that it must function as a potent B cell immunogen during natural canine visceral leishmaniasis. However, heterogeneity in the level of response was observed. Fine mapping of the antigenic determinants was performed by means of 6 overlapping subfragments of the gp63 protein and by the use of a library of synthetic peptides. The data showed that there is some degree of immunological restriction in the recognition of the protein since reactivity was observed preferentially against the most divergent region. The epitope mapping of this region showed 2 immunodominant peptides the response to which seems to be preferentially of the IgG2 type.

Cloning and characterization of GETS-1, a goldfish Ets family member that functions as a transcriptional repressor in muscle.

PubMed

Goldman, D; Sapru, M K; Stewart, S; Plotkin, J; Libermann, T A; Wasylyk, B; Guan, K

1998-10-15

An Ets transcription factor family member, GETS-1, was cloned from a goldfish retina cDNA library. GETS-1 contains a conserved Ets DNA-binding domain at its N-terminus and is most similar to ternary complex factor (TCF) serum-response-factor protein-1a (SAP-1a). GETS-1 is expressed in many tissues, but is enriched in retina and brain. As with the TCFs SAP-1a and ets-related protein (ERP), overexpression of the GETS-1 promoter suppresses nicotinic acetylcholine receptor epsilon-subunit gene expression in cultured muscle cells. A consensus Ets binding site sequence in the promoter of the epsilon-subunit gene is required for GETS-1-mediated repression. GETS-1 repressor activity is abrogated by overexpression of an activated Ras/mitogen-activated protein kinase (MAP kinase) or by mutation of Ser-405, a MAP kinase phosphorylation site in GETS-1. Fusion proteins created between GETS-1 and the Gal4 DNA-binding domain show that, like other TCFs, GETS-1 contains a C-terminal activation domain that is activated by a Ras/MAP kinase signalling cascade. Interestingly, mutation of Ser-405 located within this activation domain abrogated transcriptional activation of the fusion protein.

Genome-wide analysis of the TPX2 family proteins in Eucalyptus grandis.

PubMed

Du, Pingzhou; Kumar, Manoj; Yao, Yuan; Xie, Qiaoli; Wang, Jinyan; Zhang, Baolong; Gan, Siming; Wang, Yuqi; Wu, Ai-Min

2016-11-24

The Xklp2 (TPX2) proteins belong to the microtubule-associated (MAP) family of proteins. All members of the family contain the conserved TPX2 motif, which can interact with microtubules, regulate microtubule dynamics or assist with different microtubule functions, for example, maintenance of cell morphology or regulation of cell growth and development. However, the role of members of the TPX family have not been studied in the model tree species Eucalyptus to date. Here, we report the identification of the members of the TPX2 family in Eucalyptus grandis (Eg) and analyse the expression patterns and functions of these genes. In present study, a comprehensive analysis of the plant TPX2 family proteins was performed. Phylogenetic analyses indicated that the genes can be classified into 6 distinct subfamilies. A genome-wide survey identified 12 members of the TPX2 family in the sequenced genome of Eucalyptus grandis. The basic genetic properties of the TPX2 family in Eucalyptus were analysed. Our results suggest that the TPX2 family proteins within different sub-groups are relatively conserved but there are important differences between groups. Quantitative real-time PCR (qRT-PCR) was performed to confirm the expression levels of the genes in different tissues. The results showed that in the whole plant, the levels of EgWDL5 transcript are the highest, followed by those of EgWDL4. Compared with other tissues, the level of the EgMAP20 transcript is the highest in the root. Over-expression of EgMAP20 in Arabidopsis resulted in organ twisting. The cotyledon petioles showed left-handed twisting while the hypocotyl epidermal cells produced right-handed helical twisting. Finally, EgMAP20, EgWDL3 and EgWDL3L were all able to decorate microtubules. Plant TPX2 family proteins were systematically analysed using bioinformatics methods. There are 12 TPX2 family proteins in Eucalyptus. We have performed an initial characterization of the functions of several members of the TPX2 family. We found that the gene products are localized to the microtubule cytoskeleton. Our results lay the foundation for future efforts to reveal the biological significance of TPX2 family proteins in Eucalyptus.

Effects of gonadectomy and testosterone treatment on aquaporin expression in the kidney of normotensive and hypertensive rats.

PubMed

Loh, Su Yi; Giribabu, Nelli; Salleh, Naguib

2017-07-01

We tested the hypothesis that testosterone-induced increase in blood pressure was due to changes in aquaporin (AQP) expression in kidneys. In this study, expression level of kidney AQPs was investigated under testosterone influence. Adult normotensive Wistar Kyoto (WKY) and hypertensive SHR male and female rats underwent gonadectomy. For female rats, testosterone was given for six weeks duration, two weeks following ovariectomy via subcutaneous silastic implant. Mean arterial pressure (MAP) was measured in all the rats after eight weeks via carotid artery cannulation and the rats were then sacrificed and kidneys were harvested for analyses of AQP-1, 2, 3, 4, 6, and 7 mRNA and protein expressions by quantitative real-time PCR and Western blotting, respectively. Distribution of AQP subunits' protein in kidneys was observed by immunofluorescence. In male WKY rats, MAP, AQP-1, 2, 4, and 7 protein; and mRNA expression decreased however AQP-3 protein and mRNA expression increased following orchidectomy. The vice versa effects were observed in testosterone-treated ovariectomized female WKY rats. However, no changes in AQP-6 expression were observed. Meanwhile, in adult male SHR rats, MAP and expression level of all AQP subunits decreased following orchidectomy. The opposite effects were seen in ovariectomized female SHR rats following testosterone treatment. Immunofluorescence study showed AQP-1 and AQP-7 were distributed in the proximal convoluted tubules (PCT) while AQP-2, AQP-4, and AQP-6 were distributed in the collecting ducts (CDs). AQP-3 was distributed in the PCT and CD. In conclusion, changes in AQP subunit expression in kidneys could explain changes in blood pressure under testosterone influence. Impact statement This study provides fundamental understanding on the mechanisms underlying testosterone-induced increase in blood pressure which involve regulation of aquaporin channel subunits in the kidneys. A better understanding of this issue can help to explain the reason for higher blood pressure in males as compared to females and may explain the reason for higher blood pressure in females after menopause than females before menopause, the former most probably related to the changes in female androgen.

The Arabidopsis microtubule-associated protein MAP65-3 supports infection by filamentous biotrophic pathogens by down-regulating salicylic acid-dependent defenses.

PubMed

Quentin, Michaël; Baurès, Isabelle; Hoefle, Caroline; Caillaud, Marie-Cécile; Allasia, Valérie; Panabières, Franck; Abad, Pierre; Hückelhoven, Ralph; Keller, Harald; Favery, Bruno

2016-03-01

The oomycete Hyaloperonospora arabidopsidis and the ascomycete Erysiphe cruciferarum are obligate biotrophic pathogens causing downy mildew and powdery mildew, respectively, on Arabidopsis. Upon infection, the filamentous pathogens induce the formation of intracellular bulbous structures called haustoria, which are required for the biotrophic lifestyle. We previously showed that the microtubule-associated protein AtMAP65-3 plays a critical role in organizing cytoskeleton microtubule arrays during mitosis and cytokinesis. This renders the protein essential for the development of giant cells, which are the feeding sites induced by root knot nematodes. Here, we show that AtMAP65-3 expression is also induced in leaves upon infection by the downy mildew oomycete and the powdery mildew fungus. Loss of AtMAP65-3 function in the map65-3 mutant dramatically reduced infection by both pathogens, predominantly at the stages of leaf penetration. Whole-transcriptome analysis showed an over-represented, constitutive activation of genes involved in salicylic acid (SA) biosynthesis, signaling, and defense execution in map65-3, whereas jasmonic acid (JA)-mediated signaling was down-regulated. Preventing SA synthesis and accumulation in map65-3 rescued plant susceptibility to pathogens, but not the developmental phenotype caused by cytoskeleton defaults. AtMAP65-3 thus has a dual role. It positively regulates cytokinesis, thus plant growth and development, and negatively interferes with plant defense against filamentous biotrophs. Our data suggest that downy mildew and powdery mildew stimulate AtMAP65-3 expression to down-regulate SA signaling for infection. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The protein expression landscape of the Arabidopsis root

PubMed Central

Petricka, Jalean J.; Schauer, Monica A.; Megraw, Molly; Breakfield, Natalie W.; Thompson, J. Will; Georgiev, Stoyan; Soderblom, Erik J.; Ohler, Uwe; Moseley, Martin Arthur; Grossniklaus, Ueli; Benfey, Philip N.

2012-01-01

Because proteins are the major functional components of cells, knowledge of their cellular localization is crucial to gaining an understanding of the biology of multicellular organisms. We have generated a protein expression map of the Arabidopsis root providing the identity and cell type-specific localization of nearly 2,000 proteins. Grouping proteins into functional categories revealed unique cellular functions and identified cell type-specific biomarkers. Cellular colocalization provided support for numerous protein–protein interactions. With a binary comparison, we found that RNA and protein expression profiles are weakly correlated. We then performed peak integration at cell type-specific resolution and found an improved correlation with transcriptome data using continuous values. We performed GeLC-MS/MS (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry) proteomic experiments on mutants with ectopic and no root hairs, providing complementary proteomic data. Finally, among our root hair-specific proteins we identified two unique regulators of root hair development. PMID:22447775

PodNet, a protein-protein interaction network of the podocyte.

PubMed

Warsow, Gregor; Endlich, Nicole; Schordan, Eric; Schordan, Sandra; Chilukoti, Ravi K; Homuth, Georg; Moeller, Marcus J; Fuellen, Georg; Endlich, Karlhans

2013-07-01

Interactions between proteins crucially determine cellular structure and function. Differential analysis of the interactome may help elucidate molecular mechanisms during disease development; however, this analysis necessitates mapping of expression data on protein-protein interaction networks. These networks do not exist for the podocyte; therefore, we built PodNet, a literature-based mouse podocyte network in Cytoscape format. Using database protein-protein interactions, we expanded PodNet to XPodNet with enhanced connectivity. In order to test the performance of XPodNet in differential interactome analysis, we examined podocyte developmental differentiation and the effect of cell culture. Transcriptomes of podocytes in 10 different states were mapped on XPodNet and analyzed with the Cytoscape plugin ExprEssence, based on the law of mass action. Interactions between slit diaphragm proteins are most significantly upregulated during podocyte development and most significantly downregulated in culture. On the other hand, our analysis revealed that interactions lost during podocyte differentiation are not regained in culture, suggesting a loss rather than a reversal of differentiation for podocytes in culture. Thus, we have developed PodNet as a valuable tool for differential interactome analysis in podocytes, and we have identified established and unexplored regulated interactions in developing and cultured podocytes.

DMP1 mutations in autosomal recessive hypophosphatemia implicate a bone matrix protein in the regulation of phosphate homeostasis

PubMed Central

Lorenz-Depiereux, Bettina; Bastepe, Murat; Benet-Pagès, Anna; Amyere, Mustapha; Wagenstaller, Janine; Müller-Barth, Ursula; Badenhoop, Klaus; Kaiser, Stephanie M; Rittmaster, Roger S; Shlossberg, Alan H; Olivares, José L; Loris, César; Ramos, Feliciano J; Glorieux, Francis; Vikkula, Miikka; Jüppner, Harald; Strom, Tim M

2018-01-01

Hypophosphatemia is a genetically heterogeneous disease. Here, we mapped an autosomal recessive form (designated ARHP) to chromosome 4q21 and identified homozygous mutations in DMP1 (dentin matrix protein 1), which encodes a non-collagenous bone matrix protein expressed in osteoblasts and osteocytes. Intact plasma levels of the phosphaturic protein FGF23 were clearly elevated in two of four affected individuals, providing a possible explanation for the phosphaturia and inappropriately normal 1,25(OH)2D levels and suggesting that DMP1 may regulate FGF23 expression. PMID:17033625

DMP1 mutations in autosomal recessive hypophosphatemia implicate a bone matrix protein in the regulation of phosphate homeostasis.

PubMed

Lorenz-Depiereux, Bettina; Bastepe, Murat; Benet-Pagès, Anna; Amyere, Mustapha; Wagenstaller, Janine; Müller-Barth, Ursula; Badenhoop, Klaus; Kaiser, Stephanie M; Rittmaster, Roger S; Shlossberg, Alan H; Olivares, José L; Loris, César; Ramos, Feliciano J; Glorieux, Francis; Vikkula, Miikka; Jüppner, Harald; Strom, Tim M

2006-11-01

Hypophosphatemia is a genetically heterogeneous disease. Here, we mapped an autosomal recessive form (designated ARHP) to chromosome 4q21 and identified homozygous mutations in DMP1 (dentin matrix protein 1), which encodes a non-collagenous bone matrix protein expressed in osteoblasts and osteocytes. Intact plasma levels of the phosphaturic protein FGF23 were clearly elevated in two of four affected individuals, providing a possible explanation for the phosphaturia and inappropriately normal 1,25(OH)2D levels and suggesting that DMP1 may regulate FGF23 expression.

Proteome map of Aspergillus nidulans during osmoadaptation.

PubMed

Kim, Yonghyun; Nandakumar, M P; Marten, Mark R

2007-09-01

The model filamentous fungus Aspergillus nidulans, when grown in a moderate level of osmolyte (+0.6M KCl), was previously found to have a significantly reduced cell wall elasticity (Biotech Prog, 21:292, 2005). In this study, comparative proteomic analysis via two-dimensional gel electrophoresis (2de) and matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry was used to assess molecular level events associated with this phenomenon. Thirty of 90 differentially expressed proteins were identified. Sequence homology and conserved domains were used to assign probable function to twenty-one proteins currently annotated as "hypothetical." In osmoadapted cells, there was an increased expression of glyceraldehyde-3-phosphate dehydrogenase and aldehyde dehydrogenase, as well as a decreased expression of enolase, suggesting an increased glycerol biosynthesis and decreased use of the TCA cycle. There also was an increased expression of heat shock proteins and Shp1-like protein degradation protein, implicating increased protein turnover. Five novel osmoadaptation proteins of unknown functions were also identified.

Identification of three critical regions within mouse interleukin 2 by fine structural deletion analysis.

PubMed Central

Zurawski, S M; Zurawski, G

1988-01-01

We have analyzed structure--function relationships of the protein hormone murine interleukin 2 by fine structural deletion mapping. A total of 130 deletion mutant proteins, together with some substitution and insertion mutant proteins, was expressed in Escherichia coli and analyzed for their ability to sustain the proliferation of a cloned murine T cell line. This analysis has permitted a functional map of the protein to be drawn and classifies five segments of the protein, which together contain 48% of the sequence, as unessential to the biological activity of the protein. A further 26% of the protein is classified as important, but not crucial, for the activity. Three regions, consisting of amino acids 32-35, 66-77 and 119-141 contain the remaining 26% of the protein and are critical to the biological activity of the protein. The functional map is discussed in the context of the possible role of the identified critical regions in the structure of the hormone and its binding to the interleukin 2 receptor complex. Images PMID:3261239

Analysis of Nuclear Lamina Proteins in Myoblast Differentiation by Functional Complementation.

PubMed

Tapia, Olga; Gerace, Larry

2016-01-01

We describe straightforward methodology for structure-function mapping of nuclear lamina proteins in myoblast differentiation, using populations of C2C12 myoblasts in which the endogenous lamina components are replaced with ectopically expressed mutant versions of the proteins. The procedure involves bulk isolation of C2C12 cell populations expressing the ectopic proteins by lentiviral transduction, followed by depletion of the endogenous proteins using siRNA, and incubation of cells under myoblast differentiation conditions. Similar methodology may be applied to mouse embryo fibroblasts or to other cell types as well, for the identification and characterization of sequences of lamina proteins involved in functions that can be measured biochemically or cytologically.

MAP17 Is a Necessary Activator of Renal Na+/Glucose Cotransporter SGLT2

PubMed Central

Coady, Michael J.; El Tarazi, Abdulah; Santer, René; Bissonnette, Pierre; Sasseville, Louis J.; Calado, Joaquim; Lussier, Yoann; Dumayne, Christopher; Bichet, Daniel G.

2017-01-01

The renal proximal tubule reabsorbs 90% of the filtered glucose load through the Na+-coupled glucose transporter SGLT2, and specific inhibitors of SGLT2 are now available to patients with diabetes to increase urinary glucose excretion. Using expression cloning, we identified an accessory protein, 17 kDa membrane-associated protein (MAP17), that increased SGLT2 activity in RNA-injected Xenopus oocytes by two orders of magnitude. Significant stimulation of SGLT2 activity also occurred in opossum kidney cells cotransfected with SGLT2 and MAP17. Notably, transfection with MAP17 did not change the quantity of SGLT2 protein at the cell surface in either cell type. To confirm the physiologic relevance of the MAP17–SGLT2 interaction, we studied a cohort of 60 individuals with familial renal glucosuria. One patient without any identifiable mutation in the SGLT2 coding gene (SLC5A2) displayed homozygosity for a splicing mutation (c.176+1G>A) in the MAP17 coding gene (PDZK1IP1). In the proximal tubule and in other tissues, MAP17 is known to interact with PDZK1, a scaffolding protein linked to other transporters, including Na+/H+ exchanger 3, and to signaling pathways, such as the A-kinase anchor protein 2/protein kinase A pathway. Thus, these results provide the basis for a more thorough characterization of SGLT2 which would include the possible effects of its inhibition on colocalized renal transporters. PMID:27288013

Spectra-first feature analysis in clinical proteomics - A case study in renal cancer.

PubMed

Goh, Wilson Wen Bin; Wong, Limsoon

2016-10-01

In proteomics, useful signal may be unobserved or lost due to the lack of confident peptide-spectral matches. Selection of differential spectra, followed by associative peptide/protein mapping may be a complementary strategy for improving sensitivity and comprehensiveness of analysis (spectra-first paradigm). This approach is complementary to the standard approach where functional analysis is performed only on the finalized protein list assembled from identified peptides from the spectra (protein-first paradigm). Based on a case study of renal cancer, we introduce a simple spectra-binning approach, MZ-bin. We demonstrate that differential spectra feature selection using MZ-bin is class-discriminative and can trace relevant proteins via spectra associative mapping. Moreover, proteins identified in this manner are more biologically coherent than those selected directly from the finalized protein list. Analysis of constituent peptides per protein reveals high expression inconsistency, suggesting that the measured protein expressions are in fact, poor approximations of true protein levels. Moreover, analysis at the level of constituent peptides may provide higher resolution insight into the underlying biology: Via MZ-bin, we identified for the first time differential splice forms for the known renal cancer marker MAPT. We conclude that the spectra-first analysis paradigm is a complementary strategy to the traditional protein-first paradigm and can provide deeper level insight.

Transcriptional enhancer from milk protein genes

DOEpatents

Casperson, Gerald F.; Schmidhauser, Christian T.; Bissell, Mina J.

1999-01-01

The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

Mapping the Extracellular and Membrane Proteome Associated with the Vasculature and the Stroma in the Embryo*

PubMed Central

Soulet, Fabienne; Kilarski, Witold W.; Roux-Dalvai, Florence; Herbert, John M. J.; Sacewicz, Izabela; Mouton-Barbosa, Emmanuelle; Bicknell, Roy; Lalor, Patricia; Monsarrat, Bernard; Bikfalvi, Andreas

2013-01-01

In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments. PMID:23674615

Primary structure, expression and chromosomal locus of a human homolog of rat ERK3.

PubMed

Meloche, S; Beatty, B G; Pellerin, J

1996-10-03

We report the cloning and characterization of a human cDNA encoding a novel homolog of rat extracellular signal-regulated kinase 3 (ERK3). The cDNA encodes a predicted protein of 721 amino acids which shares 92% amino acid identity with rat ERK3 over their shared length. Interestingly, the human protein contains a unique extension of 178 amino acids at its carboxy terminal extremity. The human ERK3 protein also displays various degrees of homology to other members of the MAP kinases family, but does not contain the typical TXY regulatory motif between subdomains VII and VIII. Northern blot analysis revealed that ERK3 mRNA is widely distributed in human tissues, with the highest expression detected in skeletal muscle. The human ERK3 gene was mapped by fluorescence in situ hybridization to chromosome 15q21, a region associated with chromosomal abnormalities in acute nonlymphoblastic leukemias. This information should prove valuable in designing studies to define the cellular function of the ERK3 protein kinase.

Identification and molecular characterization of MYB Transcription Factor Superfamily in C4 model plant foxtail millet (Setaria italica L.).

PubMed

Muthamilarasan, Mehanathan; Khandelwal, Rohit; Yadav, Chandra Bhan; Bonthala, Venkata Suresh; Khan, Yusuf; Prasad, Manoj

2014-01-01

MYB proteins represent one of the largest transcription factor families in plants, playing important roles in diverse developmental and stress-responsive processes. Considering its significance, several genome-wide analyses have been conducted in almost all land plants except foxtail millet. Foxtail millet (Setaria italica L.) is a model crop for investigating systems biology of millets and bioenergy grasses. Further, the crop is also known for its potential abiotic stress-tolerance. In this context, a comprehensive genome-wide survey was conducted and 209 MYB protein-encoding genes were identified in foxtail millet. All 209 S. italica MYB (SiMYB) genes were physically mapped onto nine chromosomes of foxtail millet. Gene duplication study showed that segmental- and tandem-duplication have occurred in genome resulting in expansion of this gene family. The protein domain investigation classified SiMYB proteins into three classes according to number of MYB repeats present. The phylogenetic analysis categorized SiMYBs into ten groups (I-X). SiMYB-based comparative mapping revealed a maximum orthology between foxtail millet and sorghum, followed by maize, rice and Brachypodium. Heat map analysis showed tissue-specific expression pattern of predominant SiMYB genes. Expression profiling of candidate MYB genes against abiotic stresses and hormone treatments using qRT-PCR revealed specific and/or overlapping expression patterns of SiMYBs. Taken together, the present study provides a foundation for evolutionary and functional characterization of MYB TFs in foxtail millet to dissect their functions in response to environmental stimuli.

Poorly expressed endogenous ecotropic provirus of DBA/2 mice encodes a mutant Pr65gag protein that is not myristylated.

PubMed Central

Copeland, N G; Jenkins, N A; Nexø, B; Schultz, A M; Rein, A; Mikkelsen, T; Jørgensen, P

1988-01-01

DBA/2 mice carry a single endogenous ecotropic murine leukemia provirus designated Emv-3. Although this provirus appears to be nondefective by genomic restriction enzyme mapping, weanling mice do not produce virus and only about one-third of adult mice ever express virus. 5-Iododeoxyuridine and 5-azacytidine, two potent inducers of ecotropic virus expression, are relatively ineffective at inducing Emv-3 expression. However, the chemical carcinogen 7,12-dimethylbenz(a)anthracene can induce ecotropic virus expression in approximately 95% of treated DBA/2 mice. Previous experiments involving DNA transfection and marker rescue analysis of molecularly cloned Emv-3 DNA suggested that Emv-3 carries a small defect(s) in the gag gene, not detectable by restriction enzyme mapping, that inhibits virus expression in vivo and in vitro. Using a combination of approaches, including DNA sequencing, peptide mapping, and metabolic labeling of cells with [3H]myristate, we have demonstrated that the defect in Emv-3 most likely results from a single nucleotide substitution within the gene for p15gag that inhibits myristylation of the Pr65gag N terminus. Myristylation of Pr65gag is thought to be required for this protein to associate with the plasma membrane and is essential for virus particle formation. These results provide a conceptual framework for understanding how Emv-3 expression is regulated during development and after chemical induction. Images PMID:2826810

Reduced transcript stabilization restricts TNF-alpha expression in RAW264.7 macrophages infected with pathogenic mycobacteria: evidence for an involvement of lipomannan.

PubMed

Basler, Tina; Holtmann, Helmut; Abel, Jens; Eckstein, Torsten; Baumer, Wolfgang; Valentin-Weigand, Peter; Goethe, Ralph

2010-01-01

Despite the critical role that TNF-alpha plays in the containment of mycobacterial infection, the mechanisms involved in regulation of its expression by mycobacteria are poorly defined. We addressed this question by studying MAP, which causes a chronic enteritis in ruminants and is linked to human Crohn's disease. We found that in MAP infected macrophages, TNF-alpha gene expression was substantially lower than in macrophages infected with nonpathogenic MS or stimulated with LPS. TNF-alpha transcriptional one could not fully explain the differential TNF-alpha mRNA expression, suggesting that there must be a substantial contribution by post-transcriptional mechanisms.Accordingly, we found reduced TNF-alpha mRNA stability in MAP-infected macrophages. Further comparison of MAP- and MS-infected macrophages revealed that lower TNF-alpha mRNA stability combined with lower mRNA and protein expression in MAP-infected macrophages correlated with lower p38 MAPK phosphorylation. These findings were independent of viability of MAP and MS. We demonstrate that the major mycobacterial cell-wall lipoglycan LM of MAP and MS induced TNF-alpha mRNA transcription,but only the MS-LM induced p38 MAPK-dependent transcript stabilization. Overall, our data suggest that pathogenic mycobacteria cause weak p38 and TNF-alpha mRNA stabilization as a result of their structural cell-wall components such as LM and thereby, restrict TNF-alpha expression in macrophages.

Specificity protein 1 regulates topoisomerase IIβ expression in SH-SY5Y cells during neuronal differentiation.

PubMed

Guo, Hui; Cao, Cuili; Chi, Xueqian; Zhao, Junxia; Liu, Xia; Zhou, Najing; Han, Shuo; Yan, Yongxin; Wang, Yanling; Xu, Yannan; Yan, Yunli; Cui, Huixian; Sun, Hongxia

2014-10-01

Topoisomerase IIβ (top IIβ) is a nuclear enzyme with an essential role in neural development. The regulation of top IIβ gene expression during neural differentiation is poorly understood. Functional analysis of top IIβ gene structure displayed a GC box sequence in its transcription promoter, which binds the nuclear transcription factor specificity protein 1 (Sp1). Sp1 regulates gene expression via multiple mechanisms and is essential for early embryonic development. This study seeks to determine whether Sp1 regulates top IIβ gene expression during neuronal differentiation. For this purpose, human neuroblastoma SH-SY5Y cells were induced to neuronal differentiation in the presence of all-trans retinoic acid (RA) for 5 days. After incubation with 10 μM RA for 3-5 days, a majority of the cells exited the cell cycle to become postmitotic neurons, characterized by the presence of longer neurite outgrowths and expression of the neuronal marker microtubule-associated protein-2 (MAP2). Elevated Sp1 and top IIβ mRNA and protein levels were detected and found to be positively correlated with the differentiation stage. Chromatin immunoprecipitation assay demonstrated an increased recruitment of Sp1 to the top IIβ promoter after RA treatment. Mithramycin A, a compound that interferes with Sp1 binding to GC-rich DNA sequences, downregulated the expression of top IIβ, resulting in reduced expression of MAP2 and decreased neurite length compared with the control group. Our results indicate that Sp1 regulates top IIβ expression by binding to the GC box of the gene promoter during neuronal differentiation in SH-SY5Y cells. © 2014 Wiley Periodicals, Inc.

The FTMap family of web servers for determining and characterizing ligand binding hot spots of proteins

PubMed Central

Kozakov, Dima; Grove, Laurie E.; Hall, David R.; Bohnuud, Tanggis; Mottarella, Scott; Luo, Lingqi; Xia, Bing; Beglov, Dmitri; Vajda, Sandor

2016-01-01

FTMap is a computational mapping server that identifies binding hot spots of macromolecules, i.e., regions of the surface with major contributions to the ligand binding free energy. To use FTMap, users submit a protein, DNA, or RNA structure in PDB format. FTMap samples billions of positions of small organic molecules used as probes and scores the probe poses using a detailed energy expression. Regions that bind clusters of multiple probe types identify the binding hot spots, in good agreement with experimental data. FTMap serves as basis for other servers, namely FTSite to predict ligand binding sites, FTFlex to account for side chain flexibility, FTMap/param to parameterize additional probes, and FTDyn to map ensembles of protein structures. Applications include determining druggability of proteins, identifying ligand moieties that are most important for binding, finding the most bound-like conformation in ensembles of unliganded protein structures, and providing input for fragment based drug design. FTMap is more accurate than classical mapping methods such as GRID and MCSS, and is much faster than the more recent approaches to protein mapping based on mixed molecular dynamics. Using 16 probe molecules, the FTMap server finds the hot spots of an average size protein in less than an hour. Since FTFlex performs mapping for all low energy conformers of side chains in the binding site, its completion time is proportionately longer. PMID:25855957

The X-linked juvenile retinoschisis protein retinoschisin is a novel regulator of mitogen-activated protein kinase signalling and apoptosis in the retina.

PubMed

Plössl, Karolina; Weber, Bernhard H F; Friedrich, Ulrike

2017-04-01

X-linked juvenile retinoschisis (XLRS) is a hereditary retinal dystrophy in young males, caused by mutations in the RS1 gene. The function of the encoded protein, termed retinoschisin, and the molecular mechanisms underlying XLRS pathogenesis are still unresolved, although a direct interaction partner of the secreted retinoschisin, the retinal Na/K-ATPase, was recently identified. Earlier gene expression studies in retinoschisin-deficient (Rs1h -/Y ) mice provided a first indication of pathological up-regulation of mitogen-activated protein (MAP) kinase signalling in disease pathogenesis. To further investigate the role for retinoschisin in MAP kinase regulation, we exposed Y-79 cells and murine Rs1h -/Y retinae to recombinant retinoschisin and the XLRS-associated mutant RS1-C59S. Although normal retinoschisin stably bound to retinal cells, RS1-C59S exhibited a strongly reduced binding affinity. Simultaneously, exposure to normal retinoschisin significantly reduced phosphorylation of C-RAF and MAP kinases ERK1/2 in Y-79 cells and murine Rs1h -/Y retinae. Expression of MAP kinase target genes C-FOS and EGR1 was also down-regulated in both model systems. Finally, retinoschisin treatment decreased pro-apoptotic BAX-2 transcript levels in Y-79 cells and Rs1h -/Y retinae. Upon retinoschisin treatment, these cells showed increased resistance against apoptosis, reflected by decreased caspase-3 activity (in Y-79 cells) and increased photoreceptor survival (in Rs1h -/Y retinal explants). RS1-C59S did not influence C-RAF or ERK1/2 activation, C-FOS or EGR1 expression, or apoptosis. Our data imply that retinoschisin is a novel regulator of MAP kinase signalling and exerts an anti-apoptotic effect on retinal cells. We therefore discuss that disturbances of MAP kinase signalling by retinoschisin deficiency could be an initial step in XLRS pathogenesis. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

NASA Technical Reports Server (NTRS)

Safadi, F.; Reddy, V. S.; Reddy, A. S.

2000-01-01

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

An alternative methionine aminopeptidase, MAP-A, is required for nitrogen starvation and high-light acclimation in the cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Drath, Miriam; Baier, Kerstin; Forchhammer, Karl

2009-05-01

Methionine aminopeptidases (MetAPs or MAPs, encoded by map genes) are ubiquitous and pivotal enzymes for protein maturation in all living organisms. Whereas most bacteria harbour only one map gene, many cyanobacterial genomes contain two map paralogues, the genome of Synechocystis sp. PCC 6803 even three. The physiological function of multiple map paralogues remains elusive so far. This communication reports for the first time differential MetAP function in a cyanobacterium. In Synechocystis sp. PCC 6803, the universally conserved mapC gene (sll0555) is predominantly expressed in exponentially growing cells and appears to be a housekeeping gene. By contrast, expression of mapA (slr0918) and mapB (slr0786) genes increases during stress conditions. The mapB paralogue is only transiently expressed, whereas the widely distributed mapA gene appears to be the major MetAP during stress conditions. A mapA-deficient Synechocystis mutant shows a subtle impairment of photosystem II properties even under non-stressed conditions. In particular, the binding site for the quinone Q(B) is affected, indicating specific N-terminal methionine processing requirements of photosystem II components. MAP-A-specific processing becomes essential under certain stress conditions, since the mapA-deficient mutant is severely impaired in surviving conditions of prolonged nitrogen starvation and high light exposure.

Receptor-like Molecules on Human Intestinal Epithelial Cells Interact with an Adhesion Factor from Lactobacillus reuteri.

PubMed

Matsuo, Yosuke; Miyoshi, Yukihiro; Okada, Sanae; Satoh, Eiichi

2012-01-01

A surface protein of Lactobacillus reuteri, mucus adhesion-promoting protein (MapA), is considered to be an adhesion factor. MapA is expressed in L. reuteri strains and adheres to piglet gastric mucus, collagen type I, and human intestinal epithelial cells such as Caco-2. The aim of this study was to identify molecules that mediate the attachment of MapA from L. reuteri to the intestinal epithelial cell surface by investigating the adhesion of MapA to receptor-like molecules on Caco-2 cells. MapA-binding receptor-like molecules were detected in Caco-2 cell lysates by 2D-PAGE. Two proteins, annexin A13 (ANXA13) and paralemmin (PALM), were identified by MALDI TOF-MS. The results of a pull-down assay showed that MapA bound directly to ANXA13 and PALM. Fluorescence microscopy studies confirmed that MapA binding to ANXA13 and PALM was colocalized on the Caco-2 cell membrane. To evaluate whether ANXA13 and PALM are important for MapA adhesion, ANXA13 and PALM knockdown cell lines were established. The adhesion of MapA to the abovementioned cell lines was reduced compared with that to wild-type Caco-2 cells. These knockdown experiments established the importance of these receptor-like molecules in MapA adhesion.

Amitriptyline induces early growth response-1 gene expression via ERK and JNK mitogen-activated protein kinase pathways in rat C6 glial cells.

PubMed

Chung, Eun Young; Shin, Soon Young; Lee, Young Han

2007-07-05

Astrocytes play important roles in guiding the construction of the nervous system, controlling extracellular ions and neurotransmitters, and regulating CNS synaptogenesis. Egr-1 is a transcription factor involved in neuronal differentiation and astrocyte cell proliferation. In this study, we investigated whether the tricyclic antidepressant (TCA) amitriptyline induces Egr-1 expression in astrocytes using rat C6 glioma cells as a model. We found that amitriptyline increased the expression of Egr-1 in a dose- and time-dependent manner. The amitriptyline-induced Egr-1 expression was mediated through serum response elements (SREs) in the Egr-1 promoter. SREs were activated by the Ets-domain transcription factor Elk-1 through the ERK and JNK mitogen-activated protein (MAP) kinase pathways. The inhibition of the ERK and JNK MAP kinase signals attenuated amitriptyline-induced transactivation of Gal4-Elk-1 and Egr-1 promoter activity. Our findings suggest that the induction of Egr-1 expression in astrocytes may be required to attain the therapeutic effects of antidepressant drugs.

Spatially resolved proteome mapping of laser capture microdissected tissue with automated sample transfer to nanodroplets.

PubMed

Zhu, Ying; Dou, Maowei; Piehowski, Paul D; Liang, Yiran; Wang, Fangjun; Chu, Rosalie K; Chrisler, Will; Smith, Jordan N; Schwarz, Kaitlynn C; Shen, Yufeng; Shukla, Anil K; Moore, Ronald J; Smith, Richard D; Qian, Wei-Jun; Kelly, Ryan T

2018-06-24

Current mass spectrometry (MS)-based proteomics approaches are ineffective for mapping protein expression in tissue sections with high spatial resolution due to the limited overall sensitivity of conventional workflows. Here we report an integrated and automated method to advance spatially resolved proteomics by seamlessly coupling laser capture microdissection (LCM) with a recently developed nanoliter-scale sample preparation system termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples). The workflow is enabled by prepopulating nanowells with DMSO, which serves as a sacrificial capture liquid for microdissected tissues. The DMSO droplets efficiently collect laser-pressure catapulted LCM tissues as small as 20 µm in diameter with success rates >87%. We also demonstrate that tissue treatment with DMSO can significantly improve proteome coverage, likely due to its ability to dissolve lipids from tissue and enhance protein extraction efficiency. The LCM-nanoPOTS platform was able to identify 180, 695, and 1827 protein groups on average from 12-µm-thick rat brain cortex tissue sections with diameters of 50, 100, and 200 µm, respectively. We also analyzed 100-µm-diameter sections corresponding to 10-18 cells from three different regions of rat brain and comparatively quantified ~1000 proteins, demonstrating the potential utility for high-resolution spatially resolved mapping of protein expression in tissues. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Comparative 2D-DIGE proteomic analysis of bovine mammary epithelial cells during lactation reveals protein signatures for lactation persistency and milk yield.

PubMed

Janjanam, Jagadeesh; Singh, Surender; Jena, Manoj K; Varshney, Nishant; Kola, Srujana; Kumar, Sudarshan; Kaushik, Jai K; Grover, Sunita; Dang, Ajay K; Mukesh, Manishi; Prakash, B S; Mohanty, Ashok K

2014-01-01

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

Fine mapping of regulatory loci for mammalian gene expression using radiation hybrids

PubMed Central

Park, Christopher C; Ahn, Sangtae; Bloom, Joshua S; Lin, Andy; Wang, Richard T; Wu, Tongtong; Sekar, Aswin; Khan, Arshad H; Farr, Christine J; Lusis, Aldons J; Leahy, Richard M; Lange, Kenneth; Smith, Desmond J

2010-01-01

We mapped regulatory loci for nearly all protein-coding genes in mammals using comparative genomic hybridization and expression array measurements from a panel of mouse–hamster radiation hybrid cell lines. The large number of breaks in the mouse chromosomes and the dense genotyping of the panel allowed extremely sharp mapping of loci. As the regulatory loci result from extra gene dosage, we call them copy number expression quantitative trait loci, or ceQTLs. The −2log10P support interval for the ceQTLs was <150 kb, containing an average of <2–3 genes. We identified 29,769 trans ceQTLs with −log10P > 4, including 13 hotspots each regulating >100 genes in trans. Further, this work identifies 2,761 trans ceQTLs harboring no known genes, and provides evidence for a mode of gene expression autoregulation specific to the X chromosome. PMID:18362883

Analytical results for a stochastic model of gene expression with arbitrary partitioning of proteins

NASA Astrophysics Data System (ADS)

Tschirhart, Hugo; Platini, Thierry

2018-05-01

In biophysics, the search for analytical solutions of stochastic models of cellular processes is often a challenging task. In recent work on models of gene expression, it was shown that a mapping based on partitioning of Poisson arrivals (PPA-mapping) can lead to exact solutions for previously unsolved problems. While the approach can be used in general when the model involves Poisson processes corresponding to creation or degradation, current applications of the method and new results derived using it have been limited to date. In this paper, we present the exact solution of a variation of the two-stage model of gene expression (with time dependent transition rates) describing the arbitrary partitioning of proteins. The methodology proposed makes full use of the PPA-mapping by transforming the original problem into a new process describing the evolution of three biological switches. Based on a succession of transformations, the method leads to a hierarchy of reduced models. We give an integral expression of the time dependent generating function as well as explicit results for the mean, variance, and correlation function. Finally, we discuss how results for time dependent parameters can be extended to the three-stage model and used to make inferences about models with parameter fluctuations induced by hidden stochastic variables.

Regulation of the grapevine polygalacturonase-inhibiting protein encoding gene: expression pattern, induction profile and promoter analysis.

PubMed

Joubert, D Albert; de Lorenzo, Giulia; Vivier, Melané A

2013-03-01

Regulation of defense in plants is a complex process mediated by various signaling pathways. Promoter analysis of defense-related genes is useful to understand these signaling pathways involved in regulation. To this end, the regulation of the polygalacturonase-inhibiting protein encoding gene from Vitis vinifera L. (Vvpgip1) was analyzed with regard to expression pattern and induction profile as well as the promoter in terms of putative regulatory elements present, core promoter size and the start of transcription. Expression of Vvpgip1 is tissue-specific and developmentally regulated. Vvpgip1 expression was induced in response to auxin, salicylic acid and sugar treatment, wounding and pathogen infection. The start of transcription was mapped to 17 bp upstream of the ATG and the core promoter was mapped to the 137 bp upstream of the ATG. Fructose- and Botrytis responsiveness were identified in the region between positions -3.1 and -1.5 kb. The analyses showed induction in water when the leaves were submersed and this response and the response to wounding mapped to the region between positions -1.1 and -0.1 kb. In silico analyses revealed putative cis-acting elements in these areas that correspond well to the induction stimuli tested.

Expression of klotho mRNA and protein in rat brain parenchyma from early postnatal development into adulthood

PubMed Central

Clinton, Sarah M.; Glover, Matthew E.; Maltare, Astha; Laszczyk, Ann M.; Mehi, Stephen J.; Simmons, Rebecca K.; King, Gwendalyn D.

2013-01-01

Without the age-regulating protein klotho, mouse lifespan is shortened and the rapid onset of age-related disorders occurs. Conversely, overexpression of klotho extends mouse lifespan. Klotho is most abundant in kidney and expressed in a limited number of other organs, including the brain, where klotho levels are highest in choroid plexus. Reports vary on where klotho is expressed within the brain parenchyma, and no data is available as to whether klotho levels change across postnatal development. We used in situ hybridization to map klotho mRNA expression in the developing and adult rat brain and report moderate, widespread expression across grey matter regions. mRNA expression levels in cortex, hippocampus, caudate putamen, and amygdala decreased during the second week of life and then gradually rose to adult levels by postnatal day 21. Immunohistochemistry revealed a protein expression pattern similar to the mRNA results, with klotho protein expressed widely throughout the brain. Klotho protein co-localized with both the neuronal marker NeuN, as well as, oligodendrocyte marker olig2. These results provide the first anatomical localization of klotho mRNA and protein in rat brain parenchyma and demonstrate that klotho levels vary during early postnatal development. PMID:23838326

A Conserved p38 Mitogen-Activated Protein Kinase Pathway Regulates Drosophila Immunity Gene Expression

PubMed Central

Han, Zhiqiang Stanley; Enslen, Hervé; Hu, Xiaodi; Meng, Xiangjun; Wu, I-Huan; Barrett, Tamera; Davis, Roger J.; Ip, Y. Tony

1998-01-01

Accumulating evidence suggests that the insect and mammalian innate immune response is mediated by homologous regulatory components. Proinflammatory cytokines and bacterial lipopolysaccharide stimulate mammalian immunity by activating transcription factors such as NF-κB and AP-1. One of the responses evoked by these stimuli is the initiation of a kinase cascade that leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase on Thr and Tyr within the motif Thr-Gly-Tyr, which is located within subdomain VIII. We have investigated the possible involvement of the p38 MAP kinase pathway in the Drosophila immune response. Two genes that are highly homologous to the mammalian p38 MAP kinase were molecularly cloned and characterized. Furthermore, genes that encode two novel Drosophila MAP kinase kinases, D-MKK3 and D-MKK4, were identified. D-MKK3 is an efficient activator of both Drosophila p38 MAP kinases, while D-MKK4 is an activator of D-JNK but not D-p38. These data establish that Drosophila indeed possesses a conserved p38 MAP kinase signaling pathway. We have examined the role of the D-p38 MAP kinases in the regulation of insect immunity. The results revealed that one of the functions of D-p38 is to attenuate antimicrobial peptide gene expression following exposure to lipopolysaccharide. PMID:9584193

A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection

NASA Astrophysics Data System (ADS)

Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

2000-05-01

RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

PubMed

Hayakawa-Yano, Yoshika; Suyama, Satoshi; Nogami, Masahiro; Yugami, Masato; Koya, Ikuko; Furukawa, Takako; Zhou, Li; Abe, Manabu; Sakimura, Kenji; Takebayashi, Hirohide; Nakanishi, Atsushi; Okano, Hideyuki; Yano, Masato

2017-09-15

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states. © 2017 Hayakawa-Yano et al.; Published by Cold Spring Harbor Laboratory Press.

Changes in inflorescence protein during advanced stages of floret development in Buchloe dactyloides (Poaceae).

PubMed

Zhou, Y-J; Xue, J-G; Wang, X-G; Zhang, X-Q

2012-11-12

Buffalograss, Buchloe dactyloides, is a dioecious species native to the Great Plains of North America. The florets at the early stages of development possess both gynoecium and androecium organ primordia but later become unisexual. Very little is known about the proteomic changes that occur when the florets change from hermaphroditism to unisexuality. We compared the protein composition of florets at the hermaphroditic stage with that at the unisexual stage. The development stage of the floret was determined by stereomicroscopic observation. Two-dimensional gel electrophoresis was used to separate the proteins extracted from female and male inflorescences. Stage- specific protein maps, with an average of about 400 spots per map, were analyzed with the protein analysis software. Eighteen spots were found to be differentially expressed between the hermaphrodite and unisexual stages. Of these, 12 were present at both stages but with a different expression value. Four specific spots appeared at the hermaphrodite stage and disappeared at the unisexual stage. Two specific protein spots were associated with female and male floret differentiation. One appears to be associated with contabescence in the female floret and the final protein appears to lead to the abortion of gynoecium in the male floret. The MALDI TOF/TOF technique was used for peptide mass fingerprinting of the differentially expressed proteins and the MASCOT software was used to search the protein database. However, only two protein spots were identified from the database. These were aldolase1 and Os05g0574400 (similar to malate dehydrogenase). This type of proteomic study can help to identify novel protein products and determine the mechanisms involved in the floral sex differentiation process in buffalo grass.

Staufen 2 regulates mGluR long-term depression and Map1b mRNA distribution in hippocampal neurons.

PubMed

Lebeau, Geneviève; Miller, Linda C; Tartas, Maylis; McAdam, Robyn; Laplante, Isabel; Badeaux, Frédérique; DesGroseillers, Luc; Sossin, Wayne S; Lacaille, Jean-Claude

2011-01-01

The two members of the Staufen family of RNA-binding proteins, Stau1 and Stau2, are present in distinct ribonucleoprotein complexes and associate with different mRNAs. Stau1 is required for protein synthesis-dependent long-term potentiation (L-LTP) in hippocampal pyramidal cells. However, the role of Stau2 in synaptic plasticity remains unexplored. We found that unlike Stau1, Stau2 is not required for L-LTP. In contrast, Stau2, but not Stau1, is necessary for DHPG-induced protein synthesis-dependent long-term depression (mGluR-LTD). While Stau2 is involved in early development of spines, its down-regulation does not alter spine morphology or spontaneous miniature synaptic activity in older cultures where LTD occurs. In addition, Stau2, but not Stau1, knockdown reduces the dendritic localization of Map1b mRNA, a specific transcript involved in mGluR-LTD. Moreover, mGluR stimulation with DHPG induces Map1b, but not Map2, mRNA dissociation from mRNA granules containing Stau2 and the ribosomal protein P0. This dissociation was not observed in cells in which Stau2 was depleted. Finally, Stau2 knockdown reduces basal Map1b protein expression in dendrites and prevents DHPG-induced increases in dendritic Map1b protein level. We suggest a role for Stau2 in the generation and regulation of Map1b mRNA containing granules that are required for mGluR-LTD.

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells.

PubMed

Fiume, Giuseppe; Scialdone, Annarita; Rizzo, Francesca; De Filippo, Maria Rosaria; Laudanna, Carmelo; Albano, Francesco; Golino, Gaetanina; Vecchio, Eleonora; Pontoriero, Marilena; Mimmi, Selena; Ceglia, Simona; Pisano, Antonio; Iaccino, Enrico; Palmieri, Camillo; Paduano, Sergio; Viglietto, Giuseppe; Weisz, Alessandro; Scala, Giuseppe; Quinto, Ileana

2016-11-07

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK -shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK -shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3'- and 5'-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

PubMed Central

Fiume, Giuseppe; Scialdone, Annarita; Rizzo, Francesca; De Filippo, Maria Rosaria; Laudanna, Carmelo; Albano, Francesco; Golino, Gaetanina; Vecchio, Eleonora; Pontoriero, Marilena; Mimmi, Selena; Ceglia, Simona; Pisano, Antonio; Iaccino, Enrico; Palmieri, Camillo; Paduano, Sergio; Viglietto, Giuseppe; Weisz, Alessandro; Scala, Giuseppe; Quinto, Ileana

2016-01-01

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein. PMID:27827994

Use of enoyl coenzyme A hydratase of Mycobacterium avium subsp. paratuberculosis for the serological diagnosis of Johne's disease.

PubMed

Nagata, Reiko; Kawaji, Satoko; Mori, Yasuyuki

2013-10-01

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), remains difficult to control because of the lack of specific and sensitive diagnostic tests. In order to improve the specificity of sero-diagnosis for JD, the phage display library derived from genomic DNA of MAP was immunoscreened to identify novel antigenic targets. We selected a clone using antibodies from MAP experimentally infected cattle, and annotated its coding sequence as MAP1197 in the MAP genome, which encoded "echA12_2" in the MAP protein (Map-echA) belonging to Enoyl-CoA hydratase, known as a crotonase enzyme. The Map-echA was expressed in Esherichia coli and purified as a histidine-tag recombinant protein (rMap-echA), and the diagnostic potential of the protein was further evaluated by enzyme-linked immunosorbent assays (ELISA). Antibody responses to rMap-echA were higher in MAP-infected cattle than in uninfected cattle. The specificity of the Map-echA ELISA was also confirmed by evaluation with hyper-immune sera against various kinds of Mycobacterium species. Furthermore, in all experimentally infected cattle the antibody against rMap-echA was detected 2-7months earlier than by a commercially available ELISA kit. These results suggested that Map-echA can be used as a specific and sensitive serological diagnostic antigen for the detection of MAP infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

PubMed

Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

2015-11-20

The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.

Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors

PubMed Central

Whisenant, Thomas C.; Ho, David T.; Benz, Ryan W.; Rogers, Jeffrey S.; Kaake, Robyn M.; Gordon, Elizabeth A.; Huang, Lan; Baldi, Pierre; Bardwell, Lee

2010-01-01

In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases, especially for weakly expressed proteins. Here we have developed a hybrid computational search algorithm that combines machine learning and expert knowledge to identify kinase docking sites, and used this algorithm to search the human genome for novel MAP kinase substrates and regulators focused on the JNK family of MAP kinases. Predictions were tested by peptide array followed by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and mutant proteins. Using this procedure, we found new ‘D-site’ class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). Finally, we identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, suggesting that a direct connection between MAP kinase and hedgehog signaling may occur at the level of these key regulators. These results demonstrate that a genome-wide search for MAP kinase docking sites can be used to find new docking sites and substrates. PMID:20865152

Identification and Molecular Characterization of MYB Transcription Factor Superfamily in C4 Model Plant Foxtail Millet (Setaria italica L.)

PubMed Central

Muthamilarasan, Mehanathan; Khandelwal, Rohit; Yadav, Chandra Bhan; Bonthala, Venkata Suresh; Khan, Yusuf; Prasad, Manoj

2014-01-01

MYB proteins represent one of the largest transcription factor families in plants, playing important roles in diverse developmental and stress-responsive processes. Considering its significance, several genome-wide analyses have been conducted in almost all land plants except foxtail millet. Foxtail millet (Setaria italica L.) is a model crop for investigating systems biology of millets and bioenergy grasses. Further, the crop is also known for its potential abiotic stress-tolerance. In this context, a comprehensive genome-wide survey was conducted and 209 MYB protein-encoding genes were identified in foxtail millet. All 209 S. italica MYB (SiMYB) genes were physically mapped onto nine chromosomes of foxtail millet. Gene duplication study showed that segmental- and tandem-duplication have occurred in genome resulting in expansion of this gene family. The protein domain investigation classified SiMYB proteins into three classes according to number of MYB repeats present. The phylogenetic analysis categorized SiMYBs into ten groups (I - X). SiMYB-based comparative mapping revealed a maximum orthology between foxtail millet and sorghum, followed by maize, rice and Brachypodium. Heat map analysis showed tissue-specific expression pattern of predominant SiMYB genes. Expression profiling of candidate MYB genes against abiotic stresses and hormone treatments using qRT-PCR revealed specific and/or overlapping expression patterns of SiMYBs. Taken together, the present study provides a foundation for evolutionary and functional characterization of MYB TFs in foxtail millet to dissect their functions in response to environmental stimuli. PMID:25279462

Off-line monitoring of bacterial stress response during recombinant protein production using an optical biosensor.

PubMed

Vostiar, Igor; Tkac, Jan; Mandenius, Carl-Fredrik

2004-07-15

A surface plasmon resonance (SPR) biosensor was used to monitor the profiles of the heat-shock protein (DnaK) and the expression of a heterologous protein to map the dynamics of the cellular stress response in Escherichia coli. As expression system was used an E. coli strain overproducing human recombinant superoxide dismutase (rhSOD). Expression of DnaK showed complex patterns differing with strength of induction. The strong up-regulation of DnaK expression was observed in all cultivations which over-produced of rhSOD. Similar patterns were not observed in non-induced reference cultures. Differences in DnaK concentration profiles were correlated with induction strength. Presented data, carried out in shake flask and glucose limited fed-batch cultivation, show a good consistency with previously published transcriptional profiling results and provide complementary information to understand stress response related to overproduction of recombinant protein. The study also demonstrates the feasibility of using the SPR as a two channel protein array for monitoring of intracellular components.

Independent localization of MAP2, CaMKIIα and β-actin RNAs in low copy numbers.

PubMed

Mikl, Martin; Vendra, Georgia; Kiebler, Michael A

2011-09-30

Messenger RNA localization involves the assembly of ribonucleoprotein particles (RNPs) and their subsequent transport along the cytoskeleton to their final destination. Here, we provide new evidence that microtubule-associated protein 2 (MAP2), calcium/calmodulin-dependent protein kinase II (CaMKIIα) and β-actin RNAs localize to dendrites in distinct RNPs, which contain--unexpectedly--very few RNA molecules. The number of MAP2 molecules per particle is affected by synaptic activity and Staufen 2, indicating that RNP composition is tightly controlled. Our data suggest that the independent localization of individual RNAs in low copy numbers could contribute to tighter temporal and spatial control of expression in neurons and synapse-specific plasticity.

Effects of Cot expression on the nuclear translocation of NF-kappaB in RBL-2H3 cells.

PubMed

Chikamatsu, Satomi; Furuno, Tadahide; Kinoshita, Yosuke; Inoh, Yoshikazu; Hirashima, Naohide; Teshima, Reiko; Nakanishi, Mamoru

2007-03-01

Cot is a serine/threonine protein kinase and is classified as a mitogen-activated protein (MAP) kinase kinase kinase. Overexpression of this protein has been shown to activate the extracellular signal-regulated kinase, the c-Jun N-terminal kinase, and the p38 MAP kinase pathways and to stimulate NF-AT and NF-kappaB-dependent transcription. Here we have shown that Cot kinase activity is intimately involved in the high affinity receptor for IgE (FcvarepsilonRI)-mediated nuclear translocation of NF-kappaB1 independent of NF-kappaB-inducing kinase (NIK) in rat basophilic leukemia (RBL-2H3) cells. A transfected green fluorescent protein-tagged NF-kappaB1 (GFP-NF-kappaB1) resided in the cytoplasm in RBL-2H3 cells and it remained in the cytoplasm even when Cot tagged with red fluorescent protein (Cot-RFP) was co-expressed. Western blotting analysis showed that IkappaB kinases (IKKs) were expressed in RBL-2H3 cells but NIK was not. GFP-NF-kappaB1 translocated from the cytoplasm to the nucleus after the aggregation of FcvarepsilonRI in Cot-transfected cells but not in kinase-deficient Cot-transfected cells. This finding gives a new insight into the role of Cot in the FcvarepsilonRI-mediated NF-kappaB activation in mast cells.

Broad Neutralization of Ebolaviruses via a Fusion Loop Epitope Elicited by Immunization

DTIC Science & Technology

2017-03-31

overnight. After incubation with blocking buffer (BB, 2% non- fat milk , 5% FBS in PBS), the WT or mutant supernatant in five-fold serial dilution in BB was...replication competent rVSV pseudotyped with filovirus GP, which also expressed the reporter protein GFP (rVSV-GP-GFP) (Miller et al., 2012). CA45 potently...for proper protein folding and expression. The epitope mapping identified EBOV GP residues R64 within the N-terminus of GP1 in addition to Y517

Automated Purification of Recombinant Proteins: Combining High-throughput with High Yield

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

Lin, Chiann Tso; Moore, Priscilla A.; Auberry, Deanna L.

2006-05-01

Protein crystallography, mapping protein interactions and other approaches of current functional genomics require not only purifying large numbers of proteins but also obtaining sufficient yield and homogeneity for downstream high-throughput applications. There is a need for the development of robust automated high-throughput protein expression and purification processes to meet these requirements. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli: First - a filtration separation protocol based on expression of 800 ml E. coli cultures followed by filtration purification using Ni2+-NTATM Agarose (Qiagen). Second - a smallermore » scale magnetic separation method based on expression in 25 ml cultures of E.coli followed by 96-well purification on MagneHisTM Ni2+ Agarose (Promega). Both workflows provided comparable average yields of proteins about 8 ug of purified protein per unit of OD at 600 nm of bacterial culture. We discuss advantages and limitations of the automated workflows that can provide proteins more than 90 % pure in the range of 100 ug – 45 mg per purification run as well as strategies for optimization of these protocols.« less

Alterations in the nuclear proteome of HIV-1 infected T-cells

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

DeBoer, Jason; Jagadish, Teena; Haverland, Nicole A.

Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified fourmore » clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines.« less

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

PubMed

Buszczak, Michael; Paterno, Shelley; Lighthouse, Daniel; Bachman, Julia; Planck, Jamie; Owen, Stephenie; Skora, Andrew D; Nystul, Todd G; Ohlstein, Benjamin; Allen, Anna; Wilhelm, James E; Murphy, Terence D; Levis, Robert W; Matunis, Erika; Srivali, Nahathai; Hoskins, Roger A; Spradling, Allan C

2007-03-01

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

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

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

Higgins, G.A.; Lewis, D.A.; Bahmanyar, S.

1988-02-01

The authors have mapped the neuroanatomical distribution of amyloid-..beta..-protein mRNA within neuronal subpopulations of the hippocampal formation in the cynomolgus monkey (Macaca fascicularis), normal aged human, and patients with Alzheimer disease. Amyloid-..beta..-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-..beta..-protein mRNAmore » in the cornu Ammonis field 3 and parasubiculum are equivalent. These findings suggest that within certain neuronal subpopulations cell type-specific regulation of amyloid-..beta..-protein gene expression may be altered in Alzheimer disease.« less

Proteomic analysis of the nuclear matrix in the early stages of rat liver carcinogenesis: Identification of differentially expressed and MAR-binding proteins

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

Barboro, Paola; D'Arrigo, Cristina; Repaci, Erica

Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentiallymore » expressed; among these, 15 spots were under-expressed and 8 spots were over-expressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7-7.0 and 6.2-7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure.« less

Receptor-like Molecules on Human Intestinal Epithelial Cells Interact with an Adhesion Factor from Lactobacillus reuteri

PubMed Central

MATSUO, Yosuke; MIYOSHI, Yukihiro; OKADA, Sanae; SATOH, Eiichi

2012-01-01

A surface protein of Lactobacillus reuteri, mucus adhesion-promoting protein (MapA), is considered to be an adhesion factor. MapA is expressed in L. reuteri strains and adheres to piglet gastric mucus, collagen type I, and human intestinal epithelial cells such as Caco-2. The aim of this study was to identify molecules that mediate the attachment of MapA from L. reuteri to the intestinal epithelial cell surface by investigating the adhesion of MapA to receptor-like molecules on Caco-2 cells. MapA-binding receptor-like molecules were detected in Caco-2 cell lysates by 2D-PAGE. Two proteins, annexin A13 (ANXA13) and paralemmin (PALM), were identified by MALDI TOF-MS. The results of a pull-down assay showed that MapA bound directly to ANXA13 and PALM. Fluorescence microscopy studies confirmed that MapA binding to ANXA13 and PALM was colocalized on the Caco-2 cell membrane. To evaluate whether ANXA13 and PALM are important for MapA adhesion, ANXA13 and PALM knockdown cell lines were established. The adhesion of MapA to the abovementioned cell lines was reduced compared with that to wild-type Caco-2 cells. These knockdown experiments established the importance of these receptor-like molecules in MapA adhesion. PMID:24936355

Human retina-specific amine oxidase (RAO): cDNA cloning, tissue expression, and chromosomal mapping

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

Imamura, Yutaka; Kubota, Ryo; Wang, Yimin

In search of candidate genes for hereditary retinal disease, we have employed a subtractive and differential cDNA cloning strategy and isolated a novel retina-specific cDNA. Nucleotide sequence analysis revealed an open reading frame of 2187 bp, which encodes a 729-amino-acid protein with a calculated molecular mass of 80,644 Da. The putative protein contained a conserved domain of copper amine oxidase, which is found in various species from bacteria to mammals. It showed the highest homology to bovine serum amine oxidase, which is believed to control the level of serum biogenic amines. Northern blot analysis of human adult and fetal tissuesmore » revealed that the protein is expressed abundantly and specifically in retina as a 2.7-kb transcript. Thus, we considered this protein a human retina-specific amine oxidase (RAO). The RAO gene (AOC2) was mapped by fluorescence in situ hybridization to human chromosome 17q21. We propose that AOC2 may be a candidate gene for hereditary ocular diseases. 38 refs., 4 figs.« less

Proteome Analyses of Staphylococcus aureus Biofilm at Elevated Levels of NaCl

PubMed Central

Islam, Nazrul; Ross, Julia M; Marten, Mark R

2016-01-01

Our studies demonstrate that sodium chloride (NaCl) induces changes in biofilm, mediated by increased production of polysaccharides intercellular adhesion (PIA). We identified 12 proteins that showed higher abundance in increased level of NaCl. This includes one important protein (IsaA) known to be associated with biofilm stability. In addition, we also found higher abundance of a cold shock protein, CspA, at higher NaCl. We have also identified several other proteins that are differentially expressed to the elevated levels of NaCl and mapped them in the regulatory pathways of PIA. The majority of proteins are involved with various aspects bacterial metabolic function. Our results demonstrated that NaCl influences gene regulatory networks controlling exopolysaccharide expression. PMID:26973848

Low cross-reactivity of T-cell responses against lipids from Mycobacterium bovis and M. avium paratuberculosis during natural infection

PubMed Central

Van Rhijn, Ildiko; Nguyen, Thi Kim Anh; Michel, Anita; Cooper, Dave; Govaerts, Marc; Cheng, Tan-Yun; van Eden, Willem; Moody, D. Branch; Coetzer, Jacobus A. W.; Rutten, Victor; Koets, Ad P.

2011-01-01

Although CD1 proteins are known to present mycobacterial lipid antigens to T cells, there is little understanding of the in vivo behavior of T cells restricted by CD1a, CD1b and CD1c, and the relative immunogenicity and immunodominance of individual lipids within the total array of lipids that comprise a bacterium. Because bovines express multiple CD1 proteins and are natural hosts of Mycobacterium bovis and Mycobacterium avium paratuberculosis (MAP), we used them as a new animal model of CD1 function. Here, we report the surprisingly divergent responses against lipids produced by these two pathogens during infection. Despite considerable overlap in lipid content, only three out of 69 animals cross-react with M. bovis and MAP total lipid preparations. The unidentified immunodominant compound of M. bovis is a hydrophilic compound, whereas the immunodominant lipid of MAP is presented by CD1b and was identified as glucose monomycolate (GMM). The preferential recognition of GMM antigen by MAP-infected cattle may be explained by the higher expression of GMM by MAP than by M. bovis. The bacterial species-specific nature of the CD1-restricted, adaptive T-cell response affects the approach to development of lipid based immunodiagnostic tests. PMID:19688747

Arabidopsis Microtubule-Associated Protein MAP65-3 Cross-Links Antiparallel Microtubules toward Their Plus Ends in the Phragmoplast via Its Distinct C-Terminal Microtubule Binding Domain[W

PubMed Central

Ho, Chin-Min Kimmy; Lee, Yuh-Ru Julie; Kiyama, Lindsay D.; Dinesh-Kumar, Savithramma P.; Liu, Bo

2012-01-01

Plant cytokinesis is brought about by the phragmoplast, which contains an antiparallel microtubule (MT) array. The MT-associated protein MAP65-3 acts as an MT-bundling factor that specifically cross-links antiparallel MTs near their plus ends. MAP65 family proteins contain an N-terminal dimerization domain and C-terminal MT interaction domain. Compared with other MAP65 isoforms, MAP65-3 contains an extended C terminus. A MT binding site was discovered in the region between amino acids 496 and 588 and found to be essential for the organization of phragmoplast MTs. The frequent cytokinetic failure caused by loss of MAP65-3 was not rescued by ectopic expression of MAP65-1 under the control of the MAP65-3 promoter, indicating nonoverlapping functions between the two isoforms. In the presence of MAP65-3, however, ectopic MAP65-1 appeared in the phragmoplast midline. We show that MAP65-1 could acquire the function of MAP65-3 when the C terminus of MAP65-3, which contains the MT binding site, was grafted to it. Our results also show that MAP65-1 and MAP65-3 may share redundant functions in MT stabilization. Such a stabilization effect was likely brought about by MT binding and bundling. We conclude that MAP65-3 contains a distinct C-terminal MT binding site with a specific role in cross-linking antiparallel MTs toward their plus ends in the phragmoplast. PMID:22570443

Rapid analysis of protein interactions: On-chip micropurification of recombinant protein expressed in Esherichia coli.

PubMed

Natsume, Tohru; Taoka, Masato; Manki, Hiroshi; Kume, Shouen; Isobe, Toshiaki; Mikoshiba, Katsuhiko

2002-09-01

We describe a rapid analysis of interactions between antibodies and a recombinant protein present in total cell lysates. Using a surface plasmon resonance biosensor, a low concentration of glutathione-S-transferase (GST) fused protein expressed in small scale Esherichia coli culture was purified on an anti-GST antibody immobilized sensor chip. The 'on-chip purification' was verified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry by measuring the molecular masses of recombinant proteins purified on the sensor chip. The specific binding of monoclonal antibodies for the on-chip micropurified recombinant proteins can then be monitored, thus enabling kinetic analysis and epitope mapping of the bound antibodies. This approach reduced time, resources and sample consumption by avoiding conventional steps related to concentration and purification.

Hepatitis C virus core protein activates autophagy through EIF2AK3 and ATF6 UPR pathway-mediated MAP1LC3B and ATG12 expression

PubMed Central

Wang, Ji; Kang, Rongyan; Huang, He; Xi, Xueyan; Wang, Bei; Wang, Jianwei; Zhao, Zhendong

2014-01-01

HCV infection induces autophagy, but how this occurs is unclear. Here, we report the induction of autophagy by the structural HCV core protein and subsequent endoplasmic reticular (ER) stress in Huh7 hepatoma cells. During ER stress, both the EIF2AK3 and ATF6 pathways of the unfolded protein response (UPR) were activated by HCV core protein. Then, these pathways upregulated transcription factors ATF4 and DDIT3. The ERN1-XBP1 pathway was not activated. Through ATF4 in the EIF2AK3 pathway, the autophagy gene ATG12 was upregulated. DDIT3 upregulated the transcription of autophagy gene MAP1LC3B (LC3B) by directly binding to the –253 to –99 base region of the LC3B promoter, contributing to the development of autophagy. Collectively, these data suggest not only a novel role for the HCV core protein in autophagy but also offer new insight into detailed molecular mechanisms with respect to HCV-induced autophagy, specifically how downstream UPR molecules regulate key autophagic gene expression. PMID:24589849

Regulation of Hsp27 and Hsp70 expression in human and mouse skin construct models by caveolae following exposure to the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide

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

Black, Adrienne T.; Hayden, Patrick J.; Casillas, Robert P.

Dermal exposure to the vesicant sulfur mustard causes marked inflammation and tissue damage. Basal keratinocytes appear to be a major target of sulfur mustard. In the present studies, mechanisms mediating skin toxicity were examined using a mouse skin construct model and a full-thickness human skin equivalent (EpiDerm-FT{sup TM}). In both systems, administration of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide (CEES, 100-1000 {mu}M) at the air surface induced mRNA and protein expression of heat shock proteins 27 and 70 (Hsp27 and Hsp70). CEES treatment also resulted in increased expression of caveolin-1, the major structural component of caveolae. Immunohistochemistry revealedmore » that Hsp27, Hsp70 and caveolin-1 were localized in basal and suprabasal layers of the epidermis. Caveolin-1 was also detected in fibroblasts in the dermal component of the full thickness human skin equivalent. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that Hsp27 and Hsp70 were localized in caveolae. Treatment of mouse keratinocytes with filipin III or methyl-{beta}-cyclodextrin, which disrupt caveolar structure, markedly suppressed CEES-induced Hsp27 and Hsp70 mRNA and protein expression. CEES treatment is known to activate JNK and p38 MAP kinases; in mouse keratinocytes, inhibition of these enzymes suppressed CEES-induced expression of Hsp27 and Hsp70. These data suggest that MAP kinases regulate Hsp 27 and Hsp70; moreover, caveolae-mediated regulation of heat shock protein expression may be important in the pathophysiology of vesicant-induced skin toxicity.« less

Regulation of Hsp27 and Hsp70 expression in human and mouse skin construct models by caveolae following exposure to the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide

PubMed Central

Black, Adrienne T.; Hayden, Patrick J.; Casillas, Robert P.; Heck, Diane E.; Gerecke, Donald R.; Sinko, Patrick J.; Laskin, Debra L.; Laskin, Jeffrey D.

2012-01-01

Dermal exposure to the vesicant sulfur mustard causes marked inflammation and tissue damage. Basal keratinocytes appear to be a major target of sulfur mustard. In the present studies, mechanisms mediating skin toxicity were examined using a mouse skin construct model and a full-thickness human skin equivalent (EpiDerm-FTTM). In both systems, administration of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide (CEES, 100–1000 µM) at the air surface induced mRNA and protein expression of heat shock proteins 27 and 70 (Hsp27 and Hsp70). CEES treatment also resulted in increased expression of caveolin-1, the major structural component of caveolae. Immunohistochemistry revealed that Hsp27, Hsp70 and caveolin-1 were localized in basal and suprabasal layers of the epidermis. Caveolin-1 was also detected in fibroblasts in the dermal component of the full thickness human skin equivalent. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that Hsp27 and Hsp70 were localized in caveolae. Treatment of mouse keratinocytes with filipin III or methyl-β-cyclodextrin, which disrupt caveolar structure, markedly suppressed CEES-induced Hsp27 and Hsp70 mRNA and protein expression. CEES treatment is known to activate JNK and p38 MAP kinases; in mouse keratinocytes, inhibition of these enzymes suppressed CEES-induced expression of Hsp27 and Hsp70. These data suggest that MAP kinases regulate Hsp 27 and Hsp70; moreover, caveolae-mediated regulation of heat shock protein expression may be important in the pathophysiology of vesicant-induced skin toxicity. PMID:21457723

Regulation of Hsp27 and Hsp70 expression in human and mouse skin construct models by caveolae following exposure to the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide.

PubMed

Black, Adrienne T; Hayden, Patrick J; Casillas, Robert P; Heck, Diane E; Gerecke, Donald R; Sinko, Patrick J; Laskin, Debra L; Laskin, Jeffrey D

2011-06-01

Dermal exposure to the vesicant sulfur mustard causes marked inflammation and tissue damage. Basal keratinocytes appear to be a major target of sulfur mustard. In the present studies, mechanisms mediating skin toxicity were examined using a mouse skin construct model and a full-thickness human skin equivalent (EpiDerm-FT™). In both systems, administration of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide (CEES, 100-1000μM) at the air surface induced mRNA and protein expression of heat shock proteins 27 and 70 (Hsp27 and Hsp70). CEES treatment also resulted in increased expression of caveolin-1, the major structural component of caveolae. Immunohistochemistry revealed that Hsp27, Hsp70 and caveolin-1 were localized in basal and suprabasal layers of the epidermis. Caveolin-1 was also detected in fibroblasts in the dermal component of the full thickness human skin equivalent. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that Hsp27 and Hsp70 were localized in caveolae. Treatment of mouse keratinocytes with filipin III or methyl-β-cyclodextrin, which disrupt caveolar structure, markedly suppressed CEES-induced Hsp27 and Hsp70 mRNA and protein expression. CEES treatment is known to activate JNK and p38 MAP kinases; in mouse keratinocytes, inhibition of these enzymes suppressed CEES-induced expression of Hsp27 and Hsp70. These data suggest that MAP kinases regulate Hsp 27 and Hsp70; moreover, caveolae-mediated regulation of heat shock protein expression may be important in the pathophysiology of vesicant-induced skin toxicity. Copyright © 2011 Elsevier Inc. All rights reserved.

A mutation in a new gene bglJ, activates the bgl operon in Escherichia coli K-12

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

Giel, M.; Desnoyer, M.; Lopilato, J.

1996-06-01

A new mutation , bglJ4, has been characterized that results in the expression of the silent bgl operon. The bgl operon encodes proteins necessary for the transport and utilization of the aromatic {beta}-glucosides arbutin and salicin. A variety of mutations activate the operon and result in a Bgl{sup +} phenotype. Activating mutations are located upstream of the bgl promoter and in genes located elsewhere on the chromosome. Mutations outside of the bgl operon occur in the genes encoding DNA gyrase and in the gene encoding the nucleoid associated protein H-NS. The mutation described here, bglJ4, has been mapped to amore » new locus at min 99 on the Escherichia coli K-12 genetic map. The putative protein encoded by the bglJ gene has homology to a family of transcriptional activators. Evidence is presented that increased expression of the bglJ product is needed for activation of the bgl operon. 56 refs., 3 figs., 3 tabs.« less

Alpha-linolenic acid regulates Cox2/VEGF/MAP kinase pathway and decreases the expression of HPV oncoproteins E6/E7 through restoration of p53 and Rb expression in human cervical cancer cell lines.

PubMed

Deshpande, Rashmi; Mansara, Prakash; Kaul-Ghanekar, Ruchika

2016-03-01

Cervical cancer represents the largest cause of mortality in women worldwide. In our previous report, we have shown how alpha-linolenic acid (ALA), an omega-3 fatty acid, regulated the growth of cervical cancer cells. The present study aimed to explore mechanistic details for the anticancer activity of ALA in cervical cancer cell lines, SiHa and HeLa. ALA significantly modulated the growth kinetics of the cells and reduced cell migration with concomitant decrease in the expression of VEGF, MMP-2, and MMP-9 proteins. Besides this, ALA significantly decreased the expression of phosphorylated p38, pERK1/2, c-JUN, NFκB, and COX2, proteins. Most importantly, ALA reduced the expression of HPV onco-proteins E6 and E7, resulting into restoration of expression of tumor suppressor proteins, p53 and Rb. These results suggested that ALA could be explored for its therapeutic potential in cervical cancer.

Expression of Antisense Long Noncoding RNAs as Potential Regulators in Rainbow Trout with Different Tolerance to Plant-Based Diets.

PubMed

Abernathy, Jason; Overturf, Ken

2018-01-04

Reformulation of aquafeeds in salmonid diets to include more plant proteins is critical for sustainable aquaculture. However, increasing plant proteins can lead to stunted growth and enteritis. Toward an understanding of the regulatory mechanisms behind plant protein utilization, directional RNA sequencing of liver tissues from a rainbow trout strain selected for growth on an all plant-protein diet and a control strain, both fed a plant diet for 12 weeks, were utilized to construct long noncoding RNAs. Antisense long noncoding RNAs were selected for differential expression and functional analyses since they have been shown to have regulatory actions within a genome. A total of 142 unique antisense long noncoding RNAs were differentially expressed between strains, 60 of which could be mapped to a gene. Genes underlying these noncoding RNAs are indicated in lipid metabolism and immunity. Six noncoding transcripts were also found to overlap with differentially expressed protein-coding genes, all of which were co-expressed. Associating variation in regulatory elements between rainbow trout strains with differing tolerance to plant-protein diets will assist in future studies toward increased gains throughout carnivorous aquaculture.

Comparative 2D-DIGE Proteomic Analysis of Bovine Mammary Epithelial Cells during Lactation Reveals Protein Signatures for Lactation Persistency and Milk Yield

PubMed Central

Janjanam, Jagadeesh; Singh, Surender; Jena, Manoj K.; Varshney, Nishant; Kola, Srujana; Kumar, Sudarshan; Kaushik, Jai K.; Grover, Sunita; Dang, Ajay K.; Mukesh, Manishi; Prakash, B. S.; Mohanty, Ashok K.

2014-01-01

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling. PMID:25111801

N-Caffeoyltryptamine, a Potent Anti-Inflammatory Phenolic Amide, Suppressed MCP-1 Expression in LPS-stimulated THP-1 Cells and Rats Fed a High-Fat Diet.

PubMed

Park, Jae B

2017-05-27

Monocyte chemoattractant protein-1 (MCP-1) is a well-known chemokine critically involved in the pathophysiological progression of several inflammatory diseases including arthrosclerosis. N -caffeoyltryptamine is a phenolic amide with strong anti-inflammatory effects. Therefore, in this paper, the potential effect of N -caffeoyltryptamine on MCP-1 expression was investigated as a potential p38 mitogen-activated protein (MAP) kinase inhibitor in vitro and in vivo. At the concentration of 20 μM, N -caffeoyltryptamine significantly inhibited p38 MAP kinase α, β, γ and δ by 15-50% ( p < 0.05), particularly p38 MAP kinase α (IC 50 = 16.7 μM) and β (IC 50 = 18.3 μM). Also, the pretreatment of the lipopolysaccharide (LPS)-stimulated THP-1 cells with N -caffeoyltryptamine (10, 20 and 40 μM) led to significant suppression of MCP-1 production by 10-45% ( p < 0.05) in the cells. Additionally, N -caffeoyltryptamine was also able to significantly downregulate MCP-1 mRNA expression in the THP-1 cells ( p < 0.05). On the basis of this strong inhibition in vitro, an animal study was conducted to confirm this inhibitory effect in vivo. Rats were divided into three groups ( n = 8): a normal control diet (C), a high-fat diet (HF), or a high-fat diet supplemented with N -caffeoyltryptamine (2 mg per day) (HFS). After 16 weeks, blood samples were collected from the rats in each group, and MCP-1 levels were determined in plasma with other atherogenic markers (C-reactive protein and soluble E-selectin (sE-selectin)). As expected, the average MCP-1 levels of the HF group were found to be higher than those of the C group ( p < 0.05). However, the MCP-1 levels of the HFS group were significantly lower than those of the HF group ( p < 0.05), suggesting that N -caffeoyltryptamine could decrease MCP-1 expression in vivo. Related to other atherogenic markers such as C-reactive protein and sE-selectin, there was no significant difference in their levels between the HF and HFS groups. These data suggest that N -caffeoyltryptamine may specifically suppress MCP-1 expression in vitro and in vivo, possibly by inhibiting p38 MAP kinase.

High humidity suppresses ssi4-mediated cell death and disease resistance upstream of MAP kinase activation, H2O2 production and defense gene expression.

PubMed

Zhou, Fasong; Menke, Frank L H; Yoshioka, Keiko; Moder, Wolfgang; Shirano, Yumiko; Klessig, Daniel F

2004-09-01

The Arabidopsis ssi4 mutant, which exhibits spontaneous lesion formation, constitutive expression of pathogenesis-related (PR) genes and enhanced resistance to virulent bacterial and oomycete pathogens, contains a gain-of-function mutation in a TIR-NBS-LRR type R gene. Epistatic analyses revealed that both PR gene expression and disease resistance are activated via a salicylic acid (SA)- and EDS1-dependent, but NPR1- and NDR1-independent signaling pathway. In this study, we demonstrate that in moderate relative humidity (RH; 60%), the ssi4 mutant accumulates H(2)O(2) and SA prior to lesion formation and displays constitutive activation of the MAP kinases AtMPK6 and AtMPK3. It also constitutively expresses a variety of defense-associated genes, including those encoding the WRKY transcription factors AtWRKY29 and AtWRKY6, the MAP kinases AtMPK6 and AtMPK3, the powdery mildew R proteins RPW8.1 and RPW8.2, EDS1 and PR proteins. All of these ssi4-induced responses, as well as the chlorotic, stunted morphology and enhanced disease resistance phenotype, are suppressed by high RH (95%) growth conditions. Thus, a humidity sensitive factor (HSF) appears to function at an early point in the ssi4 signaling pathway. All ssi4 phenotypes, except for MAP kinase activation, also were suppressed by the eds1-1 mutation. Thus, ssi4-induced MAP kinase activation occurs downstream of the HSF but either upstream of EDS1 or on a separate branch of the ssi4 signaling pathway. SA is a critical signaling component in ssi4-mediated defense responses. However, exogenously supplied SA failed to restore lesion formation in high RH-grown ssi4 plants, although it induced defense gene expression. Thus, additional signals also are involved.

Synchrotron radiation based STXM analysis and micro-XRF mapping of differential expression of extracellular thiol groups by Acidithiobacillus ferrooxidans grown on Fe(2+) and S(0).

PubMed

Xia, Jin-Lan; Liu, Hong-Chang; Nie, Zhen-Yuan; Peng, An-An; Zhen, Xiang-Jun; Yang, Yun; Zhang, Xiu-Li

2013-09-01

The differential expression of extracellular thiol groups by Acidithiobacillus ferrooxidans grown on substrates Fe(2+) and S(0) was investigated by using synchrotron radiation based scanning transmission X-ray microscopy (STXM) imaging and microbeam X-ray fluorescence (μ-XRF) mapping. The extracellular thiol groups (SH) were first alkylated by iodoacetic acid forming Protein-SCH2COOH and then the P-SCH2COOH was marked by calcium ions forming P-SCH2COOCa. The STXM imaging and μ-XRF mapping of SH were based on analysis of SCH2COO-bonded Ca(2+). The results indicated that the thiol group content of A. ferrooxidans grown on S(0) is 3.88 times to that on Fe(2+). Combined with selective labeling of SH by Ca(2+), the STXM imaging and μ-XRF mapping provided an in situ and rapid analysis of differential expression of extracellular thiol groups. © 2013.

Amygdala stimulation promotes recovery of behavioral performance in a spatial memory task and increases GAP-43 and MAP-2 in the hippocampus and prefrontal cortex of male rats.

PubMed

Mercerón-Martínez, D; Almaguer-Melian, W; Alberti-Amador, E; Bergado, J A

2018-06-19

The relationships between affective and cognitive processes are an important issue of present neuroscience. The amygdala, the hippocampus and the prefrontal cortex appear as main players in these mechanisms. We have shown that post-training electrical stimulation of the basolateral amygdala (BLA) speeds the acquisition of a motor skill, and produces a recovery in behavioral performance related to spatial memory in fimbria-fornix (FF) lesioned animals. BLA electrical stimulation rises bdnf RNA expression, BDNF protein levels, and arc RNA expression in the hippocampus. In the present paper we have measured the levels of one presynaptic protein (GAP-43) and one postsynaptic protein (MAP-2) both involved in synaptogenesis to assess whether structural neuroplastic mechanisms are involved in the memory enhancing effects of BLA stimulation. A single train of BLA stimulation produced in healthy animals an increase in the levels of GAP-43 and MAP-2 that lasted days in the hippocampus and the prefrontal cortex. In FF-lesioned rats, daily post-training stimulation of the BLA ameliorates the memory deficit of the animals and induces an increase in the level of both proteins. These results support the hypothesis that the effects of amygdala stimulation on memory recovery are sustained by an enhanced formation of new synapses. Copyright © 2018. Published by Elsevier Inc.

Identification and Characterization of Three Differentially Expressed Genes, Encoding S-Adenosylhomocysteine Hydrolase, Methionine Aminopeptidase, and a Histone-Like Protein, in the Toxic Dinoflagellate Alexandrium fundyense†

PubMed Central

Taroncher-Oldenburg, Gaspar; Anderson, Donald M.

2000-01-01

Genes showing differential expression related to the early G1 phase of the cell cycle during synchronized circadian growth of the toxic dinoflagellate Alexandrium fundyense were identified and characterized by differential display (DD). The determination in our previous work that toxin production in Alexandrium is relegated to a narrow time frame in early G1 led to the hypothesis that transcriptionally up- or downregulated genes during this subphase of the cell cycle might be related to toxin biosynthesis. Three genes, encoding S-adenosylhomocysteine hydrolase (Sahh), methionine aminopeptidase (Map), and a histone-like protein (HAf), were isolated. Sahh was downregulated, while Map and HAf were upregulated, during the early G1 phase of the cell cycle. Sahh and Map encoded amino acid sequences with about 90 and 70% similarity to those encoded by several eukaryotic and prokaryotic Sahh and Map genes, respectively. The partial Map sequence also contained three cobalt binding motifs characteristic of all Map genes. HAf encoded an amino acid sequence with 60% similarity to those of two histone-like proteins from the dinoflagellate Crypthecodinium cohnii Biecheler. This study documents the potential of applying DD to the identification of genes that are related to physiological processes or cell cycle events in phytoplankton under conditions where small sample volumes represent an experimental constraint. The identification of an additional 21 genes with various cell cycle-related DD patterns also provides evidence for the importance of pretranslational or transcriptional regulation in dinoflagellates, contrary to previous reports suggesting the possibility that translational mechanisms are the primary means of circadian regulation in this group of organisms. PMID:10788388

Mouse scrapie responsive gene 1 (Scrg1): genomic organization, physical linkage to sap30, genetic mapping on chromosome 8, and expression in neuronal primary cell cultures.

PubMed

Dron, M; Tartare, X; Guillo, F; Haik, S; Barbin, G; Maury, C; Tovey, M; Dandoy-Dron, F

2000-11-15

We have previously reported a transcript of a novel mouse gene (Scrg1) with increased expression in transmissible spongiform encephalopathies and the cloning of the human mRNA analogue. In this paper, we present the genomic organization of the mouse and human SCRG1 loci, which exhibit a high degree of conservation. The genes are composed of three exons; the two downstream exons contain the protein coding region. The mouse gene is expressed in brain tissue essentially as a 0.7-kb message but also as a minor 2.6-kb mRNA. We have sequenced 20 kb of DNA at the mouse Scrg1 locus and found that the longer transcript is the prolongation of the 0.7-kb mRNA to a polyadenylation site located about 2 kb further downstream. Sequencing revealed that the mouse Scrg1 gene is physically linked to Sap30, a gene that encodes a protein of the histone deacetylase complex, and genetic linkage mapping assigned the localization of Scrg1 to chromosome 8 between Ant1 and Hmg2. Northern blot analysis showed that Scrg1 is under strict developmental control in mouse embryo and is expressed by cells of neuronal origin in vitro. Comparison of the rat, mouse, and human SCRG1 proteins identified a box of 35 identical contiguous amino acids and a characteristic cysteine distribution pattern defining a new protein signature. Copyright 2000 Academic Press.

Development and validation of a whole-cell inhibition assay for bacterial methionine aminopeptidase by surface-enhanced laser desorption ionization-time of flight mass spectrometry.

PubMed

Greis, Kenneth D; Zhou, Songtao; Siehnel, Richard; Klanke, Chuck; Curnow, Alan; Howard, Jeremy; Layh-Schmitt, Gerlinde

2005-08-01

Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP.

Identification of two functional nuclear localization signals in the capsid protein of duck circovirus

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

Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin

The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNAmore » binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.« less

The Xanthomonas euvesicatoria type III effector XopAU is an active protein kinase that manipulates plant MAP kinase signaling.

PubMed

Teper, Doron; Girija, Anil Madhusoodana; Bosis, Eran; Popov, Georgy; Savidor, Alon; Sessa, Guido

2018-01-01

The Gram-negative bacterium Xanthomonas euvesicatoria (Xe) is the causal agent of bacterial spot disease of pepper and tomato. Xe delivers effector proteins into host cells through the type III secretion system to promote disease. Here, we show that the Xe effector XopAU, which is conserved in numerous Xanthomonas species, is a catalytically active protein kinase and contributes to the development of disease symptoms in pepper plants. Agrobacterium-mediated expression of XopAU in host and non-host plants activated typical defense responses, including MAP kinase phosphorylation, accumulation of pathogenesis-related (PR) proteins and elicitation of cell death, that were dependent on the kinase activity of the effector. XopAU-mediated cell death was not dependent on early signaling components of effector-triggered immunity and was also observed when the effector was delivered into pepper leaves by Xanthomonas campestris pv. campestris, but not by Xe. Protein-protein interaction studies in yeast and in planta revealed that XopAU physically interacts with components of plant immunity-associated MAP kinase cascades. Remarkably, XopAU directly phosphorylated MKK2 in vitro and enhanced its phosphorylation at multiple sites in planta. Consistent with the notion that MKK2 is a target of XopAU, silencing of the MKK2 homolog or overexpression of the catalytically inactive mutant MKK2K99R in N. benthamiana plants reduced XopAU-mediated cell death and MAPK phosphorylation. Furthermore, yeast co-expressing XopAU and MKK2 displayed reduced growth and this phenotype was dependent on the kinase activity of both proteins. Together, our results support the conclusion that XopAU contributes to Xe disease symptoms in pepper plants and manipulates host MAPK signaling through phosphorylation and activation of MKK2.

Proteomic response of mouse pituitary gland under heat stress revealed active regulation of stress responsive proteins.

PubMed

Memon, Shahar Bano; Lian, Li; Gadahi, Javaid Ali; Genlin, Wang

2016-10-01

The mapping of tissue proteomes can identify the molecular regulators and effectors of their physiological activity. However, proteomic response of a mammalian tissue against heat stress (HS) particularly of the pituitary gland has not yet been resolved. The proteomic response of the mouse pituitary gland against HS at 40 o C was evaluated by iTRAQ. We found that, HS actively regulates stress-related proteins. Among 375 differentially expressed proteins, 26 up and 46 downregulated proteins were found as stress responsive proteins. Two proteins belonging to the HSP70 and one to HSP90 family were found upregulated. Meanwhile, the expression of HSP90α (Cytosolic), HSP60, and HSP84b were observed to be downregulated. A neuroprotective enzyme Nmnat3 was observed to be significantly upregulated. Three proteins related to the intermediate filament (IF) proteins (lamins, vimentin and keratins) were also found to be upregulated. We reported, an association between the IF proteins and HSPs as a biological marker of HS. The expression of Apo A-IV was upregulated and might be one explanation for low food intake during HS. Our findings indicated that, differentially expressed proteins might be played important roles in combating HS. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparative Proteomic Analysis of Yak Follicular Fluid during Estrus

PubMed Central

Guo, Xian; Pei, Jie; Ding, Xuezhi; Chu, Min; Bao, Pengjia; Wu, Xiaoyun; Liang, Chunnian; Yan, Ping

2016-01-01

The breeding of yaks is highly seasonal, there are many crucial proteins involved in the reproduction control program, especially in follicular development. In order to isolate differential proteins between mature and immature follicular fluid (FF) of yak, the FF from yak follicles with different sizes were sampled respectively, and two-dimensional gel electrophoresis (2-DE) of the proteins was carried out. After silver staining, the Image Master 2D platinum software was used for protein analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was performed for differential protein identification. The expression level of transferrin and enolase superfamily member 1 (ENOSF1) was determined by Western blotting for verification analysis. The results showed that 2-DE obtained an electrophoresis map of proteins from mature and immature yak FF with high resolution and repeatability. A comparison of protein profiles identified 12 differently expressed proteins, out of which 10 of them were upregulated while 2 were downregulated. Western blotting showed that the expression of transferrin and ENOSF1 was enhanced with follicular development. Both the obtained protein profiles and the differently expressed proteins identified in this study provided experimental data related to follicular development during yak breeding seasons. This study also laid the foundation for understanding the microenvironment during oocyte development. PMID:26954118

HITS-CLIP yields genome-wide insights into brain alternative RNA processing

NASA Astrophysics Data System (ADS)

Licatalosi, Donny D.; Mele, Aldo; Fak, John J.; Ule, Jernej; Kayikci, Melis; Chi, Sung Wook; Clark, Tyson A.; Schweitzer, Anthony C.; Blume, John E.; Wang, Xuning; Darnell, Jennifer C.; Darnell, Robert B.

2008-11-01

Protein-RNA interactions have critical roles in all aspects of gene expression. However, applying biochemical methods to understand such interactions in living tissues has been challenging. Here we develop a genome-wide means of mapping protein-RNA binding sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova revealed extremely reproducible RNA-binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova-RNA interactions in 3' untranslated regions, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein-RNA interactions in vivo.

Independent localization of MAP2, CaMKIIα and β-actin RNAs in low copy numbers

PubMed Central

Mikl, Martin; Vendra, Georgia; Kiebler, Michael A

2011-01-01

Messenger RNA localization involves the assembly of ribonucleoprotein particles (RNPs) and their subsequent transport along the cytoskeleton to their final destination. Here, we provide new evidence that microtubule-associated protein 2 (MAP2), calcium/calmodulin-dependent protein kinase II (CaMKIIα) and β-actin RNAs localize to dendrites in distinct RNPs, which contain—unexpectedly—very few RNA molecules. The number of MAP2 molecules per particle is affected by synaptic activity and Staufen 2, indicating that RNP composition is tightly controlled. Our data suggest that the independent localization of individual RNAs in low copy numbers could contribute to tighter temporal and spatial control of expression in neurons and synapse-specific plasticity. PMID:21869818

The Histamine H1 Receptor Participates in the Increased Dorsal Telencephalic Neurogenesis in Embryos from Diabetic Rats.

PubMed

Solís, Karina H; Méndez, Laura I; García-López, Guadalupe; Díaz, Néstor F; Portillo, Wendy; De Nova-Ocampo, Mónica; Molina-Hernández, Anayansi

2017-01-01

Increased neuron telencephalic differentiation during deep cortical layer formation has been reported in embryos from diabetic mice. Transitory histaminergic neurons within the mesencephalon/rhombencephalon are responsible for fetal histamine synthesis during development, fibers from this system arrives to the frontal and parietal cortex at embryo day (E) 15. Histamine is a neurogenic factor for cortical neural stem cells in vitro through H 1 receptor (H 1 R) which is highly expressed during corticogenesis in rats and mice. Furthermore, in utero administration of an H 1 R antagonist, chlorpheniramine, decreases the neuron markers microtubuline associated protein 2 (MAP2) and forkhead box protein 2. Interestingly, in the diabetic mouse model of diabetes induced with streptozotocin, an increase in fetal neurogenesis in terms of MAP2 expression in the telencephalon is reported at E11.5. Because of the reported effects on cortical neuron differentiation of maternal diabetes in one hand and of histamine in the other, here the participation of histamine and H 1 R on the increased dorsal telencephalic neurogenesis was explored. First, the increased neurogenesis in the dorsal telencephalon at E14 in diabetic rats was corroborated by immunohistochemistry and Western blot. Then, changes during corticogenesis in the level of histamine was analyzed by ELISA and in H 1 R expression by qRT-PCR and Western blot and, finally, we tested H 1 R participation in the increased dorsal telencephalic neurogenesis by the systemic administration of chlorpheniramine. Our results showed a significant increase of histamine at E14 and in the expression of the receptor at E12. The administration of chlorpheniramine to diabetic rats at E12 prevented the increased expression of βIII-tubulin and MAP2 mRNAs (neuron markers) and partially reverted the increased level of MAP2 protein at E14, concluding that H 1 R have an important role in the increased neurogenesis within the dorsal telencephalon of embryos from diabetic rats. This study opens new perspective on the participation of HA and H 1 R receptor in early corticogenesis in health and disease.

The Histamine H1 Receptor Participates in the Increased Dorsal Telencephalic Neurogenesis in Embryos from Diabetic Rats

PubMed Central

Solís, Karina H.; Méndez, Laura I.; García-López, Guadalupe; Díaz, Néstor F.; Portillo, Wendy; De Nova-Ocampo, Mónica; Molina-Hernández, Anayansi

2017-01-01

Increased neuron telencephalic differentiation during deep cortical layer formation has been reported in embryos from diabetic mice. Transitory histaminergic neurons within the mesencephalon/rhombencephalon are responsible for fetal histamine synthesis during development, fibers from this system arrives to the frontal and parietal cortex at embryo day (E) 15. Histamine is a neurogenic factor for cortical neural stem cells in vitro through H1 receptor (H1R) which is highly expressed during corticogenesis in rats and mice. Furthermore, in utero administration of an H1R antagonist, chlorpheniramine, decreases the neuron markers microtubuline associated protein 2 (MAP2) and forkhead box protein 2. Interestingly, in the diabetic mouse model of diabetes induced with streptozotocin, an increase in fetal neurogenesis in terms of MAP2 expression in the telencephalon is reported at E11.5. Because of the reported effects on cortical neuron differentiation of maternal diabetes in one hand and of histamine in the other, here the participation of histamine and H1R on the increased dorsal telencephalic neurogenesis was explored. First, the increased neurogenesis in the dorsal telencephalon at E14 in diabetic rats was corroborated by immunohistochemistry and Western blot. Then, changes during corticogenesis in the level of histamine was analyzed by ELISA and in H1R expression by qRT-PCR and Western blot and, finally, we tested H1R participation in the increased dorsal telencephalic neurogenesis by the systemic administration of chlorpheniramine. Our results showed a significant increase of histamine at E14 and in the expression of the receptor at E12. The administration of chlorpheniramine to diabetic rats at E12 prevented the increased expression of βIII-tubulin and MAP2 mRNAs (neuron markers) and partially reverted the increased level of MAP2 protein at E14, concluding that H1R have an important role in the increased neurogenesis within the dorsal telencephalon of embryos from diabetic rats. This study opens new perspective on the participation of HA and H1R receptor in early corticogenesis in health and disease. PMID:29311766

The Carboxyl Terminus of v-Abl Protein Can Augment SH2 Domain Function

PubMed Central

Warren, David; Heilpern, Andrew J.; Berg, Kent; Rosenberg, Naomi

2000-01-01

Abelson murine leukemia virus (Ab-MLV) transforms NIH 3T3 and pre-B cells via expression of the v-Abl tyrosine kinase. Although the enzymatic activity of this molecule is absolutely required for transformation, other regions of the protein are also important for this response. Among these are the SH2 domain, involved in phosphotyrosine-dependent protein-protein interactions, and the long carboxyl terminus, which plays an important role in transformation of hematopoietic cells. Important signals are sent from each of these regions, and transformation is most likely orchestrated by the concerted action of these different parts of the protein. To explore this idea, we compared the ability of the v-Src SH2 domain to substitute for that of v-Abl in the full-length P120 v-Abl protein and in P70 v-Abl, a protein that lacks the carboxyl terminus characteristic of Abl family members. Ab-MLV strains expressing P70/S2 failed to transform NIH 3T3 cells and demonstrated a greatly reduced capacity to mediate signaling events associated with the Ras-dependent mitogen-activated protein (MAP) kinase pathway. In contrast, Ab-MLV strains expressing P120/S2 were indistinguishable from P120 with respect to these features. Analyses of additional mutants demonstrated that the last 162 amino acids of the carboxyl terminus were sufficient to restore transformation. These data demonstrate that an SH2 domain with v-Abl substrate specificity is required for NIH 3T3 transformation in the absence of the carboxyl terminus and suggest that cooperativity between the extreme carboxyl terminus and the SH2 domain facilitates the transmission of transforming signals via the MAP kinase pathway. PMID:10775585

Effects of SNPs from the differentially expressed swine odorant binding protein (OBP) gene on average daily gain

USDA-ARS?s Scientific Manuscript database

The objective of this study was to analyze genetic mutations in the swine odorant binding protein (OBP), map them on the swine genome, and to investigate the association between genetic variants and growth traits. The tissue sample of intramuscular fat from 10 individuals for each growth stage (0, 9...

Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors*

PubMed Central

Deshmukh, Atul S.; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T.; Cox, Jürgen; Mann, Matthias

2015-01-01

Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. PMID:25616865

TGFβ1-mediated PI3K/Akt and p38 MAP kinase dependent alternative splicing of fibronectin extra domain A in human podocyte culture.

PubMed

Madne, Tarunkumar Hemraj; Dockrell, Mark Edward Carl

2018-04-30

Alternative splicing is an important gene regulation process to distribute proteins in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin (Fn) protein, present in the extra cellular matrix (ECM) and a recognised marker of various pathologies. TGFβ1 has been shown to induce alternative splicing of EDA+Fn in many cell types. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. In our previous study we have demonstrated expression and alternative splicing of EDA+Fn in basal condition in human podocytes culture. TGFβ1 further induced the basal expression and alternative splicing of EDA+Fn through Alk5 receptor and SR proteins. In this study, we have investigated TGFβ1 mediated signalling involved in alternative splicing of EDA+Fn in human podocytes. We have performed western blotting to characterise the expression of the EDA+Fn protein and other signalling proteins and RT-PCR to look for signalling pathways involved in regulation of alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We have used TGFβ1 as a stimulator and SB431542, SB202190 and LY294002 for inhibitory studies. In this work, we have demonstrated in human podocytes culture TGFβ1 2.5ng/ml induced phosphorylation of Smad1/5/8, Smad2 and Smad3 via the ALK5 receptor. TGFβ1 significantly induced the PI3K/Akt pathway and the PI3K/Akt pathway inhibitor LY294002 significantly downregulated basal as well as TGFβ1 induced alternative splicing of EDA+Fn in human podocytes. In addition to this, TGFβ1 significantly induced the p38 MAP kinase signalling pathway and p38 MAP kinase signalling pathway inhibitor SB202190 downregulated the TGFβ1-mediated alternative splicing of EDA+Fn in human podocytes. The results with PI3K and p38 MAP kinase signalling pathway suggest that inhibiting PI3K signalling pathway downregulated the basal alternative splicing of EDA+Fn in human podocytes and its the inhibition of p38 Map Kinase signalling pathway which had specifically downregulated the TGFβ1 mediated alternative splicing of EDA+Fn in human podocytes culture. Activation of TGFβ1-mediated Smad1/5/8 via Alk5 receptor suggests that TGFβ1 signalling pathway involved Alk5/Alk1 receptor axis signalling in human podocytes.

Dissecting Pistil Responses to Incompatible and Compatible Pollen in Self-Incompatibility Brassica oleracea Using Comparative Proteomics.

PubMed

Zeng, Jing; Gao, Qiguo; Shi, Songmei; Lian, Xiaoping; Converse, Richard; Zhang, Hecui; Yang, Xiaohong; Ren, Xuesong; Chen, Song; Zhu, Liquan

2017-04-01

Angiosperms have developed self-incompatibility (SI) systems to reject self-pollen, thereby promoting outcrossing. The Brassicaceae belongs to typical sporophytic system, having a single S-locus controlled SI response, and was chosen as a model system to study SI-related intercellular signal transduction. In this regard, the downstream factor of EXO70A1 was unknown. Here, protein two-dimensional electrophoresis (2-DE) method and coupled with matrix-assisted laser desorption ionization/time of flight of flight mass spectrometry (MALDI-TOF -MS) and peptide mass fingerprinting (PMF) was used to further explore the mechanism of SI responses in Brassica oleracea L. var. capitata L. at protein level. To further confirm the time point of protein profile change, total proteins were collected from B. oleracea pistils at 0 min, 1 h, and 2 h after self-pollination. In total 902, 1088 and 1023 protein spots were separated in 0 min, 1 h and 2 h 2-DE maps, respectively. Our analyses of self-pollination profiles indicated that proteins mainly changed at 1 h post-pollination in B. oleracea. Moreover, 1077 protein spots were separated in cross-pollinated 1 h (CP) pistil 2-DE map. MALDI-TOF-MS and PMF successfully identified 34 differentially-expressed proteins (DEPs) in SP and CP 1 h 2-DE maps. Gene ontology and KEGG analysis revealed an array of proteins grouped in the following categories: stress and defense response (35%), protein metabolism (18%), carbohydrate and energy metabolism (12%), regulation of translation (9%), pollen tube development (12%), transport (9%) and cytoskeletal (6%). Sets of DEPs identified specifically in SP or only up-regulated expressed in CP pistils were chosen for funther investigating in floral organs and during the process of self- and cross-pollination. The function of these DEPs in terms of their potential involvement in SI in B. oleracea is discussed.

Integrative transcriptome, proteome, phosphoproteome and genetic mapping reveals new aspects in a fiberless mutant of cotton

PubMed Central

Ma, Qi-Feng; Wu, Chun-Hui; Wu, Man; Pei, Wen-Feng; Li, Xing-Li; Wang, Wen-Kui; Zhang, Jinfa; Yu, Ji-Wen; Yu, Shu-Xun

2016-01-01

To investigate the molecular mechanisms of fiber initiation in cotton (Gossypium spp.), an integrated approach combining transcriptome, iTRAQ-based proteome and genetic mapping was taken to compare the ovules of the Xuzhou 142 wild type (WT) with its fuzzless-lintless (fl) mutant at −3 and 0 day post-anthesis. A total of 1,953 mRNAs, 187 proteins, and 131 phosphoproteins were differentially expressed (DE) between WT and fl, and the levels of transcripts and their encoded proteins and phosphoproteins were highly congruent. A functional analysis suggested that the abundance of proteins were mainly involved in amino sugar, nucleotide sugar and fatty acid metabolism, one carbon pool for folate metabolism and flavonoid biosynthesis. qRT-PCR, Western blotting, and enzymatic assays were performed to confirm the regulation of these transcripts and proteins. A molecular mapping located the lintless gene li3 in the fl mutant on chromosome 26 for the first time. A further in-silico physical mapping of DE genes with sequence variations between fl and WT identified one and four candidate genes in the li3 and n2 regions, respectively. Taken together, the transcript abundance, phosphorylation status of proteins at the fiber initiation stage and candidate genes have provided insights into regulatory processes underlying cotton fiber initiation. PMID:27075604

Mitogen-Activated Protein Kinase 8 (MAP3K8) Mediates the Signaling Pathway of Estradiol Stimulating Progesterone Production Through G Protein-Coupled Receptor 30 (GPR30) in Mouse Corpus Luteum.

PubMed

Liu, Ying; Li, Yueqin; Zhang, Di; Liu, Jiali; Gou, Kemian; Cui, Sheng

2015-05-01

The corpus luteum (CL) is a transient endocrine gland developed from the ovulated follicles, and the most important function is to synthesize and secrete progesterone (P(4)), a key hormone to maintain normal pregnancy and estrous cycle in most mammals. It is known that estrogen has a vital role in stimulating P(4) synthesis in CL, but it still remains unclear about the mechanism of estradiol (E(2)) regulating P(4) production in CL. Our results here first show that all of the CL cells express MAPK 8 (MAP3K8), and the MAP3K8 level is much higher at the midstage than at the early and late stages during CL development. The further functional studies show that the forced inhibition of endogenous MAP3K8 by using MAP3K8 small interfering RNA and MAP3K8 signaling inhibitor (MAP3K8i) in the luteal cells significantly block the P(4) synthesis and neutralize the enhancing effect of E(2) on P(4) production in the CL. In addition, our results here demonstrate that the stimulating effect of E(2) on P(4) synthesis relies on the estrogen no-classical protein-coupled receptor 30, and MAP3K8 is involved in mediating the protein-coupled receptor 30signaling of E(2) affecting P(4) synthesis via stimulating ERK phosphorylation. These novel findings are critical for our understanding the ovary physiology and pathological mechanism.

Calcium: A novel and efficient inducer of differentiation of adipose-derived stem cells into neuron-like cells.

PubMed

Goudarzi, Farjam; Tayebinia, Heidar; Karimi, Jamshid; Habibitabar, Elahe; Khodadadi, Iraj

2018-06-05

This study comparatively investigated the effectiveness of calcium and other well-known inducers such as isobutylmethylxanthine (IBMX) and insulin in differentiating human adipose-derived stem cells (ADSCs) into neuronal-like cells. ADSCs were immunophenotyped and differentiated into neuron-like cells with different combinations of calcium, IBMX, and insulin. Calcium mobilization across the membrane was determined. Differentiated cells were characterized by cell cycle profiling, staining of Nissl bodies, detecting the gene expression level of markers such as neuronal nuclear antigen (NeuN), microtubule associated protein 2 (MAP2), neuron-specific enolase (NSE), doublecortin, synapsin I, glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) by quantitative real-time polymerase chain reaction (quantitative real-time polymerase chain reaction (qRT-PCR) and protein level by the immunofluorescence technique. Treatment with Ca + IBMX + Ins induced neuronal appearance and projection of neurite-like processes in the cells, accompanied with inhibition of proliferation and halt in the cell cycle. A significantly higher expression of MBP, GFAP, NeuN, NSE, synapsin 1, doublecortin, and MAP2 was detected in differentiated cells, confirming the advantages of Ca + IBMX + Ins to the other combinations of inducers. Here, we showed an efficient protocol for neuronal differentiation of ADSCs, and calcium fostered differentiation by augmenting the number of neuron-like cells and instantaneous increase in the expression of neuronal markers. © 2018 Wiley Periodicals, Inc.

A Pilot Proteogenomic Study with Data Integration Identifies MCT1 and GLUT1 as Prognostic Markers in Lung Adenocarcinoma.

PubMed

Stewart, Paul A; Parapatics, Katja; Welsh, Eric A; Müller, André C; Cao, Haoyun; Fang, Bin; Koomen, John M; Eschrich, Steven A; Bennett, Keiryn L; Haura, Eric B

2015-01-01

We performed a pilot proteogenomic study to compare lung adenocarcinoma to lung squamous cell carcinoma using quantitative proteomics (6-plex TMT) combined with a customized Affymetrix GeneChip. Using MaxQuant software, we identified 51,001 unique peptides that mapped to 7,241 unique proteins and from these identified 6,373 genes with matching protein expression for further analysis. We found a minor correlation between gene expression and protein expression; both datasets were able to independently recapitulate known differences between the adenocarcinoma and squamous cell carcinoma subtypes. We found 565 proteins and 629 genes to be differentially expressed between adenocarcinoma and squamous cell carcinoma, with 113 of these consistently differentially expressed at both the gene and protein levels. We then compared our results to published adenocarcinoma versus squamous cell carcinoma proteomic data that we also processed with MaxQuant. We selected two proteins consistently overexpressed in squamous cell carcinoma in all studies, MCT1 (SLC16A1) and GLUT1 (SLC2A1), for further investigation. We found differential expression of these same proteins at the gene level in our study as well as in other public gene expression datasets. These findings combined with survival analysis of public datasets suggest that MCT1 and GLUT1 may be potential prognostic markers in adenocarcinoma and druggable targets in squamous cell carcinoma. Data are available via ProteomeXchange with identifier PXD002622.

The C-Type Lectin OCILRP2 Costimulates EL4 T Cell Activation via the DAP12-Raf-MAP Kinase Pathway

PubMed Central

Lou, Qiang; Zhang, Wei; Liu, Guangchao; Ma, Yuanfang

2014-01-01

OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation. PMID:25411776

The C-type lectin OCILRP2 costimulates EL4 T cell activation via the DAP12-Raf-MAP kinase pathway.

PubMed

Lou, Qiang; Zhang, Wei; Liu, Guangchao; Ma, Yuanfang

2014-01-01

OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation.

A Unique Procedure to Identify Cell Surface Markers Through a Spherical Self-Organizing Map Applied to DNA Microarray Analysis.

PubMed

Sugii, Yuh; Kasai, Tomonari; Ikeda, Masashi; Vaidyanath, Arun; Kumon, Kazuki; Mizutani, Akifumi; Seno, Akimasa; Tokutaka, Heizo; Kudoh, Takayuki; Seno, Masaharu

2016-01-01

To identify cell-specific markers, we designed a DNA microarray platform with oligonucleotide probes for human membrane-anchored proteins. Human glioma cell lines were analyzed using microarray and compared with normal and fetal brain tissues. For the microarray analysis, we employed a spherical self-organizing map, which is a clustering method suitable for the conversion of multidimensional data into two-dimensional data and displays the relationship on a spherical surface. Based on the gene expression profile, the cell surface characteristics were successfully mirrored onto the spherical surface, thereby distinguishing normal brain tissue from the disease model based on the strength of gene expression. The clustered glioma-specific genes were further analyzed by polymerase chain reaction procedure and immunocytochemical staining of glioma cells. Our platform and the following procedure were successfully demonstrated to categorize the genes coding for cell surface proteins that are specific to glioma cells. Our assessment demonstrates that a spherical self-organizing map is a valuable tool for distinguishing cell surface markers and can be employed in marker discovery studies for the treatment of cancer.

Proteome reference map and regulation network of neonatal rat cardiomyocyte

PubMed Central

Li, Zi-jian; Liu, Ning; Han, Qi-de; Zhang, You-yi

2011-01-01

Aim: To study and establish a proteome reference map and regulation network of neonatal rat cardiomyocyte. Methods: Cultured cardiomyocytes of neonatal rats were used. All proteins expressed in the cardiomyocytes were separated and identified by two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Biological networks and pathways of the neonatal rat cardiomyocytes were analyzed using the Ingenuity Pathway Analysis (IPA) program (www.ingenuity.com). A 2-DE database was made accessible on-line by Make2ddb package on a web server. Results: More than 1000 proteins were separated on 2D gels, and 148 proteins were identified. The identified proteins were used for the construction of an extensible markup language-based database. Biological networks and pathways were constructed to analyze the functions associate with cardiomyocyte proteins in the database. The 2-DE database of rat cardiomyocyte proteins can be accessed at http://2d.bjmu.edu.cn. Conclusion: A proteome reference map and regulation network of the neonatal rat cardiomyocytes have been established, which may serve as an international platform for storage, analysis and visualization of cardiomyocyte proteomic data. PMID:21841810

Six stroma-based RNA markers diagnostic for prostate cancer in European-Americans validated at the RNA and protein levels in patients in China

PubMed Central

Zhu, Jianguo; Pan, Cong; Jiang, Jun; Deng, Mingsen; Gao, Hengjun; Men, Bozhao; McClelland, Michael; Mercola, Dan; Zhong, Wei-De; Jia, Zhenyu

2015-01-01

We previously analyzed human prostate tissue containing stroma near to tumor and from cancer-negative tissues of volunteers. Over 100 candidate gene expression differences were identified and used to develop a classifier that could detect nearby tumor with an accuracy of 97% (sensitivity = 98% and specificity = 88%) based on 364 independent test cases from primarily European American cases. These stroma-based gene signatures have the potential to identify cancer patients among those with negative biopsies. In this study, we used prostate tissues from Chinese cases to validate six of these markers (CAV1, COL4A2, HSPB1, ITGB3, MAP1A and MCAM). In validation by real-time PCR, four genes (COL4A2, HSPB1, ITGB3, and MAP1A) demonstrated significantly lower expression in tumor-adjacent stroma compared to normal stroma (p value ≤ 0.05). Next, we tested whether these expression differences could be extended to the protein level. In IHC assays, all six selected proteins showed lower expression in tumor-adjacent stroma compared to the normal stroma, of which COL4A2, HSPB1 and ITGB3 showed significant differences (p value ≤ 0.05). These results suggest that biomarkers for diagnosing prostate cancer based on tumor microenvironment may be applicable across multiple racial groups. PMID:26158290

Motor Skills Training Improves Sensorimotor Dysfunction and Increases Microtubule-Associated Protein 2 mRNA Expression in Rats with Intracerebral Hemorrhage.

PubMed

Tamakoshi, Keigo; Kawanaka, Kentaro; Onishi, Hideaki; Takamatsu, Yasuyuki; Ishida, Kazuto

2016-08-01

In this study, we examined the effects of motor skills training on the sensorimotor function and the expression of genes associated with synaptic plasticity after intracerebral hemorrhage (ICH) in rats. Male Wistar rats were subjected to ICH or sham operation. ICH was caused by the injection of collagenase into the left striatum. Rats were randomly assigned to no training, acrobatic training, and sham groups. The acrobatic group performed 5 types of acrobatic tasks from 4 to 28 days after surgery. The forelimb sensorimotor function was evaluated over time using forepaw grasping, forelimb placing, and postural instability tests. At 14 and 29 days after the lesion, we analyzed the mRNA expression levels of microtubule-associated protein 2 (MAP2), brain-derived neurotrophic factor, and growth-associated protein 43 in the bilateral sensorimotor cortex (forelimb area) by real-time reverse transcription-polymerase chain reaction. Motor skills training in ICH rats improved the sensorimotor dysfunction significantly from the early phase. The mRNA expression level of MAP2 was upregulated in the ipsilesional sensorimotor cortex by motor skills training at 29 days after the lesion. Our results suggest that sensorimotor functional recovery following motor skills training after ICH is promoted by dendritic growth in the ipsilesional sensorimotor cortex. Copyright © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved.

[Multiplexing mapping of human cDNAs]. Final report, September 1, 1991--February 28, 1994

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

Not Available

Using PCR with automated product analysis, 329 human brain cDNA sequences have been assigned to individual human chromosomes. Primers were designed from single-pass cDNA sequences expressed sequence tags (ESTs). Primers were used in PCR reactions with DNA from somatic cell hybrid mapping panels as templates, often with multiplexing. Many ESTs mapped match sequence database records. To evaluate of these matches, the position of the primers relative to the matching region (In), the BLAST scores and the Poisson probability values of the EST/sequence record match were determined. In cases where the gene product was stringently identified by the sequence match hadmore » already been mapped, the gene locus determined by EST was consistent with the previous position which strongly supports the validity of assigning unknown genes to human chromosomes based on the EST sequence matches. In the present cases mapping the ESTs to a chromosome can also be considered to have mapped the known gene product: rolipram-sensitive cAMP phosphodiesterase, chromosome 1; protein phosphatase 2A{beta}, chromosome 4; alpha-catenin, chromosome 5; the ELE1 oncogene, chromosome 10q11.2 or q2.1-q23; MXII protein, chromosome l0q24-qter; ribosomal protein L18a homologue, chromosome 14; ribosomal protein L3, chromosome 17; and moesin, Xp11-cen. There were also ESTs mapped that were closely related to non-human sequence records. These matches therefore can be considered to identify human counterparts of known gene products, or members of known gene families. Examples of these include membrane proteins, translation-associated proteins, structural proteins, and enzymes. These data then demonstrate that single pass sequence information is sufficient to design PCR primers useful for assigning cDNA sequences to human chromosomes. When the EST sequence matches previous sequence database records, the chromosome assignments of the EST can be used to make preliminary assignments of the human gene to a chromosome.« less

Mapping the hemagglutination domain of rotaviruses.

PubMed Central

Fuentes-Pananá, E M; López, S; Gorziglia, M; Arias, C F

1995-01-01

Most strains of animal rotaviruses are able to agglutinate erythrocytes, and the surface protein VP4 is the virus hemagglutinin. To map the hemagglutination domain on VP4 while preserving the conformation of the protein, we constructed full-length chimeras between the VP4 genes of hemagglutinating (YM) and nonhemagglutinating (KU) rotavirus strains. The parental and chimeric genes were expressed in insect cells, and the recombinant VP4 proteins were evaluated for their capacity to agglutinate human type O erythrocytes. Three chimeric genes, encoding amino acids 1 to 208 (QKU), 93 to 208 (QC), and 93 to 776 (QYM) of the YM VP4 protein in a KU VP4 background, were constructed. YM VP4 and chimeras QKU and QC were shown to specifically hemagglutinate, indicating that the region between amino acids 93 and 208 of YM VP4 is sufficient to determine the hemagglutination activity of the protein. PMID:7884915

Arginine-vasopressin directly promotes a thermogenic and pro-inflammatory adipokine expression profile in brown adipocytes.

PubMed

Küchler, Sebastian; Perwitz, Nina; Schick, Rafael Reinhold; Klein, Johannes; Westphal, Sören

2010-09-24

Arginine-vasopressin (AVP) - via activation of the hypothalamic-pituitary-adrenal (HPA) axis - may play a role in the regulation of energy homeostasis and related cardiovascular complications. Brown adipose tissue (BAT) - via dissipation of energy in the form of heat - contributes to whole body energy balance. BAT expresses vasopressin receptors. We investigated direct effects of AVP on brown adipose endocrine and metabolic functions. UCP-1 protein expression in differentiated brown adipocytes was induced after acute exposure of adipocytes to AVP. This effect was time-dependent with a maximum increase after 8h. AVP also induced a time- and dose-dependent increase in p38 MAP kinase phosphorylation. Pharmacological inhibition of p38 MAP kinase with SB 202190 abolished the induction of UCP-1 protein expression. Furthermore, while acute AVP treatment enhanced mRNA expression of MCP-1 and IL-6, adiponectin mRNA expression was reduced. Yet, on the level of intracellular glucose uptake, there was no AVP-induced change of adipose insulin-induced glucose uptake. Finally, there was no difference in lipid accumulation between control and AVP-treated cells. Taken together, our data demonstrate direct effects of AVP on thermogenic, inflammatory, and glucoregulatory gene expression in brown adipocytes, thus expanding the hitherto known spectrum of this neuropeptides's biological effects and suggesting a direct adipotropic role as a stress-promoting factor. Copyright 2010 Elsevier B.V. All rights reserved.

The expression ratio of Map7/B2M is prognostic for survival in patients with stage II colon cancer.

PubMed

Blum, Craig; Graham, Amanda; Yousefzadeh, Matt; Shrout, Jessica; Benjamin, Katie; Krishna, Murli; Hoda, Raza; Hoda, Rana; Cole, David J; Garrett-Mayer, Elizabeth; Reed, Carolyn; Wallace, Michael; Mitas, Michael

2008-09-01

Colorectal cancer (CRC) is the second most frequent cause of cancer-related death in the United States. To determine whether certain molecular markers might be prognostic for survival, we measured by quantitative real-time RT-PCR the expression levels of 15 previously studied genes that are known to be up-regulated or down-regulated in the progression of epithelial cancers. The tumor samples were extracted from formalin-fixed paraffin-embedded primary tissues derived from patients with Stage II CRC who developed disease recurrence within two years (n=10), or were disease-free for at least 4 years (n=12). We were able to determine, by AUC curve analysis, that the ratio of microtubule associated protein 7 (Map7)/B2M was predictive of outcome in our sample set. Further, using Kaplan-Meier survival analysis, we observed significantly different curves as a function of marker positivity for the Map7/B2M (p=0.0001; HR=11) expression ratio. This suggests that the expression ratio of Map7/B2M may serve as a valuable prognostic marker in patients with Stage II colon cancer, and potentially guide therapeutic decision making.

The expression ratio of Map7/B2M is prognostic for survival in patients with stage II colon cancer

PubMed Central

BLUM, CRAIG; GRAHAM, AMANDA; YOUSEFZADEH, MATT; SHROUT, JESSICA; BENJAMIN, KATIE; KRISHNA, MURLI; HODA, RAZA; HODA, RANA; COLE, DAVID J.; GARRETT-MAYER, ELIZABETH; REED, CAROLYN; WALLACE, MICHAEL; MITAS, MICHAEL

2012-01-01

Colorectal cancer (CRC) is the second most frequent cause of cancer-related death in the United States. To determine whether certain molecular markers might be prognostic for survival, we measured by quantitative real-time RT-PCR the expression levels of 15 previously studied genes that are known to be up-regulated or down-regulated in the progression of epithelial cancers. The tumor samples were extracted from formalin-fixed paraffin-embedded primary tissues derived from patients with Stage II CRC who developed disease recurrence within two years (n=10), or were disease-free for at least 4 years (n=12). We were able to determine, by AUC curve analysis, that the ratio of microtubule associated protein 7 (Map7)/B2M was predictive of outcome in our sample set. Further, using Kaplan-Meier survival analysis, we observed significantly different curves as a function of marker positivity for the Map7/B2M (p=0.0001; HR=11) expression ratio. This suggests that the expression ratio of Map7/B2M may serve as a valuable prognostic marker in patients with Stage II colon cancer, and potentially guide therapeutic decision making. PMID:18695889

Physical structure and chromosomal localization of a gene encoding human p58[sup clk-1], a cell division control related protein kinase

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

Eipers, P.G.

1992-01-01

The gene for the human p58[sup clk[minus]1] protein kinase, a cell division control-related gene, has been mapped by somatic cell hybrid analyses, in situ localization with the chromosomal gene, and nested polymerase chain reaction amplification of microdissected chromosomes. These studies indicate that the expressed p58[sup clk[minus]1] chromosomal gene maps to 1p36, while a highly related p58[sup clk[minus]1] sequence of unknown nature maps to chromosome 15. Assignment of a p34[sup cdc2]-related gene to 1p36 region, including neuroblastoma, ductal carcinoma of the breast, malignant melanoma, Merkel cell carcinoma and endocrine neoplasia among others. Aberrant expression of this protein kinase negatively regulates normalmore » cellular growth. The p58[sup clk[minus]1] protein contains a central domain of 299 amino acids that is 46% identical to human p34[sup cdc2], the master mitotic protein kinase. This dissertation details the complete structure of the p58[sup clk[minus]1] chromosomal gene, including its putative promoter region, transcriptional start sites, exonic sequences, and intron/exon boundary sequences. The gene is 10 kb in size and contains 12 exons and 11 introns. Interestingly, the rather large 2.0 kb 3[prime] untranslated region is interrupted by an intron that separates a region containing numerous AUUUA destabilization motifs from the coding region. Furthermore, the expression of this gene in normal human tissues, as well as several human tumor cell samples and lines, is examined. The origin of multiple human transcripts from the same chromosomal gene, and the possible differential stability of these various transcripts, is discussed with regard to the transcriptional and post-transcriptional regulation of this gene. This is the first report of the chromosomal gene structure of a member of the p34[sup cdc2] supergene family.« less

The Carnegie Protein Trap Library: A Versatile Tool for Drosophila Developmental Studies

PubMed Central

Buszczak, Michael; Paterno, Shelley; Lighthouse, Daniel; Bachman, Julia; Planck, Jamie; Owen, Stephenie; Skora, Andrew D.; Nystul, Todd G.; Ohlstein, Benjamin; Allen, Anna; Wilhelm, James E.; Murphy, Terence D.; Levis, Robert W.; Matunis, Erika; Srivali, Nahathai; Hoskins, Roger A.; Spradling, Allan C.

2007-01-01

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600–900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions. PMID:17194782

Regulation of MMP-3 expression and secretion by the chemokine eotaxin-1 in human chondrocytes.

PubMed

Chao, Pin-Zhir; Hsieh, Ming-Shium; Cheng, Chao-Wen; Lin, Yung-Feng; Chen, Chien-Ho

2011-11-25

Osteoarthritis (OA) is characterized by the degradation of articular cartilage, marked by the breakdown of matrix proteins. Studies demonstrated the involvement of chemokines in this process, and some may potentially serve as diagnostic markers and therapeutic targets; however, the underlying signal transductions are not well understood. We investigated the effects of the CC chemokine eotaxin-1 (CCL11) on the matrix metalloproteinase (MMP) expression and secretion in the human chondrocyte cell line SW1353 and primary chondrocytes. Eotaxin-1 significantly induced MMP-3 mRNA expression in a dose-dependent manner. Inhibitors of extracellular signal-regulated kinase (ERK) and p38 kinase were able to repress eotaxin-1-induced MMP-3 expression. On the contrary, Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPs), a competitive cAMP antagonist for cAMP receptors, and H-89, a protein kinase A (PKA) inhibitor, markedly enhanced eotaxin-1-induced MMP-3 expression. These results suggest that MMP-3 expression is specifically mediated by the G protein-coupled eotaxin-1 receptor activities. Interestingly, little amount of MMP-3 protein was detected in the cell lysates of eotaxin-1-treated SW1353 cells, and most of MMP-3 protein was in the culture media. Furthermore we found that the eotaxin-1-dependent MMP-3 protein secretion was regulated by phospholipase C (PLC)-protein kinase C (PKC) cascade and c-Jun N-terminal kinase (JNK)/mitogen-activated protein (MAP) kinase pathways. These data indicate a specific regulation of MMP-3 secretion also by eotaxin-1 receptor activities. Eotaxin-1 not only induces MMP-3 gene expression but also promotes MMP-3 protein secretion through G protein-coupled eotaxin-1 receptor activities. Chemokines, such as eotaxin-1, could be a potential candidate in the diagnosis and treatment of arthritis.

Prediction of Heterodimeric Protein Complexes from Weighted Protein-Protein Interaction Networks Using Novel Features and Kernel Functions

PubMed Central

Ruan, Peiying; Hayashida, Morihiro; Maruyama, Osamu; Akutsu, Tatsuya

2013-01-01

Since many proteins express their functional activity by interacting with other proteins and forming protein complexes, it is very useful to identify sets of proteins that form complexes. For that purpose, many prediction methods for protein complexes from protein-protein interactions have been developed such as MCL, MCODE, RNSC, PCP, RRW, and NWE. These methods have dealt with only complexes with size of more than three because the methods often are based on some density of subgraphs. However, heterodimeric protein complexes that consist of two distinct proteins occupy a large part according to several comprehensive databases of known complexes. In this paper, we propose several feature space mappings from protein-protein interaction data, in which each interaction is weighted based on reliability. Furthermore, we make use of prior knowledge on protein domains to develop feature space mappings, domain composition kernel and its combination kernel with our proposed features. We perform ten-fold cross-validation computational experiments. These results suggest that our proposed kernel considerably outperforms the naive Bayes-based method, which is the best existing method for predicting heterodimeric protein complexes. PMID:23776458

R Script Approach to Infer Toxoplasma Infection Mechanisms From Microarrays and Domain-Domain Protein Interactions

PubMed Central

Arenas, Ailan F; Salcedo, Gladys E; Gomez-Marin, Jorge E

2017-01-01

Pathogen-host protein-protein interaction systems examine the interactions between the protein repertoires of 2 distinct organisms. Some of these pathogen proteins interact with the host protein system and may manipulate it for their own advantages. In this work, we designed an R script by concatenating 2 functions called rowDM and rowCVmed to infer pathogen-host interaction using previously reported microarray data, including host gene enrichment analysis and the crossing of interspecific domain-domain interactions. We applied this script to the Toxoplasma-host system to describe pathogen survival mechanisms from human, mouse, and Toxoplasma Gene Expression Omnibus series. Our outcomes exhibited similar results with previously reported microarray analyses, but we found other important proteins that could contribute to toxoplasma pathogenesis. We observed that Toxoplasma ROP38 is the most differentially expressed protein among toxoplasma strains. Enrichment analysis and KEGG mapping indicated that the human retinal genes most affected by Toxoplasma infections are those related to antiapoptotic mechanisms. We suggest that proteins PIK3R1, PRKCA, PRKCG, PRKCB, HRAS, and c-JUN could be the possible substrates for differentially expressed Toxoplasma kinase ROP38. Likewise, we propose that Toxoplasma causes overexpression of apoptotic suppression human genes. PMID:29317802

ERK1/2 and p38 MAP kinases control prion protein fragment 90-231-induced astrocyte proliferation and microglia activation.

PubMed

Thellung, Stefano; Villa, Valentina; Corsaro, Alessandro; Pellistri, Francesca; Venezia, Valentina; Russo, Claudio; Aceto, Antonio; Robello, Mauro; Florio, Tullio

2007-11-01

Astrogliosis and microglial activation are a common feature during prion diseases, causing the release of chemoattractant and proinflammatory factors as well as reactive free radicals, involved in neuronal degeneration. The recombinant protease-resistant domain of the prion protein (PrP90-231) displays in vitro neurotoxic properties when refolded in a beta-sheet-rich conformer. Here, we report that PrP90-231 induces the secretion of several cytokines, chemokines, and nitric oxide (NO) release, in both type I astrocytes and microglial cells. PrP90-231 elicited in both cell types the activation of ERK1/2 MAP kinase that displays, in astrocytes, a rapid kinetics and a proliferative response. Conversely, in microglia, PrP90-231-dependent MAP kinase activation was delayed and long lasting, inducing functional activation and growth arrest. In microglial cells, NO release, dependent on the expression of the inducible NO synthase (iNOS), and the secretion of the chemokine CCL5 were Ca(2+) dependent and under the control of the MAP kinases ERK1/2 and p38: ERK1/2 inhibition, using PD98059, reduced iNOS expression, while p38 blockade by PD169316 inhibited CCL5 release. In summary, we demonstrate that glial cells are activated by extracellular misfolded PrP90-231 resulting in a proliferative/secretive response of astrocytes and functional activation of microglia, both dependent on MAP kinase activation. In particular, in microglia, PrP90-231 activated a complex signalling cascade involved in the regulation of NO and chemokine release. These data argue in favor of a causal role for misfolded prion protein in sustaining glial activation and, possibly, glia-mediated neuronal death.

2',5'-Dihydroxychalcone down-regulates endothelial connexin43 gap junctions and affects MAP kinase activation.

PubMed

Lee, Yi-Nan; Yeh, Hung-I; Tian, Tin-Yi; Lu, Wen-Wei; Ko, Yu-Shien; Tsai, Cheng-Ho

2002-09-30

We examined the effect of 2',5'-dihydroxychalcone on connexin43 (Cx43) expression and gap-junctional communication in human umbilical vein endothelial cells (HUVEC). The result showed that expression of Cx43 is rapidly reduced by 2',5'-dihydroxychalcone in a dose-dependent manner, Concomitantly, the communication function, determined by fluorescence recovery after photobleaching (FRAP), is decreased. We further investigated whether the mitogen-activated protein (MAP) kinase and the degradation pathway of gap junctions are involved in these processes. Although the change of Cx43 is not affected by the level of fetal calf serum (FCS) used in the medium, activation of MAP kinase varies, depending on the FCS level. At a low level (0.5%), the chalcone inhibits the activation, like PD98059, a specific inhibitor of MAP kinase kinase. However, at a high level (20%), MAP kinase is activated. On the other hand, the chalcone's down-regulating effect on Cx43, while is totally blocked by protease inhibitors leupeptin and N-acetyl-leucyl-norleucinal (ALLN), persists in the presence of PD98059, We concluded that 2',5'-dihydroxychalcone down-regulates Cx43 expression and gap-junctional communication in the HUVEC via enhancement of the proteolysis pathway, and this compound possesses dual effects on MAP kinase activation.

Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal, a lipid peroxidation end product

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

Zheng, Ruijin; Heck, Diane E.; Mishin, Vladimir

2014-03-01

4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86–98 fold within 6 h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependentmore » increases in mRNA and protein expression which were maximum after 6 h with 30 μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2 −/− mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress. - Highlights: • Lipid peroxidation generates 4-hydroxynonenal, a reactive aldehyde. • 4-HNE induces antioxidant proteins in mouse keratinocytes. • Induction of antioxidant proteins is regulated via MAP kinases, Nrf2 and caveolae. • 4-HNE is an effective signaling molecule in keratinocytes.« less

Purα Repaired Expanded Hexanucleotide GGGGCC Repeat Noncoding RNA-Caused Neuronal Toxicity in Neuro-2a Cells.

PubMed

Shen, Jianying; Zhang, Yu; Zhao, Shi; Mao, Hong; Wang, Zhongjing; Li, Honglian; Xu, Zihui

2018-05-01

Expanded hexanucleotide GGGGCC repeat in a noncoding region of C9ORF72 is the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). However, its molecular pathogenesis remains unclear. In our previous study, the expanded GGGGCC repeats have been shown to be sufficient to cause neurodegeneration. In order to investigate the further role of expanded GGGGCC repeats in the neuron, the normal r(GGGGCC) 3 and mutant-type expanded r(GGGGCC) 30 expression vectors were transfected into Neuro-2a cells. Cell proliferation, dendrite development, and the proteins' levels of microtubule-associated protein-2 (MAP2) and cyclin-dependent kinase-5 (CDK5) were used to evaluate the cell toxicity of GGGGCC repeats on Neuro-2a cells. The results were shown that expression of expanded GGGGCC repeats caused neuronal cell toxicity in Neuro-2a cells, enhanced the expression of pMAP2 and pCDK5. Moreover, overexpression of Purα repaired expanded GGGGCC repeat-inducing neuronal toxicity in Neuro-2a cells and reduced the expression of pMAP2 and pCDK5. In all, our findings suggested that the expanded GGGGCC repeats might cause neurodegeneration through destroyed neuron cells. And the GGGGCC repeat-induced neuronal cell toxicity was inhibited by upregulation of Purα. We inferred that Purα inhibits expanded GGGGCC repeat-inducing neurodegeneration, which might reveal a novel mechanism of neurodegenerative diseases ALS and FTD.

Color threshold and ratio of S100 beta, MAP5, NF68/200, GABA & GAD. I. Distribution in inner ear afferents

NASA Technical Reports Server (NTRS)

Fermin, C. D.; Martin, D. S.; Hara, H.

1997-01-01

Afferents of chick embryos (Gallus domesticus) VIIIth nerve were examined at E3, E6, E9, E13, El7, and hatching (NH) for anti-S100 beta, anti-MAP5, anti-GABA, anti-GAD and anti-NF68/200 stain. Different ages were processed together to determine if the distribution of these antibodies changed during synaptogenesis and myelination. Color thresholding showed that saturation of pixels changed for S100 beta only 5%, for NF68/200 10%, and for MAP5, 10%, between E9-NH. Color ratio of NF68/200 over MAP5 was 1.00 at E13 and 0.25 at E16 and NH. S100 beta, GABA and GAD were co-expressed on nerve endings at the edge of the maculae and center of the cristae, whereas hair cells in the center of the maculae expressed either S100 beta or GABA, but not both. S100 beta/NF68/200 shared antigenic sites on the chalices, but NF68/200 expression was higher than S100 beta in the chalices at hatching. MAP5 was expressed in more neurons than NF68/200 at E11, whereas NF68/200 was more abundant than MAP5 at hatching. The results suggest that: 1) the immunoexpression of these neuronal proteins is modulated concomitantly with the establishment of afferent synapses and myelination; 2) S100 beta may serve a neurotrophic function in the chalices where it is co-expressed with the neurotransmitter GABA and its synthesizing enzyme GAD.

Regulation of the expression of plant resistance gene SNC1 by a protein with a conserved BAT2 domain.

PubMed

Li, Yingzhong; Tessaro, Mark J; Li, Xin; Zhang, Yuelin

2010-07-01

Plant Resistance (R) genes encode immune receptors that recognize pathogens and activate defense responses. Because of fitness costs associated with maintaining R protein-mediated resistance, expression levels of R genes have to be tightly regulated. However, mechanisms on how R-gene expression is regulated are poorly understood. Here we show that MODIFIER OF snc1, 1 (MOS1) regulates the expression of SUPPRESSOR OF npr1-1, CONSTITUTIVE1 (SNC1), which encodes a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat type of R protein in Arabidopsis (Arabidopsis thaliana). In the mos1 loss-of-function mutant plants, snc1 expression is repressed and constitutive resistance responses mediated by snc1 are lost. The repression of snc1 expression in mos1 is released by knocking out DECREASE IN DNA METHYLATION1. In mos1 mutants, DNA methylation in a region upstream of SNC1 is altered. Furthermore, expression of snc1 transgenes using the native promoter does not require MOS1, indicating that regulation of SNC1 expression by MOS1 is at the chromatin level. Map-based cloning of MOS1 revealed that it encodes a novel protein with a HLA-B ASSOCIATED TRANSCRIPT2 (BAT2) domain that is conserved in plants and animals. Our study on MOS1 suggests that BAT2 domain-containing proteins may function in regulation of gene expression at chromatin level.

The Application of an Emerging Technique for Protein–Protein Interaction Interface Mapping: The Combination of Photo-Initiated Cross-Linking Protein Nanoprobes with Mass Spectrometry

PubMed Central

Ptáčková, Renata; Ječmen, Tomáš; Novák, Petr; Hudeček, Jiří; Stiborová, Marie; Šulc, Miroslav

2014-01-01

Protein–protein interaction was investigated using a protein nanoprobe capable of photo-initiated cross-linking in combination with high-resolution and tandem mass spectrometry. This emerging experimental approach introduces photo-analogs of amino acids within a protein sequence during its recombinant expression, preserves native protein structure and is suitable for mapping the contact between two proteins. The contact surface regions involved in the well-characterized interaction between two molecules of human 14-3-3ζ regulatory protein were used as a model. The employed photo-initiated cross-linking techniques extend the number of residues shown to be within interaction distance in the contact surface of the 14-3-3ζ dimer (Gln8–Met78). The results of this study are in agreement with our previously published data from molecular dynamic calculations based on high-resolution chemical cross-linking data and Hydrogen/Deuterium exchange mass spectrometry. The observed contact is also in accord with the 14-3-3ζ X-ray crystal structure (PDB 3dhr). The results of the present work are relevant to the structural biology of transient interaction in the 14-3-3ζ protein, and demonstrate the ability of the chosen methodology (the combination of photo-initiated cross-linking protein nanoprobes and mass spectrometry analysis) to map the protein-protein interface or regions with a flexible structure. PMID:24865487

Functional characterization of a multi-cancer risk locus on chr5p15.33 reveals regulation of TERT by ZNF148.

PubMed

Fang, Jun; Jia, Jinping; Makowski, Matthew; Xu, Mai; Wang, Zhaoming; Zhang, Tongwu; Hoskins, Jason W; Choi, Jiyeon; Han, Younghun; Zhang, Mingfeng; Thomas, Janelle; Kovacs, Michael; Collins, Irene; Dzyadyk, Marta; Thompson, Abbey; O'Neill, Maura; Das, Sudipto; Lan, Qi; Koster, Roelof; Stolzenberg-Solomon, Rachael S; Kraft, Peter; Wolpin, Brian M; Jansen, Pascal W T C; Olson, Sara; McGlynn, Katherine A; Kanetsky, Peter A; Chatterjee, Nilanjan; Barrett, Jennifer H; Dunning, Alison M; Taylor, John C; Newton-Bishop, Julia A; Bishop, D Timothy; Andresson, Thorkell; Petersen, Gloria M; Amos, Christopher I; Iles, Mark M; Nathanson, Katherine L; Landi, Maria Teresa; Vermeulen, Michiel; Brown, Kevin M; Amundadottir, Laufey T

2017-05-02

Genome wide association studies (GWAS) have mapped multiple independent cancer susceptibility loci to chr5p15.33. Here, we show that fine-mapping of pancreatic and testicular cancer GWAS within one of these loci (Region 2 in CLPTM1L) focuses the signal to nine highly correlated SNPs. Of these, rs36115365-C associated with increased pancreatic and testicular but decreased lung cancer and melanoma risk, and exhibited preferred protein-binding and enhanced regulatory activity. Transcriptional gene silencing of this regulatory element repressed TERT expression in an allele-specific manner. Proteomic analysis identifies allele-preferred binding of Zinc finger protein 148 (ZNF148) to rs36115365-C, further supported by binding of purified recombinant ZNF148. Knockdown of ZNF148 results in reduced TERT expression, telomerase activity and telomere length. Our results indicate that the association with chr5p15.33-Region 2 may be explained by rs36115365, a variant influencing TERT expression via ZNF148 in a manner consistent with elevated TERT in carriers of the C allele.

Determining Optimal Microwave Antigen Retrieval Conditions for Microtubule-Associated Protein 2 Immunohistochemistry in the Guinea Pig Brain

DTIC Science & Technology

2002-12-01

sections of formalin-fixed guinea pig brains using different MAP-2 monoclonal antibodies. Brain sections were boiled in sodium citrate, citric acid...citric acid solution at pH 6.0 is the optimal microwave-assisted AR method for immunolabeling MAP-2 in formalin-fixed, paraffin-processed guinea pig brain...studies on archival guinea pig brain paraffin blocks, ultimately relaxing the use of additional animals to evaluate changes in MAP-2 expression between chemical warfare nerve agent-treated and control samples.

PRAK, a novel protein kinase regulated by the p38 MAP kinase.

PubMed Central

New, L; Jiang, Y; Zhao, M; Liu, K; Zhu, W; Flood, L J; Kato, Y; Parry, G C; Han, J

1998-01-01

We have identified and cloned a novel serine/ threonine kinase, p38-regulated/activated protein kinase (PRAK). PRAK is a 471 amino acid protein with 20-30% sequence identity to the known MAP kinase-regulated protein kinases RSK1/2/3, MNK1/2 and MAPKAP-K2/3. PRAK was found to be expressed in all human tissues and cell lines examined. In HeLa cells, PRAK was activated in response to cellular stress and proinflammatory cytokines. PRAK activity was regulated by p38alpha and p38beta both in vitro and in vivo and Thr182 was shown to be the regulatory phosphorylation site. Activated PRAK in turn phosphorylated small heat shock protein 27 (HSP27) at the physiologically relevant sites. An in-gel kinase assay demonstrated that PRAK is a major stress-activated kinase that can phosphorylate small heat shock protein, suggesting a potential role for PRAK in mediating stress-induced HSP27 phosphorylation in vivo. PMID:9628874

N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

PubMed Central

Tirumuru, Nagaraja; Zhao, Boxuan Simen; Lu, Wuxun; Lu, Zhike; He, Chuan; Wu, Li

2016-01-01

The internal N6-methyladenosine (m6A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1–3) bind to m6A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1–3 proteins recognize m6A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4+ T-cells. We further mapped the YTHDF1–3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1–3 in cells had the opposite effects. Moreover, silencing the m6A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m6A erasers increased Gag expression. Our findings suggest an important role of m6A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis. DOI: http://dx.doi.org/10.7554/eLife.15528.001 PMID:27371828

Cloning of cDNA of major antigen of foot and mouth disease virus and expression in E. coli

NASA Astrophysics Data System (ADS)

Küpper, Hans; Keller, Walter; Kurz, Christina; Forss, Sonja; Schaller, Heinz

1981-02-01

Double-stranded DNA copies of the single-stranded genomic RNA of foot and mouth disease virus have been cloned into the Escherichia coli plasmid pBR322. A restriction map of the viral genome was established and aligned with the biochemical map of foot and mouth disease virus. The coding sequence for structural protein VP1, the major antigen of the virus, was identified and inserted into a plasmid vector where the expression of this sequence is under control of the phage λ PL promoter. In an appropriate host the synthesis of antigenic polypeptide can be demonstrated by radioimmunoassay.

Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication.

PubMed

Courtney, David G; Kennedy, Edward M; Dumm, Rebekah E; Bogerd, Hal P; Tsai, Kevin; Heaton, Nicholas S; Cullen, Bryan R

2017-09-13

Many viral RNAs are modified by methylation of the N 6 position of adenosine (m 6 A). m 6 A is thought to regulate RNA splicing, stability, translation, and secondary structure. Influenza A virus (IAV) expresses m 6 A-modified RNAs, but the effects of m 6 A on this segmented RNA virus remain unclear. We demonstrate that global inhibition of m 6 A addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular m 6 A "reader" protein YTHDF2 increases IAV gene expression and replication. To address whether m 6 A residues modulate IAV RNA function in cis, we mapped m 6 A residues on the IAV plus (mRNA) and minus (vRNA) strands and used synonymous mutations to ablate m 6 A on both strands of the hemagglutinin (HA) segment. These mutations inhibited HA mRNA and protein expression while leaving other IAV mRNAs and proteins unaffected, and they also resulted in reduced IAV pathogenicity in mice. Thus, m 6 A residues in IAV transcripts enhance viral gene expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Reverse chemical ecology: Olfactory proteins from the giant panda and their interactions with putative pheromones and bamboo volatiles

PubMed Central

Zhu, Jiao; Arena, Simona; Spinelli, Silvia; Zhang, Guiquan; Wei, Rongping; Cambillau, Christian; Scaloni, Andrea; Wang, Guirong; Pelosi, Paolo

2017-01-01

The giant panda Ailuropoda melanoleuca belongs to the family of Ursidae; however, it is not carnivorous, feeding almost exclusively on bamboo. Being equipped with a typical carnivorous digestive apparatus, the giant panda cannot get enough energy for an active life and spends most of its time digesting food or sleeping. Feeding and mating are both regulated by odors and pheromones; therefore, a better knowledge of olfaction at the molecular level can help in designing strategies for the conservation of this species. In this context, we have identified the odorant-binding protein (OBP) repertoire of the giant panda and mapped the protein expression in nasal mucus and saliva through proteomics. Four OBPs have been identified in nasal mucus, while the other two were not detected in the samples examined. In particular, AimelOBP3 is similar to a subset of OBPs reported as pheromone carriers in the urine of rodents, saliva of the boar, and seminal fluid of the rabbit. We expressed this protein, mapped its binding specificity, and determined its crystal structure. Structural data guided the design and preparation of three protein mutants bearing single-amino acid replacements in the ligand-binding pocket, for which the corresponding binding affinity spectra were measured. We also expressed AimelOBP5, which is markedly different from AimelOBP3 and complementary in its binding spectrum. By comparing our binding data with the structures of bamboo volatiles and those of typical mammalian pheromones, we formulate hypotheses on which may be the most relevant semiochemicals for the giant panda. PMID:29078359

Reverse chemical ecology: Olfactory proteins from the giant panda and their interactions with putative pheromones and bamboo volatiles.

PubMed

Zhu, Jiao; Arena, Simona; Spinelli, Silvia; Liu, Dingzhen; Zhang, Guiquan; Wei, Rongping; Cambillau, Christian; Scaloni, Andrea; Wang, Guirong; Pelosi, Paolo

2017-11-14

The giant panda Ailuropoda melanoleuca belongs to the family of Ursidae; however, it is not carnivorous, feeding almost exclusively on bamboo. Being equipped with a typical carnivorous digestive apparatus, the giant panda cannot get enough energy for an active life and spends most of its time digesting food or sleeping. Feeding and mating are both regulated by odors and pheromones; therefore, a better knowledge of olfaction at the molecular level can help in designing strategies for the conservation of this species. In this context, we have identified the odorant-binding protein (OBP) repertoire of the giant panda and mapped the protein expression in nasal mucus and saliva through proteomics. Four OBPs have been identified in nasal mucus, while the other two were not detected in the samples examined. In particular, AimelOBP3 is similar to a subset of OBPs reported as pheromone carriers in the urine of rodents, saliva of the boar, and seminal fluid of the rabbit. We expressed this protein, mapped its binding specificity, and determined its crystal structure. Structural data guided the design and preparation of three protein mutants bearing single-amino acid replacements in the ligand-binding pocket, for which the corresponding binding affinity spectra were measured. We also expressed AimelOBP5, which is markedly different from AimelOBP3 and complementary in its binding spectrum. By comparing our binding data with the structures of bamboo volatiles and those of typical mammalian pheromones, we formulate hypotheses on which may be the most relevant semiochemicals for the giant panda.

Development and Validation of a Whole-Cell Inhibition Assay for Bacterial Methionine Aminopeptidase by Surface-Enhanced Laser Desorption Ionization-Time of Flight Mass Spectrometry

PubMed Central

Greis, Kenneth D.; Zhou, Songtao; Siehnel, Richard; Klanke, Chuck; Curnow, Alan; Howard, Jeremy; Layh-Schmitt, Gerlinde

2005-01-01

Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP. PMID:16048957

The mapping of yeast's G-protein coupled receptor with an atomic force microscope

NASA Astrophysics Data System (ADS)

Takenaka, Musashi; Miyachi, Yusuke; Ishii, Jun; Ogino, Chiaki; Kondo, Akihiko

2015-03-01

An atomic force microscope (AFM) can measure the adhesion force between a sample and a cantilever while simultaneously applying a rupture force during the imaging of a sample. An AFM should be useful in targeting specific proteins on a cell surface. The present study proposes the use of an AFM to measure the adhesion force between targeting receptors and their ligands, and to map the targeting receptors. In this study, Ste2p, one of the G protein-coupled receptors (GPCRs), was chosen as the target receptor. The specific force between Ste2p on a yeast cell surface and a cantilever modified with its ligand, α-factor, was measured and found to be approximately 250 pN. In addition, through continuous measuring of the cell surface, a mapping of the receptors on the cell surface could be performed, which indicated the differences in the Ste2p expression levels. Therefore, the proposed AFM system is accurate for cell diagnosis.

Proteomic analysis of Lupinus angustifolius (var. Zeus and Bojar) and Lupinus luteus (var. Lord and Parys) seed proteins and their hydrolysates.

PubMed

Czubinski, Jaroslaw; Montowska, Magdalena; Pospiech, Edward; Lampart-Szczapa, Eleonora

2017-12-01

Proteins enzymatic digestion is a very complex process, during which some components are degraded, whereas others remain in an unchanged form. Moreover, enzymatic hydrolysis is one of the most popular methods used to reduce the allergenicity of food proteins. In the present study, the efficiency of enzymatic hydrolysis of lupin seed proteins was assessed by proteomic analysis as performed by two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry identification. Two digestion systems were used: oriented digestion carried out by trypsin and model in vitro digestion mimicking the conditions present in the gastrointestinal tract. The comparisons of 2-DE maps of proteins isolated form different lupin seed species revealed that the differences in proteins expression were observed mainly in the central parts of gels (i.e. in the molecular weight range from 20 to 70 kDa, and the pH range 5-7). In total, 27 differentially expressed proteins spots were successfully identified by mass spectrometry analysis. An important reduction in the number of proteins spots on 2-DE maps was observed when trypsin and the in vitro digestion model were applied. The protein spot insensitive to digestion in both hydrolysis systems was identified as β-conglutin. The results of the present study provide insight into the nature of the digestion process that may take place after lupin seed protein intake and highlight the important fact that some of the proteins are insensitive to digestive enzyme activity. Moreover, evaluation of digestion activity of trypsin towards lupin seed proteins may be used for the development of specific processes with respect to hypoallergenic food production. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Small Molecular Weight Soybean Protein-Derived Peptides Nutriment Attenuates Rat Burn Injury-Induced Muscle Atrophy by Modulation of Ubiquitin-Proteasome System and Autophagy Signaling Pathway.

PubMed

Zhao, Fen; Yu, Yonghui; Liu, Wei; Zhang, Jian; Liu, Xinqi; Liu, Lingying; Yin, Huinan

2018-03-21

This article describes results of the effect of dietary supplementation with small molecular weight soybean protein-derived peptides on major rat burn injury-induced muscle atrophy. As protein nutrients have been previously implicated to play an important role in improving burn injury outcomes, optimized more readily absorbed small molecular weight soybean protein-derived peptides were evaluated. Thus, the quantity, sodium dodecyl sulfate polyacrylamide-gel electrophoresis patterns, molecular weight distribution, and composition of amino acids of the prepared peptides were analyzed, and a major full-thickness 30% total body surface area burn-injury rat model was utilized to assess the impact of supplementation with soybean protein-derived peptides on initial systemic inflammatory responses as measured by interferon-gamma (IFN-γ), chemokine (C-C motif) ligand 2 (CCL2, also known as MCP-1), chemokine (C-C motif) ligand 7 (CCL7, also known as MCP-3), and generation of muscle atrophy as measured by tibialis anterior muscle (TAM) weight relative to total body weight. Induction of burn injury-induced muscle atrophy ubiquitin-proteasome system (UPS) signaling pathways in effected muscle tissues was determined by Western blot protein expression measurements of E3 ubiquitin-protein ligase TRIM-63 (TRIM63, also known as MuRF1) and F-box only protein 32 (FBXO32, also known as atrogin-1 or MAFbx). In addition, induction of burn injury-induced autophagy signaling pathways associated with muscle atrophy in effected muscle tissues was assessed by immunohistochemical analysis as measured by microtubule-associated proteins 1 light chain 3 (MAP1LC3, or commonly abbreviated as LC3) and beclin-1 (BECN1) expression, as well as relative induction of cytoplasmic-liberated form of MAP1LC3 (LC3-I) and phagophore and autophagosome membrane-bound form of MAP1LC3 (LC3-II), and BECN1 protein expression by Western blot analysis. Nutrient supplementation with small molecular weight soybean protein-derived peptides resulted a significant reduction in burn injury-induced inflammatory markers, muscle atrophy, induction of TRIM63 and FBXO32 muscle atrophy signaling pathways, and induction of autophagy signaling pathways LC3 and BECN1 associated with muscle atrophy. These results implicated that small molecular weight soybean-derived peptides dietary supplementation could be used as an adjunct therapy in burn injury management to reduce the development or severity of muscle atrophy for improved burn patient outcomes.

Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

PubMed

Deshmukh, Atul S; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T; Cox, Jürgen; Mann, Matthias

2015-04-01

Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture.

PubMed

Wagner, Mary Ann; Eschenbrenner, Michel; Horn, Troy A; Kraycer, Jo Ann; Mujer, Cesar V; Hagius, Sue; Elzer, Philip; DelVecchio, Vito G

2002-08-01

Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.

Massive Collection of Full-Length Complementary DNA Clones and Microarray Analyses:. Keys to Rice Transcriptome Analysis

NASA Astrophysics Data System (ADS)

Kikuchi, Shoshi

2009-02-01

Completion of the high-precision genome sequence analysis of rice led to the collection of about 35,000 full-length cDNA clones and the determination of their complete sequences. Mapping of these full-length cDNA sequences has given us information on (1) the number of genes expressed in the rice genome; (2) the start and end positions and exon-intron structures of rice genes; (3) alternative transcripts; (4) possible encoded proteins; (5) non-protein-coding (np) RNAs; (6) the density of gene localization on the chromosome; (7) setting the parameters of gene prediction programs; and (8) the construction of a microarray system that monitors global gene expression. Manual curation for rice gene annotation by using mapping information on full-length cDNA and EST assemblies has revealed about 32,000 expressed genes in the rice genome. Analysis of major gene families, such as those encoding membrane transport proteins (pumps, ion channels, and secondary transporters), along with the evolution from bacteria to higher animals and plants, reveals how gene numbers have increased through adaptation to circumstances. Family-based gene annotation also gives us a new way of comparing organisms. Massive amounts of data on gene expression under many kinds of physiological conditions are being accumulated in rice oligoarrays (22K and 44K) based on full-length cDNA sequences. Cluster analyses of genes that have the same promoter cis-elements, that have similar expression profiles, or that encode enzymes in the same metabolic pathways or signal transduction cascades give us clues to understanding the networks of gene expression in rice. As a tool for that purpose, we recently developed "RiCES", a tool for searching for cis-elements in the promoter regions of clustered genes.

The interaction and integration of auxin signaling components.

PubMed

Hayashi, Ken-ichiro

2012-06-01

IAA, a naturally occurring auxin, is a simple signaling molecule that regulates many diverse steps of plant development. Auxin essentially coordinates plant development through transcriptional regulation. Auxin binds to TIR1/AFB nuclear receptors, which are F-box subunits of the SCF ubiquitin ligase complex. The auxin signal is then modulated by the quantitative and qualitative responses of the Aux/IAA repressors and the auxin response factor (ARF) transcription factors. The specificity of the auxin-regulated gene expression profile is defined by several factors, such as the expression of these regulatory proteins, their post-transcriptional regulation, their stability and the affinity between these regulatory proteins. Auxin-binding protein 1 (ABP1) is a candidate protein for an auxin receptor that is implicated in non-transcriptional auxin signaling. ABP1 also affects TIR1/AFB-mediated auxin-responsive gene expression, implying that both the ABP1 and TIR1/AFB signaling machineries coordinately control auxin-mediated physiological events. Systematic approaches using the comprehensive mapping of the expression and interaction of signaling modules and computational modeling would be valuable for integrating our knowledge of auxin signals and responses.

Expression of Ambra1 in mouse brain during physiological and Alzheimer type aging.

PubMed

Sepe, Sara; Nardacci, Roberta; Fanelli, Francesca; Rosso, Pamela; Bernardi, Cinzia; Cecconi, Francesco; Mastroberardino, Pier G; Piacentini, Mauro; Moreno, Sandra

2014-01-01

Autophagy is a major protein degradation pathway, essential for stress-induced and constitutive protein turnover. In nervous tissue, autophagy is constitutively active and crucial to neuronal survival. The efficiency of the autophagic pathway reportedly undergoes age-related decline, and autophagy defects are observed in neurodegenerative diseases. Since Ambra1 plays a fundamental role in regulating the autophagic process in developing nervous tissue, we investigated the expression of this protein in mature mouse brain and during physiological and Alzheimer type aging. The present study accomplished the first complete map of Ambra1 protein distribution in the various brain areas, and highlights differential expression in neuronal/glial cell populations. Differences in Ambra1 content are possibly related to specific neuronal features and properties, particularly concerning susceptibility to neurodegeneration. Furthermore, the analysis of Ambra1 expression in physiological and pathological brain aging supports important, though conflicting, functions of autophagy in neurodegenerative processes. Thus, novel therapeutic approaches, based on autophagy modulation, should also take into account the age-dependent roles of this mechanism in establishing, promoting, or counteracting neurodegeneration. Copyright © 2014 Elsevier Inc. All rights reserved.

The gene for fibroblast activation protein {alpha} (FAP), a putative cell surface-bound serine protease expressed in cancer stroma and wound healing, maps to chromosome band 2q23

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

Mathew, S.; Murty, V.V.V.S.; Chaganti, R.S.K.

The human fibroblast activation protein {alpha} (FAP{alpha}) is an inducible cell surface glycoprotein of M{sub r} 95,000 recognized by a number of monoclonal antibodies (mAbs), including the prototype mAb F19. Immunohistochemical studies have shown that FAP{alpha} expression in vivo is tightly regulated, with transient expression in some fetal mesenchymal tissues but absence of expression in most normal adult tissues. Reexpression of FAP{alpha} is observed in the reactive stromal fibroblasts of several common types of epithelial cancers, including >90% of breast, colorectal, and lung carcinomas and healing wounds. Cloning and sequence analysis of an FAP{alpha}-specific cDNA has revealed that the moleculemore » is encoded by a novel gene, FAP, which shows sequence similarity to members of the serine protease family of integral membrane proteins, namely dipeptidyl peptidase IV (DPPIV, also known as lymphocyte activation antigen, CD26, or adenosine dearoinase binding protein) and DPPX, a DPPIV-related molecule of unknown function. 15 refs., 1 fig.« less

Mapping the binding domain of the F18 fimbrial adhesin.

PubMed

Smeds, A; Pertovaara, M; Timonen, T; Pohjanvirta, T; Pelkonen, S; Palva, A

2003-04-01

F18 fimbrial Esherichia coli strains are associated with porcine postweaning diarrhea and pig edema disease. Recently, the FedF subunit was identified as the adhesin of the F18 fimbriae. In this study, adhesion domains of FedF were further studied by constructing deletions within the fedF gene and expressing FedF proteins with deletions either together with the other F18 fimbrial subunits or as fusion proteins tagged with maltose binding protein. The region essential for adhesion to porcine intestinal epithelial cells was mapped between amino acid residues 60 and 109 of FedF. To map the binding domain even more closely, all eight charged amino acid residues within this region were independently replaced by alanine. Three of these single point mutants expressing F18 fimbriae exhibited significantly diminished capabilities to adhere to porcine epithelial cells in vitro. In addition, a triple point mutation and a double point mutation completely abolished receptor adhesiveness. The result further confirmed that the region between amino acid residues 60 and 109 is essential for the binding of F18 fimbriae to their receptor. In addition, the adhesion capability of the binding domain was eliminated after treatment with iodoacetamide, suggesting the formation of a disulfide bridge between Cys-63 and Cys-83, whereas Cys-111 and Cys-116 could be deleted without affecting the binding ability of FedF.

Intracerebroventricular oxytocin administration in rats enhances object recognition and increases expression of neurotrophins, microtubule-associated protein 2, and synapsin I.

PubMed

Havranek, Tomas; Zatkova, Martina; Lestanova, Zuzana; Bacova, Zuzana; Mravec, Boris; Hodosy, Julius; Strbak, Vladimir; Bakos, Jan

2015-06-01

Brain oxytocin regulates a variety of social and affiliative behaviors and affects also learning and memory. However, mechanisms of its action at the level of neuronal circuits are not fully understood. The present study tests the hypothesis that molecular factors required for memory formation and synaptic plasticity, including brain-derived neurotrophic factor, neural growth factor, nestin, microtubule-associated protein 2 (MAP2), and synapsin I, are enhanced by central administration of oxytocin. We also investigated whether oxytocin enhances object recognition and acts as anxiolytic agent. Therefore, male Wistar rats were infused continuously with oxytocin (20 ng/µl) via an osmotic minipump into the lateral cerebral ventricle for 7 days; controls were infused with vehicle. The object recognition test, open field test, and elevated plus maze test were performed on the sixth, seventh, and eighth days from starting the infusion. No significant effects of oxytocin on anxious-like behavior were observed. The object recognition test showed that oxytocin-treated rats significantly preferred unknown objects. Oxytocin treatment significantly increased gene expression and protein levels of neurotrophins, MAP2, and synapsin I in the hippocampus. No changes were observed in nestin expression. Our results provide the first direct evidence implicating oxytocin as a regulator of brain plasticity at the level of changes of neuronal growth factors, cytoskeletal proteins, and behavior. The data support assumption that oxytocin is important for short-term hippocampus-dependent memory. © 2015 Wiley Periodicals, Inc.

The cld mutation: narrowing the critical chromosomal region and selecting candidate genes.

PubMed

Péterfy, Miklós; Mao, Hui Z; Doolittle, Mark H

2006-10-01

Combined lipase deficiency (cld) is a recessive, lethal mutation specific to the tw73 haplotype on mouse Chromosome 17. While the cld mutation results in lipase proteins that are inactive, aggregated, and retained in the endoplasmic reticulum (ER), it maps separately from the lipase structural genes. We have narrowed the gene critical region by about 50% using the tw18 haplotype for deletion mapping and a recombinant chromosome used originally to map cld with respect to the phenotypic marker tf. The region now extends from 22 to 25.6 Mbp on the wild-type chromosome, currently containing 149 genes and 50 expressed sequence tags (ESTs). To identify the affected gene, we have selected candidates based on their known role in associated biological processes, cellular components, and molecular functions that best fit with the predicted function of the cld gene. A secondary approach was based on differences in mRNA levels between mutant (cld/cld) and unaffected (+/cld) cells. Using both approaches, we have identified seven functional candidates with an ER localization and/or an involvement in protein maturation and folding that could explain the lipase deficiency, and six expression candidates that exhibit large differences in mRNA levels between mutant and unaffected cells. Significantly, two genes were found to be candidates with regard to both function and expression, thus emerging as the strongest candidates for cld. We discuss the implications of our mapping results and our selection of candidates with respect to other genes, deletions, and mutations occurring in the cld critical region.

Regulation of cotton (Gossypium hirsutum) drought responses by mitogen-activated protein (MAP) kinase cascade-mediated phosphorylation of GhWRKY59.

PubMed

Li, Fangjun; Li, Maoying; Wang, Ping; Cox, Kevin L; Duan, Liusheng; Dever, Jane K; Shan, Libo; Li, Zhaohu; He, Ping

2017-09-01

Drought is a key limiting factor for cotton (Gossypium spp.) production, as more than half of the global cotton supply is grown in regions with high water shortage. However, the underlying mechanism of the response of cotton to drought stress remains elusive. By combining genome-wide transcriptome profiling and a loss-of-function screen using virus-induced gene silencing, we identified Gossypium hirsutum GhWRKY59 as an important transcription factor that regulates the drought stress response in cotton. Biochemical and genetic analyses revealed a drought stress-activated mitogen-activated protein (MAP) kinase cascade consisting of GhMAP3K15-Mitogen-activated Protein Kinase Kinase 4 (GhMKK4)-Mitogen-activated Protein Kinase 6 (GhMPK6) that directly phosphorylates GhWRKY59 at residue serine 221. Interestingly, GhWRKY59 is required for dehydration-induced expression of GhMAPK3K15, constituting a positive feedback loop of GhWRKY59-regulated MAP kinase activation in response to drought stress. Moreover, GhWRKY59 directly binds to the W-boxes of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2 (GhDREB2), which encodes a dehydration-inducible transcription factor regulating the plant hormone abscisic acid (ABA)-independent drought response. Our study identified a complete MAP kinase cascade that phosphorylates and activates a key WRKY transcription factor, and elucidated a regulatory module, consisting of GhMAP3K15-GhMKK4-GhMPK6-GhWRKY59-GhDREB2, that is involved in controlling the cotton drought response. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Proteomic approaches in brain research and neuropharmacology.

PubMed

Vercauteren, Freya G G; Bergeron, John J M; Vandesande, Frans; Arckens, Lut; Quirion, Rémi

2004-10-01

Numerous applications of genomic technologies have enabled the assembly of unprecedented inventories of genes, expressed in cells under specific physiological and pathophysiological conditions. Complementing the valuable information generated through functional genomics with the integrative knowledge of protein expression and function should enable the development of more efficient diagnostic tools and therapeutic agents. Proteomic analyses are particularly suitable to elucidate posttranslational modifications, expression levels and protein-protein interactions of thousands of proteins at a time. In this review, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) investigations of brain tissues in neurodegenerative diseases such as Alzheimer's disease, Down syndrome and schizophrenia, and the construction of 2D-PAGE proteome maps of the brain are discussed. The role of the Human Proteome Organization (HUPO) as an international coordinating organization for proteomic efforts, as well as challenges for proteomic technologies and data analysis are also addressed. It is expected that the use of proteomic strategies will have significant impact in neuropharmacology over the coming decade.

larvalign: Aligning Gene Expression Patterns from the Larval Brain of Drosophila melanogaster.

PubMed

Muenzing, Sascha E A; Strauch, Martin; Truman, James W; Bühler, Katja; Thum, Andreas S; Merhof, Dorit

2018-01-01

The larval brain of the fruit fly Drosophila melanogaster is a small, tractable model system for neuroscience. Genes for fluorescent marker proteins can be expressed in defined, spatially restricted neuron populations. Here, we introduce the methods for 1) generating a standard template of the larval central nervous system (CNS), 2) spatial mapping of expression patterns from different larvae into a reference space defined by the standard template. We provide a manually annotated gold standard that serves for evaluation of the registration framework involved in template generation and mapping. A method for registration quality assessment enables the automatic detection of registration errors, and a semi-automatic registration method allows one to correct registrations, which is a prerequisite for a high-quality, curated database of expression patterns. All computational methods are available within the larvalign software package: https://github.com/larvalign/larvalign/releases/tag/v1.0.

Heritability and genetic basis of protein level variation in an outbred population

PubMed Central

Liu, Yi-Chun; Tekkedil, Manu M.; Steinmetz, Lars M.; Caudy, Amy A.; Fraser, Andrew G.

2014-01-01

The genetic basis of heritable traits has been studied for decades. Although recent mapping efforts have elucidated genetic determinants of transcript levels, mapping of protein abundance has lagged. Here, we analyze levels of 4084 GFP-tagged yeast proteins in the progeny of a cross between a laboratory and a wild strain using flow cytometry and high-content microscopy. The genotype of trans variants contributed little to protein level variation between individual cells but explained >50% of the variance in the population’s average protein abundance for half of the GFP fusions tested. To map trans-acting factors responsible, we performed flow sorting and bulk segregant analysis of 25 proteins, finding a median of five protein quantitative trait loci (pQTLs) per GFP fusion. Further, we find that cis-acting variants predominate; the genotype of a gene and its surrounding region had a large effect on protein level six times more frequently than the rest of the genome combined. We present evidence for both shared and independent genetic control of transcript and protein abundance: More than half of the expression QTLs (eQTLs) contribute to changes in protein levels of regulated genes, but several pQTLs do not affect their cognate transcript levels. Allele replacements of genes known to underlie trans eQTL hotspots confirmed the correlation of effects on mRNA and protein levels. This study represents the first genome-scale measurement of genetic contribution to protein levels in single cells and populations, identifies more than a hundred trans pQTLs, and validates the propagation of effects associated with transcript variation to protein abundance. PMID:24823668

Plant Genome Resources at the National Center for Biotechnology Information

PubMed Central

Wheeler, David L.; Smith-White, Brian; Chetvernin, Vyacheslav; Resenchuk, Sergei; Dombrowski, Susan M.; Pechous, Steven W.; Tatusova, Tatiana; Ostell, James

2005-01-01

The National Center for Biotechnology Information (NCBI) integrates data from more than 20 biological databases through a flexible search and retrieval system called Entrez. A core Entrez database, Entrez Nucleotide, includes GenBank and is tightly linked to the NCBI Taxonomy database, the Entrez Protein database, and the scientific literature in PubMed. A suite of more specialized databases for genomes, genes, gene families, gene expression, gene variation, and protein domains dovetails with the core databases to make Entrez a powerful system for genomic research. Linked to the full range of Entrez databases is the NCBI Map Viewer, which displays aligned genetic, physical, and sequence maps for eukaryotic genomes including those of many plants. A specialized plant query page allow maps from all plant genomes covered by the Map Viewer to be searched in tandem to produce a display of aligned maps from several species. PlantBLAST searches against the sequences shown in the Map Viewer allow BLAST alignments to be viewed within a genomic context. In addition, precomputed sequence similarities, such as those for proteins offered by BLAST Link, enable fluid navigation from unannotated to annotated sequences, quickening the pace of discovery. NCBI Web pages for plants, such as Plant Genome Central, complete the system by providing centralized access to NCBI's genomic resources as well as links to organism-specific Web pages beyond NCBI. PMID:16010002

Genetic dissection of seed oil and protein content and identification of networks associated with oil content in Brassica napus.

PubMed

Chao, Hongbo; Wang, Hao; Wang, Xiaodong; Guo, Liangxing; Gu, Jianwei; Zhao, Weiguo; Li, Baojun; Chen, Dengyan; Raboanatahiry, Nadia; Li, Maoteng

2017-04-10

High-density linkage maps can improve the precision of QTL localization. A high-density SNP-based linkage map containing 3207 markers covering 3072.7 cM of the Brassica napus genome was constructed in the KenC-8 × N53-2 (KNDH) population. A total of 67 and 38 QTLs for seed oil and protein content were identified with an average confidence interval of 5.26 and 4.38 cM, which could explain up to 22.24% and 27.48% of the phenotypic variation, respectively. Thirty-eight associated genomic regions from BSA overlapped with and/or narrowed the SOC-QTLs, further confirming the QTL mapping results based on the high-density linkage map. Potential candidates related to acyl-lipid and seed storage underlying SOC and SPC, respectively, were identified and analyzed, among which six were checked and showed expression differences between the two parents during different embryonic developmental periods. A large primary carbohydrate pathway based on potential candidates underlying SOC- and SPC-QTLs, and interaction networks based on potential candidates underlying SOC-QTLs, was constructed to dissect the complex mechanism based on metabolic and gene regulatory features, respectively. Accurate QTL mapping and potential candidates identified based on high-density linkage map and BSA analyses provide new insights into the complex genetic mechanism of oil and protein accumulation in the seeds of rapeseed.

Direct Correlation between Motile Behavior and Protein Abundance in Single Cells

PubMed Central

Gillet, Sébastien; Frankel, Nicholas W.; Weibel, Douglas B.

2016-01-01

Understanding how stochastic molecular fluctuations affect cell behavior requires the quantification of both behavior and protein numbers in the same cells. Here, we combine automated microscopy with in situ hydrogel polymerization to measure single-cell protein expression after tracking swimming behavior. We characterized the distribution of non-genetic phenotypic diversity in Escherichia coli motility, which affects single-cell exploration. By expressing fluorescently tagged chemotaxis proteins (CheR and CheB) at different levels, we quantitatively mapped motile phenotype (tumble bias) to protein numbers using thousands of single-cell measurements. Our results disagreed with established models until we incorporated the role of CheB in receptor deamidation and the slow fluctuations in receptor methylation. Beyond refining models, our central finding is that changes in numbers of CheR and CheB affect the population mean tumble bias and its variance independently. Therefore, it is possible to adjust the degree of phenotypic diversity of a population by adjusting the global level of expression of CheR and CheB while keeping their ratio constant, which, as shown in previous studies, confers functional robustness to the system. Since genetic control of protein expression is heritable, our results suggest that non-genetic diversity in motile behavior is selectable, supporting earlier hypotheses that such diversity confers a selective advantage. PMID:27599206

Exploring the Genomic Roadmap and Molecular Phylogenetics Associated with MODY Cascades Using Computational Biology.

PubMed

Chakraborty, Chiranjib; Bandyopadhyay, Sanghamitra; Doss, C George Priya; Agoramoorthy, Govindasamy

2015-04-01

Maturity onset diabetes of the young (MODY) is a metabolic and genetic disorder. It is different from type 1 and type 2 diabetes with low occurrence level (1-2%) among all diabetes. This disorder is a consequence of β-cell dysfunction. Till date, 11 subtypes of MODY have been identified, and all of them can cause gene mutations. However, very little is known about the gene mapping, molecular phylogenetics, and co-expression among MODY genes and networking between cascades. This study has used latest servers and software such as VarioWatch, ClustalW, MUSCLE, G Blocks, Phylogeny.fr, iTOL, WebLogo, STRING, and KEGG PATHWAY to perform comprehensive analyses of gene mapping, multiple sequences alignment, molecular phylogenetics, protein-protein network design, co-expression analysis of MODY genes, and pathway development. The MODY genes are located in chromosomes-2, 7, 8, 9, 11, 12, 13, 17, and 20. Highly aligned block shows Pro, Gly, Leu, Arg, and Pro residues are highly aligned in the positions of 296, 386, 437, 455, 456 and 598, respectively. Alignment scores inform us that HNF1A and HNF1B proteins have shown high sequence similarity among MODY proteins. Protein-protein network design shows that HNF1A, HNF1B, HNF4A, NEUROD1, PDX1, PAX4, INS, and GCK are strongly connected, and the co-expression analyses between MODY genes also show distinct association between HNF1A and HNF4A genes. This study has used latest tools of bioinformatics to develop a rapid method to assess the evolutionary relationship, the network development, and the associations among eleven MODY genes and cascades. The prediction of sequence conservation, molecular phylogenetics, protein-protein network and the association between the MODY cascades enhances opportunities to get more insights into the less-known MODY disease.

The quantitative and condition-dependent Escherichia coli proteome

PubMed Central

Schmidt, Alexander; Kochanowski, Karl; Vedelaar, Silke; Ahrné, Erik; Volkmer, Benjamin; Callipo, Luciano; Knoops, Kèvin; Bauer, Manuel; Aebersold, Ruedi; Heinemann, Matthias

2016-01-01

Measuring precise concentrations of proteins can provide insights into biological processes. Here, we use efficient protein extraction and sample fractionation and state-of-the-art quantitative mass spectrometry techniques to generate a comprehensive, condition-dependent protein abundance map of Escherichia coli. We measure cellular protein concentrations for 55% of predicted E. coli genes (>2300 proteins) under 22 different experimental conditions and identify methylation and N-terminal protein acetylations previously not known to be prevalent in bacteria. We uncover system-wide proteome allocation, expression regulation, and post-translational adaptations. These data provide a valuable resource for the systems biology and broader E. coli research communities. PMID:26641532

Identification of the subgenomic promoter of the coat protein gene of cucumber fruit mottle mosaic virus and development of a heterologous expression vector.

PubMed

Rhee, Sun-Ju; Jang, Yoon Jeong; Lee, Gung Pyo

2016-06-01

Heterologous gene expression using plant virus vectors enables research on host-virus interactions and the production of useful proteins, but the host range of plant viruses limits the practical applications of such vectors. Here, we aimed to develop a viral vector based on cucumber fruit mottle mosaic virus (CFMMV), a member of the genus Tobamovirus, whose members infect cucurbits. The subgenomic promoter (SGP) in the coat protein (CP) gene, which was used to drive heterologous expression, was mapped by analyzing deletion mutants from a CaMV 35S promoter-driven infectious CFMMV clone. The region from nucleotides (nt) -55 to +160 relative to the start codon of the open reading frame (ORF) of CP was found to be a fully active promoter, and the region from nt -55 to +100 was identified as the active core promoter. Based on these SGPs, we constructed a cloning site in the CFMMV vector and successfully expressed enhanced green fluorescent protein (EGFP) in Nicotiana benthamiana and watermelon (Citrullus lanatus). Co-inoculation with the P19 suppressor increased EGFP expression and viral replication by blocking degradation of the viral genome. Our CFMMV vector will be useful as an expression vector in cucurbits.

Silibinin induces apoptosis of HT29 colon carcinoma cells through early growth response-1 (EGR-1)-mediated non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) up-regulation.

PubMed

Woo, Seon Min; Min, Kyoung-Jin; Kim, Shin; Park, Jong-Wook; Kim, Dong Eun; Chun, Kyung-Soo; Kim, Young Ho; Lee, Tae-Jin; Kim, Sang Hyun; Choi, Yung Hyun; Chang, Jong-Soo; Kwon, Taeg Kyu

2014-03-25

Silibinin, an effective anti-cancer and chemopreventive agent, has been shown to exert multiple effects on cancer cells, including inhibition of both cell proliferation and migration. However, the molecular mechanisms responsible for these effects are not fully understood. We observed that silibinin significantly induced the expression of the non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) in both p53 wild-type and p53-null cancer cell lines, suggesting that silibinin-induced NAG-1 up-regulation is p53-independent manner. Silibinin up-regulates early growth response-1 (EGR-1) expression. The ectopic expression of EGR-1 significantly increased NAG-1 promoter activity and NAG-1 protein expression in a dose-dependent manner. Furthermore, down-regulation of EGR-1 expression using siRNA markedly reduced silibinin-mediated NAG-1 expression, suggesting that the expression of EGR-1 is critical for silibinin-induced NAG-1 expression. We also observed that reactive oxygen species (ROS) are generated by silibinin; however, ROS did not affect silibinin-induced NAG-1 expression and apoptosis. In addition, we demonstrated that the mitogen-activated protein kinase (MAP kinase) signal transduction pathway is involved in silibinin-induced NAG-1 expression. Inhibitors of p38 MAP kinase (SB203580) attenuated silibinin-induced NAG-1 expression. Furthermore, we found that siRNA-mediated knockdown of NAG-1 attenuated silibinin-induced apoptosis. Collectively, the results of this study demonstrate for the first time that up-regulation of NAG-1 contributes to silibinin-induced apoptosis in cancer cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Tumor necrosis factor-alpha stimulation of calcitonin gene-related peptide expression and secretion from rat trigeminal ganglion neurons.

PubMed

Bowen, Elizabeth J; Schmidt, Thomas W; Firm, Christina S; Russo, Andrew F; Durham, Paul L

2006-01-01

Expression of the neuropeptide calcitonin gene-related peptide (CGRP) in trigeminal ganglion is implicated in neurovascular headaches and temporomandibular joint disorders. Elevation of cytokines contributes to the pathology of these diseases. However, a connection between cytokines and CGRP gene expression in trigeminal ganglion nerves has not been established. We have focused on the effects of the cytokine tumor necrosis factor-alpha (TNF-alpha). TNFR1 receptors were found on the majority of CGRP-containing rat trigeminal ganglion neurons. Treatment of cultures with TNF-alpha stimulated CGRP secretion. In addition, the intracellular signaling intermediate from the TNFR1 receptor, ceramide, caused a similar increase in CGRP release. TNF-alpha caused a coordinate increase in CGRP promoter activity. TNF-alpha treatment activated the transcription factor NF-kappaB, as well as the Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways. The importance of TNF-alpha induction of MAP kinase pathways was demonstrated by inhibiting MAP kinases with pharmacological reagents and gene transfer with an adenoviral vector encoding MAP kinase phosphatase-1 (MKP-1). We propose that selective and regulated inhibition of MAP kinases in trigeminal neurons may be therapeutically beneficial for inflammatory disorders involving elevated CGRP levels.

Tumor necrosis factor-α stimulation of calcitonin gene-related peptide expression and secretion from rat trigeminal ganglion neurons

PubMed Central

Bowen, Elizabeth J.; Schmidt, Thomas W.; Firm, Christina S.; Russo, Andrew F.; Durham, Paul L.

2006-01-01

Expression of the neuropeptide calcitonin gene-related peptide (CGRP) in trigeminal ganglion is implicated in neurovascular headaches and temporomandibular joint disorders. Elevation of cytokines contributes to the pathology of these diseases. However, a connection between cytokines and CGRP gene expression in trigeminal ganglion nerves has not been established. We have focused on the effects of the cytokine tumor necrosis factorα (TNFα). TNFR1 receptors were found on the majority of CGRP-containing rat trigeminal ganglion neurons. Treatment of cultures with TNFα stimulated CGRP secretion. In addition, the intracellular signaling intermediate from the TNFR1 receptor, ceramide, caused a similar increase in CGRP release. TNFα caused a coordinate increase in CGRP promoter activity. TNFα treatment activated the transcription factor NF-κB, as well as the Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways. The importance of TNFα induction of MAP kinase pathways was demonstrated by inhibiting MAP kinases with pharmacological reagents and gene transfer with an adenoviral vector encoding MAP kinase phosphatase-1 (MKP-1). We propose that selective and regulated inhibition of MAP kinases in trigeminal neurons may be therapeutically beneficial for inflammatory disorders involving elevated CGRP levels. PMID:16277606

Activity-dependent and graded BACE1 expression in the olfactory epithelium is mediated by the retinoic acid metabolizing enzyme CYP26B1.

PubMed

Login, Hande; Butowt, Rafal; Bohm, Staffan

2015-07-01

It is well established that environmental influences play a key role in sculpting neuronal connectivity in the brain. One example is the olfactory sensory map of topographic axonal connectivity. While intrinsic odorant receptor signaling in olfactory sensory neurons (OSN) determines anterior-posterior counter gradients of the axonal guidance receptors Neuropilin-1 and Plexin-A1, little is known about stimulus-dependent gradients of protein expression, which correlates with the functional organization of the olfactory sensory map along its dorsomedial (DM)-ventrolateral (VL) axis. Deficiency of the Alzheimer's β-secretase BACE1, which is expressed in a DM(low)-VL(high) gradient, results in OSN axon targeting errors in a DM > VL and gene dose-dependent manner. We show that expression of BACE1 and the all-trans retinoic acid (RA)-degrading enzyme Cyp26B1 form DM-VL counter gradients in the olfactory epithelium. Analyses of mRNA and protein levels in OSNs after naris occlusion, in mice deficient in the olfactory cyclic nucleotide-gated channel and in relation to onset of respiration, show that BACE1 and Cyp26B1 expression in OSNs inversely depend on neuronal activity. Overexpression of a Cyp26B1 or presence of a dominant negative RA receptor transgene selectively in OSNs, inhibit BACE1 expression while leaving the DM(low)-VL(high) gradient of the axonal guidance protein Neuropilin-2 intact. We conclude that stimulus-dependent neuronal activity can control the expression of the RA catabolic enzyme Cyp26B1 and downstream genes such as BACE1. This result is pertinent to an understanding of the mechanisms by which a topographic pattern of connectivity is achieved and modified as a consequence of graded gene expression and sensory experience.

Overexpression of tissue-nonspecific alkaline phosphatase increases the expression of neurogenic differentiation markers in the human SH-SY5Y neuroblastoma cell line.

PubMed

Graser, Stephanie; Mentrup, Birgit; Schneider, Doris; Klein-Hitpass, Ludger; Jakob, Franz; Hofmann, Christine

2015-10-01

Patients suffering from the rare hereditary disease hypophosphatasia (HPP), which is based on mutations in the ALPL gene, tend to develop central nervous system (CNS) related issues like epileptic seizures and neuropsychiatric illnesses such as anxiety and depression, in addition to well-known problems with the mineralization of bones and teeth. Analyses of the molecular role of tissue-nonspecific alkaline phosphatase (TNAP) in transgenic SH-SY5Y(TNAPhigh) neuroblastoma cells compared to SH-SY5Y(TNAPlow) cells indicate that the enzyme influences the expression levels of neuronal marker genes like RNA-binding protein, fox-1 homolog 3 (NEUN) and enolase 2, gamma neuronal (NSE) as well as microtubule-binding proteins like microtubule-associated protein 2 (MAP2) and microtubule-associated protein tau (TAU) during neurogenic differentiation. Fluorescence staining of SH-SY5Y(TNAPhigh) cells reveals TNAP localization throughout the whole length of the developed projection network and even synapsin Ι co-localization with strong TNAP signals at some spots at least at the early time points of differentiation. Additional immunocytochemical staining shows higher MAP2 expression in SH-SY5Y(TNAPhigh) cells and further a distinct up-regulation of tau and MAP2 in the course of neurogenic differentiation. Interestingly, transgenic SH-SY5Y(TNAPhigh) cells are able to develop longer cellular processes compared to control cells after stimulation with all-trans retinoic acid (RA). Current therapies for HPP prioritize improvement of the bone phenotype. Unraveling the molecular role of TNAP in extraosseous tissues, like in the CNS, will help to improve treatment strategies for HPP patients. Taking this rare disease as a model may also help to dissect TNAP's role in neurodegenerative diseases and even improve future treatment of common pathologies. Copyright © 2015 Elsevier Inc. All rights reserved.

The Reg1-interacting proteins, Bmh1, Bmh2, Ssb1, and Ssb2, have roles in maintaining glucose repression in Saccharomyces cerevisiae.

PubMed

Dombek, Kenneth M; Kacherovsky, Nataly; Young, Elton T

2004-09-10

In Saccharomyces cerevisiae, a type 1 protein phosphatase complex composed of the Glc7 catalytic subunit and the Reg1 regulatory subunit represses expression of many glucose-regulated genes. Here we show that the Reg1-interacting proteins Bmh1, Bmh2, Ssb1, and Ssb2 have roles in glucose repression. Deleting both BMH genes causes partially constitutive ADH2 expression without significantly increasing the level of Adr1 protein, the major activator of ADH2 expression. Adr1 and Bcy1, the regulatory subunit of cAMP-dependent protein kinase, are both required for this effect indicating that constitutive expression in Deltabmh1Deltabmh2 cells uses the same activation pathway that operates in Deltareg1 cells. Deletion of both BMH genes and REG1 causes a synergistic relief from repression, suggesting that Bmh proteins also act independently of Reg1 during glucose repression. A two-hybrid interaction with the Bmh proteins was mapped to amino acids 187-232, a region of Reg1 that is conserved in different classes of fungi. Deleting this region partially releases SUC2 from glucose repression. This indicates a role for the Reg1-Bmh interaction in glucose repression and also suggests a broad role for Bmh proteins in this process. An in vivo Reg1-Bmh interaction was confirmed by copurification of Bmh proteins with HA(3)-TAP-tagged Reg1. The nonconventional heat shock proteins Ssb1 and Ssb2 are also copurified with HA(3)-TAP-tagged Reg1. Deletion of both SSB genes modestly decreases repression of ADH2 expression in the presence of glucose, suggesting that Ssb proteins, perhaps through their interaction with Reg1, play a minor role in glucose repression.

Solenopsis invicta virus 3: mapping of structural proteins, ribosomal frameshifting, and similarities to Acyrthosiphon pisum virus and Kelp fly virus.

PubMed

Valles, Steven M; Bell, Susanne; Firth, Andrew E

2014-01-01

Solenopsis invicta virus 3 (SINV-3) is a positive-sense single-stranded RNA virus that infects the red imported fire ant, Solenopsis invicta. We show that the second open reading frame (ORF) of the dicistronic genome is expressed via a frameshifting mechanism and that the sequences encoding the structural proteins map to both ORF2 and the 3' end of ORF1, downstream of the sequence that encodes the RNA-dependent RNA polymerase. The genome organization and structural protein expression strategy resemble those of Acyrthosiphon pisum virus (APV), an aphid virus. The capsid protein that is encoded by the 3' end of ORF1 in SINV-3 and APV is predicted to have a jelly-roll fold similar to the capsid proteins of picornaviruses and caliciviruses. The capsid-extension protein that is produced by frameshifting, includes the jelly-roll fold domain encoded by ORF1 as its N-terminus, while the C-terminus encoded by the 5' half of ORF2 has no clear homology with other viral structural proteins. A third protein, encoded by the 3' half of ORF2, is associated with purified virions at sub-stoichiometric ratios. Although the structural proteins can be translated from the genomic RNA, we show that SINV-3 also produces a subgenomic RNA encoding the structural proteins. Circumstantial evidence suggests that APV may also produce such a subgenomic RNA. Both SINV-3 and APV are unclassified picorna-like viruses distantly related to members of the order Picornavirales and the family Caliciviridae. Within this grouping, features of the genome organization and capsid domain structure of SINV-3 and APV appear more similar to caliciviruses, perhaps suggesting the basis for a "Calicivirales" order.

Neonatal manipulation of oxytocin prevents lipopolysaccharide-induced decrease in gene expression of growth factors in two developmental stages of the female rat.

PubMed

Bakos, Jan; Lestanova, Zuzana; Strbak, Vladimir; Havranek, Tomas; Bacova, Zuzana

2014-10-01

Oxytocin production and secretion is important for early development of the brain. Long-term consequences of manipulation of oxytocin system might include changes in markers of brain plasticity - cytoskeletal proteins and neurotrophins. The aim of the present study was (1) to determine whether neonatal oxytocin administration affects gene expression of nestin, microtubule-associated protein-2 (MAP-2), brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the brain of two developmental stages of rat and (2) to evaluate whether neonatal oxytocin administration protects against lipopolysaccharide (LPS) induced inflammation. Neonatal oxytocin did not prevent a decrease of body weight in the LPS treated animals. Oxytocin significantly increased gene expression of BDNF in the right hippocampus in 21-day and 2-month old rats of both sexes. Gene expression of NGF and MAP-2 significantly increased in males treated with oxytocin. Both, growth factors and intermediate filament-nestin mRNA levels, were reduced in females exposed to LPS. Oxytocin treatment prevented a decrease in the gene expression of only growth factors. In conclusion, neonatal manipulation of oxytocin has developmental and sex-dependent effect on markers of brain plasticity. These results also indicate, that oxytocin may be protective against inflammation particularly in females. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mining Missing Membrane Proteins by High-pH Reverse-Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry.

PubMed

Kitata, Reta Birhanu; Dimayacyac-Esleta, Baby Rorielyn T; Choong, Wai-Kok; Tsai, Chia-Feng; Lin, Tai-Du; Tsou, Chih-Chiang; Weng, Shao-Hsing; Chen, Yi-Ju; Yang, Pan-Chyr; Arco, Susan D; Nesvizhskii, Alexey I; Sung, Ting-Yi; Chen, Yu-Ju

2015-09-04

Despite significant efforts in the past decade toward complete mapping of the human proteome, 3564 proteins (neXtProt, 09-2014) are still "missing proteins". Over one-third of these missing proteins are annotated as membrane proteins, owing to their relatively challenging accessibility with standard shotgun proteomics. Using nonsmall cell lung cancer (NSCLC) as a model study, we aim to mine missing proteins from disease-associated membrane proteome, which may be still largely under-represented. To increase identification coverage, we employed Hp-RP StageTip prefractionation of membrane-enriched samples from 11 NSCLC cell lines. Analysis of membrane samples from 20 pairs of tumor and adjacent normal lung tissue was incorporated to include physiologically expressed membrane proteins. Using multiple search engines (X!Tandem, Comet, and Mascot) and stringent evaluation of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66% membrane proteins) and 178 missing proteins (74 membrane proteins) with PSM-, peptide-, and protein-level FDR of 1%. Through multiple reaction monitoring using synthetic peptides, we provided additional evidence of eight missing proteins including seven with transmembrane helix domains. This study demonstrates that mining missing proteins focused on cancer membrane subproteome can greatly contribute to map the whole human proteome. All data were deposited into ProteomeXchange with the identifier PXD002224.

Proteomic analysis by iTRAQ in red claw crayfish, Cherax quadricarinatus, hematopoietic tissue cells post white spot syndrome virus infection.

PubMed

Jeswin, Joseph; Xie, Xiao-lu; Ji, Qiao-lin; Wang, Ke-jian; Liu, Hai-peng

2016-03-01

To elucidate proteomic changes of Hpt cells from red claw crayfish, Cherax quadricarinatus, we have carried out isobaric tags for relative and absolute quantitation (iTRAQ) of cellular proteins at both early (1 hpi) and late stage (12 hpi) post white spot syndrome virus (WSSV) infection. Protein database search revealed 594 protein hits by Mascot, in which 17 and 30 proteins were present as differentially expressed proteins at early and late viral infection, respectively. Generally, these differentially expressed proteins include: 1) the metabolic process related proteins in glycolysis and glucogenesis, DNA replication, nucleotide/amino acid/fatty acid metabolism and protein biosynthesis; 2) the signal transduction related proteins like small GTPases, G-protein-alpha stimulatory subunit, proteins bearing PDZ- or 14-3-3-domains that help holding together and organize signaling complexes, casein kinase I and proteins of the MAP-kinase signal transduction pathway; 3) the immune defense related proteins such as α-2 macroglobulin, transglutaminase and trans-activation response RNA-binding protein 1. Taken together, these protein information shed new light on the host cellular response against WSSV infection in a crustacean cell culture. Copyright © 2016 Elsevier Ltd. All rights reserved.

Insights into bird wing evolution and digit specification from polarizing region fate maps.

PubMed

Towers, Matthew; Signolet, Jason; Sherman, Adrian; Sang, Helen; Tickle, Cheryll

2011-08-09

The proposal that birds descended from theropod dinosaurs with digits 2, 3 and 4 was recently given support by short-term fate maps, suggesting that the chick wing polarizing region-a group that Sonic hedgehog-expressing cells-gives rise to digit 4. Here we show using long-term fate maps that Green fluorescent protein-expressing chick wing polarizing region grafts contribute only to soft tissues along the posterior margin of digit 4, supporting fossil data that birds descended from theropods that had digits 1, 2 and 3. In contrast, digit IV of the chick leg with four digits (I-IV) arises from the polarizing region. To determine how digit identity is specified over time, we inhibited Sonic hedgehog signalling. Fate maps show that polarizing region and adjacent cells are specified in parallel through a series of anterior to posterior digit fates-a process of digit specification that we suggest is involved in patterning all vertebrate limbs with more than three digits.

Regulation of MMP-3 expression and secretion by the chemokine eotaxin-1 in human chondrocytes

PubMed Central

2011-01-01

Background Osteoarthritis (OA) is characterized by the degradation of articular cartilage, marked by the breakdown of matrix proteins. Studies demonstrated the involvement of chemokines in this process, and some may potentially serve as diagnostic markers and therapeutic targets; however, the underlying signal transductions are not well understood. Methods We investigated the effects of the CC chemokine eotaxin-1 (CCL11) on the matrix metalloproteinase (MMP) expression and secretion in the human chondrocyte cell line SW1353 and primary chondrocytes. Results Eotaxin-1 significantly induced MMP-3 mRNA expression in a dose-dependent manner. Inhibitors of extracellular signal-regulated kinase (ERK) and p38 kinase were able to repress eotaxin-1-induced MMP-3 expression. On the contrary, Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPs), a competitive cAMP antagonist for cAMP receptors, and H-89, a protein kinase A (PKA) inhibitor, markedly enhanced eotaxin-1-induced MMP-3 expression. These results suggest that MMP-3 expression is specifically mediated by the G protein-coupled eotaxin-1 receptor activities. Interestingly, little amount of MMP-3 protein was detected in the cell lysates of eotaxin-1-treated SW1353 cells, and most of MMP-3 protein was in the culture media. Furthermore we found that the eotaxin-1-dependent MMP-3 protein secretion was regulated by phospholipase C (PLC)-protein kinase C (PKC) cascade and c-Jun N-terminal kinase (JNK)/mitogen-activated protein (MAP) kinase pathways. These data indicate a specific regulation of MMP-3 secretion also by eotaxin-1 receptor activities. Conclusions Eotaxin-1 not only induces MMP-3 gene expression but also promotes MMP-3 protein secretion through G protein-coupled eotaxin-1 receptor activities. Chemokines, such as eotaxin-1, could be a potential candidate in the diagnosis and treatment of arthritis. PMID:22114952

Proteomic analysis of papaya (Carica papaya L.) displaying typical sticky disease symptoms.

PubMed

Rodrigues, Silas P; Ventura, José A; Aguilar, Clemente; Nakayasu, Ernesto S; Almeida, Igor C; Fernandes, Patricia M B; Zingali, Russolina B

2011-07-01

Papaya (Carica papaya L.) hosts the only described laticifer-infecting virus (Papaya meleira virus, PMeV), which is the causal agent of papaya sticky disease. To understand the systemic effects of PMeV in papaya, we conducted a comprehensive proteomic analysis of leaf samples from healthy and diseased plants grown under field conditions. First, a reference 2-DE map was established for proteins from healthy samples. A total of 486 reproducible spots were identified, and MALDI-TOF-MS/MS data identified 275 proteins accounting for 159 distinct proteins from 231 spots that were annotated. Second, the differential expression of proteins from healthy and diseased leaves was determined through parallel experiments, using 2-DE and DIGE followed by MALDI-TOF-MS/MS and LC-IonTrap-MS/MS, respectively. Conventional 2-DE analysis revealed 75 differentially expressed proteins. Of those, 48 proteins were identified, with 26 being upregulated (U) and 22 downregulated (D). In general, metabolism-related proteins were downregulated, and stress-responsive proteins were upregulated. This expression pattern was corroborated by the results of the DIGE analysis, which identified 79 differentially expressed proteins, with 23 identified (17 U and 6 D). Calreticulin and the proteasome subunits 20S and RPT5a were shown to be upregulated during infection by both 2-DE and DIGE analyses. These data may help shed light on plant responses against stresses and viral infections. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The role of MAP kinases in the induction of iNOS expression in neutrophils exposed to NDMA: the involvement transcription factors.

PubMed

Ratajczak-Wrona, W; Jablonska, E; Garley, M; Jablonski, J; Radziwon, P; Iwaniuk, A

2013-01-01

The role of MAP kinases in the activation of AP-1 (c-Jun, c-Fos) and NF-κB p65 engaged in the regulation of iNOS expression in human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was analyzed in the study. The study included a group of 20 healthy individuals. Isolated human PMN were incubated in the presence of NDMA. Selective MAP kinases inhibitors were used. The expression of proteins in the cytoplasmic and nuclear fractions was assessed using Western blot method. The results show that NDMA intensifies iNOS, c-Jun, NF-κB p65 and IκB-α expression in the analyzed PMNs. The blocking of the p38 pathway led to lower iNOS expression, and higher expression of c-Jun and c-Fos in the cytoplasmic fraction, and also lower c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. A decrease in iNOS expression in the cytoplasmic fraction, and also c-Jun in both fractions of the examined cells, was observed as a result of JNK pathway inhibition. The blocking of the ERK5 pathway led to higher iNOS, c-Jun and c-Fos expression in the cytoplasmic fraction, and higher c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. The study also demonstrated that blocking of the p38 and JNK pathways resulted in higher expression of NF-κB p65 and IκB-α in the cytoplasmic fraction and their lower expression in the nuclear fraction of these cells. Our data indicate the role of MAP kinases p38 and JNK in the activation of c-Jun and NF-κB p65 transcription factors engaged in the regulation of iNOS expression in human neutrophils exposed to NDMA. However ERK5 kinase is not involved in the regulation of iNOS and NO production by those cells.

Functions of bromodomain-containing proteins and their roles in homeostasis and cancer.

PubMed

Fujisawa, Takao; Filippakopoulos, Panagis

2017-04-01

Bromodomains (BRDs) are evolutionarily conserved protein-protein interaction modules that are found in a wide range of proteins with diverse catalytic and scaffolding functions and are present in most tissues. BRDs selectively recognize and bind to acetylated Lys residues - particularly in histones - and thereby have important roles in the regulation of gene expression. BRD-containing proteins are frequently dysregulated in cancer, they participate in gene fusions that generate diverse, frequently oncogenic proteins, and many cancer-causing mutations have been mapped to the BRDs themselves. Importantly, BRDs can be targeted by small-molecule inhibitors, which has stimulated many translational research projects that seek to attenuate the aberrant functions of BRD-containing proteins in disease.

Comparative bioinformatics analyses and profiling of lysosome-related organelle proteomes

NASA Astrophysics Data System (ADS)

Hu, Zhang-Zhi; Valencia, Julio C.; Huang, Hongzhan; Chi, An; Shabanowitz, Jeffrey; Hearing, Vincent J.; Appella, Ettore; Wu, Cathy

2007-01-01

Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for seven lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles.

Metabolic Genetic Screens Reveal Multidimensional Regulation of Virulence Gene Expression in Listeria monocytogenes and an Aminopeptidase That Is Critical for PrfA Protein Activation.

PubMed

Friedman, Sivan; Linsky, Marika; Lobel, Lior; Rabinovich, Lev; Sigal, Nadejda; Herskovits, Anat A

2017-06-01

Listeria monocytogenes is an environmental saprophyte and intracellular bacterial pathogen. Upon invading mammalian cells, the bacterium senses abrupt changes in its metabolic environment, which are rapidly transduced to regulation of virulence gene expression. To explore the relationship between L. monocytogenes metabolism and virulence, we monitored virulence gene expression dynamics across a library of genetic mutants grown under two metabolic conditions known to activate the virulent state: charcoal-treated rich medium containing glucose-1-phosphate and minimal defined medium containing limiting concentrations of branched-chain amino acids (BCAAs). We identified over 100 distinct mutants that exhibit aberrant virulence gene expression profiles, the majority of which mapped to nonessential metabolic genes. Mutants displayed enhanced, decreased, and early and late virulence gene expression profiles, as well as persistent levels, demonstrating a high plasticity in virulence gene regulation. Among the mutants, one was noteworthy for its particularly low virulence gene expression level and mapped to an X-prolyl aminopeptidase (PepP). We show that this peptidase plays a role in posttranslational activation of the major virulence regulator, PrfA. Specifically, PepP mediates recruitment of PrfA to the cytoplasmic membrane, a step identified as critical for PrfA protein activation. This study establishes a novel step in the complex mechanism of PrfA activation and further highlights the cross regulation of metabolism and virulence. Copyright © 2017 American Society for Microbiology.

The Human Pancreas Proteome Defined by Transcriptomics and Antibody-Based Profiling

PubMed Central

Fagerberg, Linn; Hallström, Björn M.; Schwenk, Jochen M.; Uhlén, Mathias; Korsgren, Olle; Lindskog, Cecilia

2014-01-01

The pancreas is composed of both exocrine glands and intermingled endocrine cells to execute its diverse functions, including enzyme production for digestion of nutrients and hormone secretion for regulation of blood glucose levels. To define the molecular constituents with elevated expression in the human pancreas, we employed a genome-wide RNA sequencing analysis of the human transcriptome to identify genes with elevated expression in the human pancreas. This quantitative transcriptomics data was combined with immunohistochemistry-based protein profiling to allow mapping of the corresponding proteins to different compartments and specific cell types within the pancreas down to the single cell level. Analysis of whole pancreas identified 146 genes with elevated expression levels, of which 47 revealed a particular higher expression as compared to the other analyzed tissue types, thus termed pancreas enriched. Extended analysis of in vitro isolated endocrine islets identified an additional set of 42 genes with elevated expression in these specialized cells. Although only 0.7% of all genes showed an elevated expression level in the pancreas, this fraction of transcripts, in most cases encoding secreted proteins, constituted 68% of the total mRNA in pancreas. This demonstrates the extreme specialization of the pancreas for production of secreted proteins. Among the elevated expression profiles, several previously not described proteins were identified, both in endocrine cells (CFC1, FAM159B, RBPJL and RGS9) and exocrine glandular cells (AQP12A, DPEP1, GATM and ERP27). In summary, we provide a global analysis of the pancreas transcriptome and proteome with a comprehensive list of genes and proteins with elevated expression in pancreas. This list represents an important starting point for further studies of the molecular repertoire of pancreatic cells and their relation to disease states or treatment effects. PMID:25546435

Chimeras taking shape: Potential functions of proteins encoded by chimeric RNA transcripts

PubMed Central

Frenkel-Morgenstern, Milana; Lacroix, Vincent; Ezkurdia, Iakes; Levin, Yishai; Gabashvili, Alexandra; Prilusky, Jaime; del Pozo, Angela; Tress, Michael; Johnson, Rory; Guigo, Roderic; Valencia, Alfonso

2012-01-01

Chimeric RNAs comprise exons from two or more different genes and have the potential to encode novel proteins that alter cellular phenotypes. To date, numerous putative chimeric transcripts have been identified among the ESTs isolated from several organisms and using high throughput RNA sequencing. The few corresponding protein products that have been characterized mostly result from chromosomal translocations and are associated with cancer. Here, we systematically establish that some of the putative chimeric transcripts are genuinely expressed in human cells. Using high throughput RNA sequencing, mass spectrometry experimental data, and functional annotation, we studied 7424 putative human chimeric RNAs. We confirmed the expression of 175 chimeric RNAs in 16 human tissues, with an abundance varying from 0.06 to 17 RPKM (Reads Per Kilobase per Million mapped reads). We show that these chimeric RNAs are significantly more tissue-specific than non-chimeric transcripts. Moreover, we present evidence that chimeras tend to incorporate highly expressed genes. Despite the low expression level of most chimeric RNAs, we show that 12 novel chimeras are translated into proteins detectable in multiple shotgun mass spectrometry experiments. Furthermore, we confirm the expression of three novel chimeric proteins using targeted mass spectrometry. Finally, based on our functional annotation of exon organization and preserved domains, we discuss the potential features of chimeric proteins with illustrative examples and suggest that chimeras significantly exploit signal peptides and transmembrane domains, which can alter the cellular localization of cognate proteins. Taken together, these findings establish that some chimeric RNAs are translated into potentially functional proteins in humans. PMID:22588898

Integrative Transcriptome Profiling of Cognitive Aging and Its Preservation through Ser/Thr Protein Phosphatase Regulation.

PubMed

Park, C Sehwan; Valomon, Amandine; Welzl, Hans

2015-01-01

Environmental enrichment has been reported to delay or restore age-related cognitive deficits, however, a mechanism to account for the cause and progression of normal cognitive decline and its preservation by environmental enrichment is lacking. Using genome-wide SAGE-Seq, we provide a global assessment of differentially expressed genes altered with age and environmental enrichment in the hippocampus. Qualitative and quantitative proteomics in naïve young and aged mice was used to further identify phosphorylated proteins differentially expressed with age. We found that increased expression of endogenous protein phosphatase-1 inhibitors in aged mice may be characteristic of long-term environmental enrichment and improved cognitive status. As such, hippocampus-dependent performances in spatial, recognition, and associative memories, which are sensitive to aging, were preserved by environmental enrichment and accompanied by decreased protein phosphatase activity. Age-associated phosphorylated proteins were also found to correspond to the functional categories of age-associated genes identified through transcriptome analysis. Together, this study provides a comprehensive map of the transcriptome and proteome in the aging brain, and elucidates endogenous protein phosphatase-1 inhibition as a potential means through which environmental enrichment may ameliorate age-related cognitive deficits.

Expression and signaling of G protein-coupled estrogen receptor 1 (GPER) in rat sertoli cells.

PubMed

Lucas, Thaís F G; Royer, Carine; Siu, Erica R; Lazari, Maria Fatima M; Porto, Catarina S

2010-08-01

The aim of the present study was to investigate the expression and signaling of the G protein-coupled estrogen receptor 1 (GPER) in cultured immature rat Sertoli cells--in which we have previously described the classical estrogen receptors (ESR1 and ESR2). Expression of GPER in cultured Sertoli cells from 15-day-old rats was detected by RT-PCR and immunoassays. Gper transcripts also were present in testes from 5-, 15-, and 120-day-old rats. Short-term treatment of Sertoli cells with 17beta-estradiol (E2), the GPER agonist G-1, or the ESR antagonist ICI 182,780 (ICI) rapidly activated MAPK3/1 (ERK1/2), even after down-regulation of ESR1 and ESR2, suggesting a role for GPER in the rapid E2 action in these cells. MAPK3/1 phosphorylation induced by ICI or G-1 was blocked by pertussis toxin, selective inhibitor of the SRC family of protein tyrosine kinases, metalloprotease inhibitor, MAP2K1/2 inhibitor, and epidermal growth factor receptor (EGFR) kinase inhibitor. Furthermore, E2, but not G-1, induced up-regulation of cyclin D1 in the Sertoli cells. This effect was blocked by ICI. E2 and G-1 decreased BAX and increased BCL2 expression and these effects were blocked by MAP2K1/2 inhibitor and EGFR kinase inhibitor. The pretreatment with ICI did not block the effect of E2. Taken together, these results indicate that in Sertoli cells 1) GPER-mediated MAPK3/1 activation occurs via EGFR transactivation through G protein beta gamma subunits that promote SRC-mediated metalloprotease-dependent release of EGFR ligands, which bind to EGFR and lead to MAPK3/1 phosphorylation; 2) E2-ESRs play a role in Sertoli cell proliferation; and 3) E2-GPER may regulate gene expression involved with apoptosis. ESR and GPER may mediate actions important for Sertoli cell function and maintenance of normal testis development and homeostasis.

Early transcutaneous electrical nerve stimulation reduces hyperalgesia and decreases activation of spinal glial cells in mice with neuropathic pain.

PubMed

Matsuo, Hideaki; Uchida, Kenzo; Nakajima, Hideaki; Guerrero, Alexander Rodriguez; Watanabe, Shuji; Takeura, Naoto; Sugita, Daisuke; Shimada, Seiichiro; Nakatsuka, Terumasa; Baba, Hisatoshi

2014-09-01

Although transcutaneous electrical nerve stimulation (TENS) is widely used for the treatment of neuropathic pain, its effectiveness and mechanism of action in reducing neuropathic pain remain uncertain. We investigated the effects of early TENS (starting from the day after surgery) in mice with neuropathic pain, on hyperalgesia, glial cell activation, pain transmission neuron sensitization, expression of proinflammatory cytokines, and opioid receptors in the spinal dorsal horn. Following nerve injury, TENS and behavioral tests were performed every day. Immunohistochemical, immunoblot, and flow cytometric analysis of the lumbar spinal cord were performed after 8 days. Early TENS reduced mechanical and thermal hyperalgesia and decreased the activation of microglia and astrocytes (P<0.05). In contrast, the application of TENS at 1 week (TENS-1w) or 2 weeks (TENS-2w) after injury was ineffective in reducing hyperalgesia (mechanical and thermal) or activation of microglia and astrocytes. Early TENS decreased p-p38 within microglia (P<0.05), the expression levels of protein kinase C (PKC-γ), and phosphorylated anti-phospho-cyclic AMP response element-binding protein (p-CREB) in the superficial spinal dorsal horn neurons (P<0.05), mitogen-activated protein (MAP) kinases, and proinflammatory cytokines, and increased the expression levels of opioid receptors (P<0.05). The results suggested that the application of early TENS relieved hyperalgesia in our mouse model of neuropathic pain by inhibiting glial activation, MAP kinase activation, PKC-γ, and p-CREB expression, and proinflammatory cytokines expression, as well as maintenance of spinal opioid receptors. The findings indicate that TENS treatment is more effective when applied as early after nerve injury as possible. Copyright © 2014 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Alternative Polyadenylation Directs Tissue-Specific miRNA Targeting in Caenorhabditis elegans Somatic Tissues

PubMed Central

Blazie, Stephen M.; Geissel, Heather C.; Wilky, Henry; Joshi, Rajan; Newbern, Jason; Mangone, Marco

2017-01-01

mRNA expression dynamics promote and maintain the identity of somatic tissues in living organisms; however, their impact in post-transcriptional gene regulation in these processes is not fully understood. Here, we applied the PAT-Seq approach to systematically isolate, sequence, and map tissue-specific mRNA from five highly studied Caenorhabditis elegans somatic tissues: GABAergic and NMDA neurons, arcade and intestinal valve cells, seam cells, and hypodermal tissues, and studied their mRNA expression dynamics. The integration of these datasets with previously profiled transcriptomes of intestine, pharynx, and body muscle tissues, precisely assigns tissue-specific expression dynamics for 60% of all annotated C. elegans protein-coding genes, providing an important resource for the scientific community. The mapping of 15,956 unique high-quality tissue-specific polyA sites in all eight somatic tissues reveals extensive tissue-specific 3′untranslated region (3′UTR) isoform switching through alternative polyadenylation (APA) . Almost all ubiquitously transcribed genes use APA and harbor miRNA targets in their 3′UTRs, which are commonly lost in a tissue-specific manner, suggesting widespread usage of post-transcriptional gene regulation modulated through APA to fine tune tissue-specific protein expression. Within this pool, the human disease gene C. elegans orthologs rack-1 and tct-1 use APA to switch to shorter 3′UTR isoforms in order to evade miRNA regulation in the body muscle tissue, resulting in increased protein expression needed for proper body muscle function. Our results highlight a major positive regulatory role for APA, allowing genes to counteract miRNA regulation on a tissue-specific basis. PMID:28348061

Alternative Polyadenylation Directs Tissue-Specific miRNA Targeting in Caenorhabditis elegans Somatic Tissues.

PubMed

Blazie, Stephen M; Geissel, Heather C; Wilky, Henry; Joshi, Rajan; Newbern, Jason; Mangone, Marco

2017-06-01

mRNA expression dynamics promote and maintain the identity of somatic tissues in living organisms; however, their impact in post-transcriptional gene regulation in these processes is not fully understood. Here, we applied the PAT-Seq approach to systematically isolate, sequence, and map tissue-specific mRNA from five highly studied Caenorhabditis elegans somatic tissues: GABAergic and NMDA neurons, arcade and intestinal valve cells, seam cells, and hypodermal tissues, and studied their mRNA expression dynamics. The integration of these datasets with previously profiled transcriptomes of intestine, pharynx, and body muscle tissues, precisely assigns tissue-specific expression dynamics for 60% of all annotated C. elegans protein-coding genes, providing an important resource for the scientific community. The mapping of 15,956 unique high-quality tissue-specific polyA sites in all eight somatic tissues reveals extensive tissue-specific 3'untranslated region (3'UTR) isoform switching through alternative polyadenylation (APA) . Almost all ubiquitously transcribed genes use APA and harbor miRNA targets in their 3'UTRs, which are commonly lost in a tissue-specific manner, suggesting widespread usage of post-transcriptional gene regulation modulated through APA to fine tune tissue-specific protein expression. Within this pool, the human disease gene C. elegans orthologs rack-1 and tct-1 use APA to switch to shorter 3'UTR isoforms in order to evade miRNA regulation in the body muscle tissue, resulting in increased protein expression needed for proper body muscle function. Our results highlight a major positive regulatory role for APA, allowing genes to counteract miRNA regulation on a tissue-specific basis. Copyright © 2017 Blazie et al.

MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia.

PubMed

Rowe, P S; de Zoysa, P A; Dong, R; Wang, H R; White, K E; Econs, M J; Oudet, C L

2000-07-01

Oncogenic hypophosphatemic osteomalacia (OHO) is characterized by a renal phosphate leak, hypophosphatemia, low-serum calcitriol (1,25-vitamin-D3), and abnormalities in skeletal mineralization. Resection of OHO tumors results in remission of the symptoms, and there is evidence that a circulating phosphaturic factor plays a role in the bone disease. This paper describes the characterization and cloning of a gene that is a candidate for the tumor-secreted phosphaturic factor. This new gene has been named MEPE (matrix extracellular phosphoglycoprotein) and has major similarities to a group of bone-tooth mineral matrix phospho-glycoproteins (osteopontin (OPN; HGMW-approved symbol SPP1), dentin sialo phosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein II (IBSP), and bone morphogenetic proteins (BMP). All the proteins including MEPE contain RGD sequence motifs that are proposed to be essential for integrin-receptor interactions. Of further interest is the finding that MEPE, OPN, DSPP, DMP1, IBSP, and BMP3 all map to a defined region in chromosome 4q. Refined mapping localizes MEPE to 4q21.1 between ESTs D4S2785 (WI-6336) and D4S2844 (WI-3770). MEPE is 525 residues in length with a short N-terminal signal peptide. High-level expression of MEPE mRNA occurred in all four OHO tumors screened. Three of 11 non-OHO tumors screened contained trace levels of MEPE expression (detected only after RT-PCR and Southern 32P analysis). Normal tissue expression was found in bone marrow and brain with very-low-level expression found in lung, kidney, and human placenta. Evidence is also presented for the tumor secretion of clusterin (HGMW-approved symbol CLU) and its possible role as a cytotoxic factor in one of the OHO patients described.

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase.

PubMed

Hatahet, Feras; Blazyk, Jessica L; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E; Beckwith, Jonathan; Boyd, Dana

2015-12-08

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase

PubMed Central

Hatahet, Feras; Blazyk, Jessica L.; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E.; Beckwith, Jonathan; Boyd, Dana

2015-01-01

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants. PMID:26598701

Up-regulated BAFF and BAFF receptor expression in patients with intractable temporal lobe epilepsy and a pilocarpine-induced epilepsy rat model.

PubMed

Ma, Limin; Li, Ruohan; Huang, Hao; Yuan, Jinxian; Ou, Shu; Xu, Tao; Yu, Xinyuan; Liu, Xi; Chen, Yangmei

2017-05-01

Some studies have suggested that BAFF and BAFFR are highly expressed in the central nervous system (CNS) and participate in inflammatory and immune associated diseases. However, whether BAFF and BAFFR are involved in the pathogenesis of epilepsy remains unknown. This study aimed to investigate the expression of BAFF and BAFFR proteins in the brains of patients with temporal lobe epilepsy (TLE) and in a pilocarpine-induced rat model of TLE to identify possible roles of the BAFF-BAFFR signaling pathway in epileptogenesis. Real-time quantitative polymerase chain reaction (RT-qPCR), western blot, immunohistochemistry, and double-immunofluorescence were performed in this study. The results showed that BAFF and BAFFR expression levels were markedly up-regulated in intractable TLE patients and TLE rats. Moreover, BAFF and BAFFR proteins mainly highly expressed in the membranes and cytoplasms of the dendritic marker MAP2 in the cortex and hippocampus. Therefore, the significant increased in BAFF and BAFFR protein expression in both TLE patients and rats suggest that BAFF and BAFFR may play important roles in regulating the pathogenesis of epilepsy. Copyright © 2017 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

Global analysis of WRKY transcription factor superfamily in Setaria identifies potential candidates involved in abiotic stress signaling

PubMed Central

Muthamilarasan, Mehanathan; Bonthala, Venkata S.; Khandelwal, Rohit; Jaishankar, Jananee; Shweta, Shweta; Nawaz, Kashif; Prasad, Manoj

2015-01-01

Transcription factors (TFs) are major players in stress signaling and constitute an integral part of signaling networks. Among the major TFs, WRKY proteins play pivotal roles in regulation of transcriptional reprogramming associated with stress responses. In view of this, genome- and transcriptome-wide identification of WRKY TF family was performed in the C4model plants, Setaria italica (SiWRKY) and S. viridis (SvWRKY), respectively. The study identified 105 SiWRKY and 44 SvWRKY proteins that were computationally analyzed for their physicochemical properties. Sequence alignment and phylogenetic analysis classified these proteins into three major groups, namely I, II, and III with majority of WRKY proteins belonging to group II (53 SiWRKY and 23 SvWRKY), followed by group III (39 SiWRKY and 11 SvWRKY) and group I (10 SiWRKY and 6 SvWRKY). Group II proteins were further classified into 5 subgroups (IIa to IIe) based on their phylogeny. Domain analysis showed the presence of WRKY motif and zinc finger-like structures in these proteins along with additional domains in a few proteins. All SiWRKY genes were physically mapped on the S. italica genome and their duplication analysis revealed that 10 and 8 gene pairs underwent tandem and segmental duplications, respectively. Comparative mapping of SiWRKY and SvWRKY genes in related C4 panicoid genomes demonstrated the orthologous relationships between these genomes. In silico expression analysis of SiWRKY and SvWRKY genes showed their differential expression patterns in different tissues and stress conditions. Expression profiling of candidate SiWRKY genes in response to stress (dehydration and salinity) and hormone treatments (abscisic acid, salicylic acid, and methyl jasmonate) suggested the putative involvement of SiWRKY066 and SiWRKY082 in stress and hormone signaling. These genes could be potential candidates for further characterization to delineate their functional roles in abiotic stress signaling. PMID:26635818

Global analysis of WRKY transcription factor superfamily in Setaria identifies potential candidates involved in abiotic stress signaling.

PubMed

Muthamilarasan, Mehanathan; Bonthala, Venkata S; Khandelwal, Rohit; Jaishankar, Jananee; Shweta, Shweta; Nawaz, Kashif; Prasad, Manoj

2015-01-01

Transcription factors (TFs) are major players in stress signaling and constitute an integral part of signaling networks. Among the major TFs, WRKY proteins play pivotal roles in regulation of transcriptional reprogramming associated with stress responses. In view of this, genome- and transcriptome-wide identification of WRKY TF family was performed in the C4model plants, Setaria italica (SiWRKY) and S. viridis (SvWRKY), respectively. The study identified 105 SiWRKY and 44 SvWRKY proteins that were computationally analyzed for their physicochemical properties. Sequence alignment and phylogenetic analysis classified these proteins into three major groups, namely I, II, and III with majority of WRKY proteins belonging to group II (53 SiWRKY and 23 SvWRKY), followed by group III (39 SiWRKY and 11 SvWRKY) and group I (10 SiWRKY and 6 SvWRKY). Group II proteins were further classified into 5 subgroups (IIa to IIe) based on their phylogeny. Domain analysis showed the presence of WRKY motif and zinc finger-like structures in these proteins along with additional domains in a few proteins. All SiWRKY genes were physically mapped on the S. italica genome and their duplication analysis revealed that 10 and 8 gene pairs underwent tandem and segmental duplications, respectively. Comparative mapping of SiWRKY and SvWRKY genes in related C4 panicoid genomes demonstrated the orthologous relationships between these genomes. In silico expression analysis of SiWRKY and SvWRKY genes showed their differential expression patterns in different tissues and stress conditions. Expression profiling of candidate SiWRKY genes in response to stress (dehydration and salinity) and hormone treatments (abscisic acid, salicylic acid, and methyl jasmonate) suggested the putative involvement of SiWRKY066 and SiWRKY082 in stress and hormone signaling. These genes could be potential candidates for further characterization to delineate their functional roles in abiotic stress signaling.

Adenosine-Induced Atrial Fibrillation: Localized Reentrant Drivers in Lateral Right Atria due to Heterogeneous Expression of Adenosine A1 Receptors and GIRK4 Subunits in the Human Heart.

PubMed

Li, Ning; Csepe, Thomas A; Hansen, Brian J; Sul, Lidiya V; Kalyanasundaram, Anuradha; Zakharkin, Stanislav O; Zhao, Jichao; Guha, Avirup; Van Wagoner, David R; Kilic, Ahmet; Mohler, Peter J; Janssen, Paul M L; Biesiadecki, Brandon J; Hummel, John D; Weiss, Raul; Fedorov, Vadim V

2016-08-09

Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor and its downstream GIRK (G protein-coupled inwardly rectifying potassium channels) channels (IK,Ado). We applied biatrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and nonfailing human hearts (n=37). Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10-100 µmol/L) produced more significant shortening of action potential durations in RA (from 290±45 to 239±41 ms, 17.3±10.4%; P<0.01) than LA (from 307±24 to 286±23 ms, 6.7±6.6%; P<0.01). In 10 hearts, adenosine induced AF (317±116 s) that, when sustained (≥2 minutes), was primarily maintained by 1 to 2 localized reentrant drivers in lateral RA. Tertiapin (10-100 nmol/L), a selective GIRK channel blocker, counteracted adenosine-induced action potential duration shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher adenosine A1 receptor (2.7±1.7-fold; P<0.01) and GIRK4 (1.7±0.8-fold; P<0.05) protein expression than lateral/posterior LA. This study revealed a 3-fold RA-to-LA adenosine A1 receptor protein expression gradient in the human heart, leading to significantly greater RA versus LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest adenosine A1 receptor/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine. © 2016 American Heart Association, Inc.

Constructing Proteome Reference Map of the Porcine Jejunal Cell Line (IPEC-J2) by Label-Free Mass Spectrometry.

PubMed

Kim, Sang Hoon; Pajarillo, Edward Alain B; Balolong, Marilen P; Lee, Ji Yoon; Kang, Dae-Kyung

2016-06-28

In this study, the global proteome of the IPEC-J2 cell line was evaluated using ultra-high performance liquid chromatography coupled to a quadrupole Q Exactive™ Orbitrap mass spectrometer. Proteins were isolated from highly confluent IPEC-J2 cells in biological replicates and analyzed by label-free mass spectrometry prior to matching against a porcine genomic dataset. The results identified 1,517 proteins, accounting for 7.35% of all genes in the porcine genome. The highly abundant proteins detected, such as actin, annexin A2, and AHNAK nucleoprotein, are involved in structural integrity, signaling mechanisms, and cellular homeostasis. The high abundance of heat shock proteins indicated their significance in cellular defenses, barrier function, and gut homeostasis. Pathway analysis and annotation using the Kyoto Encyclopedia of Genes and Genomes database resulted in a putative protein network map of the regulation of immunological responses and structural integrity in the cell line. The comprehensive proteome analysis of IPEC-J2 cells provides fundamental insights into overall protein expression and pathway dynamics that might be useful in cell adhesion studies and immunological applications.

Fine mapping of Restorer-of-fertility in pepper (Capsicum annuum L.) identified a candidate gene encoding a pentatricopeptide repeat (PPR)-containing protein.

PubMed

Jo, Yeong Deuk; Ha, Yeaseong; Lee, Joung-Ho; Park, Minkyu; Bergsma, Alex C; Choi, Hong-Il; Goritschnig, Sandra; Kloosterman, Bjorn; van Dijk, Peter J; Choi, Doil; Kang, Byoung-Cheorl

2016-10-01

Using fine mapping techniques, the genomic region co-segregating with Restorer - of - fertility ( Rf ) in pepper was delimited to a region of 821 kb in length. A PPR gene in this region, CaPPR6 , was identified as a strong candidate for Rf based on expression pattern and characteristics of encoding sequence. Cytoplasmic-genic male sterility (CGMS) has been used for the efficient production of hybrid seeds in peppers (Capsicum annuum L.). Although the mitochondrial candidate genes that might be responsible for cytoplasmic male sterility (CMS) have been identified, the nuclear Restorer-of-fertility (Rf) gene has not been isolated. To identify the genomic region co-segregating with Rf in pepper, we performed fine mapping using an Rf-segregating population consisting of 1068 F2 individuals, based on BSA-AFLP and a comparative mapping approach. Through six cycles of chromosome walking, the co-segregating region harboring the Rf locus was delimited to be within 821 kb of sequence. Prediction of expressed genes in this region based on transcription analysis revealed four candidate genes. Among these, CaPPR6 encodes a pentatricopeptide repeat (PPR) protein with PPR motifs that are repeated 14 times. Characterization of the CaPPR6 protein sequence, based on alignment with other homologs, showed that CaPPR6 is a typical Rf-like (RFL) gene reported to have undergone diversifying selection during evolution. A marker developed from a sequence near CaPPR6 showed a higher prediction rate of the Rf phenotype than those of previously developed markers when applied to a panel of breeding lines of diverse origin. These results suggest that CaPPR6 is a strong candidate for the Rf gene in pepper.

Direct integrin alphavbeta6-ERK binding: implications for tumour growth.

PubMed

Ahmed, Nuzhat; Niu, Jun; Dorahy, Douglas J; Gu, Xinhua; Andrews, Sarah; Meldrum, Cliff J; Scott, Rodney J; Baker, Mark S; Macreadie, Ian G; Agrez, Michael V

2002-02-21

Blockade of the mitogen-activated protein (MAP) kinase pathway suppresses growth of colon cancer in vivo. Here we demonstrate a direct link between the extracellular signal-regulated kinase ERK2 and the growth-promoting cell adhesion molecule, integrin alphavbeta6, in colon cancer cells. Down-regulation of beta6 integrin subunit expression inhibits tumour growth in vivo and MAP kinase activity in response to serum stimulation. In alphavbeta6-expressing cells ERK2 is bound only to the beta6 subunit. The increase in cytosolic MAP kinase activity upon epidermal growth factor stimulation is all accounted for by beta6-bound ERK. Deletion of the ERK2 binding site on the beta6 cytoplasmic domain inhibits tumour growth and leads to an association between ERK and the beta5 subunit. The physical interaction between integrin alphavbeta6 and ERK2 defines a novel paradigm of integrin-mediated signalling and provides a therapeutic target for cancer treatment.

Expression and purification of active mouse and human NEIL3 proteins

PubMed Central

Liu, Minmin; Bandaru, Viswanath; Holmes, Alicia; Averill, April M.; Cannan, Wendy

2012-01-01

Endonuclease VIII-like 3 (Neil3) is one of the five DNA glycosylases found in mammals that recognize and remove oxidized bases, and initiate the Base Excision Repair (BER) pathway. Previous attempts to express and purify the mouse and human orthologs of Neil3 in their active form have not been successful. Here we report the construction of bicistronic expression vectors for expressing in Escherichia coli the full-length mouse Neil3 (MmuNeil3), its glycosylase domain (MmuNeil3Δ324), as well as the glycosylase domain of human Neil3 (NEIL3Δ324). The purified Neil3 proteins are all active, and NEIL3Δ324 exhibits similar glycosylase/lyase activity as MmuNeil3Δ324 on both single-stranded and double-stranded substrates containing thymine glycol (Tg), spiroiminodihydantoin (Sp) or an abasic site (AP). We show that N-terminal initiator methionine processing is critical for the activity of both mouse and human Neil3 proteins. Co-expressing an E. coli methionine aminopeptidase (EcoMap) Y168A variant with MmuNeil3, MmuNeil3Δ324 and NEIL3Δ324 improves the N-terminal methionine processing and increases the percentage of active Neil3 proteins in the preparation. The purified Neil3 proteins are suitable for biochemical, structural and functional studies. PMID:22569481

Array-Based Gene Discovery with Three Unrelated Subjects Shows SCARB2/LIMP-2 Deficiency Causes Myoclonus Epilepsy and Glomerulosclerosis

PubMed Central

Berkovic, Samuel F.; Dibbens, Leanne M.; Oshlack, Alicia; Silver, Jeremy D.; Katerelos, Marina; Vears, Danya F.; Lüllmann-Rauch, Renate; Blanz, Judith; Zhang, Ke Wei; Stankovich, Jim; Kalnins, Renate M.; Dowling, John P.; Andermann, Eva; Andermann, Frederick; Faldini, Enrico; D'Hooge, Rudi; Vadlamudi, Lata; Macdonell, Richard A.; Hodgson, Bree L.; Bayly, Marta A.; Savige, Judy; Mulley, John C.; Smyth, Gordon K.; Power, David A.; Saftig, Paul; Bahlo, Melanie

2008-01-01

Action myoclonus-renal failure syndrome (AMRF) is an autosomal-recessive disorder with the remarkable combination of focal glomerulosclerosis, frequently with glomerular collapse, and progressive myoclonus epilepsy associated with storage material in the brain. Here, we employed a novel combination of molecular strategies to find the responsible gene and show its effects in an animal model. Utilizing only three unrelated affected individuals and their relatives, we used homozygosity mapping with single-nucleotide polymorphism chips to localize AMRF. We then used microarray-expression analysis to prioritize candidates prior to sequencing. The disorder was mapped to 4q13-21, and microarray-expression analysis identified SCARB2/Limp2, which encodes a lysosomal-membrane protein, as the likely candidate. Mutations in SCARB2/Limp2 were found in all three families used for mapping and subsequently confirmed in two other unrelated AMRF families. The mutations were associated with lack of SCARB2 protein. Reanalysis of an existing Limp2 knockout mouse showed intracellular inclusions in cerebral and cerebellar cortex, and the kidneys showed subtle glomerular changes. This study highlights that recessive genes can be identified with a very small number of subjects. The ancestral lysosomal-membrane protein SCARB2/LIMP-2 is responsible for AMRF. The heterogeneous pathology in the kidney and brain suggests that SCARB2/Limp2 has pleiotropic effects that may be relevant to understanding the pathogenesis of other forms of glomerulosclerosis or collapse and myoclonic epilepsies. PMID:18308289

Identification of a novel human kinase supporter of Ras (hKSR-2) that functions as a negative regulator of Cot (Tpl2) signaling.

PubMed

Channavajhala, Padma L; Wu, Leeying; Cuozzo, John W; Hall, J Perry; Liu, Wei; Lin, Lih-Ling; Zhang, Yuhua

2003-11-21

Kinase suppressor of Ras (KSR) is an integral and conserved component of the Ras signaling pathway. Although KSR is a positive regulator of the Ras/mitogen-activated protein (MAP) kinase pathway, the role of KSR in Cot-mediated MAPK activation has not been identified. The serine/threonine kinase Cot (also known as Tpl2) is a member of the MAP kinase kinase kinase (MAP3K) family that is known to regulate oncogenic and inflammatory pathways; however, the mechanism(s) of its regulation are not precisely known. In this report, we identify an 830-amino acid novel human KSR, designated hKSR-2, using predictions from genomic data base mining based on the structural profile of the KSR kinase domain. We show that, similar to the known human KSR, hKSR-2 co-immunoprecipitates with many signaling components of the Ras/MAPK pathway, including Ras, Raf, MEK-1, and ERK-1/2. In addition, we demonstrate that hKSR-2 co-immunoprecipitates with Cot and that co-expression of hKSR-2 with Cot significantly reduces Cot-mediated MAPK and NF-kappaB activation. This inhibition is specific to Cot, because Ras-induced ERK and IkappaB kinase-induced NF-kappaB activation are not significantly affected by hKSR-2 co-expression. Moreover, Cot-induced interleukin-8 production in HeLa cells is almost completely inhibited by the concurrent expression of hKSR-2, whereas transforming growth factor beta-activated kinase 1 (TAK1)/TAK1-binding protein 1 (TAB1)-induced interleukin-8 production is not affected by hKSR-2 co-expression. Taken together, these results indicate that hKSR-2, a new member of the KSR family, negatively regulates Cot-mediated MAP kinase and NF-kappaB pathway signaling.

Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants.

PubMed

Wistow, Graeme; Bernstein, Steven L; Wyatt, M Keith; Fariss, Robert N; Behal, Amita; Touchman, Jeffrey W; Bouffard, Gerald; Smith, Don; Peterson, Katherine

2002-06-15

The retinal pigment epithelium (RPE) and choroid comprise a functional unit of the eye that is essential to normal retinal health and function. Here we describe expressed sequence tag (EST) analysis of human RPE/choroid as part of a project for ocular bioinformatics. A cDNA library (cs) was made from human RPE/choroid and sequenced. Data were analyzed and assembled using the program GRIST (GRouping and Identification of Sequence Tags). Complete sequencing, Northern and Western blots, RH mapping, peptide antibody synthesis and immunofluorescence (IF) have been used to examine expression patterns and genome location for selected transcripts and proteins. Ten thousand individual sequence reads yield over 6300 unique gene clusters of which almost half have no matches with named genes. One of the most abundant transcripts is from a gene (named "alpha") that maps to the BBS1 region of chromosome 11. A number of tissue preferred transcripts are common to both RPE/choroid and iris. These include oculoglycan/opticin, for which an alternative splice form is detected in RPE/choroid, and "oculospanin" (Ocsp), a novel tetraspanin that maps to chromosome 17q. Antiserum to Ocsp detects expression in RPE, iris, ciliary body, and retinal ganglion cells by IF. A newly identified gene for a zinc-finger protein (TIRC) maps to 19q13.4. Variant transcripts of several genes were also detected. Most notably, the predominant form of Bestrophin represented in cs contains a longer open reading frame as a result of splice junction skipping. The unamplified cs library gives a view of the transcriptional repertoire of the adult RPE/choroid. A large number of potentially novel genes and splice forms and candidates for genetic diseases are revealed. Clones from this collection are being included in a large, nonredundant set for cDNA microarray construction.

Comparative proteomic analysis of differentially expressed proteins in the early milky stage of rice grains during high temperature stress

PubMed Central

Liao, Jiang-Lin; Zhou, Hui-Wen; Huang, Ying-Jin

2014-01-01

Rice yield and quality are adversely affected by high temperatures, and these effects are more pronounced at the ‘milky stage’ of the rice grain ripening phase. Identifying the functional proteins involved in the response of rice to high temperature stress may provide the basis for improving heat tolerance in rice. In the present study, a comparative proteomic analysis of paired, genetically similar heat-tolerant and heat-sensitive rice lines was conducted. Two-dimensional electrophoresis (2-DE) revealed a total of 27 differentially expressed proteins in rice grains, predominantly from the heat-tolerant lines. The protein profiles clearly indicated variations in protein expression between the heat-tolerant and heat-sensitive rice lines. Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis revealed that 25 of the 27 differentially displayed proteins were homologous to known functional proteins. These homologous proteins were involved in biosynthesis, energy metabolism, oxidation, heat shock metabolism, and the regulation of transcription. Seventeen of the 25 genes encoding the differentially displayed proteins were mapped to rice chromosomes according to the co-segregating conditions between the simple sequence repeat (SSR) markers and the target genes in recombinant inbred lines (RILs). The proteins identified in the present study provide a basis to elucidate further the molecular mechanisms underlying the adaptation of rice to high temperature stress. PMID:24376254

Identification of sequences in herpes simplex virus type 1 ICP22 that influence RNA polymerase II modification and viral late gene expression.

PubMed

Bastian, Thomas W; Rice, Stephen A

2009-01-01

Previous studies have shown that the herpes simplex virus type 1 (HSV-1) immediate-early protein ICP22 alters the phosphorylation of the host cell RNA polymerase II (Pol II) during viral infection. In this study, we have engineered several ICP22 plasmid and virus mutants in order to map the ICP22 sequences that are involved in this function. We identify a region in the C-terminal half of ICP22 (residues 240 to 340) that is critical for Pol II modification and further show that the N-terminal half of the protein (residues 1 to 239) is not required. However, immunofluorescence analysis indicates that the N-terminal half of ICP22 is needed for its localization to nuclear body structures. These results demonstrate that ICP22's effects on Pol II do not require that it accumulate in nuclear bodies. As ICP22 is known to enhance viral late gene expression during infection of certain cultured cells, including human embryonic lung (HEL) cells, we used our engineered viral mutants to map this function of ICP22. It was found that mutations in both the N- and C-terminal halves of ICP22 result in similar defects in viral late gene expression and growth in HEL cells, despite having distinctly different effects on Pol II. Thus, our results genetically uncouple ICP22's effects on Pol II from its effects on viral late gene expression. This suggests that these two functions of ICP22 may be due to distinct activities of the protein.

Integrative analysis and expression profiling of secondary cell wall genes in C4 biofuel model Setaria italica reveals targets for lignocellulose bioengineering

PubMed Central

Muthamilarasan, Mehanathan; Khan, Yusuf; Jaishankar, Jananee; Shweta, Shweta; Lata, Charu; Prasad, Manoj

2015-01-01

Several underutilized grasses have excellent potential for use as bioenergy feedstock due to their lignocellulosic biomass. Genomic tools have enabled identification of lignocellulose biosynthesis genes in several sequenced plants. However, the non-availability of whole genome sequence of bioenergy grasses hinders the study on bioenergy genomics and their genomics-assisted crop improvement. Foxtail millet (Setaria italica L.; Si) is a model crop for studying systems biology of bioenergy grasses. In the present study, a systematic approach has been used for identification of gene families involved in cellulose (CesA/Csl), callose (Gsl) and monolignol biosynthesis (PAL, C4H, 4CL, HCT, C3H, CCoAOMT, F5H, COMT, CCR, CAD) and construction of physical map of foxtail millet. Sequence alignment and phylogenetic analysis of identified proteins showed that monolignol biosynthesis proteins were highly diverse, whereas CesA/Csl and Gsl proteins were homologous to rice and Arabidopsis. Comparative mapping of foxtail millet lignocellulose biosynthesis genes with other C4 panicoid genomes revealed maximum homology with switchgrass, followed by sorghum and maize. Expression profiling of candidate lignocellulose genes in response to different abiotic stresses and hormone treatments showed their differential expression pattern, with significant higher expression of SiGsl12, SiPAL2, SiHCT1, SiF5H2, and SiCAD6 genes. Further, due to the evolutionary conservation of grass genomes, the insights gained from the present study could be extrapolated for identifying genes involved in lignocellulose biosynthesis in other biofuel species for further characterization. PMID:26583030

Integrative analysis and expression profiling of secondary cell wall genes in C4 biofuel model Setaria italica reveals targets for lignocellulose bioengineering.

PubMed

Muthamilarasan, Mehanathan; Khan, Yusuf; Jaishankar, Jananee; Shweta, Shweta; Lata, Charu; Prasad, Manoj

2015-01-01

Several underutilized grasses have excellent potential for use as bioenergy feedstock due to their lignocellulosic biomass. Genomic tools have enabled identification of lignocellulose biosynthesis genes in several sequenced plants. However, the non-availability of whole genome sequence of bioenergy grasses hinders the study on bioenergy genomics and their genomics-assisted crop improvement. Foxtail millet (Setaria italica L.; Si) is a model crop for studying systems biology of bioenergy grasses. In the present study, a systematic approach has been used for identification of gene families involved in cellulose (CesA/Csl), callose (Gsl) and monolignol biosynthesis (PAL, C4H, 4CL, HCT, C3H, CCoAOMT, F5H, COMT, CCR, CAD) and construction of physical map of foxtail millet. Sequence alignment and phylogenetic analysis of identified proteins showed that monolignol biosynthesis proteins were highly diverse, whereas CesA/Csl and Gsl proteins were homologous to rice and Arabidopsis. Comparative mapping of foxtail millet lignocellulose biosynthesis genes with other C4 panicoid genomes revealed maximum homology with switchgrass, followed by sorghum and maize. Expression profiling of candidate lignocellulose genes in response to different abiotic stresses and hormone treatments showed their differential expression pattern, with significant higher expression of SiGsl12, SiPAL2, SiHCT1, SiF5H2, and SiCAD6 genes. Further, due to the evolutionary conservation of grass genomes, the insights gained from the present study could be extrapolated for identifying genes involved in lignocellulose biosynthesis in other biofuel species for further characterization.

A Method for Comprehensive Glycosite-Mapping and Direct Quantitation of Serum Glycoproteins.

PubMed

Hong, Qiuting; Ruhaak, L Renee; Stroble, Carol; Parker, Evan; Huang, Jincui; Maverakis, Emanual; Lebrilla, Carlito B

2015-12-04

A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and nonspecific enzyme digestions followed by nano liquid chromatography (LC)-chip/quadrupole time-of-flight mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20 min ultra high performance (UP)LC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantitation method that involves protein enrichments. The absolute protein quantitation has a wide linear range (3-4 orders of magnitude) and low limit of quantitation (femtomole level). This rapid and robust quantitation technique, which provides quantitative information for both proteins and glycosylation, will further facilitate disease biomarker discoveries.

Contrasting modes and tempos of venom expression evolution in two snake species.

PubMed

Margres, Mark J; McGivern, James J; Seavy, Margaret; Wray, Kenneth P; Facente, Jack; Rokyta, Darin R

2015-01-01

Selection is predicted to drive diversification within species and lead to local adaptation, but understanding the mechanistic details underlying this process and thus the genetic basis of adaptive evolution requires the mapping of genotype to phenotype. Venom is complex and involves many genes, but the specialization of the venom gland toward toxin production allows specific transcripts to be correlated with specific toxic proteins, establishing a direct link from genotype to phenotype. To determine the extent of expression variation and identify the processes driving patterns of phenotypic diversity, we constructed genotype-phenotype maps and compared range-wide toxin-protein expression variation for two species of snake with nearly identical ranges: the eastern diamondback rattlesnake (Crotalus adamanteus) and the eastern coral snake (Micrurus fulvius). We detected significant expression variation in C. adamanteus, identified the specific loci associated with population differentiation, and found that loci expressed at all levels contributed to this divergence. Contrary to expectations, we found no expression variation in M. fulvius, suggesting that M. fulvius populations are not locally adapted. Our results not only linked expression variation at specific loci to divergence in a polygenic, complex trait but also have extensive conservation and biomedical implications. C. adamanteus is currently a candidate for federal listing under the Endangered Species Act, and the loss of any major population would result in the irrevocable loss of a unique venom phenotype. The lack of variation in M. fulvius has significant biomedical application because our data will assist in the development of effective antivenom for this species. Copyright © 2015 by the Genetics Society of America.

Proteomic analysis of tardigrades: towards a better understanding of molecular mechanisms by anhydrobiotic organisms.

PubMed

Schokraie, Elham; Hotz-Wagenblatt, Agnes; Warnken, Uwe; Mali, Brahim; Frohme, Marcus; Förster, Frank; Dandekar, Thomas; Hengherr, Steffen; Schill, Ralph O; Schnölzer, Martina

2010-03-03

Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades.

Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

PubMed Central

Schokraie, Elham; Hotz-Wagenblatt, Agnes; Warnken, Uwe; Mali, Brahim; Frohme, Marcus; Förster, Frank; Dandekar, Thomas; Hengherr, Steffen; Schill, Ralph O.; Schnölzer, Martina

2010-01-01

Background Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. Principal Findings Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. Conclusions The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades. PMID:20224743

Structure and Expression of Genes for Flavivirus Immunogens.

DTIC Science & Technology

1985-09-01

the same order in YFV i.e., C-M-E-NSI--- NS3---NS5 and an open reading frame extends at least through the C-M-E-NS1 coding region, consistent with...been determined (Castle et al., 1985). Comparison of these results shows that 1) the six major JEV genes mapped thus far occur in the same order in YFV ...pre-M proteins and 3) the predicted structures of the E, NSI and ns2a proteins of JEV and YFV exhibit a high degree of relatedness. The E proteins

Mammalian enabled (Mena) is a critical regulator of cardiac function

PubMed Central

Aguilar, Frédérick; Belmonte, Stephen L.; Ram, Rashmi; Noujaim, Sami F.; Dunaevsky, Olga; Protack, Tricia L.; Jalife, Jose; Todd Massey, H.; Gertler, Frank B.

2011-01-01

Mammalian enabled (Mena) of the Drosophila enabled/vasodilator-stimulated phosphoprotein gene family is a cytoskeletal protein implicated in actin regulation and cell motility. Cardiac Mena expression is enriched in intercalated discs (ICD), the critical intercellular communication nexus between adjacent muscle cells. We previously identified Mena gene expression to be a key predictor of human and murine heart failure (HF). To determine the in vivo function of Mena in the heart, we assessed Mena protein expression in multiple HF models and characterized the effects of genetic Mena deletion on cardiac structure and function. Immunoblot analysis revealed significant upregulation of Mena protein expression in left ventricle tissue from patients with end-stage HF, calsequestrin-overexpressing mice, and isoproterenol-infused mice. Characterization of the baseline cardiac function of adult Mena knockout mice (Mena−/−) via echocardiography demonstrated persistent cardiac dysfunction, including a significant reduction in percent fractional shortening compared with wild-type littermates. Electrocardiogram PR and QRS intervals were significantly prolonged in Mena−/− mice, manifested by slowed conduction on optical mapping studies. Ultrastructural analysis of Mena−/− hearts revealed disrupted organization and widening of ICD structures, mislocalization of the gap junction protein connexin 43 (Cx43) to the lateral borders of cardiomyoycytes, and increased Cx43 expression. Furthermore, the expression of vinculin (an adherens junction protein) was significantly reduced in Mena−/− mice. We report for the first time that genetic ablation of Mena results in cardiac dysfunction, highlighted by diminished contractile performance, disrupted ICD structure, and slowed electrical conduction. PMID:21335464

Mammalian enabled (Mena) is a critical regulator of cardiac function.

PubMed

Aguilar, Frédérick; Belmonte, Stephen L; Ram, Rashmi; Noujaim, Sami F; Dunaevsky, Olga; Protack, Tricia L; Jalife, Jose; Todd Massey, H; Gertler, Frank B; Blaxall, Burns C

2011-05-01

Mammalian enabled (Mena) of the Drosophila enabled/vasodilator-stimulated phosphoprotein gene family is a cytoskeletal protein implicated in actin regulation and cell motility. Cardiac Mena expression is enriched in intercalated discs (ICD), the critical intercellular communication nexus between adjacent muscle cells. We previously identified Mena gene expression to be a key predictor of human and murine heart failure (HF). To determine the in vivo function of Mena in the heart, we assessed Mena protein expression in multiple HF models and characterized the effects of genetic Mena deletion on cardiac structure and function. Immunoblot analysis revealed significant upregulation of Mena protein expression in left ventricle tissue from patients with end-stage HF, calsequestrin-overexpressing mice, and isoproterenol-infused mice. Characterization of the baseline cardiac function of adult Mena knockout mice (Mena(-/-)) via echocardiography demonstrated persistent cardiac dysfunction, including a significant reduction in percent fractional shortening compared with wild-type littermates. Electrocardiogram PR and QRS intervals were significantly prolonged in Mena(-/-) mice, manifested by slowed conduction on optical mapping studies. Ultrastructural analysis of Mena(-/-) hearts revealed disrupted organization and widening of ICD structures, mislocalization of the gap junction protein connexin 43 (Cx43) to the lateral borders of cardiomyoycytes, and increased Cx43 expression. Furthermore, the expression of vinculin (an adherens junction protein) was significantly reduced in Mena(-/-) mice. We report for the first time that genetic ablation of Mena results in cardiac dysfunction, highlighted by diminished contractile performance, disrupted ICD structure, and slowed electrical conduction.

Xcat, a novel mouse model for Nance-Horan syndrome inhibits expression of the cytoplasmic-targeted Nhs1 isoform.

PubMed

Huang, Kristen M; Wu, Junhua; Duncan, Melinda K; Moy, Chris; Dutra, Amalia; Favor, Jack; Da, Tong; Stambolian, Dwight

2006-01-15

Nance-Horan syndrome (NHS) is an X-linked disorder characterized by congenital cataracts, dental anomalies, dysmorphic features and mental retardation. A recent report suggests that the novel gene NHS1 is involved in this disorder due to the presence of point mutations in NHS patients. A possible mouse model for NHS, Xcat, was mapped to a 2.11 Mb interval on the X-chromosome. Sequence and FISH analysis of the X-chromosome region containing the Xcat mutation reveal a large insertion between exons 1 and 2 of the mouse Nhs1 gene. The insertion inhibits the expression of the Nhs1 isoform containing exon 1 and results in exclusive expression of the alternative isoform containing exon 1A. Quantitative RT-PCR of Xcat cDNA shows reduced levels of Nhs1 transcripts. The Nhs1 protein is strongly expressed within the cytoplasm of elongating lens fiber cells from wild-type neonate lens, but is significantly reduced within the Xcat lens. Transient transfection studies of CHO cells with Nhs1-GFP fusion proteins were done to determine whether the amino acids encoded by exon 1 were critical for protein localization. We found the presence of Nhs1 exon 1 critical for localization of the fusion protein to the cytoplasm, whereas fusion proteins lacking Nhs1 exon 1 are predominantly nuclear. These results indicate that the first exon of Nhs1 contains crucial information required for the proper expression and localization of Nhs1 protein. Inhibition of expression of the exon 1 containing isoform results in the abnormal phenotype of Xcat.

Identification and Comparative Expression Profiles of Chemoreception Genes Revealed from Major Chemoreception Organs of the Rice Leaf Folder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

PubMed Central

Zeng, Fang-Fang; Zhao, Zhen-Fei; Yan, Miao-Jun; Zhou, Wen; Zhang, Zan; Zhang, Aijun; Lu, Zhong-Xian; Wang, Man-Qun

2015-01-01

To better understand the olfactory mechanisms in the rice leaf folder, Cnaphalocrocis medinalis (Guenée), a serious pest of rice in Asia, we established six partial transcriptomes from antennae, protarsus, and reproductive organs of male and female adults. A total of 102 transcripts were identified, including 29 odorant receptors (ORs), 15 ionotropic receptors (IRs), 30 odorant-binding proteins (OBPs), 26 chemosensory proteins (CSPs), and 2 sensory neuron membrane proteins (SNMPs). The expression patterns of these genes were calculated by fragments per kilobase of exon per million fragments mapped (FPKM) and validated by real-time quantitative PCR (RT-qPCR). Some transcripts were exclusively expressed in specific organs, such as female protarsus, whereas others were universally expressed, this varied expression profile may provide insights into the specific functions mediated by chemoreception proteins in insects. To the best of our knowledge, among the 102 identified transcripts, 81 are novel and have never been reported before. In addition, it also is the first time that ORs and IRs are identified in C. medinalis. Our findings significantly enhance the currently limited understanding olfactory mechanisms of the olfactory mechanisms underlying the chemoreception system in C. medinalis. PMID:26657286

GhMAP3K65, a Cotton Raf-Like MAP3K Gene, Enhances Susceptibility to Pathogen Infection and Heat Stress by Negatively Modulating Growth and Development in Transgenic Nicotiana benthamiana.

PubMed

Zhai, Na; Jia, Haihong; Liu, Dongdong; Liu, Shuchang; Ma, Manli; Guo, Xingqi; Li, Han

2017-11-21

Mitogen-activated protein kinase kinase kinases (MAP3Ks), the top components of MAPK cascades, modulate many biological processes, such as growth, development and various environmental stresses. Nevertheless, the roles of MAP3Ks remain poorly understood in cotton. In this study, GhMAP3K65 was identified in cotton, and its transcription was inducible by pathogen infection, heat stress, and multiple signalling molecules. Silencing of GhMAP3K65 enhanced resistance to pathogen infection and heat stress in cotton. In contrast, overexpression of GhMAP3K65 enhanced susceptibility to pathogen infection and heat stress in transgenic Nicotiana benthamiana . The expression of defence-associated genes was activated in transgenic N. benthamiana plants after pathogen infection and heat stress, indicating that GhMAP3K65 positively regulates plant defence responses. Nevertheless, transgenic N. benthamiana plants impaired lignin biosynthesis and stomatal immunity in their leaves and repressed vitality of their root systems. In addition, the expression of lignin biosynthesis genes and lignin content were inhibited after pathogen infection and heat stress. Collectively, these results demonstrate that GhMAP3K65 enhances susceptibility to pathogen infection and heat stress by negatively modulating growth and development in transgenic N. benthamiana plants.

Proteomic analysis of ethanol-induced embryotoxicity in cultured post-implantation rat embryos.

PubMed

Usami, Makoto; Mitsunaga, Katsuyoshi; Irie, Tomohiko; Miyajima, Atsuko; Doi, Osamu

2014-04-01

Protein expression changes were examined in day 10.5 rat embryos cultured for 24 hr in the presence of ethanol by using two-dimensional electrophoresis and mass spectrometry. Exposure to ethanol resulted in quantitative changes in many embryonic protein spots (16 decreased and 28 increased) at in vitro embryotoxic concentrations (130 and 195 mM); most changes occurred in a concentration-dependent manner. For these protein spots, 17 proteins were identified, including protein disulfide isomerase A3, alpha-fetoprotein, phosphorylated cofilin-1, and serum albumin. From the gene ontology classification and pathway mapping of the identified proteins, it was found that ethanol affected several biological processes involving oxidative stress and retinoid metabolism.

A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif

NASA Technical Reports Server (NTRS)

Reddy, A. S.; Reddy, V. S.; Golovkin, M.

2000-01-01

Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

EGFR and Ras regulate DDX59 during lung cancer development.

PubMed

Yang, Lin; Zhang, Hanyin; Chen, Dan; Ding, Peikun; Yuan, Yunchang; Zhang, Yandong

2018-02-05

Oncogenes EGFR and ras are frequently mutated and activated in human lung cancers. In this report, we found that both EGFR and Ras signaling can upregulate RNA helicase DDX59 in lung cancer cells. DDX59 can be induced through the mitogen activated protein kinase (MAPK) pathway after EGFR or Ras activation. Inhibitors for Ras/Raf/MAP pathway significantly decreased DDX59 expression at both protein and mRNA levels. Through immunohistochemistry, we found that DDX59 protein expression correlated with Ras and EGFR mutation status in human lung adenocarcinoma. Finally, through a xenograft nude mice model, we demonstrated that DDX59 is pivotal for EGFR mutated lung cancer cell growth in vivo. Our study identified a novel protein downstream of Ras and EGFR, which may serve as a potential therapeutic drug target for lung cancer patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Genetic dissection of seed oil and protein content and identification of networks associated with oil content in Brassica napus

PubMed Central

Chao, Hongbo; Wang, Hao; Wang, Xiaodong; Guo, Liangxing; Gu, Jianwei; Zhao, Weiguo; Li, Baojun; Chen, Dengyan; Raboanatahiry, Nadia; Li, Maoteng

2017-01-01

High-density linkage maps can improve the precision of QTL localization. A high-density SNP-based linkage map containing 3207 markers covering 3072.7 cM of the Brassica napus genome was constructed in the KenC-8 × N53-2 (KNDH) population. A total of 67 and 38 QTLs for seed oil and protein content were identified with an average confidence interval of 5.26 and 4.38 cM, which could explain up to 22.24% and 27.48% of the phenotypic variation, respectively. Thirty-eight associated genomic regions from BSA overlapped with and/or narrowed the SOC-QTLs, further confirming the QTL mapping results based on the high-density linkage map. Potential candidates related to acyl-lipid and seed storage underlying SOC and SPC, respectively, were identified and analyzed, among which six were checked and showed expression differences between the two parents during different embryonic developmental periods. A large primary carbohydrate pathway based on potential candidates underlying SOC- and SPC-QTLs, and interaction networks based on potential candidates underlying SOC-QTLs, was constructed to dissect the complex mechanism based on metabolic and gene regulatory features, respectively. Accurate QTL mapping and potential candidates identified based on high-density linkage map and BSA analyses provide new insights into the complex genetic mechanism of oil and protein accumulation in the seeds of rapeseed. PMID:28393910

Tissue Molecular Anatomy Project (TMAP): an expression database for comparative cancer proteomics.

PubMed

Medjahed, Djamel; Luke, Brian T; Tontesh, Tawady S; Smythers, Gary W; Munroe, David J; Lemkin, Peter F

2003-08-01

By mining publicly accessible databases, we have developed a collection of tissue-specific predictive protein expression maps as a function of cancer histological state. Data analysis is applied to the differential expression of gene products in pooled libraries from the normal to the altered state(s). We wish to report the initial results of our survey across different tissues and explore the extent to which this comparative approach may help uncover panels of potential biomarkers of tumorigenesis which would warrant further examination in the laboratory.

Linkage Map of Escherichia coli K-12, Edition 10: The Traditional Map

PubMed Central

Berlyn, Mary K. B.

1998-01-01

This map is an update of the edition 9 map by Berlyn et al. (M. K. B. Berlyn, K. B. Low, and K. E. Rudd, p. 1715–1902, in F. C. Neidhardt et al., ed., Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed., vol. 2, 1996). It uses coordinates established by the completed sequence, expressed as 100 minutes for the entire circular map, and adds new genes discovered and established since 1996 and eliminates those shown to correspond to other known genes. The latter are included as synonyms. An alphabetical list of genes showing map location, synonyms, the protein or RNA product of the gene, phenotypes of mutants, and reference citations is provided. In addition to genes known to correspond to gene sequences, other genes, often older, that are described by phenotype and older mapping techniques and that have not been correlated with sequences are included. PMID:9729611

The Effect of Selenium Enrichment on Baker s Yeast Proteome

PubMed Central

El-Bayoumy, Karam; Das, Arunangshu; Russell, Stephen; Wolfe, Steven; Jordan, Rick; Renganathan, Kutralanathan; Loughran, Thomas P.; Somiari, Richard

2011-01-01

The use of regular yeast (RY) and selenium-enriched yeast (SEY) as dietary supplement is of interest because the Nutritional Prevention of Cancer (NPC) trial revealed that SEY but not RY decreased the incidence of prostate cancer (PC). Using two-dimensional difference in gel electrophoresis (2D-DIGE) – tandem mass spectrometry (MS/MS) approach, we performed proteomic analysis of RY and SEY to identify proteins that are differentially expressed as a result of selenium enrichment. 2D-DIGE revealed 96 candidate protein spots that were differentially expressed (p≤0.05) between SEY and RY. The 96 spots were selected, sequenced by LC/MS/MS and 37 proteins were unequivocally identified. The 37 identified proteins were verified with ProteinProphet software and mapped to existing Gene Ontology categories. Furthermore, the expression profile of 5 of the proteins with validated or putative roles in the carcinogenesis process, and for which antibodies against human forms of the proteins are available commercially were verified by western analysis. This study provides evidence for the first time that SEY contains higher levels of Pyruvate Kinase, HSP70, and Elongation factor 2 and lower levels of Eukaryotic Translation Initiation Factor 5A-2 and Triosephosphate Isomerase than those found in RY. PMID:22067702

Expression of the Beet necrotic yellow vein virus p25 protein induces hormonal changes and a root branching phenotype in Arabidopsis thaliana.

PubMed

Peltier, Claire; Schmidlin, Laure; Klein, Elodie; Taconnat, Ludivine; Prinsen, Els; Erhardt, Mathieu; Heintz, Dimitri; Weyens, Guy; Lefebvre, Marc; Renou, Jean-Pierre; Gilmer, David

2011-06-01

The RNA-3-encoded p25 protein was previously characterized as one of the major symptom determinants of the Beet necrotic yellow vein virus. Previous analyses reported the influence of the p25 protein in root proliferation phenotype observed in rhizomania disease on infected sugar beets (Beta vulgaris). A transgenic approach was developed, in which the p25 protein was constitutively expressed in Arabidopsis thaliana Columbia (Col-0) ecotype in order to provide new clues as to how the p25 protein might promote alone disease development and symptom expression. Transgenic plants were characterized by Southern blot and independent lines carrying single and multiple copies of the transgene were selected. Mapping of the T-DNA insertion was performed on the monocopy homozygote lines. P25 protein was localized both in the nucleus and in the cytoplasm of epidermal and root cells of transgenic plants. Although A. thaliana was not described as a susceptible host for BNYVV infection, abnormal root branching was observed on p25 protein-expressing A. thaliana plants. Moreover, these transgenic plants were more susceptible than wild-type plants to auxin analog treatment (2,4-D) but more resistant to methyl jasmonate (MeJA), abscisic acid (ABA) and to lesser extend to salicylic acid (SA). Hormonal content assays measuring plant levels of auxin (IAA), jasmonate (JA) and ethylene precursor (ACC) revealed major hormonal changes. Global transcript profiling analyses on roots displayed differential gene expressions that could corroborate root branching phenotype and stress signaling modifications.

Stress and vascular responses: atheroprotective effect of laminar fluid shear stress in endothelial cells: possible role of mitogen-activated protein kinases.

PubMed

Yoshizumi, Masanori; Abe, Jun-Ichi; Tsuchiya, Koichiro; Berk, Bradford C; Tamaki, Toshiaki

2003-03-01

Atherosclerosis preferentially occurs in areas of turbulent blood flow and low fluid shear stress, whereas laminar blood flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent findings suggest a steady laminar blood flow decreases EC apoptosis and inhibits TNF-mediated EC activation. EC apoptosis or activation is suggested to be involved in plaque erosion, which may lead to platelet aggregation. TNF-alpha regulates gene expression in ECs, in part, by stimulating mitogen-activated protein (MAP) kinases, which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAP kinases in ECs. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm(2)) on TNF-alpha-stimulated activity of three MAP kinases in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. TNF-alpha activated ERK1/2, JNK, and p38 maximally at 15 min in HUVEC. Pre-exposing HUVEC for 10 min to flow inhibited TNF-alpha activation of JNK, but showed no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, a specific ERK1/2 inhibitor, blocked the flow-mediated inhibition of TNF activation of JNK. Transfection studies with dominant-negative constructs of the protein kinase MEK5 suggested an important role for big mitogen-activated protein kinase 1 (BMK1) in flow-mediated regulation of EC activation by TNF-alpha. Understanding the mechanisms by which steady laminar flow regulates JNK activation by cytokines may provide insight into the atheroprotective mechanisms induced by laminar blood flow.

Localized Disruption of Narp in Medial Prefrontal Cortex Blocks Reinforcer Devaluation Performance

ERIC Educational Resources Information Center

Johnson, Alexander W.; Han, Sungho; Blouin, Ashley M.; Saini, Jasjit; Worley, Paul F.; During, Matthew J.; Holland, Peter C.; Baraban, Jay M.; Reti, Irving M.

2010-01-01

Neuronal activity regulated pentraxin (Narp) is a secreted protein that regulates [alpha]-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPAR) aggregation and synaptogenesis. Mapping of Narp-positive neurons in brain has revealed it is prominently expressed in several limbic system projection pathways. Consistent with this…

Immunotargeting and cloning of two CD34 variants exhibiting restricted expression in adult rat endothelia in vivo.

PubMed

Testa, Jacqueline E; Chrastina, Adrian; Oh, Phil; Li, Yan; Witkiewicz, Halina; Czarny, Malgorzata; Buss, Tim; Schnitzer, Jan E

2009-08-01

Mapping protein expression of endothelial cells (EC) in vivo is fundamental to understanding cellular function and may yield new tissue-selective targets. We have developed a monoclonal antibody, MAb J120, to a protein expressed primarily in rat lung and heart endothelium. The antigen was identified as CD34, a marker of hematopoietic stem cells and global marker of endothelial cells in human and mouse tissues. PCR-based cloning identified two CD34 variant proteins, full length and truncated, both of which are expressed on luminal endothelial cell plasma membranes (P) isolated from lung. Truncated CD34 predominated in heart P, and neither variant was detected in P from kidney or liver. CD34 in lung was readily accessible to (125)I-J120 inoculated intravenously, and immunohistochemistry showed strong CD34 expression in lung EC. Few microvessels stained in heart and kidney, and no CD34 was detected in vessels of other organs or in lymphatics. We present herein the first complete sequence of a rat CD34 variant and show for the first time that the encoded truncated variant is endogenously expressed on EC in vivo. We also demonstrate that CD34 expression in rat EC, unlike mouse and human, is restricted in its distribution enabling quite specific lung targeting in vivo.

Porcine circovirus-2 capsid protein induces cell death in PK15 cells

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

Walia, Rupali; Dardari, Rkia, E-mail: rdardari@ucalgary.ca; Chaiyakul, Mark

Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathwaysmore » involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.« less

RIPiT-Seq: A high-throughput approach for footprinting RNA:protein complexes

PubMed Central

Singh, Guramrit; Ricci, Emiliano P.; Moore, Melissa J.

2013-01-01

Development of high-throughput approaches to map the RNA interaction sites of individual RNA binding proteins (RBPs) transcriptome-wide is rapidly transforming our understanding of post-transcriptional gene regulatory mechanisms. Here we describe a ribonucleoprotein (RNP) footprinting approach we recently developed for identifying occupancy sites of both individual RBPs and multi-subunit RNP complexes. RNA:protein immunoprecipitation in tandem (RIPiT) yields highly specific RNA footprints of cellular RNPs isolated via two sequential purifications; the resulting RNA footprints can then be identified by high-throughput sequencing (Seq). RIPiT-Seq is broadly applicable to all RBPs regardless of their RNA binding mode and thus provides a means to map the RNA binding sites of RBPs with poor inherent ultraviolet (UV) crosslinkability. Further, among current high-throughput approaches, RIPiT has the unique capacity to differentiate binding sites of RNPs with overlapping protein composition. It is therefore particularly suited for studying dynamic RNP assemblages whose composition evolves as gene expression proceeds. PMID:24096052

Analysis of the complete nucleotide sequence and functional organization of the genome of Streptococcus pneumoniae bacteriophage Cp-1.

PubMed

Martín, A C; López, R; García, P

1996-06-01

Cp-1, a bacteriophage infecting Streptococcus pneumoniae, has a linear double-stranded DNA genome, with a terminal protein covalently linked to its 5' ends, that replicates by the protein-priming mechanism. We describe here the complete DNA sequence and transcriptional map of the Cp-1 genome. These analyses have led to the firm assignment of 10 genes and the localization of 19 additional open reading frames in the 19,345-bp Cp-1 DNA. Striking similarities and differences between some of these proteins and those of the Bacillus subtilis phage phi 29, a system that also replicates its DNA by the protein-priming mechanism, have been revealed. The genes coding for structural proteins and assembly factors are located in the central part of the Cp-1 genome. Several proteins corresponding to the predicted gene products were identified by in vitro and in vivo expression of the cloned genes. Mature major head protein from the virion particles results from hydrolysis of the primary gene product at the His-49 residue, whereas the phage gene is expressed in Escherichia coli without modification. We have also identified two open reading frames coding for proteins that show high degrees of similarity to the N- and C-terminal regions, respectively, of the single tail protein identified in phi 29. Sequencing and primer extension analysis suggest transcription of a small RNA showing a secondary structure similar to that of the prohead RNA required for the ATP-dependent packaging of phi 29 DNA. On the basis of its temporal expression, transcription of the Cp-1 genome takes place in two stages, early and late. Combined Northern (RNA) blot and primer extension experiments allowed us to map the 5' initiation sites of the transcripts, and we found that only three genes were transcribed from right to left. These analyses reveal that there are also noticeable differences between Cp-l and phi 29 in transcriptional organization. Considered together, the observations reported here provide new tangible evidence on phylogenetic relationships between B. subtilis and S. pneumoniae.

Recovery from retinal lesions: molecular plasticity mechanisms in visual cortex far beyond the deprived zone.

PubMed

Hu, Tjing-Tjing; Van den Bergh, Gert; Thorrez, Lieven; Heylen, Kevin; Eysel, Ulf T; Arckens, Lutgarde

2011-12-01

In cats with central retinal lesions, deprivation of the lesion projection zone (LPZ) in primary visual cortex (area 17) induces remapping of the cortical topography. Recovery of visually driven cortical activity in the LPZ involves distinct changes in protein expression. Recent observations, about molecular activity changes throughout area 17, challenge the view that its remote nondeprived parts would not be involved in this recovery process. We here investigated the dynamics of the protein expression pattern of remote nondeprived area 17 triggered by central retinal lesions to explore to what extent far peripheral area 17 would contribute to the topographic map reorganization inside the visual cortex. Using functional proteomics, we identified 40 proteins specifically differentially expressed between far peripheral area 17 of control and experimental animals 14 days to 8 months postlesion. Our results demonstrate that far peripheral area 17 is implicated in the functional adaptation to the visual deprivation, involving a meshwork of interacting proteins, operating in diverse pathways. In particular, endocytosis/exocytosis processes appeared to be essential via their intimate correlation with long-term potentiation and neurite outgrowth mechanisms.

Molecular function of microtubule-associated protein 2 for filial imprinting in domestic chicks (Gallus gallus domesticus).

PubMed

Yamaguchi, Shinji; Katagiri, Sachiko; Aoki, Naoya; Iikubo, Eiji; Kitajima, Takaaki; Matsushima, Toshiya; Homma, Koichi J

2011-01-01

RNA interference (RNAi)-mediated gene-silencing can be a tool for elucidating the role of genes in the neural basis of behavioral plasticity. Previously, we reported that exogenous DNA could be successfully delivered into newly-hatched chick brains via electroporation. Here, we used this in vivo gene-transfer technique and showed that transfected microRNA vectors preferentially silence exogenous DNA expression in neuronal cells. Using this system, the up-regulation of microtubule-associated protein 2 (MAP2) accompanying filial imprinting was suppressed in vivo, which impaired the filial imprinting in chicks. In addition, the phosphorylation of MAP2 was found to increase in parallel with filial imprinting, and lithium chloride, an inhibitor of glycogen synthase kinase 3 (GSK3), was found to impair filial imprinting. Our results suggest that the regulation of MAP2 expression and its phosphorylation are required for filial imprinting and may modify microtubule stability, thereby leading to cytoskeletal reorganization during imprinting. This in vivo RNAi-mediated gene-silencing system will facilitate the analysis of gene function in the living chick brain and provides further clues regarding the molecular mechanisms underpinning avian learning. Copyright © 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

LipC (Rv0220) Is an Immunogenic Cell Surface Esterase of Mycobacterium tuberculosis

PubMed Central

Shen, Guomiao; Singh, Krishna; Chandra, Dinesh; Serveau-Avesque, Carole; Maurin, Damien; Canaan, Stéphane; Singla, Rupak; Behera, Digambar

2012-01-01

We have reported previously the identification of novel proteins of Mycobacterium tuberculosis by the immunoscreening of an expression library of M. tuberculosis genomic DNA with sera obtained from M. tuberculosis-infected rabbits at 5 weeks postinfection. In this study, we report the further characterization of one of these antigens, LipC (Rv0220). LipC is annotated as a member of the Lip family based on the presence of the consensus motif “GXSXG” characteristic of esterases. Although predicted to be a cytoplasmic enzyme, we provide evidence that LipC is a cell surface protein that is present in both the cell wall and the capsule of M. tuberculosis. Consistent with this localization, LipC elicits strong humoral immune responses in both HIV-negative (HIV−) and HIV-positive (HIV+) tuberculosis (TB) patients. The absence of anti-LipC antibodies in sera from purified protein derivative-positive (PPD+) healthy subjects confirms its expression only during active M. tuberculosis infection. Epitope mapping of LipC identified 6 immunodominant epitopes, 5 of which map to the exposed surface of the modeled LipC protein. The recombinant LipC (rLipC) protein also elicits proinflammatory cytokine and chemokine responses from macrophages and pulmonary epithelial cells. rLipC can hydrolyze short-chain esters with the carbon chain containing 2 to 10 carbon atoms. Together, these studies demonstrate that LipC is a novel cell surface-associated esterase of M. tuberculosis that is highly immunogenic and elicits both antibodies and cytokines/chemokines. PMID:22038913

Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases.

PubMed

Dent, P; Chow, Y H; Wu, J; Morrison, D K; Jove, R; Sturgill, T W

1994-10-01

Mitogen-activated protein (MAP) kinase kinases (MKKs) are dual-specificity protein kinases which activate p42mapk and p44mapk by phosphorylation of regulatory tyrosine and threonine residues. cDNAs for two isotypes of MKK, MKK1 and MKK2, have been isolated from several species. Here we describe construction of recombinant baculoviruses for high-level expression of histidine-tagged rat MKK1 and MKK2, and procedures for production of nearly homogeneous MKK1 and MKK2 fusion proteins, in both inactive and active forms. Co-infection of Sf9 cells with either MKK1 or MKK2 virus together with recombinant viruses for Raf-1, pp60src (Y527F) and c-Ha-Ras resulted in activations of 250-fold and 150-fold for MKK1 and MKK2 respectively. Specific activities towards kinase-defective p42mapk were of the order of several hundred nanomoles of phosphate transferred/min per mg of MKK protein. The Michaelis constants for both enzymes were approx. 1 microM. Preparations of activated MKK were apparently free of Raf-1 as assessed by Western blotting. Raf-1 phosphorylated MKK1 on one major tryptic phosphopeptide, the phosphorylation of which increased with time. This phosphopeptide contained only phosphoserine and possessed neutral overall charge at pH 1.9 on two-dimensional peptide mapping. Phosphorylation of MKK1 by Raf-1 correlated with activation and reached a plateau of approximately 2 mol/mol.

Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

PubMed

Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

2016-07-29

Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mycobacterium avium subspecies induce differential expression of pro-inflammatory mediators in a murine macrophage model: evidence for enhanced pathogenicity of Mycobacterium avium subspecies paratuberculosis.

PubMed

Basler, Tina; Geffers, Robert; Weiss, Siegfried; Valentin-Weigand, Peter; Goethe, Ralph

2008-01-01

Mycobacterium avium subspecies (ssp.) paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic, non-treatable granulomatous enteritis of ruminants. MAP is the only mycobacterium affecting the intestinal tract, which is of interest since it is presently the most favoured pathogen linked to Crohn's disease (CD) in humans due to its frequent detection in CD tissues. MAP is genetically closely related to other M. avium ssp. such as M. avium ssp. avium (MAA) and M. avium ssp. hominissuis (MAH) which can cause mycobacteriosis in animals and immunocompromised humans. We have recently shown that murine macrophage cell lines represent suitable systems to analyse M. avium ssp. patho-mechanisms and could show that MAP, but not MAA, specifically inhibited the antigen-specific stimulatory capacity for CD4(+) T-cells. In the present study, we compared gene expression profiles of murine RAW264.7 macrophages in response to infections with MAP or MAA using murine high-density oligonucleotide Affymetrix microarrays. A comparison of MAP and MAA infection revealed 17 differentially expressed genes. They were expressed at a much lower level in MAP-infected macrophages than in MAA-infected macrophages. Among these were the genes for IL-1beta, IL-1alpha, CXCL2, PTGS2 (COX2), lipocalin (LCN2) and TNF, which are important pro-inflammatory factors. The microarray data were confirmed for selected genes by quantitative real-time reverse transcription PCR and, by protein array analyses and ELISA. Similar to MAA, infection with MAH also showed robust induction of IL-1beta, CXCL2, COX2, LCN2 and TNF. Taken together, our results from M. avium ssp.-infected murine macrophages provide evidence that MAP in contrast to MAA and MAH specifically suppresses the pro-inflammatory defence mechanisms of infected macrophages.

Complementary Proteome and Transcriptome Profiling in Developing Grains of a Notched-Belly Rice Mutant Reveals Key Pathways Involved in Chalkiness Formation

PubMed Central

Lin, Zhaomiao; Wang, Zunxin; Zhang, Xincheng; Li, Ganghua; Wang, Shaohua; Ding, Yanfeng

2017-01-01

Rice grain chalkiness is a highly complex trait involved in multiple metabolic pathways and controlled by polygenes and growth conditions. To uncover novel aspects of chalkiness formation, we performed an integrated profiling of gene activity in the developing grains of a notched-belly rice mutant. Using exhaustive tandem mass spectrometry-based shotgun proteomics and whole-genome RNA sequencing to generate a nearly complete catalog of expressed mRNAs and proteins, we reliably identified 38,476 transcripts and 3,840 proteins. Comparison between the translucent part and chalky part of the notched-belly grains resulted in only a few differently express genes (240) and differently express proteins (363), thus making it possible to focus on ‘core’ genes or common pathways. Several novel key pathways were identified as of relevance to chalkiness formation, in particular the shift of C and N metabolism, the down-regulation of ribosomal proteins and the resulting low abundance of storage proteins especially the 13 kDa prolamin subunit, and the suppressed photosynthetic capacity in the pericarp of the chalky part. Further, genes and proteins as transporters for carbohydrates, amino acid/peptides, proteins, lipids and inorganic ions showed an increasing expression pattern in the chalky part of the notched-belly grains. Similarly, transcripts and proteins of receptors for auxin, ABA, ethylene and brassinosteroid were also up-regulated. In summary, this joint analysis of transcript and protein profiles provides a comprehensive reference map of gene activity regarding the physiological state in the chalky endosperm. PMID:28158863

Efficient generation of transgenic reporter strains and analysis of expression patterns in Caenorhabditis elegans using Library MosSCI

PubMed Central

Kaymak, Ebru; Farley, Brian M.; Hay, Samantha A.; Li, Chihua; Ho, Samantha; Hartman, Daniel J.; Ryder, Sean P.

2016-01-01

Background In C. elegans, germline development and early embryogenesis rely on post-transcriptional regulation of maternally transcribed mRNAs. In many cases, the 3′UTR is sufficient to govern the expression patterns of these transcripts. Several RNA-binding proteins are required to regulate maternal mRNAs through the 3′UTR. Despite intensive efforts to map RNA-binding protein-mRNA interactions in vivo, the biological impact of most binding events remains unknown. Reporter studies using single copy integrated transgenes are essential to evaluate the functional consequences of interactions between RNA-binding proteins and their associated mRNAs. Results In this report, we present an efficient method of generating reporter strains with improved throughput by using a library variant of MosSCI transgenesis. Furthermore, using RNA interference, we identify the suite of RBPs that control the expression pattern of five different maternal mRNAs. Conclusions The results provide a generalizable and efficient strategy to assess the functional relevance of protein-RNA interactions in vivo, and reveal new regulatory connections between key RNA-binding proteins and their maternal mRNA targets. PMID:27294288

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

PubMed Central

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

2007-01-01

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

Myozenin: An α-actinin- and γ-filamin-binding protein of skeletal muscle Z lines

PubMed Central

Takada, Fumio; Woude, Douglas L. Vander; Tong, Hui-Qi; Thompson, Terri G.; Watkins, Simon C.; Kunkel, Louis M.; Beggs, Alan H.

2001-01-01

To better understand the structure and function of Z lines, we used sarcomeric isoforms of α-actinin and γ-filamin to screen a human skeletal muscle cDNA library for interacting proteins by using the yeast two-hybrid system. Here we describe myozenin (MYOZ), an α-actinin- and γ-filamin-binding Z line protein expressed predominantly in skeletal muscle. Myozenin is predicted to be a 32-kDa, globular protein with a central glycine-rich domain flanked by α-helical regions with no strong homologies to any known genes. The MYOZ gene has six exons and maps to human chromosome 10q22.1-q22.2. Northern blot analysis demonstrated that this transcript is expressed primarily in skeletal muscle with significantly lower levels of expression in several other tissues. Antimyozenin antisera stain skeletal muscle in a sarcomeric pattern indistinguishable from that seen by using antibodies for α-actinin, and immunogold electron microscopy confirms localization specifically to Z lines. Thus, myozenin is a skeletal muscle Z line protein that may be a good candidate gene for limb-girdle muscular dystrophy or other neuromuscular disorders. PMID:11171996

Multiple Functional Domains and Complexes of the Two Nonstructural Proteins of Human Respiratory Syncytial Virus Contribute to Interferon Suppression and Cellular Location▿

PubMed Central

Swedan, Samer; Andrews, Joel; Majumdar, Tanmay; Musiyenko, Alla; Barik, Sailen

2011-01-01

Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKKε, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo- as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKKε reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression. PMID:21795342

Multiple functional domains and complexes of the two nonstructural proteins of human respiratory syncytial virus contribute to interferon suppression and cellular location.

PubMed

Swedan, Samer; Andrews, Joel; Majumdar, Tanmay; Musiyenko, Alla; Barik, Sailen

2011-10-01

Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKKε, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo- as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKKε reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression.

On the Extent and Origins of Genic Novelty in the Phylum Nematoda

PubMed Central

Wasmuth, James; Schmid, Ralf; Hedley, Ann; Blaxter, Mark

2008-01-01

Background The phylum Nematoda is biologically diverse, including parasites of plants and animals as well as free-living taxa. Underpinning this diversity will be commensurate diversity in expressed genes, including gene sets associated specifically with evolution of parasitism. Methods and Findings Here we have analyzed the extensive expressed sequence tag data (available for 37 nematode species, most of which are parasites) and define over 120,000 distinct putative genes from which we have derived robust protein translations. Combined with the complete proteomes of Caenorhabditis elegans and Caenorhabditis briggsae, these proteins have been grouped into 65,000 protein families that in turn contain 40,000 distinct protein domains. We have mapped the occurrence of domains and families across the Nematoda and compared the nematode data to that available for other phyla. Gene loss is common, and in particular we identify nearly 5,000 genes that may have been lost from the lineage leading to the model nematode C. elegans. We find a preponderance of novelty, including 56,000 nematode-restricted protein families and 26,000 nematode-restricted domains. Mapping of the latest time-of-origin of these new families and domains across the nematode phylogeny revealed ongoing evolution of novelty. A number of genes from parasitic species had signatures of horizontal transfer from their host organisms, and parasitic species had a greater proportion of novel, secreted proteins than did free-living ones. Conclusions These classes of genes may underpin parasitic phenotypes, and thus may be targets for development of effective control measures. PMID:18596977

Trans-ancestry Fine Mapping and Molecular Assays Identify Regulatory Variants at the ANGPTL8 HDL-C GWAS Locus

PubMed Central

Cannon, Maren E.; Duan, Qing; Wu, Ying; Zeynalzadeh, Monica; Xu, Zheng; Kangas, Antti J.; Soininen, Pasi; Ala-Korpela, Mika; Civelek, Mete; Lusis, Aldons J.; Kuusisto, Johanna; Collins, Francis S.; Boehnke, Michael; Tang, Hua; Laakso, Markku; Li, Yun; Mohlke, Karen L.

2017-01-01

Recent genome-wide association studies (GWAS) have identified variants associated with high-density lipoprotein cholesterol (HDL-C) located in or near the ANGPTL8 gene. Given the extensive sharing of GWAS loci across populations, we hypothesized that at least one shared variant at this locus affects HDL-C. The HDL-C–associated variants are coincident with expression quantitative trait loci for ANGPTL8 and DOCK6 in subcutaneous adipose tissue; however, only ANGPTL8 expression levels are associated with HDL-C levels. We identified a 400-bp promoter region of ANGPTL8 and enhancer regions within 5 kb that contribute to regulating expression in liver and adipose. To identify variants functionally responsible for the HDL-C association, we performed fine-mapping analyses and selected 13 candidate variants that overlap putative regulatory regions to test for allelic differences in regulatory function. Of these variants, rs12463177-G increased transcriptional activity (1.5-fold, P = 0.004) and showed differential protein binding. Six additional variants (rs17699089, rs200788077, rs56322906, rs3760782, rs737337, and rs3745683) showed evidence of allelic differences in transcriptional activity and/or protein binding. Taken together, these data suggest a regulatory mechanism at the ANGPTL8 HDL-C GWAS locus involving tissue-selective expression and at least one functional variant. PMID:28754724

Post-transcriptional m6A editing of HIV-1 mRNAs enhances viral gene expression

PubMed Central

Kennedy, Edward M.; Bogerd, Hal P.; Kornepati, Anand V. R.; Kang, Dong; Ghoshal, Delta; Marshall, Joy B.; Poling, Brigid C.; Tsai, Kevin; Gokhale, Nandan S.; Horner, Stacy M.; Cullen, Bryan R.

2016-01-01

Summary Covalent addition of a methyl group to the adenosine N6 (m6A) is an evolutionarily conserved and common RNA modification that is thought to modulate several aspects of RNA metabolism. While the presence of multiple m6A editing sites on diverse viral RNAs was reported starting almost 40 years ago, how m6A editing affects virus replication has remained unclear. Here, we used photo-crosslinking-assisted m6A sequencing techniques to precisely map several m6A editing sites on the HIV-1 genome and report that they cluster in the HIV-1 3’ untranslated region (3'UTR). Viral 3'UTR m6A sites or analogous cellular m6A sites strongly enhanced mRNA expression in cis by recruiting the cellular YTHDF m6A “reader” proteins. Reducing YTHDF expression inhibited, while YTHDF overexpression enhanced, HIV-1 protein and RNA expression, and virus replication in CD4+ T cells. These data identify m6A editing, and the resultant recruitment of YTHDF proteins, as major positive regulators of HIV-1 mRNA expression. PMID:27117054

Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

PubMed

Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

2016-09-01

Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Significance of aquaporins’ expression in the prognosis of gastric cancer

PubMed Central

Thapa, Saroj; Chetry, Mandika; Huang, Kaiyu; Peng, Yangpei; Wang, Jinsheng; Wang, Jiaoni; Zhou, Yingying; Shen, Yigen; Xue, Yangjing; Ji, Kangting

2018-01-01

Gastric carcinoma is one of the most lethal malignancy at present with leading cause of cancer-related deaths worldwide. Aquaporins (AQPs) are a family of small, integral membrane proteins, which have been evidenced to play a crucial role in cell migration and proliferation of different cancer cells including gastric cancers. However, the aberrant expression of specific AQPs and its correlation to detect predictive and prognostic significance in gastric cancer remains elusive. In the present study, we comprehensively explored immunohistochemistry based map of protein expression profiles in normal tissues, cancer and cell lines from publicly available Human Protein Atlas (HPA) database. Moreover, to improve our understanding of general gastric biology and guide to find novel predictive prognostic gastric cancer biomarker, we also retrieved ‘The Kaplan–Meier plotter’ (KM plotter) online database with specific AQPs mRNA to overall survival (OS) in different clinicopathological features. We revealed that ubiquitous expression of AQPs protein can be effective tools to generate gastric cancer biomarker. Furthermore, high level AQP3, AQP9, and AQP11 mRNA expression were correlated with better OS in all gastric patients, whereas AQP0, AQP1, AQP4, AQP5, AQP6, AQP8, and AQP10 mRNA expression were associated with poor OS. With regard to the clinicopathological features including Laurens classification, clinical stage, human epidermal growth factor receptor 2 (HER2) status, and different treatment strategy, we could illustrate significant role of individual AQP mRNA expression in the prognosis of gastric cancer patients. Thus, our results indicated that AQP’s protein and mRNA expression in gastric cancer patients provide effective role to predict prognosis and act as an essential agent to therapeutic strategy. PMID:29678898

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

PubMed Central

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

2017-01-01

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

An intronic ncRNA-dependent regulation of SORL1 expression affecting Aβ formation is upregulated in post-mortem Alzheimer's disease brain samples.

PubMed

Ciarlo, Eleonora; Massone, Sara; Penna, Ilaria; Nizzari, Mario; Gigoni, Arianna; Dieci, Giorgio; Russo, Claudio; Florio, Tullio; Cancedda, Ranieri; Pagano, Aldo

2013-03-01

Recent studies indicated that sortilin-related receptor 1 (SORL1) is a risk gene for late-onset Alzheimer's disease (AD), although its role in the aetiology and/or progression of this disorder is not fully understood. Here, we report the finding of a non-coding (nc) RNA (hereafter referred to as 51A) that maps in antisense configuration to intron 1 of the SORL1 gene. 51A expression drives a splicing shift of SORL1 from the synthesis of the canonical long protein variant A to an alternatively spliced protein form. This process, resulting in a decreased synthesis of SORL1 variant A, is associated with impaired processing of amyloid precursor protein (APP), leading to increased Aβ formation. Interestingly, we found that 51A is expressed in human brains, being frequently upregulated in cerebral cortices from individuals with Alzheimer's disease. Altogether, these findings document a novel ncRNA-dependent regulatory pathway that might have relevant implications in neurodegeneration.

Mining Missing Membrane Proteins by High-pH Reverse Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry

PubMed Central

Kitata, Reta Birhanu; Dimayacyac-Esleta, Baby Rorielyn T.; Choong, Wai-Kok; Tsai, Chia-Feng; Lin, Tai-Du; Tsou, Chih-Chiang; Weng, Shao-Hsing; Chen, Yi-Ju; Yang, Pan-Chyr; Arco, Susan D.; Nesvizhskii, Alexey I.; Sung, Ting-Yi; Chen, Yu-Ju

2016-01-01

Despite significant efforts in the past decade towards complete mapping of the human proteome, 3564 proteins (neXtProt, 09-2014) are still “missing proteins”. Over one-third of these missing proteins are annotated as membrane proteins, owing to their relatively challenging accessibility with standard shotgun proteomics. Using non-small cell lung cancer (NSCLC) as a model study, we aim to mine missing proteins from disease-associated membrane proteome, which may be still largely under-represented. To increase identification coverage, we employed Hp-RP StageTip pre-fractionation of membrane-enriched samples from 11 NSCLC cell lines. Analysis of membrane samples from 20 pairs of tumor and adjacent normal lung tissue were incorporated to include physiologically expressed membrane proteins. Using multiple search engines (X!Tandem, Comet and Mascot) and stringent evaluation of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66% membrane proteins) and 178 missing proteins (74 membrane proteins) with PSM-, peptide-, and protein-level FDR of 1%. Through multiple reaction monitoring (MRM) using synthetic peptides, we provided additional evidences for 8 missing proteins including 7 with transmembrane helix domains (TMH). This study demonstrates that mining missing proteins focused on cancer membrane sub-proteome can greatly contribute to map the whole human proteome. All data were deposited into ProteomeXchange with the identifier PXD002224. PMID:26202522

The PDI genes of wheat and their syntenic relationship to the esp2 locus of rice.

PubMed

Johnson, Joshua C; Appels, Rudi; Bhave, Mrinal

2006-04-01

The storage protein polymers in the endosperm, stabilised by disulphide bonds, determine a number of processing qualities of wheat dough. The enzyme protein disulphide isomerase (PDI), involved in the formation of disulphide bonds, is strongly suggested to play a role in the formation of wheat storage protein bodies. Reports of the rice mutant esp2 exhibiting aberrant storage protein deposition in conjunction with a lack of PDI expression provided strong indications of a direct role for PDI in storage protein deposition. The potential significance of wheat PDI prompted the present studies into exploring any orthology between wheat PDI genes and rice PDI and esp2 loci. By designing allele-specific (AS)-polymerase chain reaction (PCR) markers, two of the three wheat PDI genes could be genetically mapped to group 4 chromosomes and showed close association with GERMIN genes. Physical mapping led to localisation of wheat PDI genes to chromosomal "bins" on the proximal section of chromosome 4AL and distal sections of 4BS and 4DS. Identification of the putative PDI gene of rice and its comparison to the esp2 locus revealed that they were present at similar positions on the short arm of chromosome 11. Analysis of a large section of the PDI-containing section of rice chromosome 11S revealed a number of putative orthologues from The Institute for Genomic Research Triticum aestivum Gene Index database, of which five had been mapped, each localising to group 4 chromosomes, many in good agreement with our mapping results. The results strongly suggest a close linkage between the esp2 marker and the PDI gene of rice and an orthology between the PDI loci of rice and wheat and predict quantitative-trait loci involved in storage protein deposition at the PDI loci.

Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells.

PubMed

Montrose-Rafizadeh, C; Avdonin, P; Garant, M J; Rodgers, B D; Kole, S; Yang, H; Levine, M A; Schwindinger, W; Bernier, M

1999-03-01

Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gs alpha, Gq/11 alpha, and Gi1,2 alpha, but not Gi3 alpha. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046-38), respectively. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046-38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046-38 cells, respectively. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.

Use of lambdagt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins

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

Petrovskis, E.A.; Timmins, J.G.; Post, L.E.

1986-10-01

A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambdagt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambdagt11 vector, the cloned proteins were expressed in Escherichia coli as ..beta..-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of (/sup 14/C)glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX;more » gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.« less

The JNK-like MAPK KGB-1 of Caenorhabditis elegans promotes reproduction, lifespan, and gene expressions for protein biosynthesis and germline homeostasis but interferes with hyperosmotic stress tolerance.

PubMed

Gerke, Peter; Keshet, Alex; Mertenskötter, Ansgar; Paul, Rüdiger J

2014-01-01

This study focused on the role of the JNK-like MAPK (mitogen-activated protein kinase) KGB-1 (kinase, GLH-binding 1) for osmoprotection and other vital functions. We mapped KGB-1 expression patterns and determined lifespan, reproduction and survival rates as well as changes in body volume, motility, and GPDH (glycerol-3-phosphate dehydrogenase) activity for glycerol production in wildtype (WT), different signaling mutants (including a kgb-1 deletion mutant, kgb-1∆) and RNAi-treated worms under control and hyperosmotic conditions. KGB-1-mediated gene expressions were studied, for instance, by RNA Sequencing, with the resulting transcriptome data analyzed using orthology-based approaches. Surprisingly, mutation/RNAi of kgb-1 and fos-1 (gene for an AP-1, activator protein 1, element) significantly promoted hyperosmotic resistance, even though hyperosmotic GPDH activity was higher in WT than in kgb-1∆. KGB-1 and moderate hyperosmolarity promoted and severe hyperosmolarity repressed kgb-1, fos-1, and jun-1 (gene for another AP-1 element) expression. Transcriptome profiling revealed, for instance, down-regulated genes for protein biosynthesis and up-regulated genes for membrane transporters in kgb-1∆ and up-regulated genes for GPDH-1 or detoxification in WT, with the latter indicating cellular damage and less effective osmoprotection in WT. KGB-1 promotes reproduction and lifespan and fosters gene expressions for AP-1 elements, protein biosynthesis, and balanced gametogenesis, but inhibits expressions for membrane transporters perhaps in order to control energy consumption. Reduced protein biosyntheses and enhanced membrane transports in kgb-1∆ most likely contribute to the high hyperosmotic tolerance of the mutant by easing the burden of the existing chaperone machinery and promoting regulatory volume increases upon hyperosmotic stress.

Sulforaphane suppresses lipopolysaccharide-induced cyclooxygenase-2 (COX-2) expression through the modulation of multiple targets in COX-2 gene promoter.

PubMed

Woo, Kyung Jin; Kwon, Taeg Kyu

2007-12-15

Sulforaphane is a natural, biologically active compound extracted from cruciferous vegetables such as broccoli and cabbage. It possesses potent anti-inflammation and anti-cancer properties. The mechanism by which sulforaphane suppresses COX-2 expression remains poorly understood. In the present report, we investigated the effect of sulforaphane on the expression of COX-2 in lipopolysaccharide (LPS)-activated Raw 264.7 cells. Sulforaphane significantly suppressed the LPS-induced COX-2 protein and mRNA expression in a dose-dependent manner. The ability of sulforaphane to suppress the expression of the COX-2 was investigated using luciferase reporters controlled by various cis-elements in COX-2 promoter region. Electrophoretic mobility shift assay (EMSA) verified that NF-kappaB, C/EBP, CREB and AP-1 were identified as responsible for the sulforaphane-mediated COX-2 down-regulation. In addition, we demonstrated the signal transduction pathway of mitogen-activated protein kinase (MAP kinase) in LPS-induced COX-2 expression. Taken together, these results demonstrate that sulforaphane effectively suppressed the LPS-induced COX-2 protein via modulation of multiple core promoter elements (NF-kappaB, C/EBP, CREB and AP-1) in the COX-2 transcriptional regulation. These results will provide new insights into the anti-inflammatory and anti-carcinogenic properties of sulforaphane.

Domain Specificity of MAP3K Family Members, MLK and Tak1, for JNK Signaling in Drosophila

PubMed Central

Stronach, Beth; Lennox, Ashley L.; Garlena, Rebecca A.

2014-01-01

A highly diverse set of protein kinases functions as early responders in the mitogen- and stress-activated protein kinase (MAPK/SAPK) signaling pathways. For instance, humans possess 14 MAPK kinase kinases (MAP3Ks) that activate Jun kinase (JNK) signaling downstream. A major challenge is to decipher the selective and redundant functions of these upstream MAP3Ks. Taking advantage of the relative simplicity of Drosophila melanogaster as a model system, we assessed MAP3K signaling specificity in several JNK-dependent processes during development and stress response. Our approach was to generate molecular chimeras between two MAP3K family members, the mixed lineage kinase, Slpr, and the TGF-β activated kinase, Tak1, which share 32% amino acid identity across the kinase domain but otherwise differ in sequence and domain structure, and then test the contributions of various domains for protein localization, complementation of mutants, and activation of signaling. We found that overexpression of the wild-type kinases stimulated JNK signaling in alternate contexts, so cells were capable of responding to both MAP3Ks, but with distinct outcomes. Relative to wild-type, the catalytic domain swaps compensated weakly or not at all, despite having a shared substrate, the JNK kinase Hep. Tak1 C-terminal domain-containing constructs were inhibitory in Tak1 signaling contexts, including tumor necrosis factor-dependent cell death and innate immune signaling; however, depressing antimicrobial gene expression did not necessarily cause phenotypic susceptibility to infection. These same constructs were neutral in the context of Slpr-dependent developmental signaling, reflecting differential subcellular protein localization and by inference, point of activation. Altogether, our findings suggest that the selective deployment of a particular MAP3K can be attributed in part to its inherent sequence differences, cellular localization, and binding partner availability. PMID:24429281

Site-specific microtubule-associated protein 4 dephosphorylation causes microtubule network densification in pressure overload cardiac hypertrophy.

PubMed

Chinnakkannu, Panneerselvam; Samanna, Venkatesababa; Cheng, Guangmao; Ablonczy, Zsolt; Baicu, Catalin F; Bethard, Jennifer R; Menick, Donald R; Kuppuswamy, Dhandapani; Cooper, George

2010-07-09

In severe pressure overload-induced cardiac hypertrophy, a dense, stabilized microtubule network forms that interferes with cardiocyte contraction and microtubule-based transport. This is associated with persistent transcriptional up-regulation of cardiac alpha- and beta-tubulin and microtubule-stabilizing microtubule-associated protein 4 (MAP4). There is also extensive microtubule decoration by MAP4, suggesting greater MAP4 affinity for microtubules. Because the major determinant of this affinity is site-specific MAP4 dephosphorylation, we characterized this in hypertrophied myocardium and then assessed the functional significance of each dephosphorylation site found by mimicking it in normal cardiocytes. We first isolated MAP4 from normal and pressure overload-hypertrophied feline myocardium; volume-overloaded myocardium, which has an equal degree and duration of hypertrophy but normal functional and cytoskeletal properties, served as a control for any nonspecific growth-related effects. After cloning cDNA-encoding feline MAP4 and obtaining its deduced amino acid sequence, we characterized by mass spectrometry any site-specific MAP4 dephosphorylation. Solely in pressure overload-hypertrophied myocardium, we identified striking MAP4 dephosphorylation at Ser-472 in the MAP4 N-terminal projection domain and at Ser-924 and Ser-1056 in the assembly-promoting region of the C-terminal microtubule-binding domain. Site-directed mutagenesis of MAP4 cDNA was then used to switch each serine to non-phosphorylatable alanine. Wild-type and mutated cDNAs were used to construct adenoviruses; microtubule network density, stability, and MAP4 decoration were assessed in normal cardiocytes following an equivalent level of MAP4 expression. The Ser-924 --> Ala MAP4 mutant produced a microtubule phenotype indistinguishable from that seen in pressure overload hypertrophy, such that Ser-924 MAP4 dephosphorylation during pressure overload hypertrophy may be central to this cytoskeletal abnormality.

Hsp27 and F-box protein β-TrCP promote degradation of mRNA decay factor AUF1.

PubMed

Li, Mei-Ling; Defren, Jennifer; Brewer, Gary

2013-06-01

Activation of the mitogen-activated protein (MAP) pathway kinases p38 and MK2 induces phosphorylation of the chaperone Hsp27 and stabilization of mRNAs containing AU-rich elements (AREs) (ARE-mRNAs). Likewise, expression of phosphomimetic mutant forms of Hsp27 also stabilizes ARE-mRNAs. It appears to perform this function by promoting degradation of the ARE-mRNA decay factor AUF1 by proteasomes. In this study, we examined the molecular mechanism linking Hsp27 phosphorylation to AUF1 degradation by proteasomes. AUF1 is a target of β-TrCP, the substrate recognition subunit of the E3 ubiquitin ligase Skp1-cullin-F-box protein complex, SCF(β-TrCP). Depletion of β-TrCP stabilized AUF1. In contrast, overexpression of β-TrCP enhanced ubiquitination and degradation of AUF1 and led to stabilization of reporter mRNAs containing cytokine AREs. Enhanced AUF1 degradation required expression of phosphomimetic mutant forms of both Hsp27 and AUF1. Our results suggest that a signaling axis composed of p38 MAP kinase-MK2-Hsp27-β-TrCP may promote AUF1 degradation by proteasomes and stabilization of cytokine ARE-mRNAs.

Mapping the subgenomic RNA promoter of the Citrus leaf blotch virus coat protein gene by Agrobacterium-mediated inoculation.

PubMed

Renovell, Agueda; Gago, Selma; Ruiz-Ruiz, Susana; Velázquez, Karelia; Navarro, Luis; Moreno, Pedro; Vives, Mari Carmen; Guerri, José

2010-10-25

Citrus leaf blotch virus has a single-stranded positive-sense genomic RNA (gRNA) of 8747 nt organized in three open reading frames (ORFs). The ORF1, encoding a polyprotein involved in replication, is translated directly from the gRNA, whereas ORFs encoding the movement (MP) and coat (CP) proteins are expressed via 3' coterminal subgenomic RNAs (sgRNAs). We characterized the minimal promoter region critical for the CP-sgRNA expression in infected cells by deletion analyses using Agrobacterium-mediated infection of Nicotiana benthamiana plants. The minimal CP-sgRNA promoter was mapped between nucleotides -67 and +50 nt around the transcription start site. Surprisingly, larger deletions in the region between the CP-sgRNA transcription start site and the CP translation initiation codon resulted in increased CP-sgRNA accumulation, suggesting that this sequence could modulate the CP-sgRNA transcription. Site-specific mutational analysis of the transcription start site revealed that the +1 guanylate and the +2 adenylate are important for CP-sgRNA synthesis. Copyright © 2010 Elsevier Inc. All rights reserved.

SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics

PubMed Central

Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

2016-01-01

Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression. PMID:27628341

Epitope mapping of the nucleocapsid protein of European and North American isolates of porcine reproductive and respiratory syndrome virus.

PubMed

Rodriguez, M J; Sarraseca, J; Garcia, J; Sanz, A; Plana-Durán, J; Ignacio Casal, J

1997-09-01

Two major genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) have been described, which correspond to the European and North American isolates. PRRSV nucleocapsid (N) protein has been identified as the most immunodominant viral protein. The N genes from two PRRSV isolates, Olot/91 (European) and Québec 807/94 (North American), were cloned and expressed in: (i) baculovirus under the control of the polyhedrin promoter and (ii) Escherichia coli using the pET3x system. The N protein from both isolates was expressed much more efficiently in E. coli as a fusion protein than in baculovirus. The antigenicity of the protein was similar in both systems and it was recognized by a collection of 48 PRRSV-positive pig sera. The antigenic structure of the PRRSV N protein was investigated using seven monoclonal antibodies (MAbs) and overlapping fragments of the protein expressed in E. coli. Four MAbs recognized two discontinuous epitopes that were present in the partially folded protein, or at least a large fragment comprising the first 78 residues. The other three MAbs revealed the presence of a common antigenic site localized in the central region of the protein (amino acids 50-66). This region is well conserved among different isolates of European and North American origin and is the most hydrophilic region of the protein. However, this epitope, although recognized by the MAbs and many pig sera, is not useful for diagnostic purposes. Moreover, none of the N protein fragments were able to mimic the antigenicity of the entire protein.

Thioredoxin Activates MKK4-NFκB Pathway in a Redox-dependent Manner to Control Manganese Superoxide Dismutase Gene Expression in Endothelial Cells*

PubMed Central

Kundumani-Sridharan, Venkatesh; Subramani, Jaganathan; Das, Kumuda C.

2015-01-01

The mitogen-activated protein kinase kinase 4 (MKK4) is activated via phosphorylation of Ser-257 and Thr-261 by upstream MAP3Ks and activates JNK and p38 MAPKs in response to cellular stress. We show that thioredoxin (Trx), a cellular redox protein, activates MKK4 via Cys-246 and Cys-266 residues as mutation of these residues renders MKK4 insensitive to phosphorylation by MAP3Ks, TNFα, or Trx. MKK4 is activated in vitro by reduced Trx but not oxidized Trx in the absence of an upstream kinase, suggesting that autophosphorylation of this protein occurs due to reduction of Cys-246 and Cys-266 by Trx. Additionally, mutation of Cys-246 and Cys-266 resulted in loss of kinase activity suggesting that the redox state of Cys-246 and Cys-266 is a critical determinant of MKK4 activation. Trx induces manganese superoxide dismutase (MnSOD) gene transcription by activating MKK4 via redox control of Cys-246 and Cys-266, as mutation of these residues abrogates MKK4 activation and MnSOD expression. We further show that MKK4 activates NFκB for its binding to the MnSOD promoter, which leads to AP-1 dissociation followed by MnSOD transcription. Taken together, our studies show that the redox status of Cys-246 and Cys-266 in MKK4 controls its activities independent of MAP3K, demonstrating integration of the endothelial redox environment to MAPK signaling. PMID:26028649

PdSlt2 Penicillium digitatum mitogen-activated-protein kinase controls sporulation and virulence during citrus fruit infection.

PubMed

de Ramón-Carbonell, Marta; Sánchez-Torres, Paloma

2017-12-01

The Slt2 mitogen-activated protein (MAP) kinase homologue of Penicillium digitatum, the most relevant pathogen-producing citrus green mould decay during postharvest, was identified and explored. The P. digitatum Slt2-MAPK coding gene (PdSlt2) was functionally characterized by homologous gene elimination and transcriptomic evaluation. The absence of PdSlt2 gene resulted in significantly reduced virulence during citrus infection. The ΔPdSlt2 mutants were also defective in asexual reproduction, showing impairment of sporulation during citrus infection. Gene expression analysis revealed that PdSlt2 was highly induced during citrus fruit infection at early stages (1 dpi). Moreover, PdSlt2 deletion altered gene expression profiles. The relative gene expression (RGE) of fungicide resistance- and fungal virulence-related genes showed that PdSlt2 acts as negative regulator of several transporter encoding genes (ABC and MFS transporters) and a positive regulator of two sterol demethylases. This study indicates that PdSlt2 MAPK is functionally preserved in P. digitatum and highlights the relevant role of the PdSlt2 MAP kinase-mediated signalling pathway in regulating diverse genes crucial for infection and asexual reproduction. Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Mapping the co-localization of the circadian proteins PER2 and BMAL1 with enkephalin and substance P throughout the rodent forebrain.

PubMed

Frederick, Ariana; Goldsmith, Jory; de Zavalia, Nuria; Amir, Shimon

2017-01-01

Despite rhythmic expression of clock genes being found throughout the central nervous system, very little is known about their function outside of the suprachiasmatic nucleus. Determining the pattern of clock gene expression across neuronal subpopulations is a key step in understanding their regulation and how they may influence the functions of various brain structures. Using immunofluorescence and confocal microscopy, we quantified the co-expression of the clock proteins BMAL1 and PER2 with two neuropeptides, Substance P (SubP) and Enkephalin (Enk), expressed in distinct neuronal populations throughout the forebrain. Regions examined included the limbic forebrain (dorsal striatum, nucleus accumbens, amygdala, stria terminalis), thalamus medial habenula of the thalamus, paraventricular nucleus and arcuate nucleus of the hypothalamus and the olfactory bulb. In most regions examined, BMAL1 was homogeneously expressed in nearly all neurons (~90%), and PER2 was expressed in a slightly lower proportion of cells. There was no specific correlation to SubP- or Enk- expressing subpopulations. The olfactory bulb was unique in that PER2 and BMAL1 were expressed in a much smaller percentage of cells, and Enk was rarely found in the same cells that expressed the clock proteins (SubP was undetectable). These results indicate that clock genes are not unique to specific cell types, and further studies will be required to determine the factors that contribute to the regulation of clock gene expression throughout the brain.

Identifying protein biomarkers in predicting disease severity of dengue virus infection using immune-related protein microarray.

PubMed

Soe, Hui Jen; Yong, Yean K; Al-Obaidi, Mazen M Jamil; Raju, Chandramathi Samudi; Gudimella, Ranganath; Manikam, Rishya; Sekaran, Shamala Devi

2018-02-01

Dengue virus is one of the most widespread flaviviruses that re-emerged throughout recent decades. The progression from mild dengue to severe dengue (SD) with the complications such as vascular leakage and hemorrhage increases the fatality rate of dengue. The pathophysiology of SD is not entirely clear. To investigate potential biomarkers that are suggestive of pathogenesis of SD, a small panel of serum samples selected from 1 healthy individual, 2 dengue patients without warning signs (DWS-), 2 dengue patients with warning signs (DWS+), and 5 patients with SD were subjected to a pilot analysis using Sengenics Immunome protein array. The overall fold changes of protein expressions and clustering heat map revealed that PFKFB4, TPM1, PDCL3, and PTPN20A were elevated among patients with SD. Differential expression analysis identified that 29 proteins were differentially elevated greater than 2-fold in SD groups than DWS- and DWS+. From the 29 candidate proteins, pathways enrichment analysis also identified insulin signaling and cytoskeleton pathways were involved in SD, suggesting that the insulin pathway may play a pivotal role in the pathogenesis of SD.

Expression patterns of protein C inhibitor in mouse development.

PubMed

Wagenaar, Gerry T M; Uhrin, Pavel; Weipoltshammer, Klara; Almeder, Marlene; Hiemstra, Pieter S; Geiger, Margarethe; Meijers, Joost C M; Schöfer, Christian

2010-02-01

Proteolysis of extracellular matrix is an important requirement for embryonic development and is instrumental in processes such as morphogenesis, angiogenesis, and cell migration. Efficient remodeling requires controlled spatio-temporal expression of both the proteases and their inhibitors. Protein C inhibitor (PCI) effectively blocks a range of serine proteases, and recently has been suggested to play a role in cell differentiation and angiogenesis. In this study, we mapped the expression pattern of PCI throughout mouse development using in situ hybridization and immunohistochemistry. We detected a wide-spread, yet distinct expression pattern with prominent PCI levels in skin including vibrissae, and in fore- and hindgut. Further sites of PCI expression were choroid plexus of brain ventricles, heart, skeletal muscles, urogenital tract, and cartilages. A strong and stage-dependent PCI expression was observed in the developing lung. In the pseudoglandular stage, PCI expression was present in distal branching tubules whereas proximal tubules did not express PCI. Later in development, in the saccular stage, PCI expression was restricted to distal bronchioli whereas sacculi did not express PCI. PCI expression declined in postnatal stages and was not detected in adult lungs. In general, embryonic PCI expression indicates multifunctional roles of PCI during mouse development. The expression pattern of PCI during lung development suggests its possible involvement in lung morphogenesis and angiogenesis.

Comparative Study of Early Cold-Regulated Proteins by Two-Dimensional Difference Gel Electrophoresis Reveals a Key Role for Phospholipase Dα1 in Mediating Cold Acclimation Signaling Pathway in Rice.

PubMed

Huo, Chenmin; Zhang, Baowen; Wang, Hui; Wang, Fawei; Liu, Meng; Gao, Yingjie; Zhang, Wenhua; Deng, Zhiping; Sun, Daye; Tang, Wenqiang

2016-04-01

To understand the early signaling steps that regulate cold responses in rice, two-dimensional difference gel electrophoresis (2-D DIGE)(1)was used to study early cold-regulated proteins in rice seedlings. Using mass spectrometry, 32 spots, which represent 26 unique proteins that showed an altered expression level within 5 min of cold treatment were identified. Among these proteins, Western blot analyses confirmed that the cellular phospholipase D α1 (OsPLDα1) protein level was increased as early as 1 min after cold treatment. Genetic studies showed that reducing the expression ofOsPLDα1makes rice plants more sensitive to chilling stress as well as cold acclimation increased freezing tolerance. Correspondingly, cold-regulated proteomic changes and the expression of the cold-responsive C repeat/dehydration-responsive element binding 1 (OsDREB1) family of transcription factors were inhibited in thepldα1mutant. We also found that the expression ofOsPLDα1is directly regulated by OsDREB1A. This transcriptional regulation ofOsPLDα1could provide positive feedback regulation of the cold signal transduction pathway in rice. OsPLDα1 hydrolyzes phosphatidylcholine to produce the signal molecule phosphatidic acid (PA). By lipid-overlay assay, we demonstrated that the rice cold signaling proteins, MAP kinase 6 (OsMPK6) and OsSIZ1, bind directly to PA. Taken together, our results suggest that OsPLDα1 plays a key role in transducing cold signaling in rice by producing PA and regulatingOsDREB1s' expression by OsMPK6, OsSIZ1, and possibly other PA-binding proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Apoptosis-inducing signal sequence mutation in carbonic anhydrase IV identified in patients with the RP17 form of retinitis pigmentosa

PubMed Central

Rebello, George; Ramesar, Rajkumar; Vorster, Alvera; Roberts, Lisa; Ehrenreich, Liezle; Oppon, Ekow; Gama, Dumisani; Bardien, Soraya; Greenberg, Jacquie; Bonapace, Giuseppe; Waheed, Abdul; Shah, Gul N.; Sly, William S.

2004-01-01

Genetic and physical mapping of the RP17 locus on 17q identified a 3.6-megabase candidate region that includes the gene encoding carbonic anhydrase IV (CA4), a glycosylphosphatidylinositol-anchored protein that is highly expressed in the choriocapillaris of the human eye. By sequencing candidate genes in this region, we identified a mutation that causes replacement of an arginine with a tryptophan (R14W) in the signal sequence of the CA4 gene at position -5 relative to the signal sequence cleavage site. This mutation was found to cosegregate with the disease phenotype in two large families and was not found in 36 unaffected family members or 100 controls. Expression of the mutant cDNA in COS-7 cells produced several findings, suggesting a mechanism by which the mutation can explain the autosomal dominant disease. In transfected COS-7 cells, the R14W mutation (i) reduced the steady-state level of carbonic anhydrase IV activity expressed by 28% due to a combination of decreased synthesis and accelerated turnover; (ii) led to up-regulation of immunoglobulin-binding protein, double-stranded RNA-regulated protein kinase-like ER kinase, and CCAAT/enhancer-binding protein homologous protein, markers of the unfolded protein response and endoplasmic reticulum stress; and (iii) induced apoptosis, as evidenced by annexin V binding and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining, in most cells expressing the mutant, but not the WT, protein. We suggest that a high level of expression of the mutant allele in the endothelial cells of the choriocapillaris leads to apoptosis, leading in turn to ischemia in the overlying retina and producing autosomal dominant retinitis pigmentosa. PMID:15090652

Activation of the mitogen-activated protein kinase pathway by bone sialoprotein regulates osteoblast differentiation.

PubMed

Gordon, Jonathan A R; Hunter, Graeme K; Goldberg, Harvey A

2009-01-01

Bone sialoprotein (BSP) is an abundant protein in the extracellular matrix of bone that has been suggested to have several different physiological functions, including the nucleation of hydroxyapatite (HA), promotion of cell attachment and binding of collagen. Studies in our lab have demonstrated that increased expression of BSP in osteoblast cells can increase expression of the osteoblast-related genes Runx2 and Osx as well as alkaline phosphatase and osteocalcin and increase matrix mineralization. To determine the molecular mechanisms responsible for the BSP-mediated increase in osteoblastic differentiation, several functional domain mutants of BSP were expressed in primary rat bone osteoblastic cells, including the contiguous glutamic acid sequences (polyGlu) and the arginine-glycine-aspartic acid (RGD) motif. Markers of osteoblast differentiation, including matrix mineralization and alkaline phosphatase staining, were increased in cells expressing BSP mutants of the polyGlu sequences but not in cells expressing RGD-mutated BSP. We also determined the dependence on integrin-associated pathways in promoting BSP-mediated differentiation responses in osteoblasts by demonstrating the activation of focal adhesion kinase, MAP kinase-associated proteins ERK1/2, ribosomal s6 kinase 2 and the AP-1 protein cFos. Thus, the mechanism regulating osteoblast differentiation by BSP was determined to be dependent on integrin-mediated intracellular signaling pathways. Copyright 2008 S. Karger AG, Basel.

Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells.

PubMed

Sugiyama, Hayami; Takahashi, Kazutoshi; Yamamoto, Takuya; Iwasaki, Mio; Narita, Megumi; Nakamura, Masahiro; Rand, Tim A; Nakagawa, Masato; Watanabe, Akira; Yamanaka, Shinya

2017-01-10

Novel APOBEC1 target 1 (Nat1) (also known as "p97," "Dap5," and "Eif4g2") is a ubiquitously expressed cytoplasmic protein that is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (Eif4g1). We previously showed that Nat1-null mouse embryonic stem cells (mES cells) are resistant to differentiation. In the current study, we found that NAT1 and eIF4G1 share many binding proteins, such as the eukaryotic translation initiation factors eIF3 and eIF4A and ribosomal proteins. However, NAT1 did not bind to eIF4E or poly(A)-binding proteins, which are critical for cap-dependent translation initiation. In contrast, compared with eIF4G1, NAT1 preferentially interacted with eIF2, fragile X mental retardation proteins (FMR), and related proteins and especially with members of the proline-rich and coiled-coil-containing protein 2 (PRRC2) family. We also found that Nat1-null mES cells possess a transcriptional profile similar, although not identical, to the ground state, which is established in wild-type mES cells when treated with inhibitors of the ERK and glycogen synthase kinase 3 (GSK3) signaling pathways. In Nat1-null mES cells, the ERK pathway is suppressed even without inhibitors. Ribosome profiling revealed that translation of mitogen-activated protein kinase kinase kinase 3 (Map3k3) and son of sevenless homolog 1 (Sos1) is suppressed in the absence of Nat1 Forced expression of Map3k3 induced differentiation of Nat1-null mES cells. These data collectively show that Nat1 is involved in the translation of proteins that are required for cell differentiation.

ChiPPI: a novel method for mapping chimeric protein-protein interactions uncovers selection principles of protein fusion events in cancer.

PubMed

Frenkel-Morgenstern, Milana; Gorohovski, Alessandro; Tagore, Somnath; Sekar, Vaishnovi; Vazquez, Miguel; Valencia, Alfonso

2017-07-07

Fusion proteins, comprising peptides deriving from the translation of two parental genes, are produced in cancer by chromosomal aberrations. The expressed fusion protein incorporates domains of both parental proteins. Using a methodology that treats discrete protein domains as binding sites for specific domains of interacting proteins, we have cataloged the protein interaction networks for 11 528 cancer fusions (ChiTaRS-3.1). Here, we present our novel method, chimeric protein-protein interactions (ChiPPI) that uses the domain-domain co-occurrence scores in order to identify preserved interactors of chimeric proteins. Mapping the influence of fusion proteins on cell metabolism and pathways reveals that ChiPPI networks often lose tumor suppressor proteins and gain oncoproteins. Furthermore, fusions often induce novel connections between non-interactors skewing interaction networks and signaling pathways. We compared fusion protein PPI networks in leukemia/lymphoma, sarcoma and solid tumors finding distinct enrichment patterns for each disease type. While certain pathways are enriched in all three diseases (Wnt, Notch and TGF β), there are distinct patterns for leukemia (EGFR signaling, DNA replication and CCKR signaling), for sarcoma (p53 pathway and CCKR signaling) and solid tumors (FGFR and EGFR signaling). Thus, the ChiPPI method represents a comprehensive tool for studying the anomaly of skewed cellular networks produced by fusion proteins in cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

NASA Technical Reports Server (NTRS)

Ji, C.; Chen, Y.; McCarthy, T. L.; Centrella, M.

1999-01-01

Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

Hepatocytes express functional NOD1 and NOD2 receptors: A role for NOD1 in hepatocyte CC and CXC chemokine production

PubMed Central

Scott, Melanie J.; Chen, Christine; Sun, Qian; Billiar, Timothy R.

2010-01-01

Background & Aims NOD-like receptors are recently described cytosolic pattern recognition receptors. NOD1 and NOD2 are members of this family that recognize bacterial cell wall components, diaminopimelic acid and muramyl dipeptide, respectively. Both NOD1 and NOD2 have been associated with many inflammatory diseases, although their role in liver inflammation and infection has not been well studied. Materials and Methods We investigated the role of NOD receptors in mouse liver by assessing expression and activation of NOD1 and NOD2 in liver and primary isolated hepatocytes from C57BL/6 mice. Results Both NOD1 and NOD2 mRNA and protein were highly expressed in hepatocytes and liver. RIP2, the main signaling partner for NODs, was also expressed. Stimulation of hepatocytes with NOD1 ligand (C12-iEDAP) induced NFκB activation, activation of MAP kinases and expression of chemokines CCL5 (RANTES) and CXCL1 (KC). C12-iEDAP also synergized with interferon (IFN)γ to increase iNOS expression and production of nitric oxide. Despite activating NFκB, NOD1 ligand did not upregulate hepatocyte production of the acute phase proteins lipopolysaccharide binding protein, serum amyloid A, or soluble CD14 in cell culture supernatants, or upregulate mRNA expression of lipopolysaccharide binding protein, serum amyloid A, C-reactive protein, or serum amyloid P. NOD2 ligand (MDP) did not activate hepatocytes when given alone, but did synergize with Toll-like receptor ligands, lipopolysaccharide (LPS), and polyI:C to activate NFκB and MAPK. Conclusions All together these data suggest an important role for hepatocyte NOD1 in attracting leukocytes to the liver during infection and for hepatic NLRs to augment innate immune responses to pathogens. PMID:20615568

A High-Resolution Proteomic Landscaping of Primary Human Dental Stem Cells: Identification of SHED- and PDLSC-Specific Biomarkers.

PubMed

Taraslia, Vasiliki; Lymperi, Stefania; Pantazopoulou, Vasiliki; Anagnostopoulos, Athanasios K; Papassideri, Issidora S; Basdra, Efthimia K; Bei, Marianna; Kontakiotis, Evangelos G; Tsangaris, George Th; Stravopodis, Dimitrios J; Anastasiadou, Ema

2018-01-05

Dental stem cells (DSCs) have emerged as a promising tool for basic research and clinical practice. A variety of adult stem cell (ASC) populations can be isolated from different areas within the dental tissue, which, due to their cellular and molecular characteristics, could give rise to different outcomes when used in potential applications. In this study, we performed a high-throughput molecular comparison of two primary human adult dental stem cell (hADSC) sub-populations: Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) and Periodontal Ligament Stem Cells (PDLSCs). A detailed proteomic mapping of SHEDs and PDLSCs, via employment of nano-LC tandem-mass spectrometry (MS/MS) revealed 2032 identified proteins in SHEDs and 3235 in PDLSCs. In total, 1516 proteins were expressed in both populations, while 517 were unique for SHEDs and 1721 were exclusively expressed in PDLSCs. Further analysis of the recorded proteins suggested that SHEDs predominantly expressed molecules that are involved in organizing the cytoskeletal network, cellular migration and adhesion, whereas PDLSCs are highly energy-producing cells, vastly expressing proteins that are implicated in various aspects of cell metabolism and proliferation. Applying the Rho-GDI signaling pathway as a paradigm, we propose potential biomarkers for SHEDs and for PDLSCs, reflecting their unique features, properties and engaged molecular pathways.

A High-Resolution Proteomic Landscaping of Primary Human Dental Stem Cells: Identification of SHED- and PDLSC-Specific Biomarkers

PubMed Central

Taraslia, Vasiliki; Lymperi, Stefania; Pantazopoulou, Vasiliki; Anagnostopoulos, Athanasios K.; Basdra, Efthimia K.; Bei, Marianna; Kontakiotis, Evangelos G.; Tsangaris, George Th.; Stravopodis, Dimitrios J.; Anastasiadou, Ema

2018-01-01

Dental stem cells (DSCs) have emerged as a promising tool for basic research and clinical practice. A variety of adult stem cell (ASC) populations can be isolated from different areas within the dental tissue, which, due to their cellular and molecular characteristics, could give rise to different outcomes when used in potential applications. In this study, we performed a high-throughput molecular comparison of two primary human adult dental stem cell (hADSC) sub-populations: Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) and Periodontal Ligament Stem Cells (PDLSCs). A detailed proteomic mapping of SHEDs and PDLSCs, via employment of nano-LC tandem-mass spectrometry (MS/MS) revealed 2032 identified proteins in SHEDs and 3235 in PDLSCs. In total, 1516 proteins were expressed in both populations, while 517 were unique for SHEDs and 1721 were exclusively expressed in PDLSCs. Further analysis of the recorded proteins suggested that SHEDs predominantly expressed molecules that are involved in organizing the cytoskeletal network, cellular migration and adhesion, whereas PDLSCs are highly energy-producing cells, vastly expressing proteins that are implicated in various aspects of cell metabolism and proliferation. Applying the Rho-GDI signaling pathway as a paradigm, we propose potential biomarkers for SHEDs and for PDLSCs, reflecting their unique features, properties and engaged molecular pathways. PMID:29304003

Hepatic Proteome Analysis of Atlantic Salmon (Salmo salar) After Exposure to Environmental Concentrations of Human Pharmaceuticals*

PubMed Central

Hampel, Miriam; Alonso, Esteban; Aparicio, Irene; Santos, Juan Luis; Leaver, Michael

2015-01-01

Pharmaceuticals are pseudopersistent aquatic pollutants with unknown effects at environmentally relevant concentrations. Atlantic salmon (Salmo salar) were exposed to Acetaminophen: 54.77 ± 34.67; Atenolol: 11.08 ± 7.98, and Carbamazepine: 7.85 ± 0.13 μg·L−1 for 5 days. After Acetaminophen treatment, 19 proteins were differently expressed, of which 11 were significant with respect to the control group (eight up-regulated and three down-regulated). After Atenolol treatment, seven differently expressed proteins were obtained in comparison with the control, of which six could be identified (four up-regulated and two down-regulated). Carbamazepine exposure resulted in 15 differently expressed proteins compared with the control, with 10 of them identified (seven up-regulated and three down-regulated). Out of these, three features were common between Acetaminophen and Carbamazepine and one between Carbamazepine and Atenolol. One feature was common across all treatments. Principal component analysis and heat map clustering showed a clear grouping of the variability caused by the applied treatments. The obtained data suggest (1) that exposure to environmentally relevant concentrations of the pharmaceuticals alters the hepatic protein expression profile of the Atlantic salmon; and (2) the existence of treatment specific processes that may be useful for biomarker development. PMID:25394398

Genomics of a Metamorphic Timing QTL: met1 Maps to a Unique Genomic Position and Regulates Morph and Species-Specific Patterns of Brain Transcription

PubMed Central

Page, Robert B.; Boley, Meredith A.; Kump, David K.; Voss, Stephen R.

2013-01-01

Very little is known about genetic factors that regulate life history transitions during ontogeny. Closely related tiger salamanders (Ambystoma species complex) show extreme variation in metamorphic timing, with some species foregoing metamorphosis altogether, an adaptive trait called paedomorphosis. Previous studies identified a major effect quantitative trait locus (met1) for metamorphic timing and expression of paedomorphosis in hybrid crosses between the biphasic Eastern tiger salamander (Ambystoma tigrinum tigrinum) and the paedomorphic Mexican axolotl (Ambystoma mexicanum). We used existing hybrid mapping panels and a newly created hybrid cross to map the met1 genomic region and determine the effect of met1 on larval growth, metamorphic timing, and gene expression in the brain. We show that met1 maps to the position of a urodele-specific chromosome rearrangement on linkage group 2 that uniquely brought functionally associated genes into linkage. Furthermore, we found that more than 200 genes were differentially expressed during larval development as a function of met1 genotype. This list of differentially expressed genes is enriched for proteins that function in the mitochondria, providing evidence of a link between met1, thyroid hormone signaling, and mitochondrial energetics associated with metamorphosis. Finally, we found that met1 significantly affected metamorphic timing in hybrids, but not early larval growth rate. Collectively, our results show that met1 regulates species and morph-specific patterns of brain transcription and life history variation. PMID:23946331

2D DIGE Does Not Reveal all: A Scotopic Report Suggests Differential Expression of a Single "Calponin Family Member" Protein for Tetany of Sphincters!

PubMed

Chaudhury, Arun

2015-01-01

Using 2D differential gel electrophoresis (DIGE) and mass spectrometry (MS), a recent report by Rattan and Ali (2015) compared proteome expression between tonically contracted sphincteric smooth muscles of the internal anal sphincter (IAS), in comparison to the adjacent rectum [rectal smooth muscles (RSM)] that contracts in a phasic fashion. The study showed the differential expression of a single 23 kDa protein SM22, which was 1.87 fold, overexpressed in RSM in comparison to IAS. Earlier studies have shown differences in expression of different proteins like Rho-associated protein kinase II, myosin light chain kinase, myosin phosphatase, and protein kinase C between IAS and RSM. The currently employed methods, despite its high-throughput potential, failed to identify these well-characterized differences between phasic and tonic muscles. This calls into question the fidelity and validatory potential of the otherwise powerful technology of 2D DIGE/MS. These discrepancies, when redressed in future studies, will evolve this recent report as an important baseline study of "sphincter proteome." Proteomics techniques are currently underutilized in examining pathophysiology of hypertensive/hypotensive disorders involving gastrointestinal sphincters, including achalasia, gastroesophageal reflux disease (GERD), spastic pylorus, seen during diabetes or chronic chemotherapy, intestinal pseudo-obstruction, and recto-anal incontinence. Global proteome mapping may provide instant snapshot of the complete repertoire of differential proteins, thus expediting to identify the molecular pathology of gastrointestinal motility disorders currently labeled "idiopathic" and facilitating practice of precision medicine.

Proteomics Analysis of Lactobacillus casei Zhang, a New Probiotic Bacterium Isolated from Traditional Home-made Koumiss in Inner Mongolia of China*

PubMed Central

Wu, Rina; Wang, Weiwei; Yu, Dongliang; Zhang, Wenyi; Li, Yan; Sun, Zhihong; Wu, Junrui; Meng, He; Zhang, Heping

2009-01-01

Lactobacillus casei Zhang, isolated from traditional home-made koumiss in Inner Mongolia of China, was considered as a new probiotic bacterium by probiotic selection tests. We carried out a proteomics study to identify and characterize proteins expressed by L. casei Zhang in the exponential phase and stationary phase. Cytosolic proteins of the strain cultivated in de Man, Rogosa, and Sharpe broth were resolved by two-dimensional gel electrophoresis using pH 4–7 linear gradients. The number of protein spots quantified from the gels was 487 ± 21 (exponential phase) and 494 ± 13 (stationary phase) among which a total of 131 spots were identified by MALDI-TOF/MS and/or MALDI-TOF/TOF according to significant growth phase-related differences or high expression intensity proteins. Accompanied by the cluster of orthologous groups (COG), codon adaptation index (CAI), and GRAVY value analysis, the study provided a very first insight into the profile of protein expression as a reference map of L. casei. Forty-seven spots were also found in the study that showed statistically significant differences between exponential phase and stationary phase. Thirty-three of the spots increased at least 2.5-fold in the stationary phase in comparison with the exponential phase, including 19 protein spots (e.g. Hsp20, DnaK, GroEL, LuxS, pyruvate kinase, and GalU) whose intensity up-shifted above 3.0-fold. Transcriptional profiles were conducted to confirm several important differentially expressed proteins by using real time quantitative PCR. The analysis suggests that the differentially expressed proteins were mainly categorized as stress response proteins and key components of central and intermediary metabolism, indicating that these proteins might play a potential important role for the adaptation to the surroundings, especially the accumulation of lactic acid in the course of growth, and the physiological processes in bacteria cell. PMID:19508964

Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC-MS/MS. 2. Similarity search between protein maps for the analysis of protein complexes.

PubMed

Jin, Ya; Yuan, Qi; Zhang, Jun; Manabe, Takashi; Tan, Wen

2015-09-01

Human bronchial smooth muscle cell soluble proteins were analyzed by a combined method of nondenaturing micro 2DE, grid gel-cutting, and quantitative LC-MS/MS and a native protein map was prepared for each of the identified 4323 proteins [1]. A method to evaluate the degree of similarity between the protein maps was developed since we expected the proteins comprising a protein complex would be separated together under nondenaturing conditions. The following procedure was employed using Excel macros; (i) maps that have three or more squares with protein quantity data were selected (2328 maps), (ii) within each map, the quantity values of the squares were normalized setting the highest value to be 1.0, (iii) in comparing a map with another map, the smaller normalized quantity in two corresponding squares was taken and summed throughout the map to give an "overlap score," (iv) each map was compared against all the 2328 maps and the largest overlap score, obtained when a map was compared with itself, was set to be 1.0 thus providing 2328 "overlap factors," (v) step (iv) was repeated for all maps providing 2328 × 2328 matrix of overlap factors. From the matrix, protein pairs that showed overlap factors above 0.65 from both protein sides were selected (431 protein pairs). Each protein pair was searched in a database (UniProtKB) on complex formation and 301 protein pairs, which comprise 35 protein complexes, were found to be documented. These results demonstrated that native protein maps and their similarity search would enable simultaneous analysis of multiple protein complexes in cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ZikaBase: An integrated ZIKV- Human Interactome Map database.

PubMed

Gurumayum, Sanathoi; Brahma, Rahul; Naorem, Leimarembi Devi; Muthaiyan, Mathavan; Gopal, Jeyakodi; Venkatesan, Amouda

2018-01-15

Re-emergence of ZIKV has caused infections in more than 1.5 million people. The molecular mechanism and pathogenesis of ZIKV is not well explored due to unavailability of adequate model and lack of publically accessible resources to provide information of ZIKV-Human protein interactome map till today. This study made an attempt to curate the ZIKV-Human interaction proteins from published literatures and RNA-Seq data. 11 direct interaction, 12 associated genes are retrieved from literatures and 3742 Differentially Expressed Genes (DEGs) are obtained from RNA-Seq analysis. The genes have been analyzed to construct the ZIKV-Human Interactome Map. The importance of the study has been illustrated by the enrichment analysis and observed that direct interaction and associated genes are enriched in viral entry into host cell. Also, ZIKV infection modulates 32% signal and 27% immune system pathways. The integrated database, ZikaBase has been developed to help the virology research community and accessible at https://test5.bicpu.edu.in. Copyright © 2017 Elsevier Inc. All rights reserved.

Analysis of the Bovine Monocyte-Derived Macrophage Response to Mycobacterium avium Subspecies Paratuberculosis Infection Using RNA-seq.

PubMed

Casey, Maura E; Meade, Kieran G; Nalpas, Nicolas C; Taraktsoglou, Maria; Browne, John A; Killick, Kate E; Park, Stephen D E; Gormley, Eamonn; Hokamp, Karsten; Magee, David A; MacHugh, David E

2015-01-01

Johne's disease, caused by infection with Mycobacterium avium subsp. paratuberculosis, (MAP), is a chronic intestinal disease of ruminants with serious economic consequences for cattle production in the United States and elsewhere. During infection, MAP bacilli are phagocytosed and subvert host macrophage processes, resulting in subclinical infections that can lead to immunopathology and dissemination of disease. Analysis of the host macrophage transcriptome during infection can therefore shed light on the molecular mechanisms and host-pathogen interplay associated with Johne's disease. Here, we describe results of an in vitro study of the bovine monocyte-derived macrophage (MDM) transcriptome response during MAP infection using RNA-seq. MDM were obtained from seven age- and sex-matched Holstein-Friesian cattle and were infected with MAP across a 6-h infection time course with non-infected controls. We observed 245 and 574 differentially expressed (DE) genes in MAP-infected versus non-infected control samples (adjusted P value ≤0.05) at 2 and 6 h post-infection, respectively. Functional analyses of these DE genes, including biological pathway enrichment, highlighted potential functional roles for genes that have not been previously described in the host response to infection with MAP bacilli. In addition, differential expression of pro- and anti-inflammatory cytokine genes, such as those associated with the IL-10 signaling pathway, and other immune-related genes that encode proteins involved in the bovine macrophage response to MAP infection emphasize the balance between protective host immunity and bacilli survival and proliferation. Systematic comparisons of RNA-seq gene expression results with Affymetrix(®) microarray data generated from the same experimental samples also demonstrated that RNA-seq represents a superior technology for studying host transcriptional responses to intracellular infection.

Analysis of the Bovine Monocyte-Derived Macrophage Response to Mycobacterium avium Subspecies Paratuberculosis Infection Using RNA-seq

PubMed Central

Casey, Maura E.; Meade, Kieran G.; Nalpas, Nicolas C.; Taraktsoglou, Maria; Browne, John A.; Killick, Kate E.; Park, Stephen D. E.; Gormley, Eamonn; Hokamp, Karsten; Magee, David A.; MacHugh, David E.

2015-01-01

Johne’s disease, caused by infection with Mycobacterium avium subsp. paratuberculosis, (MAP), is a chronic intestinal disease of ruminants with serious economic consequences for cattle production in the United States and elsewhere. During infection, MAP bacilli are phagocytosed and subvert host macrophage processes, resulting in subclinical infections that can lead to immunopathology and dissemination of disease. Analysis of the host macrophage transcriptome during infection can therefore shed light on the molecular mechanisms and host-pathogen interplay associated with Johne’s disease. Here, we describe results of an in vitro study of the bovine monocyte-derived macrophage (MDM) transcriptome response during MAP infection using RNA-seq. MDM were obtained from seven age- and sex-matched Holstein-Friesian cattle and were infected with MAP across a 6-h infection time course with non-infected controls. We observed 245 and 574 differentially expressed (DE) genes in MAP-infected versus non-infected control samples (adjusted P value ≤0.05) at 2 and 6 h post-infection, respectively. Functional analyses of these DE genes, including biological pathway enrichment, highlighted potential functional roles for genes that have not been previously described in the host response to infection with MAP bacilli. In addition, differential expression of pro- and anti-inflammatory cytokine genes, such as those associated with the IL-10 signaling pathway, and other immune-related genes that encode proteins involved in the bovine macrophage response to MAP infection emphasize the balance between protective host immunity and bacilli survival and proliferation. Systematic comparisons of RNA-seq gene expression results with Affymetrix® microarray data generated from the same experimental samples also demonstrated that RNA-seq represents a superior technology for studying host transcriptional responses to intracellular infection. PMID:25699042

Epigallocatechin activates haem oxygenase-1 expression via protein kinase Cδ and Nrf2

PubMed Central

Ogborne, Richard M.; Rushworth, Stuart A.; O’Connell, Maria A.

2008-01-01

The Nrf2/anti-oxidant response element (ARE) pathway plays an important role in regulating cellular anti-oxidants, including haem oxygenase-1 (HO-1). Various kinases have been implicated in the pathways leading to Nrf2 activation. Here, we investigated the effect of epigallocatechin (EGC) on ARE-mediated gene expression in human monocytic cells. EGC time and dose dependently increased HO-1 mRNA and protein expression but had minimal effect on expression of other ARE-regulated genes, including NAD(P)H:quinone oxidoreductase 1, glutathione cysteine ligase and ferritin. siRNA knock down of Nrf2 significantly inhibited EGC-induced HO-1 expression. Furthermore, inhibition of PKC by Ro-31-8220 dose dependently decreased EGC-induced HO-1 mRNA expression, whereas MAP kinase and phosphatidylinositol-3-kinase pathway inhibitors had no significant effect. EGC stimulated phosphorylation of PKCαβ and δ in THP-1 cells. PKCδ inhibition significantly decreased EGC-induced HO-1 mRNA expression, whereas PKCα- and β-specific inhibitors had no significant effect. These results demonstrate for the first time that EGC-induced HO-1 expression occurs via PKCδ and Nrf2. PMID:18586007

An unusual MAP kinase is required for efficient penetration of the plant surface by Ustilago maydis

PubMed Central

Brachmann, Andreas; Schirawski, Jan; Müller, Philip; Kahmann, Regine

2003-01-01

In Ustilago maydis, pathogenic development is controlled by a heterodimer of the two homeodomain proteins bW and bE. We have identified by RNA fingerprinting a b-regulated gene, kpp6, which encodes an unusual MAP kinase. Kpp6 is similar to a number of other fungal MAP kinases involved in mating and pathogenicity, but contains an additional N-terminal domain unrelated to other proteins. Transcription of the kpp6 gene yields two transcripts differing in length, but encoding proteins of identical mass. One transcript is upregulated by the bW/bE heterodimer, while the other is induced after pheromone stimulation. kpp6 deletion mutants are attenuated in pathogenicity. kpp6T355A,Y357F mutants carrying a non-activatable allele of kpp6 are more severely compromised in pathogenesis. These strains can still form appressoria, but are defective in the subsequent penetration of the plant cuticle. Kpp6 is expressed during all stages of the sexual life cycle except mature spores. We speculate that Kpp6 may respond to a plant signal and regulate the genes necessary for efficient penetration of plant tissue. PMID:12727886

Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell

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

Krieg, A.M.; Gourley, M.F.; Steinberg, A.D.

1991-05-01

Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymicmore » epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells.« less

Molecular mechanisms of pathogenesis in hepatocellular carcinoma revealed by RNA‑sequencing.

PubMed

Liu, Yao; Yang, Zhe; Du, Feng; Yang, Qiao; Hou, Jie; Yan, Xiaohong; Geng, Yi; Zhao, Yaning; Wang, Hua

2017-11-01

The present study aimed to explore the underlying molecular mechanisms of hepatocellular carcinoma (HCC). RNA‑sequencing profiles GSM629264 and GSM629265, from the GSE25599 data set, were downloaded from the Gene Expression Omnibus database and processed by quality evaluation. GSM629264 and GSM629265 were from HCC and adjacent non‑cancerous tissues, respectively. TopHat software was used for alignment analysis, followed by the detection of novel splicing sites. In addition, the Cufflinks software package was used to analyze gene expressions, and the Cuffdiff program was used to screen for differently expressed genes (DEGs) and differentially expressed splicing variants. Gene ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of DEGs were also performed. Transcription factors (TFs) and microRNAs (miRNAs) that regulate DEGs were identified, and a protein‑protein interaction (PPI) network was constructed. The hub node in the PPI network was obtained, and the TFs and miRNAs that regulated the hub node were further predicted. The quality of the sequencing data met the standards for analysis, and the clean reads were ~65%. Most sequencing reads mapped into coding sequence exons (CDS_exons), whereas other reads mapped into exon 3' untranslated regions (UTR_Exons), 5'UTR_Exons and Introns. Upregulated and downregulated DEGs between HCC and adjacent non‑cancerous tissues were screened. Genes of differentially expressed splicing variants were identified, including vesicle‑associated membrane protein 4, phosphatidylinositol glycan anchor biosynthesis class C, protein disulfide isomerase family A member 4 and growth arrest specific 5. Screened DEGs were enriched in the complement pathway. In the PPI network, ubiquitin C (UBC) was the hub node. UBC was predicted to be regulated by several TFs, including specificity protein 1 (SP1), FBJ murine osteosarcoma viral oncogene homolog (FOS), proto‑oncogene c‑JUN (JUN), FOS‑like antigen 2 (FOSL2) and SWI/SNF‑related, matrix‑associated, actin‑dependent regulator of chromatin, subfamily A, member 4 (SMARCA4), and several miRNAs, including miR‑30 and miR‑181. Results from the present study demonstrated that UBC, SP1, FOS, JUN, FOSL2, SMARCA4, miR‑30 and miR‑181 may participate in the development of HCC.

Expression, Purification and Characterization of GMZ2'.10C, a Complex Disulphide-Bonded Fusion Protein Vaccine Candidate against the Asexual and Sexual Life-Stages of the Malaria-Causing Plasmodium falciparum Parasite.

PubMed

Mistarz, Ulrik H; Singh, Susheel K; Nguyen, Tam T T N; Roeffen, Will; Yang, Fen; Lissau, Casper; Madsen, Søren M; Vrang, Astrid; Tiendrebeogo, Régis W; Kana, Ikhlaq H; Sauerwein, Robert W; Theisen, Michael; Rand, Kasper D

2017-09-01

Production and characterization of a chimeric fusion protein (GMZ2'.10C) which combines epitopes of key malaria parasite antigens: glutamate-rich protein (GLURP), merozoite surface protein 3 (MSP3), and the highly disulphide bonded Pfs48/45 (10C). GMZ2'.10C is a potential candidate for a multi-stage malaria vaccine that targets both transmission and asexual life-cycle stages of the parasite. GMZ2'.10C was produced in Lactococcus lactis and purified using either an immunoaffinity purification (IP) or a conventional purification (CP) method. Protein purity and stability was analysed by RP-HPLC, SEC-HPLC, 2-site ELISA, gel-electrophoresis and Western blotting. Structural characterization (mass analysis, peptide mapping and cysteine connectivity mapping) was performed by LC-MS/MS. CP-GMZ2'.10C resulted in similar purity, yield, structure and stability as compared to IP-GMZ2'.10C. CP-GMZ2'.10C and IP-GMZ2'.10C both elicited a high titer of transmission blocking (TB) antibodies in rodents. The intricate disulphide-bond connectivity of C-terminus Pfs48/45 was analysed by tandem mass spectrometry and was established for GMZ2'.10C and two reference fusion proteins encompassing similar parts of Pfs48/45. GMZ2'.10C, combining GMZ2' and correctly-folded Pfs48/45 can be produced by the Lactoccus lactis P170 based expression system in purity and quality for pharmaceutical development and elicit high level of TB antibodies. The cysteine connectivity for the 10C region of Pfs48/45 was revealed experimentally, providing an important guideline for employing the Pfs48/45 antigen in vaccine design.

Differentially expressed genes in the silk gland of silkworm (Bombyx mori) treated with TiO2 NPs.

PubMed

Xue, Bin; Li, Fanchi; Hu, Jingsheng; Tian, Jianghai; Li, Jinxin; Cheng, Xiaoyu; Hu, Jiahuan; Li, Bing

2017-05-05

Silk gland is a silkworm organ where silk proteins are synthesized and secreted. Dietary supplement of TiO 2 nanoparticles (NPs) promotes silk protein synthesis in silkworms. In this study, digital gene expression (DGE) tag was used to analyze the gene expression profile of the posterior silk gland of silkworms that were fed with TiO 2 NPs. In total, 5,702,823 and 6,150,719 clean tags, 55,096 and 74,715 distinct tags were detected in TiO 2 NPs treated and control groups, respectively. Compared with the control, TiO 2 NPs treated silkworms showed 306 differentially expressed genes, including 137 upregulated genes and 169 downregulated genes. Of these differentially expressed genes, 106 genes were related to silk protein synthesis, among which 97 genes were upregulated and 9 genes were downregulated. Pathway mapping using the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that 20 pathways were significantly enriched in TiO 2 NPs treated silkworms, and the metabolic pathway-related genes were the most significantly enriched. The DGE results were verified by qRT-PCR analysis of eight differentially expressed genes. The DGE and qRT-PCR results were consistent for all three upregulated genes and three of the five downregulated genes, but the expression trends of the remaining two genes were different between qRT-PCR and DGE analysis. This study enhances our understanding of the mechanism of TiO 2 NPs promoted silk protein synthesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Se metallomics during lactic fermentation of Se-enriched yogurt.

PubMed

Palomo, María; Gutiérrez, Ana M; Pérez-Conde, M Concepción; Cámara, Carmen; Madrid, Yolanda

2014-12-01

Selenium biotransformation by lactic acid bacteria during the preparation of Se-enriched yogurt was evaluated. The study focused on the distribution of selenium in the aqueous soluble protein fraction and the detection of selenoamino acids. Screening of selenium in Tris-buffer-urea soluble fraction was carried out by sodium dodecyl sulphate polyacrylamide gel electrophoresis after pre-fractionating with asymmetric field flow fractionation using inductively coupled plasma-mass spectrometry as the detector. Selenium-containing fractions were identified by peptide mapping using nano LC-ESI/LTQMS. Proteins such as thioredoxin, glutaredoxin, albumin, β-lactoglobulin, and lactoperoxidase were identified in the selenium-containing fraction. All these proteins were detected in both the control and the selenium-enriched yogurt except chaperones, which were only detected in the control samples. Chaperones are heat-shock proteins expressed in response to elevated temperature or other cellular stresses. Selenium may have an effect on chaperones expression in Lactobacillus. For the amino acids analysis, selenocysteine was the primary seleno-containing species. Copyright © 2014 Elsevier Ltd. All rights reserved.

Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

PubMed Central

Guo, Zhong; Johnston, Wayne; Kovtun, Oleksiy; Mureev, Sergey; Bröcker, Cornelia; Ungermann, Christian; Alexandrov, Kirill

2013-01-01

Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data. PMID:24312556

Production of chimeric recombinant single domain antibody-green fluorescent fusion protein in Chinese hamster ovary cells.

PubMed

Bazl, M Rajabi; Rasaee, M J; Foruzandeh, M; Rahimpour, A; Kiani, J; Rahbarizadeh, F; Alirezapour, B; Mohammadi, M

2007-02-01

There is an increasing interest in the application of nanobodies such as VHH in the field of therapy and imaging. In the present study a stable genetically engineered cell line of Chinese hamster ovary (CHO) origin transfected using two sets of expression vectors was constructed in order to permit the cytoplasmic and extracellular expression of single domain antibody along with green fluorescent protein (GFP) as reporter gene. The quality of the constructs were examined both by the restriction map as well as sequence analysis. The gene transfection and protein expression was further examined by reverse transcription-polymerase chain reaction (RT-PCR). The transfected cells were grown in 200 microg/mL hygromycin containing media and the stable cell line obtained showed fluorescent activity for more than a period of 180 days. The production of fusion protein was also detected by fluorescent microscopy, fluorescent spectroscopy as well as by enzyme-linked immunosorbent assay (ELISA) analysis. This strategy allows a rapid production of recombinant fluobodies involving VHH, which can be used in various experiments such as imaging and detection in which a primary labeled antibody is required.

Developmental study of the distribution of hypoxia-induced factor-1 alpha and microtubule-associated protein 2 in children's brainstem: comparison between controls and cases with signs of perinatal hypoxia.

PubMed

Coveñas, R; González-Fuentes, J; Rivas-Infante, E; Lagartos-Donate, M J; Cebada-Sánchez, S; Arroyo-Jiménez, M M; Insausti, R; Marcos, P

2014-06-20

Perinatal asphyxia and hypoxia are common causes of morbidity in neonates. Prenatal birth associated with hypoxemia often results in several disorders because of the lack of oxygen in the brain. Survival rates from perinatal hypoxia have improved, but appropriate treatments for recovery are still limited, with great impact on patients, their families, society in general and health systems. The aim of this work is to contribute to a better understanding of the cellular mechanisms underlying the brainstem responses to hypoxia. For this purpose, distributions of two proteins, hypoxia-inducible factor-1 alpha (HIF-1α) and microtubule-associated protein 2 (MAP-2) were analyzed in brainstems of 11 children, four of them showing neuropathological evidence of brain hypoxia. They were included in control or hypoxic groups, and then in several subgroups according to their age. Immunohistochemical labeling for these proteins revealed only cell bodies containing HIF-1α, and both cell bodies and fibers positive for MAP-2 in the children's brainstems. The distribution of HIF-1α was more restricted than that of MAP-2, and it can be suggested that the expression of HIF-1α increased with age. The distribution pattern of MAP-2 in the medulla oblongata could be more due to age-related changes than to a response to hypoxic damage, whereas in the pons several regions, such as the nucleus ambiguus or the solitary nucleus, showed different immunolabeling patterns in controls and hypoxic cases. The distribution patterns of these two proteins suggest that some brainstem regions, such as the reticular formation or the central gray, could be less affected by conditions of hypoxia. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Down-Regulation by Resveratrol of Basic Fibroblast Growth Factor-Stimulated Osteoprotegerin Synthesis through Suppression of Akt in Osteoblasts

PubMed Central

Kuroyanagi, Gen; Otsuka, Takanobu; Yamamoto, Naohiro; Matsushima-Nishiwaki, Rie; Nakakami, Akira; Mizutani, Jun; Kozawa, Osamu; Tokuda, Haruhiko

2014-01-01

It is firmly established that resveratrol, a natural food compound abundantly found in grape skins and red wine, has beneficial properties for human health. In the present study, we investigated the effect of basic fibroblast growth factor (FGF-2) on osteoprotegerin (OPG) synthesis in osteoblast-like MC3T3-E1 cells and whether resveratrol affects the OPG synthesis. FGF-2 stimulated both the OPG release and the expression of OPG mRNA. Resveratrol significantly suppressed the FGF-2-stimulated OPG release and the mRNA levels of OPG. SRT1720, an activator of SIRT1, reduced the FGF-2-induced OPG release and the OPG mRNA expression. PD98059, an inhibitor of upstream kinase activating p44/p42 mitogen-activated protein (MAP) kinase, had little effect on the FGF-2-stimulated OPG release. On the other hand, SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and Akt inhibitor suppressed the OPG release induced by FGF-2. Resveratrol failed to affect the FGF-2-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. The phosphorylation of Akt induced by FGF-2 was significantly suppressed by resveratrol or SRT1720. These findings strongly suggest that resveratrol down-regulates FGF-2-stimulated OPG synthesis through the suppression of the Akt pathway in osteoblasts and that the inhibitory effect of resveratrol is mediated at least in part by SIRT1 activation. PMID:25290095

A Novel Malaria Vaccine Candidate Antigen Expressed in Tetrahymena thermophila

PubMed Central

Eleni-Muus, Janna; Aldag, Ingo; Samuel, Kay; Creasey, Alison M.; Hartmann, Marcus W. W.; Cavanagh, David R.

2014-01-01

Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens. PMID:24489871

Understanding sequence similarity and framework analysis between centromere proteins using computational biology.

PubMed

Doss, C George Priya; Chakrabarty, Chiranjib; Debajyoti, C; Debottam, S

2014-11-01

Certain mysteries pointing toward their recruitment pathways, cell cycle regulation mechanisms, spindle checkpoint assembly, and chromosome segregation process are considered the centre of attraction in cancer research. In modern times, with the established databases, ranges of computational platforms have provided a platform to examine almost all the physiological and biochemical evidences in disease-associated phenotypes. Using existing computational methods, we have utilized the amino acid residues to understand the similarity within the evolutionary variance of different associated centromere proteins. This study related to sequence similarity, protein-protein networking, co-expression analysis, and evolutionary trajectory of centromere proteins will speed up the understanding about centromere biology and will create a road map for upcoming researchers who are initiating their work of clinical sequencing using centromere proteins.

The expression of a naturally occuriing truncated allele of an alpha-SNAP gene suppresses plant parasitic nematode infection

USDA-ARS?s Scientific Manuscript database

rhg1, defined within a 67 kb region of DNA on chromosome 18, is a major quantitative trait locus (QTL) in Glycine max (soybean) providing defense to the soybean cyst nematode (Heterodera glycines). Transcriptional mapping experiments identified an alpha soluble NSF attachment protein (alpha-SNAP) wi...

Articular Chondrocytes Express the Receptor for Advanced Glycation End Products

PubMed Central

Loeser, Richard F.; Yammani, Raghunatha R.; Carlson, Cathy S.; Chen, Hong; Cole, Ada; Im, Hee-Jeong; Bursch, Laura S.; Yan, Shi Du

2006-01-01

Objective The receptor for advanced glycation end products (RAGE) binds multiple ligands, including S100 proteins, high mobility group box chromosomal protein 1 (HMGB-1), and AGEs, all of which are present in articular cartilage. Stimulation of RAGE signaling can lead to MAP kinase activation and increased NF-κB activity. The objective of the present study was to determine if chondrocytes express functional RAGE. Methods The presence of chondrocyte RAGE was analyzed by immunohistochemistry using normal and osteoarthritic (OA) cartilage from young and old monkeys and humans, immunoblotting of chondrocyte lysates and human cartilage extracts, and reverse transcription–polymerase chain reaction (RT-PCR) analysis of RNA from chondrocytes treated with interleukin-1 (IL-1) and fibronectin fragments. RAGE signaling was evaluated by stimulating chondrocytes with S100B and HMGB-1 and analyzing for activation of the ERK MAP kinase and NF-κB. The ability of S100B and HMGB-1 to stimulate matrix metalloproteinase 13 (MMP-13) production was also assessed. A pull-down assay using biotin-labeled S100B was used to demonstrate binding to RAGE. Results RAGE was detected in sections of monkey knee cartilage and human knee and ankle cartilage. Increased immunostaining for RAGE was noted in cartilage from older adult monkeys and humans and was further increased in OA tissue. RAGE was also detected by immunoblotting and by RT-PCR, where IL-1β and fibronectin fragments were found to stimulate RAGE expression. Stimulation of chondrocytes with S100B or HMGB-1 increased phosphorylation of the ERK MAP kinase and the p65 subunit of NF-κB and increased the production of MMP-13. This signaling was inhibited in cells pretreated with soluble RAGE, and S100B was shown to bind to chondrocyte RAGE. Conclusion Articular chondrocytes express functional RAGE. The increase in RAGE noted in OA cartilage and the ability of RAGE ligands to stimulate chondrocyte MAP kinase and NF-κB activity and to stimulate MMP-13 production suggests that chondrocyte RAGE signaling could play a role in OA. PMID:16052547

Developmental expression of the neuroligins and neurexins in fragile X mice.

PubMed

Lai, Jonathan K Y; Doering, Laurie C; Foster, Jane A

2016-03-01

Neuroligins and neurexins are transsynaptic proteins involved in the maturation of glutamatergic and GABAergic synapses. Research has identified synaptic proteins and function as primary contributors to the development of fragile X syndrome. Fragile X mental retardation protein (FMRP), the protein that is lacking in fragile X syndrome, binds neuroligin-1 and -3 mRNA. Using in situ hybridization, we examined temporal and spatial expression patterns of neuroligin (NLGN) and neurexin (NRXN) mRNAs in the somatosensory (S1) cortex and hippocampus in wild-type (WT) and fragile X knockout (FMR1-KO) mice during the first 5 weeks of postnatal life. Genotype-based differences in expression included increased NLGN1 mRNA in CA1 and S1 cortex, decreased NLGN2 mRNA in CA1 and dentate gyrus (DG) regions of the hippocampus, and increased NRXN3 mRNA in CA1, DG, and S1 cortex between female WT and FMR1-KO mice. In male mice, decreased expression of NRXN3 mRNA was observed in CA1 and DG regions of FMR1-KO mice. Sex differences in hippocampal expression of NLGN2, NRXN1, NRXN2, and NRXN3 mRNAs and in S1 cortex expression of NRXN3 mRNAs were observed WT mice, whereas sex differences in NLGN3, NRXN1, NRXN2, and NRXN3 mRNA expression in the hippocampus and in NLGN1, NRXN2 and NRXN3 mRNA expression in S1 cortex were detected in FMR1-KO mice. These results provide a neuroanatomical map of NLGN and NRXN expression patterns over postnatal development in WT and FMR1-KO mice. The differences in developmental trajectory of these synaptic proteins could contribute to long-term differences in CNS wiring and synaptic function. © 2015 Wiley Periodicals, Inc.

GREEN TEA BEVERAGE AND EPIGALLOCATECIHIN GALLATE ATTENUATE NICOTINE CARDIOCYTOTOXICITY IN RAT.

PubMed

Nacerai, Haroun; Gregory, Tufo; Sihem, Berdja; Salah, Akkal; Souhila, Aouichat-Bouguerra

2017-01-01

Nicotine, the principal alkaloid in tobacco, induces a cellular damage on heart and cardiomyocyte culture. We investigate the protective role of green tea extract (GTE) against nicotine. Male albino rats were treated by injecting nicotine (1 mg/kg b.w. for 2 months) subcutaneously and thereby supplementing GTE 2% orally to them. The levels of plasma lipids, cardiac MDA (malondialdehyde) and catalase activity Mitogen-activated proteins kinases MAPKs were measured. The expression levels of (ERK 1/2, extracellular signal - regulated kinase 1/2 and P38 MAP kinase), endoplasmic reticulum stress (ERS)-related protein (GRP78 glucose regulated protein-78, HSP70 heat shock protein-70, CHOP C/EBP homologous protein), AIF (apoptosis-inducing factor) and VDAC (voltage-dependant anion channel) were evaluated by Western blot. In the in vitro study, the cardiomyocytes were exposed to nicotine (10 μM) and major GTE polyphenol epigallocatechin gallate EGCG (50 μM). Data showed that nicotine induced a significant increase on MDA levels, LDH (lactate dehy- drogenase) and aminotransferase activity compared with control. The heart sections of nicotine exposed-rats showed severe degenerative changes. Nicotine increased the expression of P38, but not ERK 1/2, ER stress-related proteins and AIF with no changes of VDAC. Concomitant GTE treatment significantly normalized and/or improved,the levels of MDA, enzymatic activity and histological injuries. The proteins expression was attenuated by GTE co-administration without any changes for VDAC. ERK 1/2 expression enhanced in GTE- treated groups. Exposure of cardiac cells to nicotine induced the expression of ERS markers and p38; the ERK 1/2 was highly expressed only in the presence of EGCG. It was suggested that green tea beverage can protect against nicotine toxicity by attenuating oxidative stress, endoplasmic reticulum stress and apoptosis. Otherwise, our results have showed that ERK1/2 and p38 are survival signaling pathways activated by GTE and EGCG.

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

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

Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

2015-03-01

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interruptsmore » the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.« less

Lineage specific expression of Polycomb Group Proteins in human embryonic stem cells in vitro.

PubMed

Pethe, Prasad; Pursani, Varsha; Bhartiya, Deepa

2015-05-01

Human embryonic (hES) stem cells are an excellent model to study lineage specification and differentiation into various cell types. Differentiation necessitates repression of specific genes not required for a particular lineage. Polycomb Group (PcG) proteins are key histone modifiers, whose primary function is gene repression. PcG proteins form complexes called Polycomb Repressive Complexes (PRCs), which catalyze histone modifications such as H2AK119ub1, H3K27me3, and H3K9me3. PcG proteins play a crucial role during differentiation of stem cells. The expression of PcG transcripts during differentiation of hES cells into endoderm, mesoderm, and ectoderm lineage is yet to be shown. In-house derived hES cell line KIND1 was differentiated into endoderm, mesoderm, and ectoderm lineages; followed by characterization using RT-PCR for HNF4A, CDX2, MEF2C, TBX5, SOX1, and MAP2. qRT-PCR and western blotting was performed to compare expression of PcG transcripts and proteins across all the three lineages. We observed that cells differentiated into endoderm showed upregulation of RING1B, BMI1, EZH2, and EED transcripts. Mesoderm differentiation was characterized by significant downregulation of all PcG transcripts during later stages. BMI1 and RING1B were upregulated while EZH2, SUZ12, and EED remained low during ectoderm differentiation. Western blotting also showed distinct expression of BMI1 and EZH2 during differentiation into three germ layers. Our study shows that hES cells differentiating into endoderm, mesoderm, and ectoderm lineages show distinct PcG expression profile at transcript and protein level. © 2015 International Federation for Cell Biology.

Transcriptomic analysis reveals tomato genes whose expression is induced specifically during effector-triggered immunity and identifies the Epk1 protein kinase which is required for the host response to three bacterial effector proteins.

PubMed

Pombo, Marina A; Zheng, Yi; Fernandez-Pozo, Noe; Dunham, Diane M; Fei, Zhangjun; Martin, Gregory B

2014-01-01

Plants have two related immune systems to defend themselves against pathogen attack. Initially,pattern-triggered immunity is activated upon recognition of microbe-associated molecular patterns by pattern recognition receptors. Pathogenic bacteria deliver effector proteins into the plant cell that interfere with this immune response and promote disease. However, some plants express resistance proteins that detect the presence of specific effectors leading to a robust defense response referred to as effector-triggered immunity. The interaction of tomato with Pseudomonas syringae pv. tomato is an established model system for understanding the molecular basis of these plant immune responses. We apply high-throughput RNA sequencing to this pathosystem to identify genes whose expression changes specifically during pattern-triggered or effector-triggered immunity. We then develop reporter genes for each of these responses that will enable characterization of the host response to the large collection of P. s. pv. tomato strains that express different combinations of effectors. Virus-induced gene silencing of 30 of the effector-triggered immunity-specific genes identifies Epk1 which encodes a predicted protein kinase from a family previously unknown to be involved in immunity. Knocked-down expression of Epk1 compromises effector-triggered immunity triggered by three bacterial effectors but not by effectors from non-bacterial pathogens. Epistasis experiments indicate that Epk1 acts upstream of effector-triggered immunity-associated MAP kinase signaling. Using RNA-seq technology we identify genes involved in specific immune responses. A functional genomics screen led to the discovery of Epk1, a novel predicted protein kinase required for plant defense activation upon recognition of three different bacterial effectors.

Quantitative trait loci mapping and gene network analysis implicate protocadherin-15 as a determinant of brain serotonin transporter expression.

PubMed

Ye, R; Carneiro, A M D; Han, Q; Airey, D; Sanders-Bush, E; Zhang, B; Lu, L; Williams, R; Blakely, R D

2014-03-01

Presynaptic serotonin (5-hydroxytryptamine, 5-HT) transporters (SERT) regulate 5-HT signaling via antidepressant-sensitive clearance of released neurotransmitter. Polymorphisms in the human SERT gene (SLC6A4) have been linked to risk for multiple neuropsychiatric disorders, including depression, obsessive-compulsive disorder and autism. Using BXD recombinant inbred mice, a genetic reference population that can support the discovery of novel determinants of complex traits, merging collective trait assessments with bioinformatics approaches, we examine phenotypic and molecular networks associated with SERT gene and protein expression. Correlational analyses revealed a network of genes that significantly associated with SERT mRNA levels. We quantified SERT protein expression levels and identified region- and gender-specific quantitative trait loci (QTLs), one of which associated with male midbrain SERT protein expression, centered on the protocadherin-15 gene (Pcdh15), overlapped with a QTL for midbrain 5-HT levels. Pcdh15 was also the only QTL-associated gene whose midbrain mRNA expression significantly associated with both SERT protein and 5-HT traits, suggesting an unrecognized role of the cell adhesion protein in the development or function of 5-HT neurons. To test this hypothesis, we assessed SERT protein and 5-HT traits in the Pcdh15 functional null line (Pcdh15(av-) (3J) ), studies that revealed a strong, negative influence of Pcdh15 on these phenotypes. Together, our findings illustrate the power of multidimensional profiling of recombinant inbred lines in the analysis of molecular networks that support synaptic signaling, and that, as in the case of Pcdh15, can reveal novel relationships that may underlie risk for mental illness. © 2014 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Specific Distribution of the Autophagic Protein GABARAPL1/GEC1 in the Developing and Adult Mouse Brain and Identification of Neuronal Populations Expressing GABARAPL1/GEC1

PubMed Central

Le Grand, Jaclyn Nicole; Bon, Karine; Fraichard, Annick; Zhang, Jianhua; Jouvenot, Michèle; Risold, Pierre-Yves; Boyer-Guittaut, Michaël; Delage-Mourroux, Régis

2013-01-01

Macroautophagy is a highly conserved cellular degradation process, regulated by autophagy-related (atg) factors, in which a double membrane autophagosome engulfs cytoplasmic components to target them for degradation. In yeast, the Atg8 protein is indispensable for autophagosome formation. In mammals, this is complicated by the presence of six Atg8 homologues grouped into the GABARAP and MAP1LC3 subfamilies. Although these proteins share a high similarity, their transcript expression, regulation and protein interactions differ, suggesting they may display individual properties and specific functions. GABARAPL1/GEC1 is a member of the GABARAP subfamily and its mRNA is the most highly expressed Atg8 homologue in the central nervous system. Consequently, we performed an in depth study of GABARAPL1 distribution in the developing and adult murine brain. Our results show that GABARAPL1 brain expression is visible as early as embryonic day 11 and progressively increases to a maximum level in the adult. Immunohistochemical staining was detected in both fibers and immature neurons in embryos but was restrained to neurons in adult tissue. By E17, intense punctate-like structures were visible and these accumulated in cortical primary neurons treated with the autophagosome/lysosome fusion inhibitor Bafilomycin A1 (Baf A1), suggesting that they represent autophagosomes. Finally, GABARAPL1 expression was particularly intense in motoneurons in the embryo and in neurons involved in somatomotor and neuroendocrine functions in the adult, particularly in the substantia nigra pars compacta, a region affected in Parkinson's disease. Our study of cerebral GABARAPL1 protein expression provides insight into its role in the development and homeostasis of the mouse brain. PMID:23690988

P32/TAP, a cellular protein that interacts with EBNA-1 of Epstein-Barr virus.

PubMed

Wang, Y; Finan, J E; Middeldorp, J M; Hayward, S D

1997-09-15

The Epstein-Barr virus (EBV) EBNA-1 protein has a central role in the maintenance of a latent EBV infection and is the only virus-encoded protein expressed in all EBV-associated tumors. EBNA-1 is required for replication of the episomal form of the latent viral genome and transactivates the latency C and LMP-1 promoters. The mechanisms by which EBNA-1 performs these functions are not known. Here we describe the cloning, expression, and characterization of a cellular protein, P32/TAP, which strongly interacts with EBNA-1. We show that P32/TAP is expressed at high levels in Raji cells and is synthesized as a proprotein of 282 amino acids (aa) that is posttranslationally processed by a two-step cleavage process to yield a mature protein of 209 aa. It has been previously reported that P32/TAP is expressed on the cell surface. Our transient expression assays detected full-length P32/TAP (1-282 aa) in the cytoplasm while mature P32/TAP protein localized to the nucleus. Three lines of evidence support P32/TAP interaction with EBNA-1. First, in the yeast two-hybrid system we mapped two interactive N-terminal regions of EBNA-1, aa 40-60 and aa 325-376, each of which contains arginine-glycine repeats. These regions interact with the C-terminal half of P32/TAP. Second, the full-length cytoplasmic P32/TAP protein can translocate nuclear EBNA-1 into the cytoplasm. Third, P32/TAP co-immunoprecipitated with EBNA-1. We have confirmed that a Gal4 fusion protein containing the C-terminal region of P32/TAP (aa 244-282) transactivates expression from a reporter containing upstream Gal4-binding sites. Deletion of the P32/TAP interactive regions of EBNA-1 severely diminished EBNA-1 transactivation of FRTKCAT in transient expression assays. Our data suggest that interaction with P32/TAP may contribute to EBNA-1-mediated transactivation. Copyright 1997 Academic Press.

A Proteomics Analysis of the Effects of Chronic Hemiparetic Stroke on Troponin T Expression in Human Vastus Lateralis

PubMed Central

Rabek, Jeffrey P.; Hafer-Macko, Charlene E.; Amaning, James K.; DeFord, James H.; Dimayuga, Vincent L.; Madsen, Mark A.; Macko, Richard F.

2009-01-01

Stroke disability is attributed to upper motor neuron deficits resulting from ischemic brain injury. We have developed proteome maps of the Vastus lateralis to examine the effects of ischemic brain injury on paretic skeletal muscle myofilament proteins. Proteomics analyses from seven hemiparetic stroke patients have detected a decrease of three troponin T isoforms in the paretic muscle suggesting that myosin–actin interactions may be attenuated. We propose that ischemic brain injury may prevent troponin T participation in complex formation thereby affecting the protein interactions associated with excitation–contraction coupling. We have also detected a novel skeletal troponin T isoform that has a C-terminal variation. Our data suggest that the decreased slow troponin T isoform pools in the paretic limb may contribute to the gait deficit after stroke. The complexity of the neurological deficit on Vastus lateralis is suggested by the multiple changes in proteins detected by our proteomics mapping. PMID:19447848

High-resolution temporal and regional mapping of MAPT expression and splicing in human brain development.

PubMed

Hefti, Marco M; Farrell, Kurt; Kim, SoongHo; Bowles, Kathryn R; Fowkes, Mary E; Raj, Towfique; Crary, John F

2018-01-01

The microtubule associated protein tau plays a critical role in the pathogenesis of neurodegenerative disease. Recent studies suggest that tau also plays a role in disorders of neuronal connectivity, including epilepsy and post-traumatic stress disorder. Animal studies have shown that the MAPT gene, which codes for the tau protein, undergoes complex pre-mRNA alternative splicing to produce multiple isoforms during brain development. Human data, particularly on temporal and regional variation in tau splicing during development are however lacking. In this study, we present the first detailed examination of the temporal and regional sequence of MAPT alternative splicing in the developing human brain. We used a novel computational analysis of large transcriptomic datasets (total n = 502 patients), quantitative polymerase chain reaction (qPCR) and western blotting to examine tau expression and splicing in post-mortem human fetal, pediatric and adult brains. We found that MAPT exons 2 and 10 undergo abrupt shifts in expression during the perinatal period that are unique in the canonical human microtubule-associated protein family, while exon 3 showed small but significant temporal variation. Tau isoform expression may be a marker of neuronal maturation, temporally correlated with the onset of axonal growth. Immature brain regions such as the ganglionic eminence and rhombic lip had very low tau expression, but within more mature regions, there was little variation in tau expression or splicing. We thus demonstrate an abrupt, evolutionarily conserved shift in tau isoform expression during the human perinatal period that may be due to tau expression in maturing neurons. Alternative splicing of the MAPT pre-mRNA may play a vital role in normal brain development across multiple species and provides a basis for future investigations into the developmental and pathological functions of the tau protein.

Induction of rat hepatic aryl sulfotransferase (SULT1A1) gene expression by triamcinolone acetonide: impact on minoxidil-mediated hypotension.

PubMed

Duanmu, Z; Dunbar, J; Falany, C N; Runge-Morris, M

2000-05-01

The hypotensive agent minoxidil (6-imino-1, 2-dihydro-1-hydroxy-2-imino-4-piperidinopyrimidine) depends upon aryl sulfotransferase (SULT1)-catalyzed sulfation for its bioactivation. Previous reports suggest that glucocorticoids induce class-specific SULT1 and isoform-specific SULT1A1 gene expression in rat liver. In the present study, rats were treated with the glucocorticoid triamcinolone acetonide (TA, 5 mg/kg/day i.p. x 3 days) or its vehicle, 2% Tween-20, prior to minoxidil, and subsequent effects on mean arterial pressure (MAP), heart rate (HR), and hepatic SULT1 gene expression were characterized. Minoxidil treatment (1.5 mg/kg) resulted in a steady decline in MAP values of 16.3 to 18.6% relative to basal control levels at 35 to 60 min following minoxidil injection. Pentachlorophenol (PCP, 40 micromol/kg i.p.), an inhibitor of SULT1 enzyme activity, effectively ablated the hypotensive effects of minoxidil. By contrast, pretreatment with TA significantly enhanced minoxidil-induced hypotension. Relative to vehicle-treated controls, TA-treated rats displayed a steeper rate of decline in MAP and more profound levels of hypotension with decreases in MAP following minoxidil administration of 27.8%. TA also produced significant increases in hepatic SULT1 mRNA expression (of 271%) and SULT1A1 immunoreactive protein levels (of 273%), relative to vehicle-treated controls. These results provide physiological evidence to support the biological relevance of SULT1A1 induction by glucocorticoids. The data indicate that steroid treatment induces SULT1A1 gene expression and, as a consequence, accentuates the hypotensive effects of minoxidil. Copyright 2000 Academic Press.

Mitogen-Activated Protein Kinases Are Associated with the Regulation of Physiological Traits and Virulence in Fusarium oxysporum f. sp. cubense

PubMed Central

Ding, Zhaojian; Li, Minhui; Sun, Fei; Xi, Pinggen; Sun, Longhua; Zhang, Lianhui; Jiang, Zide

2015-01-01

Fusarium oxysporum f. sp. cubense (FOC) is an important soil-borne fungal pathogen causing devastating vascular wilt disease of banana plants and has become a great concern threatening banana production worldwide. However, little information is known about the molecular mechanisms that govern the expression of virulence determinants of this important fungal pathogen. In this study, we showed that null mutation of three mitogen-activated protein (MAP) kinase genes, designated as FoSlt2, FoMkk2 and FoBck1, respectively, led to substantial attenuation in fungal virulence on banana plants. Transcriptional analysis revealed that the MAP kinase signaling pathway plays a key role in regulation of the genes encoding production of chitin, peroxidase, beauvericin and fusaric acid. Biochemical analysis further confirmed the essential role of MAP kinases in modulating the production of fusaric acid, which was a crucial phytotoxin in accelerating development of Fusarium wilt symptoms in banana plants. Additionally, we found that the MAP kinase FoSlt2 was required for siderophore biosynthesis under iron-depletion conditions. Moreover, disruption of the MAP kinase genes resulted in abnormal hypha and increased sensitivity to Congo Red, Calcofluor White and H2O2. Taken together, these results depict the critical roles of MAP kinases in regulation of FOC physiology and virulence. PMID:25849862

Isolation and characterization of a novel calmodulin-binding protein from potato

NASA Technical Reports Server (NTRS)

Reddy, Anireddy S N.; Day, Irene S.; Narasimhulu, S. B.; Safadi, Farida; Reddy, Vaka S.; Golovkin, Maxim; Harnly, Melissa J.

2002-01-01

Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.

Global PROTOMAP profiling to search for biomarkers of early-recurrent hepatocellular carcinoma.

PubMed

Taoka, Masato; Morofuji, Noriaki; Yamauchi, Yoshio; Ojima, Hidenori; Kubota, Daisuke; Terukina, Goro; Nobe, Yuko; Nakayama, Hiroshi; Takahashi, Nobuhiro; Kosuge, Tomoo; Isobe, Toshiaki; Kondo, Tadashi

2014-11-07

This study used global protein expression profiling to search for biomarkers to predict early recurrent hepatocellular carcinoma (HCC). HCC tissues surgically resected from patients with or without recurrence within 2 years (early recurrent) after surgery were compared with adjacent nontumor tissue and with normal liver tissue. We used the PROTOMAP strategy for comparative profiling, which integrates denaturing polyacrylamide gel electrophoresis migratory rates and high-resolution, semiquantitative mass-spectrometry-based identification of in-gel-digested tryptic peptides. PROTOMAP allows examination of global changes in the size, topography, and abundance of proteins in complex tissue samples. This approach identified 8438 unique proteins from 45 708 nonredundant peptides and generated a proteome-wide map of changes in expression and proteolytic events potentially induced by intrinsic apoptotic/necrotic pathways. In the early recurrent HCC tissue, 87 proteins were differentially expressed (≥20-fold) relative to the other tissues, 46 of which were up-regulated or specifically proteolyzed and 41 of which were down-regulated. This data set consisted of proteins that fell into various functional categories, including signal transduction and cell organization and, notably, the major catalytic pathways responsible for liver function, such as the urea cycle and detoxification metabolism. We found that aberrant proteolysis appeared to occur frequently during recurrence of HCC in several key signal transducers, including STAT1 and δ-catenin. Further investigation of these proteins will facilitate the development of novel clinical applications.

A Cryptosporidium parvum genomic region encoding hemolytic activity.

PubMed Central

Steele, M I; Kuhls, T L; Nida, K; Meka, C S; Halabi, I M; Mosier, D A; Elliott, W; Crawford, D L; Greenfield, R A

1995-01-01

Successful parasitization by Cryptosporidium parvum requires multiple disruptions in both host and protozoan cell membranes as cryptosporidial sporozoites invade intestinal epithelial cells and subsequently develop into asexual and sexual life stages. To identify cryptosporidial proteins which may play a role in these membrane alterations, hemolytic activity was used as a marker to screen a C. parvum genomic expression library. A stable hemolytic clone (H4) containing a 5.5-kb cryptosporidial genomic fragment was identified. The hemolytic activity encoded on H4 was mapped to a 1-kb region that contained a complete 690-bp open reading frame (hemA) ending in a common stop codon. A 21-kDa plasmid-encoded recombinant protein was expressed in maxicells containing H4. Subclones of H4 which contained only a portion of hemA did not induce hemolysis on blood agar or promote expression of the recombinant protein in maxicells. Reverse transcriptase-mediated PCR analysis of total RNA isolated from excysted sporozoites and the intestines of infected adult mice with severe combined immunodeficiency demonstrated that hemA is actively transcribed during the cryptosporidial life cycle. PMID:7558289

EML proteins in microtubule regulation and human disease.

PubMed

Fry, Andrew M; O'Regan, Laura; Montgomery, Jessica; Adib, Rozita; Bayliss, Richard

2016-10-15

The EMLs are a conserved family of microtubule-associated proteins (MAPs). The founding member was discovered in sea urchins as a 77-kDa polypeptide that co-purified with microtubules. This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified microtubule preparations made from unfertilized sea urchin eggs [J. Cell Sci. (1993) 104: , 445-450; J. Cell Sci. (1987) 87: (Pt 1), 71-84]. Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like protein) has been adopted [BMC Dev. Biol. (2008) 8: , 110; Dev. Genes Evol. (2000) 210: , 2-10]. The common property of these proteins is their ability to decorate microtubules. However, whether they are associated with particular microtubule populations or exercise specific functions in different microtubule-dependent processes remains unknown. Furthermore, although there is limited evidence that they regulate microtubule dynamics, the biochemical mechanisms of their molecular activity have yet to be explored. Nevertheless, interest in these proteins has grown substantially because of the identification of EML mutations in neuronal disorders and oncogenic fusions in human cancers. Here, we summarize our current knowledge of the expression, localization and structure of what is proving to be an interesting and important class of MAPs. We also speculate about their function in microtubule regulation and highlight how the studies of EMLs in human diseases may open up novel avenues for patient therapy. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

A fresh look at the male-specific region of the human Y chromosome.

PubMed

Jangravi, Zohreh; Alikhani, Mehdi; Arefnezhad, Babak; Sharifi Tabar, Mehdi; Taleahmad, Sara; Karamzadeh, Razieh; Jadaliha, Mahdieh; Mousavi, Seyed Ahmad; Ahmadi Rastegar, Diba; Parsamatin, Pouria; Vakilian, Haghighat; Mirshahvaladi, Shahab; Sabbaghian, Marjan; Mohseni Meybodi, Anahita; Mirzaei, Mehdi; Shahhoseini, Maryam; Ebrahimi, Marzieh; Piryaei, Abbas; Moosavi-Movahedi, Ali Akbar; Haynes, Paul A; Goodchild, Ann K; Nasr-Esfahani, Mohammad Hossein; Jabbari, Esmaiel; Baharvand, Hossein; Sedighi Gilani, Mohammad Ali; Gourabi, Hamid; Salekdeh, Ghasem Hosseini

2013-01-04

The Chromosome-centric Human Proteome Project (C-HPP) aims to systematically map the entire human proteome with the intent to enhance our understanding of human biology at the cellular level. This project attempts simultaneously to establish a sound basis for the development of diagnostic, prognostic, therapeutic, and preventive medical applications. In Iran, current efforts focus on mapping the proteome of the human Y chromosome. The male-specific region of the Y chromosome (MSY) is unique in many aspects and comprises 95% of the chromosome's length. The MSY continually retains its haploid state and is full of repeated sequences. It is responsible for important biological roles such as sex determination and male fertility. Here, we present the most recent update of MSY protein-encoding genes and their association with various traits and diseases including sex determination and reversal, spermatogenesis and male infertility, cancers such as prostate cancers, sex-specific effects on the brain and behavior, and graft-versus-host disease. We also present information available from RNA sequencing, protein-protein interaction, post-translational modification of MSY protein-coding genes and their implications in biological systems. An overview of Human Y chromosome Proteome Project is presented and a systematic approach is suggested to ensure that at least one of each predicted protein-coding gene's major representative proteins will be characterized in the context of its major anatomical sites of expression, its abundance, and its functional relevance in a biological and/or medical context. There are many technical and biological issues that will need to be overcome in order to accomplish the full scale mapping.

RSRC1 mutation affects intellect and behaviour through aberrant splicing and transcription, downregulating IGFBP3.

PubMed

Perez, Yonatan; Menascu, Shay; Cohen, Idan; Kadir, Rotem; Basha, Omer; Shorer, Zamir; Romi, Hila; Meiri, Gal; Rabinski, Tatiana; Ofir, Rivka; Yeger-Lotem, Esti; Birk, Ohad S

2018-04-01

RSRC1, whose polymorphism is associated with altered brain function in schizophrenia, is a member of the serine and arginine rich-related protein family. Through homozygosity mapping and whole exome sequencing we show that RSRC1 mutation causes an autosomal recessive syndrome of intellectual disability, aberrant behaviour, hypotonia and mild facial dysmorphism with normal brain MRI. Further, we show that RSRC1 is ubiquitously expressed, and that the RSRC1 mutation triggers nonsense-mediated mRNA decay of the RSRC1 transcript in patients' fibroblasts. Short hairpin RNA (shRNA)-mediated lentiviral silencing and overexpression of RSRC1 in SH-SY5Y cells demonstrated that RSRC1 has a role in alternative splicing and transcription regulation. Transcriptome profiling of RSRC1-silenced cells unravelled specific differentially expressed genes previously associated with intellectual disability, hypotonia and schizophrenia, relevant to the disease phenotype. Protein-protein interaction network modelling suggested possible intermediate interactions by which RSRC1 affects gene-specific differential expression. Patient-derived induced pluripotent stem cells, differentiated into neural progenitor cells, showed expression dynamics similar to the RSRC1-silenced SH-SY5Y model. Notably, patient neural progenitor cells had 9.6-fold downregulated expression of IGFBP3, whose brain expression is affected by MECP2, aberrant in Rett syndrome. Interestingly, Igfbp3-null mice have behavioural impairment, abnormal synaptic function and monoaminergic neurotransmission, likely correlating with the disease phenotype.

Fine mapping and identification of candidate genes for the sy-2 locus in a temperature-sensitive chili pepper (Capsicum chinense).

PubMed

Liu, Li; Venkatesh, Jelli; Jo, Yeong Deuk; Koeda, Sota; Hosokawa, Munetaka; Kang, Jin-Ho; Goritschnig, Sandra; Kang, Byoung-Cheorl

2016-08-01

The sy - 2 temperature-sensitive gene from Capsicum chinense was fine mapped to a 138.8-kb region at the distal portion of pepper chromosome 1. Based on expression analyses, two putative F-box genes were identified as sy - 2 candidate genes. Seychelles-2 ('sy-2') is a temperature-sensitive natural mutant of Capsicum chinense, which exhibits an abnormal leaf phenotype when grown at temperatures below 24 °C. We previously showed that the sy-2 phenotype is controlled by a single recessive gene, sy-2, located on pepper chromosome 1. In this study, a high-resolution genetic and physical map for the sy-2 locus was constructed using two individual F2 mapping populations derived from a cross between C. chinense mutant 'sy-2' and wild-type 'No. 3341'. The sy-2 gene was fine mapped to a 138.8-kb region between markers SNP 5-5 and SNP 3-8 at the distal portion of chromosome 1, based on comparative genomic analysis and genomic information from pepper. The sy-2 target region was predicted to contain 27 genes. Expression analysis of these predicted genes showed a differential expression pattern for ORF10 and ORF20 between mutant and wild-type plants; with both having significantly lower expression in 'sy-2' than in wild-type plants. In addition, the coding sequences of both ORF10 and ORF20 contained single nucleotide polymorphisms (SNPs) causing amino acid changes, which may have important functional consequences. ORF10 and ORF20 are predicted to encode F-box proteins, which are components of the SCF complex. Based on the differential expression pattern and the presence of nonsynonymous SNPs, we suggest that these two putative F-box genes are most likely responsible for the temperature-sensitive phenotypes in pepper. Further investigation of these genes may enable a better understanding of the molecular mechanisms of low temperature sensitivity in plants.

Postsynaptic density protein (PSD)-95 expression is markedly decreased in the hippocampal CA1 region after experimental ischemia-reperfusion injury.

PubMed

Yan, Bing Chun; Park, Joon Ha; Ahn, Ji Hyeon; Lee, Jae-Chul; Won, Moo-Ho; Kang, Il-Jun

2013-07-15

Synaptic plasticity is important for functional recovery after cerebral ischemic injury. In the present study, we investigated chronological change in the immunoreactivity of PSD-95, a kind of postsynaptic density protein, in the hippocampus proper (CA1-3 regions) after 5 min of transient cerebral ischemia in gerbils. PSD-95 immunoreactivity was observed in MAP-2-immunoreactive dendrites in the CA1-3 regions of the sham group. The PSD-95 immunoreactivity was shown as beaded structure in the MAP-2-immunoreactive dendrites. However, PSD-95 immunoreactivity began to be dramatically decreased in MAP-2-immunoreactive dendrites in the CA1 region, not CA2-3 region, at early time after ischemia-reperfusion. At 5 days after ischemia-reperfusion, MAP-2 immunoreactivity almost disappeared in the ischemic CA1 region, and PSD-95 immunoreactivity was much lower than that in the sham group. In brief, PSD-95 immunoreactivity in the CA1 dendrites was markedly decreased at early time after ischemia-reperfusion. We suggest that decreased PSD-95 immunoreactivity in the ischemic CA1 region may lead to a deficit of postsynaptic plasticity in the brain. Copyright © 2013 Elsevier B.V. All rights reserved.

MSD-MAP: A Network-Based Systems Biology Platform for Predicting Disease-Metabolite Links.

PubMed

Wathieu, Henri; Issa, Naiem T; Mohandoss, Manisha; Byers, Stephen W; Dakshanamurthy, Sivanesan

2017-01-01

Cancer-associated metabolites result from cell-wide mechanisms of dysregulation. The field of metabolomics has sought to identify these aberrant metabolites as disease biomarkers, clues to understanding disease mechanisms, or even as therapeutic agents. This study was undertaken to reliably predict metabolites associated with colorectal, esophageal, and prostate cancers. Metabolite and disease biological action networks were compared in a computational platform called MSD-MAP (Multi Scale Disease-Metabolite Association Platform). Using differential gene expression analysis with patient-based RNAseq data from The Cancer Genome Atlas, genes up- or down-regulated in cancer compared to normal tissue were identified. Relational databases were used to map biological entities including pathways, functions, and interacting proteins, to those differential disease genes. Similar relational maps were built for metabolites, stemming from known and in silico predicted metabolite-protein associations. The hypergeometric test was used to find statistically significant relationships between disease and metabolite biological signatures at each tier, and metabolites were assessed for multi-scale association with each cancer. Metabolite networks were also directly associated with various other diseases using a disease functional perturbation database. Our platform recapitulated metabolite-disease links that have been empirically verified in the scientific literature, with network-based mapping of jointly-associated biological activity also matching known disease mechanisms. This was true for colorectal, esophageal, and prostate cancers, using metabolite action networks stemming from both predicted and known functional protein associations. By employing systems biology concepts, MSD-MAP reliably predicted known cancermetabolite links, and may serve as a predictive tool to streamline conventional metabolomic profiling methodologies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Streptococcus suis Bacterin and Subunit Vaccine Immunogenicities and Protective Efficacies against Serotypes 2 and 9▿†

PubMed Central

Baums, Christoph Georg; Kock, Christoph; Beineke, Andreas; Bennecke, Katharina; Goethe, Ralph; Schröder, Charlotte; Waldmann, Karl-Heinz; Valentin-Weigand, Peter

2009-01-01

Streptococcus suis causes numerous diseases in pigs, most importantly, meningitis, arthritis, septicemia, and bronchopneumonia. One of the major problems in modern swine production is the lack of a vaccine protecting against more than one S. suis serotype. The objective of this study was to determine the protective efficacy of a serotype 2 murein-associated protein (MAP) fraction subunit vaccine in comparison to that of a bacterin against experimental challenge with serotype 2 (containing muramidase-released protein [MRP], extracellular factor, and suilysin [SLY]) and serotype 9 (containing MRP variant MRP* and SLY) strains. MAP was shown to include different surface-associated proteins, such as the MRP and surface antigen one (SAO) expressed by both pathotypes used for challenge. The results of this study demonstrated that the serotype 2 bacterin induced protective immunity against homologous challenge. In contrast, the protective efficacy of the MAP subunit vaccine was low, though MAP immunization resulted in high serum immunoglobulin G2 titers against MRP and SAO. Importantly, immunization with bacterin but not with MAP induced opsonizing antibody titers against the serotype 2 strain, and these antibody titers were found to correlate with protection. However, after absorption with a nonencapsulated isogenic mutant, the sera from bacterin-immunized piglets failed to facilitate neutrophil killing, indicating that antibodies directed against capsule may not have been essential for opsonophagocytosis. Furthermore, induction of opsonizing antibodies against serotype 9 was not detectable in the group receiving bacterin or in the group receiving the MAP vaccine. In agreement, protection against the heterologous serotype 9 strain was low in both groups. Thus, identification of an antigen protecting against these two important S. suis pathotypes remains an important goal of future studies. PMID:19109449

Draft genome analysis provides insights into the fiber yield, crude protein biosynthesis, and vegetative growth of domesticated ramie (Boehmeria nivea L. Gaud).

PubMed

Liu, Chan; Zeng, Liangbin; Zhu, Siyuan; Wu, Lingqing; Wang, Yanzhou; Tang, Shouwei; Wang, Hongwu; Zheng, Xia; Zhao, Jian; Chen, Xiaorong; Dai, Qiuzhong; Liu, Touming

2017-11-15

Plentiful bast fiber, a high crude protein content, and vigorous vegetative growth make ramie a popular fiber and forage crop. Here, we report the draft genome of ramie, along with a genomic comparison and evolutionary analysis. The draft genome contained a sequence of approximately 335.6 Mb with 42,463 predicted genes. A high-density genetic map with 4,338 single nucleotide polymorphisms (SNPs) was developed and used to anchor the genome sequence, thus, creating an integrated genetic and physical map containing a 58.2-Mb genome sequence and 4,304 molecular markers. A genomic comparison identified 1,075 unique gene families in ramie, containing 4,082 genes. Among these unique genes, five were cellulose synthase genes that were specifically expressed in stem bark, and 3 encoded a WAT1-related protein, suggesting that they are probably related to high bast fiber yield. An evolutionary analysis detected 106 positively selected genes, 22 of which were related to nitrogen metabolism, indicating that they are probably responsible for the crude protein content and vegetative growth of domesticated varieties. This study is the first to characterize the genome and develop a high-density genetic map of ramie and provides a basis for the genetic and molecular study of this crop. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Genetic mapping of the LOBED LEAF 1 (ClLL1) gene to a 127.6-kb region in watermelon (Citrullus lanatus L.)

PubMed Central

Wei, Chunhua; Chen, Xiner; Wang, Zhongyuan; Liu, Qiyan; Li, Hao; Zhang, Yong; Ma, Jianxiang; Yang, Jianqiang

2017-01-01

The lobed leaf character is a unique morphologic trait in crops, featuring many potential advantages for agricultural productivity. Although the majority of watermelon varieties feature lobed leaves, the genetic factors responsible for lobed leaf formation remain elusive. The F2:3 leaf shape segregating population offers the opportunity to study the underlying mechanism of lobed leaf formation in watermelon. Genetic analysis revealed that a single dominant allele (designated ClLL1) controlled the lobed leaf trait. A large-sized F3:4 population derived from F2:3 individuals was used to map ClLL1. A total of 5,966 reliable SNPs and indels were identified genome-wide via a combination of BSA and RNA-seq. Using the validated SNP and indel markers, the location of ClLL1 was narrowed down to a 127.6-kb region between markers W08314 and W07061, containing 23 putative ORFs. Expression analysis via qRT-PCR revealed differential expression patterns (fold-changes above 2-fold or below 0.5-fold) of three ORFs (ORF3, ORF11, and ORF18) between lobed and non-lobed leaf plants. Based on gene annotation and expression analysis, ORF18 (encoding an uncharacterized protein) and ORF22 (encoding a homeobox-leucine zipper-like protein) were considered as most likely candidate genes. Furthermore, sequence analysis revealed no polymorphisms in cDNA sequences of ORF18; however, two notable deletions were identified in ORF22. This study is the first report to map a leaf shape gene in watermelon and will facilitate cloning and functional characterization of ClLL1 in future studies. PMID:28704497

Genetic mapping of the LOBED LEAF 1 (ClLL1) gene to a 127.6-kb region in watermelon (Citrullus lanatus L.).

PubMed

Wei, Chunhua; Chen, Xiner; Wang, Zhongyuan; Liu, Qiyan; Li, Hao; Zhang, Yong; Ma, Jianxiang; Yang, Jianqiang; Zhang, Xian

2017-01-01

The lobed leaf character is a unique morphologic trait in crops, featuring many potential advantages for agricultural productivity. Although the majority of watermelon varieties feature lobed leaves, the genetic factors responsible for lobed leaf formation remain elusive. The F2:3 leaf shape segregating population offers the opportunity to study the underlying mechanism of lobed leaf formation in watermelon. Genetic analysis revealed that a single dominant allele (designated ClLL1) controlled the lobed leaf trait. A large-sized F3:4 population derived from F2:3 individuals was used to map ClLL1. A total of 5,966 reliable SNPs and indels were identified genome-wide via a combination of BSA and RNA-seq. Using the validated SNP and indel markers, the location of ClLL1 was narrowed down to a 127.6-kb region between markers W08314 and W07061, containing 23 putative ORFs. Expression analysis via qRT-PCR revealed differential expression patterns (fold-changes above 2-fold or below 0.5-fold) of three ORFs (ORF3, ORF11, and ORF18) between lobed and non-lobed leaf plants. Based on gene annotation and expression analysis, ORF18 (encoding an uncharacterized protein) and ORF22 (encoding a homeobox-leucine zipper-like protein) were considered as most likely candidate genes. Furthermore, sequence analysis revealed no polymorphisms in cDNA sequences of ORF18; however, two notable deletions were identified in ORF22. This study is the first report to map a leaf shape gene in watermelon and will facilitate cloning and functional characterization of ClLL1 in future studies.

CNS neuroplasticity and salt-sensitive hypertension induced by prior treatment with subpressor doses of ANG II or aldosterone.

PubMed

Clayton, Sarah C; Zhang, Zhongming; Beltz, Terry; Xue, Baojian; Johnson, Alan Kim

2014-06-15

Although sensitivity to high dietary NaCl is regarded to be a risk factor for cardiovascular disease, the causes of salt-sensitive hypertension remain elusive. Previously, we have shown that rats pretreated with subpressor doses of either ANG II or aldosterone (Aldo) show sensitized hypertensive responses to a mild pressor dose of ANG II when tested after an intervening delay. The current studies investigated whether such treatments will induce salt sensitivity. In studies employing an induction-delay-expression experimental design, male rats were instrumented for chronic mean arterial pressure (MAP) recording. In separate experiments, ANG II, Aldo, or vehicle was delivered either subcutaneously or intracerebroventricularly during the induction. There were no sustained differences in BP during the delay prior to being given 2% saline. While consuming 2% saline during the expression, both ANG II- and Aldo-pretreated rats showed significantly greater hypertension. When hexamethonium was used to assess autonomic control of MAP, no differences in the decrease of MAP in response to ganglionic blockade were detected during the induction. However, during the expression, the fall was greater in sensitized rats. In separate experiments, brain tissue that was collected at the end of delay showed increases in message or activation of putative markers of neuroplasticity (i.e., brain-derived neurotrophic factor, p38 mitogen-activated protein kinase, and cAMP response element-binding protein). These experiments demonstrate that prior administration of nonpressor doses of either ANG II or Aldo will induce salt sensitivity. Collectively, our findings indicate that treatment with subpressor doses of ANG II and Aldo initiate central neuroplastic changes that are involved in hypertension of different etiologies. Copyright © 2014 the American Physiological Society.

Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor*

PubMed Central

Lievens, Sam; Gerlo, Sarah; Lemmens, Irma; De Clercq, Dries J. H.; Risseeuw, Martijn D. P.; Vanderroost, Nele; De Smet, Anne-Sophie; Ruyssinck, Elien; Chevet, Eric; Van Calenbergh, Serge; Tavernier, Jan

2014-01-01

Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method's potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges. PMID:25154561

Changes in the transcriptome of morula-stage bovine embryos caused by heat shock: relationship to developmental acquisition of thermotolerance

PubMed Central

2013-01-01

Background While initially sensitive to heat shock, the bovine embryo gains thermal resistance as it progresses through development so that physiological heat shock has little effect on development to the blastocyst stage by Day 5 after insemination. Here, experiments using 3’ tag digital gene expression (3’DGE) and real-time PCR were conducted to determine changes in the transcriptome of morula-stage bovine embryos in response to heat shock (40 degrees C for 8 h) that could be associated with thermotolerance. Results Using 3’DGE, expression of 173 genes were modified by heat shock, with 94 genes upregulated by heat shock and 79 genes downregulated by heat shock. A total of 38 differentially-regulated genes were associated with the ubiquitin protein, UBC. Heat shock increased expression of one heat shock protein gene, HSPB11, and one heat shock protein binding protein, HSPBP1, tended to increase expression of HSPA1A and HSPB1, but did not affect expression of 64 other genes encoding heat shock proteins, heat shock transcription factors or proteins interacting with heat shock proteins. Moreover, heat shock increased expression of five genes associated with oxidative stress (AKR7A2, CBR1, GGH, GSTA4, and MAP2K5), decreased expression of HIF3A, but did not affect expression of 42 other genes related to free radical metabolism. Heat shock also had little effect on genes involved in embryonic development. Effects of heat shock for 2, 4 and 8 h on selected heat shock protein and antioxidant genes were also evaluated by real-time PCR. Heat shock increased steady-state amounts of mRNA for HSPA1A (P<0.05) and tended to increase expression of HSP90AA1 (P<0.07) but had no effect on expression of SOD1 or CAT. Conclusions Changes in the transcriptome of the heat-shocked bovine morula indicate that the embryo is largely resistant to effects of heat shock. As a result, transcription of genes involved in thermal protection is muted and there is little disruption of gene networks involved in embryonic development. It is likely that the increased resistance of morula-stage embryos to heat shock as compared to embryos at earlier stages of development is due in part to developmental acquisition of mechanisms to prevent accumulation of denatured proteins and free radical damage. PMID:23320502

Plasma immune protein analysis in the orange-spotted grouper Epinephelus coioides: Evidence for altered expressions of immune factors associated with a choline-supplemented diet.

PubMed

Shiu, Ya-Li; Chiu, Kuo-Hsun; Huynh, Truong-Giang; Liu, Ping-Chung; Liu, Chun-Hung

2017-06-01

This study aimed to unravel the regulatory roles of choline in activating immune responses and disease resistance of the orange-spotted grouper Epinephelus coioides. Fish were fed a choline-supplemented diet at 1 g kg -1 of feed for 30 days. Fish fed a fish meal basal diet without choline-supplement served as controls. At the end of the feeding trial, fish were challenged with Vibrio alginolyticus. Meanwhile, plasma proteomics of fish in each group were also evaluated by two-dimensional gel electrophoresis (2-DE), and differentially expressed proteins were identified by tandem mass spectrophotometry (MS/MS), then a Western blot analysis or real-time polymerase chain reaction was used to confirm differential expressions of immune-enhancing proteins. Results showed that choline significantly increased survival of E. coioides 48 days after being injected with V. alginolyticus. From maps of plasma proteins, a comparative analysis between the control and choline groups revealed that 111 spots matched, with 26 altered expression spots in the choline group. Of these 26 spots, 16 were upregulated and 10 downregulated. After protein identification by reverse-phase nano-high-performance liquid chromatography-electrospray ionization MS/MS analysis, eight of 26 proteins were found to be immune-related proteins, all of which were upregulated, including complement 3 (C3), alpha-2-macroglobulin-P-like isoform (A2M), fibrinogen beta chain precursor (FBG), and immunoglobulin heavy constant mu (Ighm) proteins. Expression of the A2M protein and A2M enzyme activity in plasma of fish fed choline significantly increased compared to the control group. Additionally, A2M messenger (m)RNA transcripts were also upregulated in the liver and kidneys. Significantly higher C3 expressions at both the mRNA and protein levels were detected in the liver of fish in the choline group. Moreover, FBG gene expressions in the liver and kidneys significantly increased, while Ighm increased in the kidneys and spleen of fish in the choline group. Our results suggest that dietary administration of choline can protect grouper against bacterial infections through activating the complement system, thereby inducing antiprotease activity and natural antibodies that play important roles in the innate immune system of fish. Copyright © 2017 Elsevier Ltd. All rights reserved.

The NB-LRR gene Pm60 confers powdery mildew resistance in wheat.

PubMed

Zou, Shenghao; Wang, Huan; Li, Yiwen; Kong, Zhaosheng; Tang, Dingzhong

2018-04-01

Powdery mildew is one of the most devastating diseases of wheat. To date, few powdery mildew resistance genes have been cloned from wheat due to the size and complexity of the wheat genome. Triticum urartu is the progenitor of the A genome of wheat and is an important source for powdery mildew resistance genes. Using molecular markers designed from scaffolds of the sequenced T. urartu accession and standard map-based cloning, a powdery mildew resistance locus was mapped to a 356-kb region, which contains two nucleotide-binding and leucine-rich repeat domain (NB-LRR) protein-encoding genes. Virus-induced gene silencing, single-cell transient expression, and stable transformation assays demonstrated that one of these two genes, designated Pm60, confers resistance to powdery mildew. Overexpression of full-length Pm60 and two allelic variants in Nicotiana benthamiana leaves induced hypersensitive cell death response, but expression of the coiled-coil domain alone was insufficient to induce hypersensitive response. Yeast two-hybrid, bimolecular fluorescence complementation and luciferase complementation imaging assays showed that Pm60 protein interacts with its neighboring NB-containing protein, suggesting that they might be functionally related. The identification and cloning of this novel wheat powdery mildew resistance gene will facilitate breeding for disease resistance in wheat. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Map-based Cloning and Characterization of the BPH18 Gene from Wild Rice Conferring Resistance to Brown Planthopper (BPH) Insect Pest

PubMed Central

Ji, Hyeonso; Kim, Sung-Ryul; Kim, Yul-Ho; Suh, Jung-Pil; Park, Hyang-Mi; Sreenivasulu, Nese; Misra, Gopal; Kim, Suk-Man; Hechanova, Sherry Lou; Kim, Hakbum; Lee, Gang-Seob; Yoon, Ung-Han; Kim, Tae-Ho; Lim, Hyemin; Suh, Suk-Chul; Yang, Jungil; An, Gynheung; Jena, Kshirod K.

2016-01-01

Brown planthopper (BPH) is a phloem sap-sucking insect pest of rice which causes severe yield loss. We cloned the BPH18 gene from the BPH-resistant introgression line derived from the wild rice species Oryza australiensis. Map-based cloning and complementation test revealed that the BPH18 encodes CC-NBS-NBS-LRR protein. BPH18 has two NBS domains, unlike the typical NBS-LRR proteins. The BPH18 promoter::GUS transgenic plants exhibited strong GUS expression in the vascular bundles of the leaf sheath, especially in phloem cells where the BPH attacks. The BPH18 proteins were widely localized to the endo-membranes in a cell, including the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and prevacuolar compartments, suggesting that BPH18 may recognize the BPH invasion at endo-membranes in phloem cells. Whole genome sequencing of the near-isogenic lines (NILs), NIL-BPH18 and NIL-BPH26, revealed that BPH18 located at the same locus of BPH26. However, these two genes have remarkable sequence differences and the independent NILs showed differential BPH resistance with different expression patterns of plant defense-related genes, indicating that BPH18 and BPH26 are functionally different alleles. These findings would facilitate elucidation of the molecular mechanism of BPH resistance and the identified novel alleles to fast track breeding BPH resistant rice cultivars. PMID:27682162

Map-based Cloning and Characterization of the BPH18 Gene from Wild Rice Conferring Resistance to Brown Planthopper (BPH) Insect Pest.

PubMed

Ji, Hyeonso; Kim, Sung-Ryul; Kim, Yul-Ho; Suh, Jung-Pil; Park, Hyang-Mi; Sreenivasulu, Nese; Misra, Gopal; Kim, Suk-Man; Hechanova, Sherry Lou; Kim, Hakbum; Lee, Gang-Seob; Yoon, Ung-Han; Kim, Tae-Ho; Lim, Hyemin; Suh, Suk-Chul; Yang, Jungil; An, Gynheung; Jena, Kshirod K

2016-09-29

Brown planthopper (BPH) is a phloem sap-sucking insect pest of rice which causes severe yield loss. We cloned the BPH18 gene from the BPH-resistant introgression line derived from the wild rice species Oryza australiensis. Map-based cloning and complementation test revealed that the BPH18 encodes CC-NBS-NBS-LRR protein. BPH18 has two NBS domains, unlike the typical NBS-LRR proteins. The BPH18 promoter::GUS transgenic plants exhibited strong GUS expression in the vascular bundles of the leaf sheath, especially in phloem cells where the BPH attacks. The BPH18 proteins were widely localized to the endo-membranes in a cell, including the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and prevacuolar compartments, suggesting that BPH18 may recognize the BPH invasion at endo-membranes in phloem cells. Whole genome sequencing of the near-isogenic lines (NILs), NIL-BPH18 and NIL-BPH26, revealed that BPH18 located at the same locus of BPH26. However, these two genes have remarkable sequence differences and the independent NILs showed differential BPH resistance with different expression patterns of plant defense-related genes, indicating that BPH18 and BPH26 are functionally different alleles. These findings would facilitate elucidation of the molecular mechanism of BPH resistance and the identified novel alleles to fast track breeding BPH resistant rice cultivars.

Amniotic Fluid Protein Profiles of Intraamniotic Inflammatory Response to Ureaplasma spp. and Other Bacteria

PubMed Central

Kacerovsky, Marian; Celec, Peter; Vlkova, Barbora; Skogstrand, Kristin; Hougaard, David M.; Cobo, Teresa; Jacobsson, Bo

2013-01-01

Objective This study aimed to evaluate the amniotic fluid protein profiles and the intensity of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria, using the multiplex xMAP technology. Methods A retrospective cohort study was undertaken in the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic. A total of 145 pregnant women with preterm prelabor rupture of membranes between gestational age 24+0 and 36+6 weeks were included in the study. Amniocenteses were performed. The presence of Ureaplasma spp. and other bacteria was evaluated using 16S rRNA gene sequencing. The levels of specific proteins were determined using multiplex xMAP technology. Results The presence of Ureaplasma spp. and other bacteria in the amniotic fluid was associated with increased levels of interleukin (IL)-6, IL-8, IL-10, brain-derived neurotropic factor, granulocyte macrophage colony stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1, and matrix metalloproteinasis-9. Ureaplasma spp. were also associated with increased levels of neurotropin-3 and triggering receptor expressed on myeloid cells-1. Conclusions The presence of Ureaplasma spp. in the amniotic fluid is associated with a slightly different protein profile of inflammatory response, but the intensity of inflammatory response to Ureaplasma spp. is comparable with the inflammatory response to other bacteria. PMID:23555967

Amniotic fluid protein profiles of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria.

PubMed

Kacerovsky, Marian; Celec, Peter; Vlkova, Barbora; Skogstrand, Kristin; Hougaard, David M; Cobo, Teresa; Jacobsson, Bo

2013-01-01

This study aimed to evaluate the amniotic fluid protein profiles and the intensity of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria, using the multiplex xMAP technology. A retrospective cohort study was undertaken in the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic. A total of 145 pregnant women with preterm prelabor rupture of membranes between gestational age 24+0 and 36+6 weeks were included in the study. Amniocenteses were performed. The presence of Ureaplasma spp. and other bacteria was evaluated using 16S rRNA gene sequencing. The levels of specific proteins were determined using multiplex xMAP technology. The presence of Ureaplasma spp. and other bacteria in the amniotic fluid was associated with increased levels of interleukin (IL)-6, IL-8, IL-10, brain-derived neurotropic factor, granulocyte macrophage colony stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1, and matrix metalloproteinasis-9. Ureaplasma spp. were also associated with increased levels of neurotropin-3 and triggering receptor expressed on myeloid cells-1. The presence of Ureaplasma spp. in the amniotic fluid is associated with a slightly different protein profile of inflammatory response, but the intensity of inflammatory response to Ureaplasma spp. is comparable with the inflammatory response to other bacteria.

Interactions and Nuclear Import of the N and P Proteins of Sonchus Yellow Net Virus, a Plant Nucleorhabdovirus

PubMed Central

Goodin, Michael M.; Austin, Jennifer; Tobias, Renée; Fujita, Miki; Morales, Christina; Jackson, Andrew O.

2001-01-01

We have characterized the interaction and nuclear localization of the nucleocapsid (N) protein and phosphoprotein (P) of sonchus yellow net nucleorhabdovirus. Expression studies with plant and yeast cells revealed that both N and P are capable of independent nuclear import. Site-specific mutagenesis and deletion analyses demonstrated that N contains a carboxy-terminal bipartite nuclear localization signal (NLS) located between amino acids 465 and 481 and that P contains a karyophillic region between amino acids 40 and 124. The N NLS was fully capable of functioning outside of the context of the N protein and was able to direct the nuclear import of a synthetic protein fusion consisting of green fluorescent protein fused to glutathione S-transferase (GST). Expression and mapping studies suggested that the karyophillic domain in P is located within the N-binding domain. Coexpression of N and P drastically affected their localization patterns relative to those of individually expressed proteins and resulted in a shift of both proteins to a subnuclear region. Yeast two-hybrid and GST pulldown experiments verified the N-P and P-P interactions, and deletion analyses have identified the N and P interacting domains. N NLS mutants were not transported to the nucleus by import-competent P, presumably because N binding masks the P NLS. Taken together, our results support a model for independent entry of N and P into the nucleus followed by associations that mediate subnuclear localization. PMID:11533202

Time-Dependent Effect of Encapsulating Alginate Hydrogel on Neurogenic Potential

PubMed Central

Razavi, Shahnaz; Khosravizadeh, Zahra; Bahramian, Hamid; Kazemi, Mohammad

2015-01-01

Objective Due to the restricted potential of neural stem cells for regeneration of central nervous system (CNS) after injury, providing an alternative source for neural stem cells is essential. Adipose derived stem cells (ADSCs) are multipotent cells with properties suitable for tissue engineering. In addition, alginate hydrogel is a biocompatible polysaccharide polymer that has been used to encapsulate many types of cells. The aim of this study was to assess the proliferation rate and level of expression of neural markers; NESTIN, glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2) in encapsulated human ADSCs (hADSCs) 10 and14 days after neural induction. Materials and Methods In this experimental study, ADSCs isolated from human were cultured in neural induction media and seeded into alginate hydrogel. The rate of proliferation and differentiation of encapsulated cells were evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay, immunocytoflourescent and realtime reverse transcriptase polymerase chain reaction (RT-PCR) analyzes 10 and 14 days after induction. Results The rate of proliferation of encapsulated cells was not significantly changed with time passage. The expression of NESTIN and GFAP significantly decreased on day 14 relative to day 10 (P<0.001) but MAP2 expression was increased. Conclusion Alginate hydrogel can promote the neural differentiation of encapsulated hADSCs with time passage. PMID:26199909

A shifted repertoire of endocannabinoid genes in the zebrafish (Danio rerio).

PubMed

McPartland, J M; Glass, Michelle; Matias, Isabel; Norris, Ryan W; Kilpatrick, C William

2007-05-01

The zebrafish has served as a model organism for developmental biology. Sequencing its genome has expanded zebrafish research into physiology and drug-development testing. Several cannabinoid pharmaceuticals are in development, but expression of endocannabinoid receptors and enzymes remains unknown in this species. We conducted a bioinformatics analysis of the zebrafish genome using 17 human endocannabinoid genes as a reference set. Putative zebrafish orthologs were identified in filtered BLAST searches as reciprocal best hits. Orthology was confirmed by three in silico methods: phylogenetic testing, synteny analysis, and functional mapping. Zebrafish expressed orthologs of cannabinoid receptor 1, transient receptor potential channel vanilloid receptor 4, GPR55 receptor, fatty acid amide hydrolase 1, monoacylglycerol lipase, NAPE-selective phospholipase D, abhydrolase domain-containing protein 4, and diacylglycerol lipase alpha and beta; and paired paralogs of cannabinoid receptor 2, fatty acid amide hydrolase 2, peroxisome proliferator-activated receptor alpha, prostaglandin-endoperoxide synthase 2, and transient receptor potential cation channel subtype A1. Functional mapping suggested the orthologs of transient receptor potential vanilloid receptor 1 and peroxisome proliferator-activated receptor gamma lack specific amino acids critical for cannabinoid ligand binding. No orthologs of N-acylethanolamine acid amidase or protein tyrosine phosphatase, non-receptor type 22 were identified. In conclusion, the zebrafish genome expresses a shifted repertoire of endocannabinoid genes. In vitro analyses are warranted before using zebrafish for cannabinoid development testing.

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed Central

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-01-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes. PMID:9121442

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-04-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes.

Nuclear Localization of the ERK MAP Kinase Mediated by Drosophila αPS2βPS Integrin and Importin-7

PubMed Central

James, Brian P.; Bunch, Thomas A.; Krishnamoorthy, Srinivasan; Perkins, Lizabeth A.

2007-01-01

The control of gene expression by the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK) requires its translocation into the nucleus. In Drosophila S2 cells nuclear accumulation of diphospho-ERK (dpERK) is greatly reduced by interfering double-stranded RNA against Drosophila importin-7 (DIM-7) or by the expression of integrin mutants, either during active cell spreading or after stimulation by insulin. In both cases, total ERK phosphorylation (on Westerns) is not significantly affected, and ERK accumulates in a perinuclear ring. Tyrosine phosphorylation of DIM-7 is reduced in cells expressing integrin mutants, indicating a mechanistic link between these components. DIM-7 and integrins localize to the same actin-containing peripheral regions in spreading cells, but DIM-7 is not concentrated in paxillin-positive focal contacts or stable focal adhesions. The Corkscrew (SHP-2) tyrosine phosphatase binds DIM-7, and Corkscrew is required for the cortical localization of DIM-7. These data suggest a model in which ERK phosphorylation must be spatially coupled to integrin-mediated DIM-7 activation to make a complex that can be imported efficiently. Moreover, dpERK nuclear import can be restored in DIM-7–deficient cells by Xenopus Importin-7, demonstrating that ERK import is an evolutionarily conserved function of this protein. PMID:17699602

Comprehensive RNA-Seq Expression Analysis of Sensory Ganglia with a Focus on Ion Channels and GPCRs in Trigeminal Ganglia

PubMed Central

Manteniotis, Stavros; Lehmann, Ramona; Flegel, Caroline; Vogel, Felix; Hofreuter, Adrian; Schreiner, Benjamin S. P.; Altmüller, Janine; Becker, Christian; Schöbel, Nicole; Hatt, Hanns; Gisselmann, Günter

2013-01-01

The specific functions of sensory systems depend on the tissue-specific expression of genes that code for molecular sensor proteins that are necessary for stimulus detection and membrane signaling. Using the Next Generation Sequencing technique (RNA-Seq), we analyzed the complete transcriptome of the trigeminal ganglia (TG) and dorsal root ganglia (DRG) of adult mice. Focusing on genes with an expression level higher than 1 FPKM (fragments per kilobase of transcript per million mapped reads), we detected the expression of 12984 genes in the TG and 13195 in the DRG. To analyze the specific gene expression patterns of the peripheral neuronal tissues, we compared their gene expression profiles with that of the liver, brain, olfactory epithelium, and skeletal muscle. The transcriptome data of the TG and DRG were scanned for virtually all known G-protein-coupled receptors (GPCRs) as well as for ion channels. The expression profile was ranked with regard to the level and specificity for the TG. In total, we detected 106 non-olfactory GPCRs and 33 ion channels that had not been previously described as expressed in the TG. To validate the RNA-Seq data, in situ hybridization experiments were performed for several of the newly detected transcripts. To identify differences in expression profiles between the sensory ganglia, the RNA-Seq data of the TG and DRG were compared. Among the differentially expressed genes (> 1 FPKM), 65 and 117 were expressed at least 10-fold higher in the TG and DRG, respectively. Our transcriptome analysis allows a comprehensive overview of all ion channels and G protein-coupled receptors that are expressed in trigeminal ganglia and provides additional approaches for the investigation of trigeminal sensing as well as for the physiological and pathophysiological mechanisms of pain. PMID:24260241

Proteomic Analysis and Identification of the Structural and Regulatory Proteins of the Rhodobacter capsulatus Gene Transfer Agent

PubMed Central

Chen, Frank; Spano, Anthony; Goodman, Benjamin E.; Blasier, Kiev R.; Sabat, Agnes; Jeffery, Erin; Norris, Andrew; Shabanowitz, Jeffrey; Hunt, Donald F.; Lebedev, Nikolai

2010-01-01

The gene transfer agent of Rhodobacter capsulatus (GTA) is a unique phage-like particle that exchanges genetic information between members of this same species of bacterium. Besides being an excellent tool for genetic mapping, the GTA has a number of advantages for biotechnological and nanoengineering purposes. To facilitate the GTA purification and identify the proteins involved in GTA expression, assembly and regulation, in the present work we construct and transform into R. capsulatus Y262 a gene coding for a C-terminally His-tagged capsid protein. The constructed protein was expressed in the cells, assembled into chimeric GTA particles inside the cells and excreted from the cells into surrounding medium. Transmission electron micrographs of phosphotungstate-stained, NiNTA-purified chimeric GTA confirm that its structure is similar to normal GTA particles, with many particles composed both of a head and a tail. The mass spectrometric proteomic analysis of polypeptides present in the GTA recovered outside the cells shows that GTA is composed of at least 9 proteins represented in the GTA gene cluster including proteins coded for by Orf’s 3, 5, 6–9, 11, 13, and 15. PMID:19105630

Proteomic analysis and identification of the structural and regulatory proteins of the Rhodobacter capsulatus gene transfer agent.

PubMed

Chen, Frank; Spano, Anthony; Goodman, Benjamin E; Blasier, Kiev R; Sabat, Agnes; Jeffery, Erin; Norris, Andrew; Shabanowitz, Jeffrey; Hunt, Donald F; Lebedev, Nikolai

2009-02-01

The gene transfer agent of Rhodobacter capsulatus (GTA) is a unique phage-like particle that exchanges genetic information between members of this same species of bacterium. Besides being an excellent tool for genetic mapping, the GTA has a number of advantages for biotechnological and nanoengineering purposes. To facilitate the GTA purification and identify the proteins involved in GTA expression, assembly and regulation, in the present work we construct and transform into R. capsulatus Y262 a gene coding for a C-terminally His-tagged capsid protein. The constructed protein was expressed in the cells, assembled into chimeric GTA particles inside the cells and excreted from the cells into surrounding medium. Transmission electron micrographs of phosphotungstate-stained, NiNTA-purified chimeric GTA confirm that its structure is similar to normal GTA particles, with many particles composed both of a head and a tail. The mass spectrometric proteomic analysis of polypeptides present in the GTA recovered outside the cells shows that GTA is composed of at least 9 proteins represented in the GTA gene cluster including proteins coded for by Orf's 3, 5, 6-9, 11, 13, and 15.

Effects of calorie restriction on the zebrafish liver proteome

PubMed Central

Jury, David R.; Kaveti, Suma; Duan, Zhong-Hui; Willard, Belinda; Kinter, Michael; Londraville, Richard

2012-01-01

A proteomic approach was taken to study how fish respond to changes in calorie availability, with the longer-term goal of understanding the evolution of lipid metabolism in vertebrates. Zebrafish (Danio rerio) were fed either high (3 rations/day) or low (1 ration/7 days) calorie diets for 5 weeks and liver proteins extracted for proteomic analyses. Proteins were separated on two-dimensional electrophoresis gels and homologous spots compared between treatments to determine which proteins were up-regulated with high-calorie diet. Fifty-five spots were excised from the gel and analyzed via LC–ESI MS/MS, which resulted in the identification of 69 unique proteins (via multiple peptides). Twenty-nine of these proteins were differentially expressed between treatments. Differentially expressed proteins were mapped to Gene Ontology (GO) terms, and these terms compared to the entire zebrafish GO annotation set by Fisher's exact test. The most significant GO terms associated with high-calorie diet are related to a decrease in oxygen-binding activity in the high-calorie treatment. This response is consistent with a well-characterized response in obese humans, indicating there may be a link between lipid storage and hypoxia sensitivity in vertebrates. PMID:20494847

Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells

PubMed Central

Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

2016-01-01

Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K-ATPase α1 and α3 subunits and increased the expression of WEE1 in HeLa cells. Antibodies against Na, K-ATPase α1 and α3 subunits alone or combinated with arenobufagin also inhibited the activity of proteasome. Furthermore, the expression of the possible intermediate proteins ataxin-1 and translationally-controlled tumor protein was increased in HeLa cells treated with arenobufagin by flow cytometry analysis, respectively. These results indicated that arenobufagin might directly bind with Na, K-ATPase α1 and α3 subunits and the inhibitive effect of arenobufagin on proteasomal activity of HeLa cells might be related to its binding with Na, K-ATPase. PMID:27428326

Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

PubMed

Yue, Qingxi; Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

2016-01-01

Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K-ATPase α1 and α3 subunits and increased the expression of WEE1 in HeLa cells. Antibodies against Na, K-ATPase α1 and α3 subunits alone or combinated with arenobufagin also inhibited the activity of proteasome. Furthermore, the expression of the possible intermediate proteins ataxin-1 and translationally-controlled tumor protein was increased in HeLa cells treated with arenobufagin by flow cytometry analysis, respectively. These results indicated that arenobufagin might directly bind with Na, K-ATPase α1 and α3 subunits and the inhibitive effect of arenobufagin on proteasomal activity of HeLa cells might be related to its binding with Na, K-ATPase.

Genome-nuclear lamina interactions and gene regulation.

PubMed

Kind, Jop; van Steensel, Bas

2010-06-01

The nuclear lamina, a filamentous protein network that coats the inner nuclear membrane, has long been thought to interact with specific genomic loci and regulate their expression. Molecular mapping studies have now identified large genomic domains that are in contact with the lamina. Genes in these domains are typically repressed, and artificial tethering experiments indicate that the lamina can actively contribute to this repression. Furthermore, the lamina indirectly controls gene expression in the nuclear interior by sequestration of certain transcription factors. A variety of DNA-binding and chromatin proteins may anchor specific loci to the lamina, while histone-modifying enzymes partly mediate the local repressive effect of the lamina. Experimental tools are now available to begin to unravel the underlying molecular mechanisms. Copyright 2010 Elsevier Ltd. All rights reserved.

Detection of RNA nucleoside modifications with the uridine-specific ribonuclease MC1 from Momordica charantia

PubMed Central

Addepalli, Balasubrahmanym; Lesner, Nicholas P.; Limbach, Patrick A.

2015-01-01

A codon-optimized recombinant ribonuclease, MC1 is characterized for its uridine-specific cleavage ability to map nucleoside modifications in RNA. The published MC1 amino acid sequence, as noted in a previous study, was used as a template to construct a synthetic gene with a natural codon bias favoring expression in Escherichia coli. Following optimization of various expression conditions, the active recombinant ribonuclease was successfully purified as a C-terminal His-tag fusion protein from E. coli [Rosetta 2(DE3)] cells. The isolated protein was tested for its ribonuclease activity against oligoribonucleotides and commercially available E. coli tRNATyr I. Analysis of MC1 digestion products by ion-pairing reverse phase liquid-chromatography coupled with mass spectrometry (IP-RP-LC-MS) revealed enzymatic cleavage of RNA at the 5′-termini of uridine and pseudouridine, but cleavage was absent if the uridine was chemically modified or preceded by a nucleoside with a bulky modification. Furthermore, the utility of this enzyme to generate complementary digestion products to other common endonucleases, such as RNase T1, which enables the unambiguous mapping of modified residues in RNA is demonstrated. PMID:26221047

cDNA cloning, tissue distribution, and chromosomal localization of myelodysplasia/myeloid leukemia factor 2 (MLF2).

PubMed

Kuefer, M U; Look, A T; Williams, D C; Valentine, V; Naeve, C W; Behm, F G; Mullersman, J E; Yoneda-Kato, N; Montgomery, K; Kucherlapati, R; Morris, S W

1996-07-15

A fusion gene between nucleophosmin (NPM) and myelodysplasia/myeloid leukemia factor 1 (MLF1) is formed by a recurrent t(3;5)(q25.1;q34) in myelodysplastic syndrome and acute myeloid leukemia. Here we report the identification of a novel gene, MLF2, which contains an open reading frame of 744 bp encoding a 248-amino-acid protein highly related to the previously identified MLF1 protein (63% similarity, 40% identity). In contrast to the tissue-restricted expression pattern of MLF1, the MLF2 messenger RNA is expressed ubiquitously. The MLF2 gene locus was mapped by fluorescence in situ hybridization to human chromosome 12p13, a chromosomal region frequently involved in translocations and deletions in acute leukemias of lymphoid or myeloid lineage. In a physical map of chromosome 12, MLF2 was found to reside on the yeast artificial chromosome clone 765b9. Southern blotting analysis of malignant cell DNAs prepared from a series of acute lymphoblastic leukemia cases with translocations involving chromosome arm 12p, as well as a group of acute myeloid leukemias with various cytogenetic abnormalities, failed to reveal MLF2 gene rearrangements.

3,5,6,7,8,3′,4′-Heptamethoxyflavone Ameliorates Depressive-Like Behavior and Hippocampal Neurochemical Changes in Chronic Unpredictable Mild Stressed Mice by Regulating the Brain-Derived Neurotrophic Factor: Requirement for ERK Activation

PubMed Central

Sawamoto, Atsushi; Okuyama, Satoshi; Amakura, Yoshiaki; Yoshimura, Morio; Yamada, Takashi; Yokogoshi, Hidehiko; Nakajima, Mitsunari

2017-01-01

We previously reported that the subcutaneous administration of 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF), a citrus polymethoxyflavone, attenuated depressive-like behavior and increased the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus of a corticosterone-induced depression-like mouse model. We herein demonstrated that (1) HMF was detectable in the brain 10 and 30 min after its oral administration, (2) orally administered HMF improved chronic unpredictable mild stress (CUMS)-induced pathological conditions, including body weight loss and depressive-like behavior, and CUMS-induced neurochemical changes, such as reduction in BDNF expression, decrease in neurogenesis, and decreased level of phosphorylated calcium-calmodulin-dependent protein kinase II in the hippocampus, and (3) these effects of HMF were inhibited by the pre-administration of U0126, a mitogen-activated protein (MAP) kinase inhibitor. These results suggest that orally administered HMF is beneficial for the upregulation of BDNF in the hippocampus via the extracellular signal-regulated kinase1/2 (ERK1/2)/MAP system, which may account for its antidepression effects. PMID:29023414

15-hydroxyprostaglandin dehydrogenase is upregulated by hydroxychloroquine in rheumatoid arthritis fibroblast-like synoviocytes.

PubMed

Kim, Hak-Jae; Lee, Sora; Lee, Haw-Yong; Won, Hansol; Chang, Sung-Hae; Nah, Seong-Su

2015-09-01

15-Hydroxyprostaglandin dehydrogenase (HPGD) is the key enzyme responsible for the metabolic inactivation of prostaglandin E2 (PGE2) catabolism. PGE2 is one of the predominant catabolic factors involved in rheumatoid arthritis (RA). However, the expression and regulation of HPGD in RA fibroblast‑like synoviocyte (FLS) remain to be elucidated. Disease‑modifying anti‑rheumatic drugs (DMARDs) are the most important anti‑arthritic drugs, which reduce the effect of joint injury. The aim of the present study was to assess the expression of HPGD in RA tissues and cells, and normal synovial tissues and cells. The effect of the most popular DMARDs, hydroxychloroquine, on the expression of HPGD in RA‑FLS was also investigated. Western blotting and immunohistochemical analysis demonstrated that the expression levels of HPGD in human synovium were lower in RA synovium compared with the normal and OA synovium. In RA‑FLS, the expression of HPGD was increased following treatment with several DMARDs, including sulfasalazine, methotrexate, and hydroxychloroquine. Hydroxychloroquine (10 µM) treatment induced the phosphorylation of ERK, SAPK/JNK and p38. Hydroxychloroquine induced a decrease in the release of PGE2, which was restored by mitogen‑activated protein (MAP) kinase pathway inhibitors. Hydroxychloroquine may therefore, affect the pathogenesis of RA through the MAP kinase pathway by regulating the expression of HPGD.

15-Hydroxyprostaglandin dehydrogenase is upregulated by hydroxychloroquine in rheumatoid arthritis fibroblast-like synoviocytes

PubMed Central

KIM, HAK-JAE; LEE, SORA; LEE, HAW-YONG; WON, HANSOL; CHANG, SUNG-HAE; NAH, SEONG-SU

2015-01-01

15-Hydroxyprostaglandin dehydrogenase (HPGD) is the key enzyme responsible for the metabolic inactivation of prostaglandin E2 (PGE2) catabolism. PGE2 is one of the predominant catabolic factors involved in rheumatoid arthritis (RA). However, the expression and regulation of HPGD in RA fibroblast-like synoviocyte (FLS) remain to be elucidated. Disease-modifying anti-rheumatic drugs (DMARDs) are the most important anti-arthritic drugs, which reduce the effect of joint injury. The aim of the present study was to assess the expression of HPGD in RA tissues and cells, and normal synovial tissues and cells. The effect of the most popular DMARDs, hydroxychloroquine, on the expression of HPGD in RA-FLS was also investigated. Western blotting and immuno-histochemical analysis demonstrated that the expression levels of HPGD in human synovium were lower in RA synovium compared with the normal and OA synovium. In RA-FLS, the expression of HPGD was increased following treatment with several DMARDs, including sulfasalazine, methotrexate, and hydroxychloroquine. Hydroxychloroquine (10 µM) treatment induced the phosphorylation of ERK, SAPK/JNK and p38. Hydroxychloroquine induced a decrease in the release of PGE2, which was restored by mitogen-activated protein (MAP) kinase pathway inhibitors. Hydroxychloroquine may therefore, affect the pathogenesis of RA through the MAP kinase pathway by regulating the expression of HPGD. PMID:26082314

Loss of MAP3K1 enhances proliferation and apoptosis during retinal development

PubMed Central

Mongan, Maureen; Wang, Jingcai; Liu, Hongshan; Fan, Yunxia; Jin, Chang; Kao, Winston Y.-W.; Xia, Ying

2011-01-01

Precise coordination of progenitor cell proliferation and differentiation is essential for proper organ morphogenesis and function during mammalian development. The mitogen-activated protein kinase kinase kinase 1 (MAP3K1) has a well-established role in anterior eyelid development, as Map3k1-knockout mice have defective embryonic eyelid closure and an `eye-open at birth' (EOB) phenotype. Here, we show that MAP3K1 is highly expressed in the posterior of the developing eye and is required for retina development. The MAP3K1-deficient mice exhibit increased proliferation and apoptosis, and Müller glial cell overproduction in the developing retinas. Consequently, the retinas of these mice show localized rosette-like arrangements in the outer nuclear layer, and develop abnormal vascularization, broken down retinal pigment epithelium, photoreceptor loss and early onset of retinal degeneration. Although the retinal defect is associated with increased cyclin D1 and CDK4/6 expression, and RB phosphorylation and E2F-target gene upregulation, it is independent of the EOB phenotype and of JNK. The retinal developmental defect still occurs in knockout mice that have undergone tarsorrhaphy, but is absent in compound mutant Map3k1+/ΔKDJnk1–/– and Map3k1+/ΔKDJnk+/–Jnk2+/– mice that have EOB and reduced JNK signaling. Our results unveil a novel role for MAP3K1 in which it crosstalks with the cell cycle regulatory pathways in the prevention of retina malformation and degeneration. PMID:21862560

Site-specific Microtubule-associated Protein 4 Dephosphorylation Causes Microtubule Network Densification in Pressure Overload Cardiac Hypertrophy*

PubMed Central

Chinnakkannu, Panneerselvam; Samanna, Venkatesababa; Cheng, Guangmao; Ablonczy, Zsolt; Baicu, Catalin F.; Bethard, Jennifer R.; Menick, Donald R.; Kuppuswamy, Dhandapani; Cooper, George

2010-01-01

In severe pressure overload-induced cardiac hypertrophy, a dense, stabilized microtubule network forms that interferes with cardiocyte contraction and microtubule-based transport. This is associated with persistent transcriptional up-regulation of cardiac α- and β-tubulin and microtubule-stabilizing microtubule-associated protein 4 (MAP4). There is also extensive microtubule decoration by MAP4, suggesting greater MAP4 affinity for microtubules. Because the major determinant of this affinity is site-specific MAP4 dephosphorylation, we characterized this in hypertrophied myocardium and then assessed the functional significance of each dephosphorylation site found by mimicking it in normal cardiocytes. We first isolated MAP4 from normal and pressure overload-hypertrophied feline myocardium; volume-overloaded myocardium, which has an equal degree and duration of hypertrophy but normal functional and cytoskeletal properties, served as a control for any nonspecific growth-related effects. After cloning cDNA-encoding feline MAP4 and obtaining its deduced amino acid sequence, we characterized by mass spectrometry any site-specific MAP4 dephosphorylation. Solely in pressure overload-hypertrophied myocardium, we identified striking MAP4 dephosphorylation at Ser-472 in the MAP4 N-terminal projection domain and at Ser-924 and Ser-1056 in the assembly-promoting region of the C-terminal microtubule-binding domain. Site-directed mutagenesis of MAP4 cDNA was then used to switch each serine to non-phosphorylatable alanine. Wild-type and mutated cDNAs were used to construct adenoviruses; microtubule network density, stability, and MAP4 decoration were assessed in normal cardiocytes following an equivalent level of MAP4 expression. The Ser-924 → Ala MAP4 mutant produced a microtubule phenotype indistinguishable from that seen in pressure overload hypertrophy, such that Ser-924 MAP4 dephosphorylation during pressure overload hypertrophy may be central to this cytoskeletal abnormality. PMID:20436166

Elevated levels of p-Mnk1, p-eIF4E and p-p70S6K proteins are associated with tumor recurrence and poor prognosis in astrocytomas.

PubMed

Fan, Weibing; Wang, Weiyuan; Mao, Xinfa; Chu, Shuzhou; Feng, Juan; Xiao, Desheng; Zhou, Jianhua; Fan, Songqing

2017-02-01

Malignant astrocytomas are able to invade neighboring and distant areas of the normal brain. Signaling pathway alterations play important role in the development of astrocytomas. Deregulation of eukaryotic translation initiation factor 4E (eIF4E) by MAP kinase-interacting kinases (Mnk) on Ser-209 directly or PI3K/mTOR/S6K pathway indirectly has a critical effect on promoting cellular proliferation, malignant transformation and metastasis. We examined and analyzed the correlation between expression of p-Mnk1, p-eIF4E and p-p70S6K proteins and clinicopathological features in 103 astrocytomas and 54 non-tumorous brain tissues. The results indicated that positive percentage of overexpression of p-Mnk1 and p-eIF4E proteins in astrocytomas were significantly higher than that of in the non-tumorous brain tissues (P < 0.05). Elevated p-Mnk1 and p-eIF4E and co-overexpressed three proteins were associated with tumor recurrence (P = 0.003, P = 0.006, P = 0.007, respectively). Overexpressed p-eIF4E significantly correlated with the tumor size (P = 0.019). In addition, overexpression of p-eIF4E and three proteins common expression were related to the WHO grade of astrocytomas (P = 0.001, P = 0.044 respectively). Spearman's rank correlation test further showed that the expression of p-Mnk1 was strongly positive correlated with the expression of p-eIF4E in astrocytomas (r = 0.294, P = 0.003). Besides, overexpression of p-eIF4E and co-expression of p-Mnk1, p-eIF4E and p-p70S6K proteins were inversely correlated with overall survival rates of astrocytomas. Multivariate Cox regression analysis further identified that the elevated p-eIF4E expression, three proteins common expression were correlated with unfavorable prognosis of astrocytomas regardless of ages and WHO grades. Taken together, overexpression of p-eIF4E and co-expression of p-Mnk1, p-eIF4E and p-p70S6K proteins could be used as novel independent poor prognostic biomarkers for patients with astrocytomas.

Enhanced expression of Gqα and PLC-β1 proteins contributes to vascular smooth muscle cell hypertrophy in SHR: role of endogenous angiotensin II and endothelin-1.

PubMed

Atef, Mohammed Emehdi; Anand-Srivastava, Madhu B

2014-07-01

Vascular Gqα signaling has been shown to contribute to cardiac hypertrophy. In addition, angiotensin II (ANG II) was shown to induce vascular smooth muscle cell (VSMC) hypertrophy through Gqα signaling; however, the studies on the role of Gqα and PLC-β1 proteins in VSMC hypertrophy in animal model are lacking. The present study was therefore undertaken to examine the role of Gqα/PLC-β1 proteins and the signaling pathways in VSMC hypertrophy using spontaneously hypertensive rats (SHR). VSMC from 16-wk-old SHR and not from 12-wk-old SHR exhibited enhanced levels of Gqα/PLC-β1 proteins compared with age-matched Wistar-Kyoto (WKY) rats as determined by Western blotting. However, protein synthesis as determined by [(3)H]leucine incorporation was significantly enhanced in VSMC from both 12- and 16-wk-old SHR compared with VSMC from age-matched WKY rats. Furthermore, the knockdown of Gqα/PLC-β1 in VSMC from 16-wk-old SHR by antisense and small interfering RNA resulted in attenuation of protein synthesis. In addition, the enhanced expression of Gqα/PLC-β1 proteins, enhanced phosphorylation of ERK1/2, and enhanced protein synthesis in VSMC from SHR were attenuated by the ANG II AT1 and endothelin-1 (ET-1) ETA receptor antagonists losartan and BQ123, respectively, but not by the ETB receptor antagonist BQ788. In addition, PD98059 decreased the enhanced expression of Gqα/PLC-β1 and protein synthesis in VSMC from SHR. These results suggest that the enhanced levels of endogenous ANG II and ET-1 through the activation of AT1 and ETA receptors, respectively, and MAP kinase signaling, enhanced the expression of Gqα/PLC-β1 proteins in VSMC from 16-wk-old SHR and result in VSMC hypertrophy. Copyright © 2014 the American Physiological Society.

MOPED 2.5—An Integrated Multi-Omics Resource: Multi-Omics Profiling Expression Database Now Includes Transcriptomics Data

PubMed Central

Montague, Elizabeth; Stanberry, Larissa; Higdon, Roger; Janko, Imre; Lee, Elaine; Anderson, Nathaniel; Choiniere, John; Stewart, Elizabeth; Yandl, Gregory; Broomall, William; Kolker, Natali

2014-01-01

Abstract Multi-omics data-driven scientific discovery crucially rests on high-throughput technologies and data sharing. Currently, data are scattered across single omics repositories, stored in varying raw and processed formats, and are often accompanied by limited or no metadata. The Multi-Omics Profiling Expression Database (MOPED, http://moped.proteinspire.org) version 2.5 is a freely accessible multi-omics expression database. Continual improvement and expansion of MOPED is driven by feedback from the Life Sciences Community. In order to meet the emergent need for an integrated multi-omics data resource, MOPED 2.5 now includes gene relative expression data in addition to protein absolute and relative expression data from over 250 large-scale experiments. To facilitate accurate integration of experiments and increase reproducibility, MOPED provides extensive metadata through the Data-Enabled Life Sciences Alliance (DELSA Global, http://delsaglobal.org) metadata checklist. MOPED 2.5 has greatly increased the number of proteomics absolute and relative expression records to over 500,000, in addition to adding more than four million transcriptomics relative expression records. MOPED has an intuitive user interface with tabs for querying different types of omics expression data and new tools for data visualization. Summary information including expression data, pathway mappings, and direct connection between proteins and genes can be viewed on Protein and Gene Details pages. These connections in MOPED provide a context for multi-omics expression data exploration. Researchers are encouraged to submit omics data which will be consistently processed into expression summaries. MOPED as a multi-omics data resource is a pivotal public database, interdisciplinary knowledge resource, and platform for multi-omics understanding. PMID:24910945

Characterization, expression profiles, intracellular distribution and association analysis of porcine PNAS-4 gene with production traits.

PubMed

Mo, Delin; Zhu, Zhengmao; te Pas, Marinus F W; Li, Xinyun; Yang, Shulin; Wang, Heng; Wang, Huanling; Li, Kui

2008-06-30

In a previous screen to identify differentially expressed genes associated with embryonic development, the porcine PNAS-4 gene had been found. Considering differentially expressed genes in early stages of muscle development are potential candidate genes to improve meat quality and production efficiency, we determined how porcine PNAS-4 gene regulates meat production. Therefore, this gene has been sequenced, expression analyzed and associated with meat production traits. We cloned the full-length cDNA of porcine PNAS-4 gene encoding a protein of 194 amino acids which was expressed in the Golgi complex. This gene was mapped to chromosome 10, q11-16, in a region of conserved synteny with human chromosome 1 where the human homologous gene was localized. Real-time PCR revealed that PNAS-4 mRNA was widely expressed with highest expression levels in skeletal muscle followed by lymph, liver and other tissues, and showed a down-regulated expression pattern during prenatal development while a up-regulated expression pattern after weaning. Association analysis revealed that allele C of SNP A1813C was prevalent in Chinese indigenous breeds whereas A was dominant allele in Landrace and Large White, and the pigs with homozygous CC had a higher fat content than those of the pigs with other genotypes (P < 0.05). Porcine PNAS-4 protein tagged with green fluorescent protein accumulated in the Golgi complex, and its mRNA showed a widespread expression across many tissues and organs in pigs. It may be an important factor affecting the meat production efficiency, because its down-regulated expression pattern during early embryogenesis suggests involvement in increase of muscle fiber number. In addition, the SNP A1813C associated with fat traits might be a genetic marker for molecular-assisted selection in animal breeding.

Heat shock proteins MoSsb1, MoSsz1, and MoZuo1 attenuate MoMkk1-mediated CWI signaling and are important for growth and pathogenicity of Magnaporthe oryzae.

PubMed

Yang, Jie; Liu, Muxing; Liu, Xinyu; Yin, Ziyi; Sun, Yi; Zhang, Haifeng; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

2018-06-05

The mitogen-activated protein (MAP) kinase MoMkk1 governs the cell wall integrity (CWI) pathway in the rice blast fungus Magnaporthe oryzae. To understand the underlying mechanism, we have identified MoSsb1 as one of the MoMkk1-interacting proteins. MoSsb1 is a stress-seventy subfamily B (Ssb) protein homolog, sharing high amino acid sequence homology with the 70-kDa heat shock proteins (Hsp70s). Hsp70 proteins are a family of conserved and ubiquitously expressed chaperones that regulate protein biogenesis by promoting protein folding, preventing protein aggregation and controlling protein degradation. We found that MoSsb1 regulates the synthesis of nascent polypeptide chains and this regulation is achieved by being in complex with other members of heat shock proteins: Hsp70 MoSsz1 and 40-kDa heat shock protein (Hsp40) MoZuo1. MoSsb1 is important for the growth, conidiation and full virulence of the blast fungus and this role is also shared by MoSsz1 and MoZuo1. Importantly, MoSsb1, MoSsz1 and MoZuo1 are all involved in the regulation of the CWI MAP kinase pathway by modulating MoMkk1 biosynthesis. Our studies reveal novel insights into how MoSsb1, MoSsz1 and MoZuo1 affect CWI signaling that is involved in regulating growth, differentiation and virulence of M. oryzae and highlight the conserved functional mechanisms of Hsp proteins in pathogenic fungi.

Mutant APP and Amyloid beta-induced defective autophagy, mitophagy, mitochondrial structural and functional changes and synaptic damage in hippocampal neurons from Alzheimer's disease.

PubMed

Reddy, P Hemachandra; Yin, XiangLin; Manczak, Maria; Kumar, Subodh; Jangampalli Adi, Pradeepkiran; Vijayan, Murali; Reddy, Arubala P

2018-04-25

The purpose of our study was to determine the toxic effects of hippocampal mutant APP and amyloid beta (Aβ) in human mutant APP (mAPP) cDNA transfected with primary mouse hippocampal neurons (HT22). Hippocampal tissues are the best source of studying learning and memory functions in patients with Alzheimer's disease (AD) and healthy controls. However, investigating immortalized hippocampal neurons that express AD proteins provide an excellent opportunity for drug testing. Using quantitative RT-PCR, immunoblotting & immunofluorescence, and transmission electron microscopy, we assessed mRNA and protein levels of synaptic, autophagy, mitophagy, mitochondrial dynamics, biogenesis, dendritic protein MAP2, and assessed mitochondrial number and length in mAPP-HT22 cells that express Swedish/Indiana mutations. Mitochondrial function was assessed by measuring the levels of hydrogen peroxide, lipid peroxidation, cytochrome c oxidase activity and mitochondrial ATP. Increased levels of mRNA and protein levels of mitochondrial fission genes, Drp1 and Fis1 and decreased levels fusion (Mfn1, Mfn2 and Opa1) biogenesis (PGC1α, NRF1, NRF2 & TFAM), autophagy (ATG5 & LC3BI, LC3BII), mitophagy (PINK1 & TERT, BCL2 & BNIPBL), synaptic (synaptophysin & PSD95) and dendritic (MAP2) genes were found in mAPP-HT22 cells relative to WT-HT22 cells. Cell survival was significantly reduced mAPP-HT22 cells. GTPase-Dp1 enzymatic activity was increased in mAPP-HT22 cells. Transmission electron microscopy revealed significantly increased mitochondrial numbers and reduced mitochondrial length in mAPP-HT22 cells. These findings suggest that hippocampal accumulation of mutant APP and Aβ is responsible for abnormal mitochondrial dynamics and defective biogenesis, reduced MAP2, autophagy, mitophagy and synaptic proteins & reduced dendritic spines and mitochondrial structural and functional changes in mutant APP hippocampal cells. These observations strongly suggest that accumulation of mAPP and Aβ causes mitochondrial, synaptic and autophagy/mitophagy abnormalities in hippocampal neurons, leading to neuronal dysfunction.

SB202190 affects cell response to hydroxyurea-induced genotoxic stress in root meristems of Vicia faba.

PubMed

Winnicki, Konrad; Maszewski, Janusz

2012-11-01

Genotoxic stress caused by a variety of chemical and physical agents may lead to DNA breaks and genome instability. Response to DNA damage depends on ATM/ATR sensor kinases and their downstream proteins, which arrange cell cycle checkpoints. Activation of ATM (ataxia-telangiectasia-mutated)/ATR (ATM and Rad 3-related) signaling pathway triggers cell cycle arrest (by keeping cyclin-Cdk complexes inactive), combined with gamma-phosphorylation of histone H2A.X and induction of DNA repair processes. However, genotoxic stress activates also mitogen-activated protein kinases (MAPKs) which may control the functions of checkpoint proteins both directly, by post-translational modifications, or indirectly, by regulation of their expression. Our results indicate that in root meristem cells of Vicia faba, MAP kinase signaling pathway takes part in response to hydroxyurea-induced genotoxic stress. It is shown that SB202190, an inhibitor of p38 MAP kinase, triggers PCC (premature chromosome condensation) more rapidly, but only if cell cycle checkpoints are alleviated by caffeine. Since SB202190 and, independently, caffeine reduces HU-mediated histone H4 Lys5 acetylation, it may be that there is a cooperation of MAP kinase signaling pathways and ATM/ATR-dependent checkpoints during response to genotoxic stress. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Membrane androgen receptor characteristics of human ZIP9 (SLC39A) zinc transporter in prostate cancer cells: Androgen-specific activation and involvement of an inhibitory G protein in zinc and MAP kinase signaling.

PubMed

Thomas, Peter; Pang, Yefei; Dong, Jing

2017-05-15

Characteristics of novel human membrane androgen receptor (mAR), ZIP9 (SLC39A9), were investigated in ZIP9-transfected PC-3 cells (PC3-ZIP9). Ligand blot analysis showed plasma membrane [ 3 H]-T binding corresponds to the position of ZIP9 on Western blots which suggests ZIP9 can bind [ 3 H]-T alone, without a protein partner. Progesterone antagonized testosterone actions, blocking increases in zinc, Erk phosphorylation and apoptosis, further evidence that ZIP9 is specifically activated by androgens. Pre-treatment with GTPγS and pertussis toxin decreased plasma membrane [ 3 H]-T binding and blocked testosterone-induced increases in Erk phosphorylation and intracellular zinc, indicating ZIP9 is coupled to an inhibitory G protein (Gi) that mediates both MAP kinase and zinc signaling. Testosterone treatment of nuclei and mitochondria which express ZIP9 decreased their zinc contents, suggesting ZIP9 also regulates free zinc through releasing it from these intracellular organelles. The results show ZIP9 is a specific Gi coupled-mAR mediating testosterone-induced MAP kinase and zinc signaling in PC3-ZIP9 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of Smoc-1 uncovers two transcript variants showing differential tissue and age specific expression in Bubalus bubalis

PubMed Central

Srivastava, Jyoti; Premi, Sanjay; Kumar, Sudhir; Parwez, Iqbal; Ali, Sher

2007-01-01

Background Secreted modular calcium binding protein-1 (Smoc-1) belongs to the BM-40 family which has been implicated with tissue remodeling, angiogenesis and bone mineralization. Besides its anticipated role in embryogenesis, Smoc-1 has been characterized only in a few mammalian species. We made use of the consensus sequence (5' CACCTCTCCACCTGCC 3') of 33.15 repeat loci to explore the buffalo transcriptome and uncovered the Smoc-1 transcript tagged with this repeat. The main objective of this study was to gain an insight into its structural and functional organization, and expressional status of Smoc-1 in water buffalo, Bubalus bubalis. Results We cloned and characterized the buffalo Smoc-1, including its copy number status, in-vitro protein expression, tissue & age specific transcription/translation, chromosomal mapping and localization to the basement membrane zone. Buffalo Smoc-1 was found to encode a secreted matricellular glycoprotein containing two EF-hand calcium binding motifs homologous to that of BM-40/SPARC family. In buffalo, this single copy gene consisted of 12 exons and was mapped onto the acrocentric chromosome 11. Though this gene was found to be evolutionarily conserved, the buffalo Smoc-1 showed conspicuous nucleotide/amino acid changes altering its secondary structure compared to that in other mammals. In silico analysis of the Smoc-1 proposed its glycoprotein nature with a calcium dependent conformation. Further, we unveiled two transcript variants of this gene, varying in their 3'UTR lengths but both coding for identical protein(s). Smoc-1 evinced highest expression of both the variants in liver and modest to negligible in other tissues. The relative expression of variant-02 was markedly higher compared to that of variant-01 in all the tissues examined. Moreover, expression of Smoc-1, though modest during the early ages, was conspicuously enhanced after 1 year and remained consistently higher during the entire life span of buffalo with gradual increment in expression of variant-02. Immunohistochemically, Smoc-1 was localized in the basement membrane zones and extracellular matrices of various tissues. Conclusion These data added to our understandings about the tissue, age and species specific functions of the Smoc-1. It also enabled us to demonstrate varying expression of the two transcript variants of Smoc-1 amongst different somatic tissues/gonads and ages, in spite of their identical coding frames. Pursuance of these variants for their roles in various disease phenotypes such as hepatocellular carcinoma and angiogenesis is envisaged to establish broader biological significance of this gene. PMID:18042303

Systems-level identification of PKA-dependent signaling in epithelial cells.

PubMed

Isobe, Kiyoshi; Jung, Hyun Jun; Yang, Chin-Rang; Claxton, J'Neka; Sandoval, Pablo; Burg, Maurice B; Raghuram, Viswanathan; Knepper, Mark A

2017-10-17

G protein stimulatory α-subunit (G αs )-coupled heptahelical receptors regulate cell processes largely through activation of protein kinase A (PKA). To identify signaling processes downstream of PKA, we deleted both PKA catalytic subunits using CRISPR-Cas9, followed by a "multiomic" analysis in mouse kidney epithelial cells expressing the G αs -coupled V2 vasopressin receptor. RNA-seq (sequencing)-based transcriptomics and SILAC (stable isotope labeling of amino acids in cell culture)-based quantitative proteomics revealed a complete loss of expression of the water-channel gene Aqp2 in PKA knockout cells. SILAC-based quantitative phosphoproteomics identified 229 PKA phosphorylation sites. Most of these PKA targets are thus far unannotated in public databases. Surprisingly, 1,915 phosphorylation sites with the motif x-(S/T)-P showed increased phosphooccupancy, pointing to increased activity of one or more MAP kinases in PKA knockout cells. Indeed, phosphorylation changes associated with activation of ERK2 were seen in PKA knockout cells. The ERK2 site is downstream of a direct PKA site in the Rap1GAP, Sipa1l1, that indirectly inhibits Raf1. In addition, a direct PKA site that inhibits the MAP kinase kinase kinase Map3k5 (ASK1) is upstream of JNK1 activation. The datasets were integrated to identify a causal network describing PKA signaling that explains vasopressin-mediated regulation of membrane trafficking and gene transcription. The model predicts that, through PKA activation, vasopressin stimulates AQP2 exocytosis by inhibiting MAP kinase signaling. The model also predicts that, through PKA activation, vasopressin stimulates Aqp2 transcription through induction of nuclear translocation of the acetyltransferase EP300, which increases histone H3K27 acetylation of vasopressin-responsive genes (confirmed by ChIP-seq).

Adenosine-Induced Atrial Fibrillation: Localized Reentrant Drivers in Lateral Right Atria due to Heterogeneous Expression of Adenosine A1 Receptors and GIRK4 Subunits in the Human Heart

PubMed Central

Li, Ning; Csepe, Thomas A.; Hansen, Brian J.; Sul, Lidiya V.; Kalyanasundaram, Anuradha; Zakharkin, Stanislav O.; Zhao, Jichao; Guha, Avirup; Van Wagoner, David R.; Kilic, Ahmet; Mohler, Peter J; Janssen, Paul ML; Biesiadecki, Brandon; Hummel, John D; Weiss, Raul; Fedorov, Vadim V.

2016-01-01

Background Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor (A1R) and its downstream GIRK channels (IK,Ado). Methods We applied bi-atrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and non-failing human hearts (n=37). Results Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10–100μM) produced more significant shortening of action potential durations (APD80) in RA (from 290±45ms to 239±41ms, 17.3±10.4%; p<0.01) than LA (from 307±24ms to 286±23ms, 6.7±6.6%; p<0.01). In ten hearts, adenosine induced AF (317±116 sec) that, when sustained (≥2 min), was primarily maintained by one/two localized reentrant drivers in lateral RA. Tertiapin (10–100nM), a selective GIRK channel blocker, counteracted adenosine-induced APD shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher A1R (2.7±1.7 fold; p<0.01) and GIRK4 (1.7±0.8 fold; p<0.05) protein expression than lateral/posterior LA. Conclusions This study revealed a three-fold RA-to-LA A1R protein expression gradient in the human heart, leading to significantly greater RA vs. LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest A1R/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine. PMID:27462069

The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions

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

Dougherty, Gerard W.; Section on Structural Cell Biology, National Institute on Deafness and Communication Disorders; Chopp, Treasa

2005-05-15

Rsu-1 is a highly conserved leucine rich repeat (LRR) protein that is expressed ubiquitously in mammalian cells. Rsu-1 was identified based on its ability to inhibit transformation by Ras, and previous studies demonstrated that ectopic expression of Rsu-1 inhibited anchorage-independent growth of Ras-transformed cells and human tumor cell lines. Using GAL4-based yeast two-hybrid screening, the LIM domain protein, PINCH1, was identified as the binding partner of Rsu-1. PINCH1 is an adaptor protein that localizes to focal adhesions and it has been implicated in the regulation of adhesion functions. Subdomain mapping in yeast revealed that Rsu-1 binds to the LIM 5more » domain of PINCH1, a region not previously identified as a specific binding domain for any other protein. Additional testing demonstrated that PINCH2, which is highly homologous to PINCH1, except in the LIM 5 domain, does not interact with Rsu-1. Glutathione transferase fusion protein binding studies determined that the LRR region of Rsu-1 interacts with PINCH1. Transient expression studies using epitope-tagged Rsu-1 and PINCH1 revealed that Rsu-1 co-immunoprecipitated with PINCH1 and colocalized with vinculin at sites of focal adhesions in mammalian cells. In addition, endogenous P33 Rsu-1 from 293T cells co-immunoprecipitated with transiently expressed myc-tagged PINCH1. Furthermore, RNAi-induced reduction in Rsu-1 RNA and protein inhibited cell attachment, and while previous studies demonstrated that ectopic expression of Rsu-1 inhibited Jun kinase activation, the depletion of Rsu-1 resulted in activation of Jun and p38 stress kinases. These studies demonstrate that Rsu-1 interacts with PINCH1 in mammalian cells and functions, in part, by altering cell adhesion.« less

Definition of IgG- and albumin-binding regions of streptococcal protein G.

PubMed

Akerström, B; Nielsen, E; Björck, L

1987-10-05

Protein G, the immunoglobin G-binding surface protein of group C and G streptococci, also binds serum albumin. The albumin-binding site on protein G is distinct from the immunoglobulin G-binding site. By mild acid hydrolysis of the papain-liberated protein G fragment (35 kDa), a 28-kDa fragment was produced which retained full immunoglobulin G-binding activity (determined by Scatchard plotting) but had lost all albumin-binding capacity. A protein G (65 kDa), isolated after cloning and expression of the protein G gene in Escherichia coli, had comparable affinity to immunoglobulin G (5-10 X 10(10)M-1), but much higher affinity to albumin than the 35- and 28-kDa protein G fragments (31, 2.6, and 0 X 10(9)M-1, respectively). The amino-terminal amino acid sequences of the 65-, 35-, and 28-kDa fragments allowed us to exactly locate the three fragments in an overall sequence map of protein G, based on the partial gene sequences published by Guss et al. (Guss, B., Eliasson, M., Olsson, A., Uhlen, M., Frej, A.-K., Jörnvall, H., Flock, J.-I., and Lindberg, M. (1986) EMBO J. 5, 1567-1575) and Fahnestock et al. (Fahnestock, S. R., Alexander, P., Nagle, J., and Filpula, D. (1986) J. Bacteriol. 167, 870-880). In this map could then be deduced the location of three homologous albumin-binding regions and three homologous immunoglobulin G-binding regions.

Cardiotrophin-1 Induces Matrix Metalloproteinase-1 in Human Aortic Endothelial Cells

PubMed Central

Tokito, Akinori; Jougasaki, Michihisa; Ichiki, Tomoko; Hamasaki, Shuichi

2013-01-01

Rupture of an atherosclerotic plaque is a key event in the development of cardiovascular disorders, in which matrix metalloproteinase-1 (MMP-1) plays a crucial role by degradation of extracellular matrix resulting in plaque instability. Cardiotrophin-1 (CT-1), a member of interleukin-6-type proinflammatory cytokines, has potent cardiovascular actions and is highly expressed in vascular endothelium, however its role in atherosclerosis has not been fully elucidated to date. The present study was designed to investigate whether CT-1 induces MMP-1 in human aortic endothelial cells (HAECs). Ribonuclease protection assay demonstrated that MMP-1 gene level in HAECs was enhanced by the treatment of CT-1 in a dose- and time-dependent manner. Immunocytochemical staining, Western immunoblot analysis and enzyme-linked immunosorbent assay revealed that CT-1 augmented MMP-1 protein synthesis and secretion. MMP-1 activity assay revealed that MMP-1 present in the supernatant of HAECs was exclusively precursor form. Casein zymography disclosed proteolytic activity in the supernatant of HAECs, which was enhanced by CT-1 treatment. Furthermore, pharmacological inhibitor study indicated the important roles of extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein (MAP) kinase, c-Jun N-terminal kinase (JNK) and Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways in mediating CT-1-induced MMP-1 gene and protein expression. These data reveal for the first time that CT-1 induces the proteolytic potential in HAECs by upregulating MMP-1 expression through ERK1/2, p38 MAP kinase, JNK and JAK/STAT pathways, and suggest that CT-1 may play an important role in the pathophysiology of atherosclerosis and plaque instability. PMID:23935888

Downregulation of toll-like receptor-mediated signalling pathways in oral lichen planus.

PubMed

Sinon, Suraya H; Rich, Alison M; Parachuru, Venkata P B; Firth, Fiona A; Milne, Trudy; Seymour, Gregory J

2016-01-01

The objective of this study was to investigate the expression of Toll-like receptors (TLR) and TLR-associated signalling pathway genes in oral lichen planus (OLP). Initially, immunohistochemistry was used to determine TLR expression in 12 formalin-fixed archival OLP tissues with 12 non-specifically inflamed oral tissues as controls. RNA was isolated from further fresh samples of OLP and non-specifically inflamed oral tissue controls (n = 6 for both groups) and used in qRT(2)-PCR focused arrays to determine the expression of TLRs and associated signalling pathway genes. Genes with a statistical significance of ±two-fold regulation (FR) and a P-value < 0.05 were considered as significantly regulated. Significantly more TLR4(+) cells were present in the inflammatory infiltrate in OLP compared with the control tissues (P < 0.05). There was no statistically significant difference in the numbers of TLR2(+) and TLR8(+) cells between the groups. TLR3 was significantly downregulated in OLP (P < 0.01). TLR8 was upregulated in OLP, but the difference between the groups was not statistically significant. The TLR-mediated signalling-associated protein genes MyD88 and TIRAP were significantly downregulated (P < 0.01 and P < 0.05), as were IRAK1 (P < 0.05), MAPK8 (P < 0.01), MAP3K1 (P < 0.05), MAP4K4 (P < 0.05), REL (P < 0.01) and RELA (P < 0.01). Stress proteins HMGB1 and the heat shock protein D1 were significantly downregulated in OLP (P < 0.01). These findings suggest a downregulation of TLR-mediated signalling pathways in OLP lesions. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Towards decrypting cryptobiosis--analyzing anhydrobiosis in the tardigrade Milnesium tardigradum using transcriptome sequencing.

PubMed

Wang, Chong; Grohme, Markus A; Mali, Brahim; Schill, Ralph O; Frohme, Marcus

2014-01-01

Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum. A prerequisite for differential gene expression analysis was the generation of a reference hybrid transcriptome atlas by assembly of Sanger, 454 and Illumina sequence data. The final assembly yielded 79,064 contigs (>100 bp) after removal of ribosomal RNAs. Around 50% of them could be annotated by SwissProt and NCBI non-redundant protein sequences. Analysis using CEGMA predicted 232 (93.5%) out of the 248 highly conserved eukaryotic genes in the assembly. We used this reference transcriptome for mapping and quantifying the expression of transcripts regulated under anhdydrobiosis in a time-series during dehydration and rehydration. 834 of the transcripts were found to be differentially expressed in a single stage (dehydration/inactive tun/rehydration) and 184 were overlapping in two stages while 74 were differentially expressed in all three stages. We have found interesting patterns of differentially expressed transcripts that are in concordance with a common hypothesis of metabolic shutdown during anhydrobiosis. This included down-regulation of several proteins of the DNA replication and translational machinery and protein degradation. Among others, heat shock proteins Hsp27 and Hsp30c were up-regulated in response to dehydration and rehydration. In addition, we observed up-regulation of ployubiquitin-B upon rehydration together with a higher expression level of several DNA repair proteins during rehydration than in the dehydration stage. Most of the transcripts identified to be differentially expressed had distinct cellular function. Our data suggest a concerted molecular adaptation in M. tardigradum that permits extreme forms of ametabolic states such as anhydrobiosis. It is temping to surmise that the desiccation tolerance of tradigrades can be achieved by a constitutive cellular protection system, probably in conjunction with other mechanisms such as rehydration-induced cellular repair.

Towards Decrypting Cryptobiosis—Analyzing Anhydrobiosis in the Tardigrade Milnesium tardigradum Using Transcriptome Sequencing

PubMed Central

Wang, Chong; Grohme, Markus A.; Mali, Brahim; Schill, Ralph O.; Frohme, Marcus

2014-01-01

Background Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum. Results A prerequisite for differential gene expression analysis was the generation of a reference hybrid transcriptome atlas by assembly of Sanger, 454 and Illumina sequence data. The final assembly yielded 79,064 contigs (>100 bp) after removal of ribosomal RNAs. Around 50% of them could be annotated by SwissProt and NCBI non-redundant protein sequences. Analysis using CEGMA predicted 232 (93.5%) out of the 248 highly conserved eukaryotic genes in the assembly. We used this reference transcriptome for mapping and quantifying the expression of transcripts regulated under anhdydrobiosis in a time-series during dehydration and rehydration. 834 of the transcripts were found to be differentially expressed in a single stage (dehydration/inactive tun/rehydration) and 184 were overlapping in two stages while 74 were differentially expressed in all three stages. We have found interesting patterns of differentially expressed transcripts that are in concordance with a common hypothesis of metabolic shutdown during anhydrobiosis. This included down-regulation of several proteins of the DNA replication and translational machinery and protein degradation. Among others, heat shock proteins Hsp27 and Hsp30c were up-regulated in response to dehydration and rehydration. In addition, we observed up-regulation of ployubiquitin-B upon rehydration together with a higher expression level of several DNA repair proteins during rehydration than in the dehydration stage. Conclusions Most of the transcripts identified to be differentially expressed had distinct cellular function. Our data suggest a concerted molecular adaptation in M. tardigradum that permits extreme forms of ametabolic states such as anhydrobiosis. It is temping to surmise that the desiccation tolerance of tradigrades can be achieved by a constitutive cellular protection system, probably in conjunction with other mechanisms such as rehydration-induced cellular repair. PMID:24651535

Mapping analysis of scaffold/matrix attachment regions (s/MARs) from two different mammalian cell lines

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

Pilus, Nur Shazwani Mohd; Ahmad, Azrin; Yusof, Nurul Yuziana Mohd

Scaffold/matrix attachment regions (S/MARs) are potential element that can be integrated into expression vector to increase expression of recombinant protein. Many studies on S/MAR have been done but none has revealed the distribution of S/MAR in a genome. In this study, we have isolated S/MAR sequences from HEK293 and Chinese hamster ovary cell lines (CHO DG44) using two different methods utilizing 2 M NaCl and lithium-3,5-diiodosalicylate (LIS). The isolated S/MARs were sequenced using Next Generation Sequencing (NGS) platform. Based on reference mapping analysis against human genome database, a total of 8,994,856 and 8,412,672 contigs of S/MAR sequences were retrieved frommore » 2M NaCl and LIS extraction of HEK293 respectively. On the other hand, reference mapping analysis of S/MAR derived from CHO DG44 against our own CHO DG44 database have generated a total of 7,204,348 and 4,672,913 contigs from 2 M NaCl and LIS extraction method respectively.« less

The product of the Saccharomyces cerevisiae cell cycle gene DBF2 has homology with protein kinases and is periodically expressed in the cell cycle.

PubMed Central

Johnston, L H; Eberly, S L; Chapman, J W; Araki, H; Sugino, A

1990-01-01

Several Saccharomyces cerevisiae dbf mutants defective in DNA synthesis have been described previously. In this paper, one of them, dbf2, is characterized in detail. The DBF2 gene has been cloned and mapped, and its nucleotide sequence has been determined. This process has identified an open reading frame capable of encoding a protein of molecular weight 64,883 (561 amino acids). The deduced amino acid sequence contains all 11 conserved domains found in various protein kinases. DBF2 was periodically expressed in the cell cycle at a time that clearly differed from the time of expression of either the histone H2A or DNA polymerase I gene. Its first function was completed very near to initiation of DNA synthesis. However, DNA synthesis in the mutant was only delayed at 37 degrees C, and the cells blocked in nuclear division. Consistent with this finding, the execution point occurred about 1 h after DNA synthesis, and the nuclear morphology of the mutant at the restrictive temperature was that of cells blocked in late nuclear division. DBF2 is therefore likely to encode a protein kinase that may function in initiation of DNA synthesis and also in late nuclear division. Images PMID:2181271

The small heat shock protein alphaA-crystallin is expressed in pancreas and acts as a negative regulator of carcinogenesis.

PubMed

Deng, Mi; Chen, Pei-Chao; Xie, Sisi; Zhao, Junqiong; Gong, Lili; Liu, Jinping; Zhang, Lan; Sun, Shuming; Liu, Jiao; Ma, Haili; Batra, Surinder K; Li, David Wan-Cheng

2010-01-01

The small heat shock protein alphaA-crystallin is a structural protein in the ocular lens. In addition, recent studies have also revealed that it is a molecular chaperone, an autokinase and a strong anti-apoptotic regulator. Besides its lenticular distribution, a previous study demonstrates that a detectable level of alphaA-crystallin is found in other tissues including thymus and spleen. In the present study, we have re-examined the distribution of alphaA-crystallin in various normal human and mouse tissues and found that the normal pancreas expresses a moderate level of alphaA-crystallin. Moreover, alphaA-crystallin is found significantly downregulated in 60 cases of pancreatic carcinoma of different types than it is in 11 normal human pancreas samples. In addition, we demonstrate that alphaA-crystallin can enhance the activity of the activating protein-1 (AP-1) through modulating the function of the MAP kinase, and also upregulates components of TGFbeta pathway. Finally, expression of alphaA-crystallin in a pancreatic cancer cell line, MiaPaCa, results in retarded cell migration. Together, these results suggest that alphaA-crystallin seems to negatively regulate pancreatic carcinogenesis. Copyright 2010 Elsevier B.V. All rights reserved.

High resolution fate map of the zebrafish diencephalon.

PubMed

Russek-Blum, Niva; Nabel-Rosen, Helit; Levkowitz, Gil

2009-07-01

The diencephalon acts as an interactive site between the sensory, central, and endocrine systems and is one of the most elaborate structures in the vertebrate brain. To better understand the embryonic development and morphogenesis of the diencephalon, we developed an improved photoactivation (uncaging)-based lineage tracing strategy. To determine the exact position of a given diencephalic progenitor domain, we used a transgenic line driving green fluorescent protein (GFP) in cells expressing the proneural protein, Neurogenin1 (Neurog1), which was used as a visible neural plate landmark. This approach facilitated precise labeling of defined groups of cells in the prospective diencephalon of the zebrafish neural plate. In this manner, we labeled multiple overlapping areas of the diencephalon, thereby ensuring both accuracy and reproducibility of our lineage tracing regardless of the dynamic changes of the developing neural plate. We present a fate map of the zebrafish diencephalon at a higher spatial resolution than previously described. (c) 2009 Wiley-Liss, Inc.

Five Xanthomonas type III effectors suppress cell death induced by components of immunity-associated MAP kinase cascades

PubMed Central

Teper, Doron; Sunitha, Sukumaran; Martin, Gregory B; Sessa, Guido

2015-01-01

Mitogen-activated protein kinase (MAPK) cascades play a fundamental role in signaling of plant immunity and mediate elicitation of cell death. Xanthomonas spp. manipulate plant signaling by using a type III secretion system to deliver effector proteins into host cells. We examined the ability of 33 Xanthomonas effectors to inhibit cell death induced by overexpression of components of MAPK cascades in Nicotiana benthamiana plants. Five effectors inhibited cell death induced by overexpression of MAPKKKα and MEK2, but not of MAP3Kϵ. In addition, expression of AvrBs1 in yeast suppressed activation of the high osmolarity glycerol MAPK pathway, suggesting that the target of this effector is conserved in eukaryotic organisms. These results indicate that Xanthomonas employs several type III effectors to suppress immunity-associated cell death mediated by MAPK cascades. PMID:26237448

MAP3K19 Is a Novel Regulator of TGF-β Signaling That Impacts Bleomycin-Induced Lung Injury and Pulmonary Fibrosis

PubMed Central

Franz-Bacon, Karin; DiTirro, Danielle N.; Ly, Tai Wei; Bacon, Kevin B.

2016-01-01

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating disease for which two medications, pirfenidone and nintedanib, have only recently been approved for treatment. The cytokine TGF-β has been shown to be a central mediator in the disease process. We investigated the role of a novel kinase, MAP3K19, upregulated in IPF tissue, in TGF-β-induced signal transduction and in bleomycin-induced pulmonary fibrosis. MAP3K19 has a very limited tissue expression, restricted primarily to the lungs and trachea. In pulmonary tissue, expression was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. MAP3K19 was also found to be overexpressed in bronchoalveolar lavage macrophages from IPF patients compared to normal patients. Treatment of A549 or THP-1 cells with either MAP3K19 siRNA or a highly potent and specific inhibitor reduced phospho-Smad2 & 3 nuclear translocation following TGF-β stimulation. TGF-β-induced gene transcription was also strongly inhibited by both the MAP3K19 inhibitor and nintedanib, whereas pirfenidone had a much less pronounced effect. In combination, the MAP3K19 inhibitor appeared to act synergistically with either pirfenidone or nintedanib, at the level of target gene transcription or protein production. Finally, in an animal model of IPF, inhibition of MAP3K19 strongly attenuated bleomycin-induced pulmonary fibrosis when administered either prophylactically ortherapeutically. In summary, these results strongly suggest that inhibition of MAP3K19 may have a beneficial therapeutic effect in the treatment of IPF and represents a novel strategy to target this disease. PMID:27144281

MAP3K19 Is a Novel Regulator of TGF-β Signaling That Impacts Bleomycin-Induced Lung Injury and Pulmonary Fibrosis.

PubMed

Boehme, Stefen A; Franz-Bacon, Karin; DiTirro, Danielle N; Ly, Tai Wei; Bacon, Kevin B

2016-01-01

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating disease for which two medications, pirfenidone and nintedanib, have only recently been approved for treatment. The cytokine TGF-β has been shown to be a central mediator in the disease process. We investigated the role of a novel kinase, MAP3K19, upregulated in IPF tissue, in TGF-β-induced signal transduction and in bleomycin-induced pulmonary fibrosis. MAP3K19 has a very limited tissue expression, restricted primarily to the lungs and trachea. In pulmonary tissue, expression was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. MAP3K19 was also found to be overexpressed in bronchoalveolar lavage macrophages from IPF patients compared to normal patients. Treatment of A549 or THP-1 cells with either MAP3K19 siRNA or a highly potent and specific inhibitor reduced phospho-Smad2 & 3 nuclear translocation following TGF-β stimulation. TGF-β-induced gene transcription was also strongly inhibited by both the MAP3K19 inhibitor and nintedanib, whereas pirfenidone had a much less pronounced effect. In combination, the MAP3K19 inhibitor appeared to act synergistically with either pirfenidone or nintedanib, at the level of target gene transcription or protein production. Finally, in an animal model of IPF, inhibition of MAP3K19 strongly attenuated bleomycin-induced pulmonary fibrosis when administered either prophylactically ortherapeutically. In summary, these results strongly suggest that inhibition of MAP3K19 may have a beneficial therapeutic effect in the treatment of IPF and represents a novel strategy to target this disease.

Genome-wide proteomics analysis on longissimus muscles in Qinchuan beef cattle.

PubMed

He, Hua; Chen, Si; Liang, Wei; Liu, Xiaolin

2017-04-01

To gain further insight into the molecular mechanism of bovine muscle development, we combined mass spectrometry characterization of proteins with Illumina deep sequencing of RNAs obtained from bovine longissimus muscle (LD) at prenatal and postnatal stages. For the proteomic study, each group of LD proteins was extracted and labeled using isobaric tags for relative and absolute quantitation (iTRAQ) method. Among the 1321 proteins identified from six samples, 390 proteins were differentially expressed in embryos at day 135 post-fertilization (Emb135d) vs. 30-month-old adult cattle (Emb135d vs. 30M) samples. Gene Ontology, Cluster of Orthologous Groups and Kyoto Encyclopedia of Genes and Genomes analyses were further conducted to better understand the different functions. Furthermore, we analyzed the relationship between transcript and protein regulation between samples by direct comparison of expression levels from transcriptomic and iTRAQ-based proteomics. Association results indicated that 1295 of 1321 proteins could be mapped to transcriptome sequencing data. This study provides the most comprehensive, targeted survey of bovine LD proteins to date and has shown the power of combining transcriptomic and proteomic approaches to provide molecular insights for understanding the developmental characteristics in bovine muscle, and even in other mammals. © 2016 Stichting International Foundation for Animal Genetics.

Bcl2-low-expressing MCF7 cells undergo necrosis rather than apoptosis upon staurosporine treatment.

PubMed Central

Poliseno, Laura; Bianchi, Laura; Citti, Lorenzo; Liberatori, Sabrina; Mariani, Laura; Salvetti, Alessandra; Evangelista, Monica; Bini, Luca; Pallini, Vitaliano; Rainaldi, Giuseppe

2004-01-01

We present a ribozyme-based strategy for studying the effects of Bcl2 down-regulation. The anti-bcl2 hammerhead ribozyme Rz-bcl2 was stably transfected into MCF7 cancer cells and the cleavage of Bcl2 mRNA was demonstrated using a new assay for cleavage product detection, while Western blot analysis showed a concomitant depletion of Bcl2 protein. Rz-bcl2-expressing cells were more sensitive to staurosporine than control cells. Moreover, both molecular and cellular read-outs indicated that staurosporine-induced cell death was necrosis rather than apoptosis in these cells. The study of the effects of Bcl2 down-regulation was extended to the global MCF7 protein expression profile, exploiting a proteomic approach. Two reference electro-pherograms of Rz-bcl2-transfected cells, one with the ribozyme in a catalytically active form and the other with the ribozyme in a catalytically inactive form, were obtained. When comparing the two-dimensional maps, 53 differentially expressed spots were found, four of which were identified by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS as calreticulin, nucleophosmin, phosphoglycerate kinase and pyruvate kinase. How the up-regulation of these proteins might help to explain the modification of Bcl2 activity is discussed. PMID:14748742

Genome-Wide Discovery of Long Non-Coding RNAs in Rainbow Trout.

PubMed

Al-Tobasei, Rafet; Paneru, Bam; Salem, Mohamed

2016-01-01

The ENCODE project revealed that ~70% of the human genome is transcribed. While only 1-2% of the RNAs encode for proteins, the rest are non-coding RNAs. Long non-coding RNAs (lncRNAs) form a diverse class of non-coding RNAs that are longer than 200 nt. Emerging evidence indicates that lncRNAs play critical roles in various cellular processes including regulation of gene expression. LncRNAs show low levels of gene expression and sequence conservation, which make their computational identification in genomes difficult. In this study, more than two billion Illumina sequence reads were mapped to the genome reference using the TopHat and Cufflinks software. Transcripts shorter than 200 nt, with more than 83-100 amino acids ORF, or with significant homologies to the NCBI nr-protein database were removed. In addition, a computational pipeline was used to filter the remaining transcripts based on a protein-coding-score test. Depending on the filtering stringency conditions, between 31,195 and 54,503 lncRNAs were identified, with only 421 matching known lncRNAs in other species. A digital gene expression atlas revealed 2,935 tissue-specific and 3,269 ubiquitously-expressed lncRNAs. This study annotates the lncRNA rainbow trout genome and provides a valuable resource for functional genomics research in salmonids.

Bioinformatics approaches for cross-species liver cancer analysis based on microarray gene expression profiling

PubMed Central

Fang, H; Tong, W; Perkins, R; Shi, L; Hong, H; Cao, X; Xie, Q; Yim, SH; Ward, JM; Pitot, HC; Dragan, YP

2005-01-01

Background The completion of the sequencing of human, mouse and rat genomes and knowledge of cross-species gene homologies enables studies of differential gene expression in animal models. These types of studies have the potential to greatly enhance our understanding of diseases such as liver cancer in humans. Genes co-expressed across multiple species are most likely to have conserved functions. We have used various bioinformatics approaches to examine microarray expression profiles from liver neoplasms that arise in albumin-SV40 transgenic rats to elucidate genes, chromosome aberrations and pathways that might be associated with human liver cancer. Results In this study, we first identified 2223 differentially expressed genes by comparing gene expression profiles for two control, two adenoma and two carcinoma samples using an F-test. These genes were subsequently mapped to the rat chromosomes using a novel visualization tool, the Chromosome Plot. Using the same plot, we further mapped the significant genes to orthologous chromosomal locations in human and mouse. Many genes expressed in rat 1q that are amplified in rat liver cancer map to the human chromosomes 10, 11 and 19 and to the mouse chromosomes 7, 17 and 19, which have been implicated in studies of human and mouse liver cancer. Using Comparative Genomics Microarray Analysis (CGMA), we identified regions of potential aberrations in human. Lastly, a pathway analysis was conducted to predict altered human pathways based on statistical analysis and extrapolation from the rat data. All of the identified pathways have been known to be important in the etiology of human liver cancer, including cell cycle control, cell growth and differentiation, apoptosis, transcriptional regulation, and protein metabolism. Conclusion The study demonstrates that the hepatic gene expression profiles from the albumin-SV40 transgenic rat model revealed genes, pathways and chromosome alterations consistent with experimental and clinical research in human liver cancer. The bioinformatics tools presented in this paper are essential for cross species extrapolation and mapping of microarray data, its analysis and interpretation. PMID:16026603

A novel isoform of MAP4 organises the paraxial microtubule array required for muscle cell differentiation

PubMed Central

Mogessie, Binyam; Roth, Daniel; Rahil, Zainab; Straube, Anne

2015-01-01

The microtubule cytoskeleton is critical for muscle cell differentiation and undergoes reorganisation into an array of paraxial microtubules, which serves as template for contractile sarcomere formation. In this study, we identify a previously uncharacterised isoform of microtubule-associated protein MAP4, oMAP4, as a microtubule organising factor that is crucial for myogenesis. We show that oMAP4 is expressed upon muscle cell differentiation and is the only MAP4 isoform essential for normal progression of the myogenic differentiation programme. Depletion of oMAP4 impairs cell elongation and cell–cell fusion. Most notably, oMAP4 is required for paraxial microtubule organisation in muscle cells and prevents dynein- and kinesin-driven microtubule–microtubule sliding. Purified oMAP4 aligns dynamic microtubules into antiparallel bundles that withstand motor forces in vitro. We propose a model in which the cooperation of dynein-mediated microtubule transport and oMAP4-mediated zippering of microtubules drives formation of a paraxial microtubule array that provides critical support for the polarisation and elongation of myotubes. DOI: http://dx.doi.org/10.7554/eLife.05697.001 PMID:25898002

Mapping of epitopes and structural analysis of antigenic sites in the nucleoprotein of rabies virus.

PubMed

Goto, H; Minamoto, N; Ito, H; Ito, N; Sugiyama, M; Kinjo, T; Kawai, A

2000-01-01

Linear epitopes on the rabies virus nucleoprotein (N) recognized by six MAbs raised against antigenic sites I (MAbs 6-4, 12-2 and 13-27) and IV (MAbs 6-9, 7-12 and 8-1) were investigated. Based on our previous studies on sites I and IV, 24 consecutively overlapping octapeptides and N- and C-terminal-deleted mutant N proteins were prepared. Results showed that all three site I epitopes studied and two site IV epitopes (for MAbs 8-1 and 6-9) mapped to aa 358-367, and that the other site IV epitope of MAb 7-12 mapped to aa 375-383. Tests using chimeric and truncated proteins showed that MAb 8-1 also requires the N-terminal sequence of the N protein to recognize its binding region more efficiently. Immunofluorescence studies demonstrated that all three site I-specific MAbs and one site IV-specific MAb (7-12) stained the N antigen that was diffusely distributed in the whole cytoplasm; the other two site IV-specific MAbs (6-9 and 8-1) detected only the N antigen in the cytoplasmic inclusion bodies (CIB). An antigenic site II-specific MAb (6-17) also detected CIB-associated N antigen alone. Furthermore, the level of diffuse N antigens decreased after treatment of infected cells with cycloheximide. These results suggest that epitopes at site I are expressed on the immature form of the N protein, but epitope structures of site IV MAbs 6-9 and 8-1 are created and/or exposed only after maturation of the N protein.

Functional and structural analysis of the sialic acid-binding domain of rotaviruses.

PubMed Central

Isa, P; López, S; Segovia, L; Arias, C F

1997-01-01

The infectivity of most animal rotaviruses is dependent on the interaction of the virus spike protein VP4 with a sialic acid (SA)-containing cell receptor, and the SA-binding domain of this protein has been mapped between amino acids 93 and 208 of its trypsin cleavage fragment VP8. To identify which residues in this region are essential for the SA-binding activity, we performed alanine mutagenesis of the rotavirus RRV VP8 expressed in bacteria as a fusion polypeptide with glutathione S-transferase. Tyrosines were primarily targeted since tyrosine has been involved in the interaction of other viral hemagglutinins with SA. Of the 15 substitutions carried out, 10 abolished the SA-dependent hemagglutination activity of the protein, as well as its ability to bind to glycophorin A in a solid-phase assay. However, only alanine substitutions for tyrosines 155 and 188 and for serine 190 did not affect the overall conformation of the protein, as judged by their interaction with a panel of conformationally sensitive neutralizing VP8 monoclonal antibodies (MAbs). These findings suggest that these three amino acids play an essential role in the SA-binding activity of the protein, presumably by interacting directly with the SA molecule. The predicted secondary structure of VP8 suggests that it is organized as 11 beta-strands separated by loops; in this model, Tyr-155 maps to loop 7 while Tyr-188 and Ser-190 map to loop 9. The close proximity of these two loops is also supported by previous results from competition experiments with neutralizing MAbs directed at RRV VP8. PMID:9261399

CR108, a novel vitamin K3 derivative induces apoptosis and breast tumor inhibition by reactive oxygen species and mitochondrial dysfunction.

PubMed

Yang, Chun-Ru; Liao, Wei-Siang; Wu, Ya-Hui; Murugan, Kaliyappan; Chen, Chinpiao; Chao, Jui-I

2013-12-15

Vitamin K3 derivatives have been shown to exert anticancer activities. Here we show a novel vitamin K3 derivative (S)-2-(2-hydroxy-3-methylbutylthio)naphthalene-1,4-dione, which is named as CR108 that induces apoptosis and tumor inhibition through reactive oxygen species (ROS) and mitochondrial dysfunction in human breast cancer. CR108 is more effective on the breast cancer cell death than other vitamin K3 derivatives. Moreover, CR108 induced apoptosis in both the non-HER-2-overexpressed MCF-7 and HER-2-overexpressed BT-474 breast cancer cells. CR108 caused the loss of mitochondrial membrane potential, cytochrome c released from mitochondria to cytosol, and cleaved PARP proteins for apoptosis induction. CR108 markedly increased ROS levels in breast cancer cells. N-acetylcysteine (NAC), a general ROS scavenger, completely blocked the CR108-induced ROS levels, mitochondrial dysfunction and apoptosis. Interestingly, CR108 increased the phosphorylation of p38 MAP kinase but conversely inhibited the survivin protein expression. NAC treatment prevented the activation of p38 MAP kinase and rescued the survivin protein levels. SB202190, a specific p38 MAP kinase inhibitor, recovered the survivin protein levels and attenuated the cytotoxicity of CR108-treated cells. Furthermore, CR108 inhibited the xenografted human breast tumor growth in nude mice. Together, we demonstrate that CR108 is a novel vitamin K3 derivative that induces apoptosis and tumor inhibition by ROS production and mitochondrial dysfunction and associates with the phosphorylation of p38 MAP kinase and the inhibition of survivin in the human breast cancer. © 2013.

Restraint of presynaptic protein levels by Wnd/DLK signaling mediates synaptic defects associated with the kinesin-3 motor Unc-104

PubMed Central

Asghari Adib, Elham; Stanchev, Doychin T; Xiong, Xin; Klinedinst, Susan; Soppina, Pushpanjali; Jahn, Thomas Robert; Hume, Richard I

2017-01-01

The kinesin-3 family member Unc-104/KIF1A is required for axonal transport of many presynaptic components to synapses, and mutation of this gene results in synaptic dysfunction in mice, flies and worms. Our studies at the Drosophila neuromuscular junction indicate that many synaptic defects in unc-104-null mutants are mediated independently of Unc-104’s transport function, via the Wallenda (Wnd)/DLK MAP kinase axonal damage signaling pathway. Wnd signaling becomes activated when Unc-104’s function is disrupted, and leads to impairment of synaptic structure and function by restraining the expression level of active zone (AZ) and synaptic vesicle (SV) components. This action concomitantly suppresses the buildup of synaptic proteins in neuronal cell bodies, hence may play an adaptive role to stresses that impair axonal transport. Wnd signaling also becomes activated when pre-synaptic proteins are over-expressed, suggesting the existence of a feedback circuit to match synaptic protein levels to the transport capacity of the axon. PMID:28925357

Comparative Transcriptome Analysis of Resistant and Susceptible Common Bean Genotypes in Response to Soybean Cyst Nematode Infection.

PubMed

Jain, Shalu; Chittem, Kishore; Brueggeman, Robert; Osorno, Juan M; Richards, Jonathan; Nelson, Berlin D

2016-01-01

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) reproduces on the roots of common bean (Phaseolus vulgaris L.) and can cause reductions in plant growth and seed yield. The molecular changes in common bean roots caused by SCN infection are unknown. Identification of genetic factors associated with SCN resistance could help in development of improved bean varieties with high SCN resistance. Gene expression profiling was conducted on common bean roots infected by SCN HG type 0 using next generation RNA sequencing technology. Two pinto bean genotypes, PI533561 and GTS-900, resistant and susceptible to SCN infection, respectively, were used as RNA sources eight days post inoculation. Total reads generated ranged between ~ 3.2 and 5.7 million per library and were mapped to the common bean reference genome. Approximately 70-90% of filtered RNA-seq reads uniquely mapped to the reference genome. In the inoculated roots of resistant genotype PI533561, a total of 353 genes were differentially expressed with 154 up-regulated genes and 199 down-regulated genes when compared to the transcriptome of non- inoculated roots. On the other hand, 990 genes were differentially expressed in SCN-inoculated roots of susceptible genotype GTS-900 with 406 up-regulated and 584 down-regulated genes when compared to non-inoculated roots. Genes encoding nucleotide-binding site leucine-rich repeat resistance (NLR) proteins, WRKY transcription factors, pathogenesis-related (PR) proteins and heat shock proteins involved in diverse biological processes were differentially expressed in both resistant and susceptible genotypes. Overall, suppression of the photosystem was observed in both the responses. Furthermore, RNA-seq results were validated through quantitative real time PCR. This is the first report describing genes/transcripts involved in SCN-common bean interaction and the results will have important implications for further characterization of SCN resistance genes in common bean.

Discovery of Possible Gene Relationships through the Application of Self-Organizing Maps to DNA Microarray Databases

PubMed Central

Chavez-Alvarez, Rocio; Chavoya, Arturo; Mendez-Vazquez, Andres

2014-01-01

DNA microarrays and cell cycle synchronization experiments have made possible the study of the mechanisms of cell cycle regulation of Saccharomyces cerevisiae by simultaneously monitoring the expression levels of thousands of genes at specific time points. On the other hand, pattern recognition techniques can contribute to the analysis of such massive measurements, providing a model of gene expression level evolution through the cell cycle process. In this paper, we propose the use of one of such techniques –an unsupervised artificial neural network called a Self-Organizing Map (SOM)–which has been successfully applied to processes involving very noisy signals, classifying and organizing them, and assisting in the discovery of behavior patterns without requiring prior knowledge about the process under analysis. As a test bed for the use of SOMs in finding possible relationships among genes and their possible contribution in some biological processes, we selected 282 S. cerevisiae genes that have been shown through biological experiments to have an activity during the cell cycle. The expression level of these genes was analyzed in five of the most cited time series DNA microarray databases used in the study of the cell cycle of this organism. With the use of SOM, it was possible to find clusters of genes with similar behavior in the five databases along two cell cycles. This result suggested that some of these genes might be biologically related or might have a regulatory relationship, as was corroborated by comparing some of the clusters obtained with SOMs against a previously reported regulatory network that was generated using biological knowledge, such as protein-protein interactions, gene expression levels, metabolism dynamics, promoter binding, and modification, regulation and transport of proteins. The methodology described in this paper could be applied to the study of gene relationships of other biological processes in different organisms. PMID:24699245

Comparative Transcriptome Analysis of Resistant and Susceptible Common Bean Genotypes in Response to Soybean Cyst Nematode Infection

PubMed Central

Jain, Shalu; Chittem, Kishore; Brueggeman, Robert; Osorno, Juan M.; Richards, Jonathan; Nelson, Berlin D.

2016-01-01

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) reproduces on the roots of common bean (Phaseolus vulgaris L.) and can cause reductions in plant growth and seed yield. The molecular changes in common bean roots caused by SCN infection are unknown. Identification of genetic factors associated with SCN resistance could help in development of improved bean varieties with high SCN resistance. Gene expression profiling was conducted on common bean roots infected by SCN HG type 0 using next generation RNA sequencing technology. Two pinto bean genotypes, PI533561 and GTS-900, resistant and susceptible to SCN infection, respectively, were used as RNA sources eight days post inoculation. Total reads generated ranged between ~ 3.2 and 5.7 million per library and were mapped to the common bean reference genome. Approximately 70–90% of filtered RNA-seq reads uniquely mapped to the reference genome. In the inoculated roots of resistant genotype PI533561, a total of 353 genes were differentially expressed with 154 up-regulated genes and 199 down-regulated genes when compared to the transcriptome of non- inoculated roots. On the other hand, 990 genes were differentially expressed in SCN-inoculated roots of susceptible genotype GTS-900 with 406 up-regulated and 584 down-regulated genes when compared to non-inoculated roots. Genes encoding nucleotide-binding site leucine-rich repeat resistance (NLR) proteins, WRKY transcription factors, pathogenesis-related (PR) proteins and heat shock proteins involved in diverse biological processes were differentially expressed in both resistant and susceptible genotypes. Overall, suppression of the photosystem was observed in both the responses. Furthermore, RNA-seq results were validated through quantitative real time PCR. This is the first report describing genes/transcripts involved in SCN-common bean interaction and the results will have important implications for further characterization of SCN resistance genes in common bean. PMID:27441552

Aldosterone Contributes to Sympathoexcitation in Renovascular Hypertension.

PubMed

Lincevicius, Gisele S; Shimoura, Caroline G; Nishi, Erika E; Perry, Juliana C; Casarini, Dulce E; Gomes, Guiomar N; Bergamaschi, Cássia T; Campos, Ruy R

2015-09-01

Although angiotensin II (Ang II) is essential to the development of renovascular hypertension, aldosterone plays a role as well. Recent studies have demonstrated a cross-talk between Ang II type 1 and mineralocorticoid receptors in the brain and kidneys. However, the role of aldosterone in the autonomic and renal dysfunction of renovascular hypertension is not well understood. The current study evaluated whether aldosterone contributes to cardiovascular and renal dysfunction in the 2 kidney-1 clip (2K1C) model. Mean arterial pressure (MAP) and baroreceptor reflex for control of the heart rate were evaluated in 2K1C treated or not treated with spironolactone (200mg/kg/day, 7 days). Tonic and reflex control of renal sympathetic nerve activity (rSNA) were assessed in urethane-anaesthetized rats. Plasma renin activity (PRA), kidney renin protein expression, renal injury, and central AT1 receptor protein expression were assessed. Spiro reduced MAP (198±4 vs. 170±9mm Hg; P < 0.05), normalized rSNA (147±9 vs. 96±10 pps; P < 0.05), and increased renal baroreceptor reflex sensitivity in the 2K1C rats. Spiro reduced α-smooth muscle actin expression in the nonclipped kidney in the 2K1C group (5±0.6 vs. 1.1±0.2%; P < 0.05). There was no change in PRA; however, a decrease in renin protein expression in the nonclipped kidney was found in the 2K1C treated group (217±30 vs. 160±19%; P < 0.05). Spiro treatment decreased AT1 receptor in the central nervous system (CNS) only in 2K1C rats (138±10 vs. 84±12%; P < 0.05). Aldosterone contributes to autonomic dysfunction and intrarenal injury in 2K1C, these effects are mediated by the CNS. © American Journal of Hypertension, Ltd 2015. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Characterization of PsMPK2, the first C1 subgroup MAP kinase from pea (Pisum sativum L.).

PubMed

Ortiz-Masia, Dolores; Perez-Amador, Miguel A; Carbonell, Pablo; Aniento, Fernando; Carbonell, Juan; Marcote, Maria J

2008-05-01

Mitogen-activated protein kinase (MAPK) cascades play a key role in plant growth and development as well as in biotic and abiotic stress responses. They are classified according to their sequence homology into four major groups (A-D). A large amount of information about MAPKs in groups A and B is available but few data of the C group have been reported. In this study, a C1 subgroup MAP kinase cDNA, PsMPK2, was isolated from Pisum sativum. PsMPK2 is expressed in vegetative (root and leaf) and reproductive (stamen, pistil and fruit) organs. Expression of PsMPK2 in Arabidopsis thaliana shows that mechanical injury and other stress signals as abscisic acid, jasmonic acid and hydrogen peroxide increase its kinase activity, extending previous results indicating that C1 subgroup MAPKs may be involved in the response to stress.

Olfactory neuroblastoma in dogs and cats--a histological and immunohistochemical analysis.

PubMed

Brosinski, K; Janik, D; Polkinghorne, A; Von Bomhard, W; Schmahl, W

2012-01-01

Olfactory neuroblastoma (ONB) was identified in 13 dogs and nine cats. The tumours were subjected to microscopical examination and were graded using a human pathological grading system. In the canine and feline tumours there was more necrosis and higher mitotic activity (mitotic index and Ki67 labelling index) than reported in human ONB. Rosettes were a common feature of feline ONBs. A significant correlation was observed between the histological grade and the Ki67 labelling index. The histopathological diagnosis of ONB was confirmed immunohistochemically by demonstration of the neuronal marker neuron-specific enolase (NSE). Two other neuron-specific antibodies specific for microtubule-associated protein-2 (MAP-2) and neuronal nuclei antigen (NeuN) were evaluated. MAP-2 expression proved to have higher specificity than labelling for NSE. NeuN expression was less sensitive and of limited practical value. Copyright © 2011 Elsevier Ltd. All rights reserved.

MALDI imaging mass spectrometry analysis-A new approach for protein mapping in multiple sclerosis brain lesions.

PubMed

Maccarrone, Giuseppina; Nischwitz, Sandra; Deininger, Sören-Oliver; Hornung, Joachim; König, Fatima Barbara; Stadelmann, Christine; Turck, Christoph W; Weber, Frank

2017-03-15

Multiple sclerosis is a disease of the central nervous system characterized by recurrent inflammatory demyelinating lesions in the early disease stage. Lesion formation and mechanisms leading to lesion remyelination are not fully understood. Matrix Assisted Laser Desorption Ionisation Mass Spectrometry imaging (MALDI-IMS) is a technology which analyses proteins and peptides in tissue, preserves their spatial localization, and generates molecular maps within the tissue section. In a pilot study we employed MALDI imaging mass spectrometry to profile and identify peptides and proteins expressed in normal-appearing white matter, grey matter and multiple sclerosis brain lesions with different extents of remyelination. The unsupervised clustering analysis of the mass spectra generated images which reflected the tissue section morphology in luxol fast blue stain and in myelin basic protein immunohistochemistry. Lesions with low remyelination extent were defined by compounds with molecular weight smaller than 5300Da, while more completely remyelinated lesions showed compounds with molecular weights greater than 15,200Da. An in-depth analysis of the mass spectra enabled the detection of cortical lesions which were not seen by routine luxol fast blue histology. An ion mass, mainly distributed at the rim of multiple sclerosis lesions, was identified by liquid chromatography and tandem mass spectrometry as thymosin beta-4, a protein known to be involved in cell migration and in restorative processes. The ion mass of thymosin beta-4 was profiled by MALDI imaging mass spectrometry in brain slides of 12 multiple sclerosis patients and validated by immunohistochemical analysis. In summary, our results demonstrate the ability of the MALDI-IMS technology to map proteins within the brain parenchyma and multiple sclerosis lesions and to identify potential markers involved in multiple sclerosis pathogenesis and/or remyelination. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteome analysis of Lactobacillus helveticus H9 during growth in skim milk.

PubMed

Chen, Y F; Zhao, W J; Wu, R N; Sun, Z H; Zhang, W Y; Wang, J C; Bilige, M; Zhang, H P

2014-12-01

Lactobacillus helveticus H9 was isolated from traditionally fermented yak milk in Tibet (China) with the ability to produce the antihypertensive peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) during milk fermentation. To understand the changes in the protein expression of L. helveticus H9, proteome analysis was performed at 3 different growth stages, lag phase (pH 6.1), log phase (pH 5.1), and stationary phase (pH 4.5) using 2-dimensional electrophoresis (2-DE). Further analysis showed that 257 differential protein spots were found and 214 protein spots were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The cellular functions of the differentially expressed proteins were complex. Interestingly, the proteolytic system-related proteins aminopeptidase N (PepN), aminopeptidase E (PepE), endopeptidase O2 (PepO2), and oligopeptide transport system permease protein (OppC) were observed only on the maps of pH 5.1 and pH 4.5, which was consistent with the presence of angiotensin I-converting enzyme (ACE)-inhibitory peptides VPP and IPP during these 2 growth stages (log phase and stationary phase). These results, combined with a previous study of gene expression of the proteolytic system, led us to conclude that the Opp transport system, pepE, and pepO2 are likely related to the production of ACE-inhibitory peptides. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

pseudoMap: an innovative and comprehensive resource for identification of siRNA-mediated mechanisms in human transcribed pseudogenes.

PubMed

Chan, Wen-Ling; Yang, Wen-Kuang; Huang, Hsien-Da; Chang, Jan-Gowth

2013-01-01

RNA interference (RNAi) is a gene silencing process within living cells, which is controlled by the RNA-induced silencing complex with a sequence-specific manner. In flies and mice, the pseudogene transcripts can be processed into short interfering RNAs (siRNAs) that regulate protein-coding genes through the RNAi pathway. Following these findings, we construct an innovative and comprehensive database to elucidate siRNA-mediated mechanism in human transcribed pseudogenes (TPGs). To investigate TPG producing siRNAs that regulate protein-coding genes, we mapped the TPGs to small RNAs (sRNAs) that were supported by publicly deep sequencing data from various sRNA libraries and constructed the TPG-derived siRNA-target interactions. In addition, we also presented that TPGs can act as a target for miRNAs that actually regulate the parental gene. To enable the systematic compilation and updating of these results and additional information, we have developed a database, pseudoMap, capturing various types of information, including sequence data, TPG and cognate annotation, deep sequencing data, RNA-folding structure, gene expression profiles, miRNA annotation and target prediction. As our knowledge, pseudoMap is the first database to demonstrate two mechanisms of human TPGs: encoding siRNAs and decoying miRNAs that target the parental gene. pseudoMap is freely accessible at http://pseudomap.mbc.nctu.edu.tw/. Database URL: http://pseudomap.mbc.nctu.edu.tw/

Genome-wide investigation and expression analyses of WD40 protein family in the model plant foxtail millet (Setaria italica L.).

PubMed

Mishra, Awdhesh Kumar; Muthamilarasan, Mehanathan; Khan, Yusuf; Parida, Swarup Kumar; Prasad, Manoj

2014-01-01

WD40 proteins play a crucial role in diverse protein-protein interactions by acting as scaffolding molecules and thus assisting in the proper activity of proteins. Hence, systematic characterization and expression profiling of these WD40 genes in foxtail millet would enable us to understand the networks of WD40 proteins and their biological processes and gene functions. In the present study, a genome-wide survey was conducted and 225 potential WD40 genes were identified. Phylogenetic analysis categorized the WD40 proteins into 5 distinct sub-families (I-V). Gene Ontology annotation revealed the biological roles of the WD40 proteins along with its cellular components and molecular functions. In silico comparative mapping with sorghum, maize and rice demonstrated the orthologous relationships and chromosomal rearrangements including duplication, inversion and deletion of WD40 genes. Estimation of synonymous and non-synonymous substitution rates revealed its evolutionary significance in terms of gene-duplication and divergence. Expression profiling against abiotic stresses provided novel insights into specific and/or overlapping expression patterns of SiWD40 genes. Homology modeling enabled three-dimensional structure prediction was performed to understand the molecular functions of WD40 proteins. Although, recent findings had shown the importance of WD40 domains in acting as hubs for cellular networks during many biological processes, it has invited a lesser research attention unlike other common domains. Being a most promiscuous interactors, WD40 domains are versatile in mediating critical cellular functions and hence this genome-wide study especially in the model crop foxtail millet would serve as a blue-print for functional characterization of WD40s in millets and bioenergy grass species. In addition, the present analyses would also assist the research community in choosing the candidate WD40s for comprehensive studies towards crop improvement of millets and biofuel grasses.

Genome-Wide Investigation and Expression Analyses of WD40 Protein Family in the Model Plant Foxtail Millet (Setaria italica L.)

PubMed Central

Mishra, Awdhesh Kumar; Muthamilarasan, Mehanathan; Khan, Yusuf; Parida, Swarup Kumar; Prasad, Manoj

2014-01-01

WD40 proteins play a crucial role in diverse protein-protein interactions by acting as scaffolding molecules and thus assisting in the proper activity of proteins. Hence, systematic characterization and expression profiling of these WD40 genes in foxtail millet would enable us to understand the networks of WD40 proteins and their biological processes and gene functions. In the present study, a genome-wide survey was conducted and 225 potential WD40 genes were identified. Phylogenetic analysis categorized the WD40 proteins into 5 distinct sub-families (I–V). Gene Ontology annotation revealed the biological roles of the WD40 proteins along with its cellular components and molecular functions. In silico comparative mapping with sorghum, maize and rice demonstrated the orthologous relationships and chromosomal rearrangements including duplication, inversion and deletion of WD40 genes. Estimation of synonymous and non-synonymous substitution rates revealed its evolutionary significance in terms of gene-duplication and divergence. Expression profiling against abiotic stresses provided novel insights into specific and/or overlapping expression patterns of SiWD40 genes. Homology modeling enabled three-dimensional structure prediction was performed to understand the molecular functions of WD40 proteins. Although, recent findings had shown the importance of WD40 domains in acting as hubs for cellular networks during many biological processes, it has invited a lesser research attention unlike other common domains. Being a most promiscuous interactors, WD40 domains are versatile in mediating critical cellular functions and hence this genome-wide study especially in the model crop foxtail millet would serve as a blue-print for functional characterization of WD40s in millets and bioenergy grass species. In addition, the present analyses would also assist the research community in choosing the candidate WD40s for comprehensive studies towards crop improvement of millets and biofuel grasses. PMID:24466268

Novel regulatory loci controlling oxygen- and pH-regulated gene expression in Salmonella typhimurium.

PubMed Central

Aliabadi, Z; Park, Y K; Slonczewski, J L; Foster, J W

1988-01-01

Three new loci were discovered, each of which participates in the regulation of anaerobic gene expression. The regulatory gene earA negatively regulates the expression of the anaerobiosis-inducible gene aniG as well as that of at least three other genes, as determined by two-dimensional polyacrylamide gel electrophoresis. The earA locus maps at 86 min. The expression of aniG was also shown to be controlled by changes in external pH under aerobic and anaerobic conditions. Maximal expression was observed under anaerobic conditions at an external pH of 6.0. Significant transcriptional activity was also observed under aerobic conditions at pH 6.0. This was in contrast to hyd, whose expression was dependent upon anaerobiosis and varied with external pH. The pH dependence disappeared under fully aerobic conditions. Mutations in earA had no effect upon hyd expression. The two other regulators identified were oxrF, which controls aniH, and oxrG, which, in concert with oxrA and oxrB, controls aniC and aniI. The oxrG locus was mapped to 88 min and appears to code for a positive regulator. Various oxr mutants were subjected to two-dimensional polyacrylamide electrophoretic analysis of anaerobiosis-inducible proteins. Several pathways of anaerobic control were observed by means of these techniques. Images PMID:3276666

Cohort-specific imputation of gene expression improves prediction of warfarin dose for African Americans.

PubMed

Gottlieb, Assaf; Daneshjou, Roxana; DeGorter, Marianne; Bourgeois, Stephane; Svensson, Peter J; Wadelius, Mia; Deloukas, Panos; Montgomery, Stephen B; Altman, Russ B

2017-11-24

Genome-wide association studies are useful for discovering genotype-phenotype associations but are limited because they require large cohorts to identify a signal, which can be population-specific. Mapping genetic variation to genes improves power and allows the effects of both protein-coding variation as well as variation in expression to be combined into "gene level" effects. Previous work has shown that warfarin dose can be predicted using information from genetic variation that affects protein-coding regions. Here, we introduce a method that improves dose prediction by integrating tissue-specific gene expression. In particular, we use drug pathways and expression quantitative trait loci knowledge to impute gene expression-on the assumption that differential expression of key pathway genes may impact dose requirement. We focus on 116 genes from the pharmacokinetic and pharmacodynamic pathways of warfarin within training and validation sets comprising both European and African-descent individuals. We build gene-tissue signatures associated with warfarin dose in a cohort-specific manner and identify a signature of 11 gene-tissue pairs that significantly augments the International Warfarin Pharmacogenetics Consortium dosage-prediction algorithm in both populations. Our results demonstrate that imputed expression can improve dose prediction and bridge population-specific compositions. MATLAB code is available at https://github.com/assafgo/warfarin-cohort.

The Expression of DJ-1 (PARK7) in Normal Human CNS and Idiopathic Parkinson's Disease

ERIC Educational Resources Information Center

Bandopadhyay, Rina; Kingsbury, Ann E.; Cookson, Mark R.; Reid, Andrew R.; Evans, Ian M.; Hope, Andrew D.; Pittman, Alan M.; Lashley, Tammaryn; Canet-Aviles, Rosa; Miller, David W.; McLendon, Chris; Strand, Catherine; Leonard, Andrew J.; Abou-Sleiman, Patrick M.; Healy, Daniel G.; Ariga, Hiroyashi; Wood, Nicholas W.; de Silva, Rohan; Revesz, Tamas; Hardy, John A.; Lees, Andrew J.

2004-01-01

Two mutations in the DJ-1 gene on chromosome1p36 have been identified recently to cause early-onset, autosomal recessive Parkinson's disease. As no information is available regarding the distribution of DJ-1 protein in the human brain, in this study we used a monoclonal antibody for DJ-1 to map its distribution in frontal cortex and substantia…

Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress

PubMed Central

Xing, Yu; Chen, Wei-hua; Jia, Wensuo; Zhang, Jianhua

2015-01-01

Superoxide dismutases (SODs) are involved in plant adaptive responses to biotic and abiotic stresses but the upstream signalling process that modulates their expression is not clear. Expression of two iron SODs, FSD2 and FSD3, was significantly increased in Arabidopsis in response to NaCl treatment but blocked in transgenic MKK5-RNAi plant, mkk5. Using an assay system for transient expression in protoplasts, it was found that mitogen-activated protein kinase kinase 5 (MKK5) was also activated in response to salt stress. Overexpression of MKK5 in wild-type plants enhanced their tolerance to salt treatments, while mkk5 mutant exhibited hypersensitivity to salt stress in germination on salt-containing media. Moreover, another kinase, MPK6, was also involved in the MKK5-mediated iron superoxide dismutase (FSD) signalling pathway in salt stress. The kinase activity of MPK6 was totally turned off in mkk5, whereas the activity of MPK3 was only partially blocked. MKK5 interacted with the MEKK1 protein that was also involved in the salt-induced FSD signalling pathway. These data suggest that salt-induced FSD2 and FSD3 expressions are influenced by MEKK1 via MKK5–MPK6-coupled signalling. This MAP kinase cascade (MEKK1, MKK5, and MPK6) mediates the salt-induced expression of iron superoxide dismutases. PMID:26136265

Identification of chromosome regions controlling seed storage proteins of narrow-leafed lupin (Lupinus angustifolius).

PubMed

Li, Xin; Islam, Shahidul; Yang, Huaan; Ma, Wujun; Yan, Guijun

2013-05-01

Narrow-leafed lupin (Lupinus angustifolius L.) is a valuable legume crop for animal feed and human health food because of its high proteins content. However, the genetics of seed storage proteins is unclear, limiting further improvement of protein quantity and quality. In this study, matrix-assisted laser desorption/ionization time of flight mass spectrometry was used for the first time to analyze lupin seed storage proteins and the spectra generated was treated as markers to investigate the chromosome locations controlling seed storage proteins in the narrow-leafed lupin. In a recombinant inbred line population of 89 individuals, 48 polymorphic protein peaks were identified and seven of which were successfully mapped onto four existing linkage groups: two on NLL-04, three on NLL-05, one on NLL-07 and one on NLL-14, with LOD values ranging from 2.6 to 7.7 confirming a significant linkage. Most protein-based markers showed distorted segregation and were failed to be integrated into the reference map. Among them, 31 were grouped into six clusters and the other ten were totally unlinked. This study provides a significant clue to study the comparative genomics/proteomics among legumes as well as for protein marker-assisted breeding. The distribution pattern of genes controlling seed storage protein revealed in this study probably exists universally among legumes or even all plants and animals. Whether genes controlling seed storage protein share the same gene expression pattern controlling other enzymes and what is the mechanism behind it are the questions which remain to be answered in the future.

The MAP kinase pathway is involved in odontoblast stimulation via p38 phosphorylation.

PubMed

Simon, Stephane; Smith, Anthony J; Berdal, Ariane; Lumley, Philip J; Cooper, Paul R

2010-02-01

We have previously shown that the p38 gene is highly expressed in odontoblasts during active primary dentinogenesis, but is drastically down-regulated as cells become quiescent in secondary dentinogenesis. Based on these observations, we hypothesized that p38 expression might be upregulated, and the protein activated by phosphorylation, when odontoblasts are stimulated such as during tertiary reactionary dentinogenesis. We stimulated immortalized, odontoblast-like MDPC-23 cells, alone or in combination, with heat-inactivated Streptococcus mutans, EDTA-extracted dentine matrix proteins (DMPs), or growth factors, including transforming growth factor (TGF)-beta1, tumor necrosis factor-alpha (TNF-alpha), and adrenomedullin (ADM). We used ELISA to measure the resulting phosphorylation of the p38 protein, as well as its degree of nuclear translocation. Our results suggest that the p38-MAPKinase pathway is activated during odontoblast stimulation in tertiary dentinogenesis by both p38 phosphorylation and enhanced nuclear translocation. Data indicate that odontoblast behaviour therefore potentially recapitulates that during active primary dentinogenesis. Copyright 2010 American Association of Endodontists. All rights reserved.

Characterization of the Eimeria maxima sporozoite surface protein IMP1.

PubMed

Jenkins, M C; Fetterer, R; Miska, K; Tuo, W; Kwok, O; Dubey, J P

2015-07-30

The purpose of this study was to characterize Eimeria maxima immune-mapped protein 1 (IMP1) that is hypothesized to play a role in eliciting protective immunity against E. maxima infection in chickens. RT-PCR analysis of RNA from unsporulated and sporulating E. maxima oocysts revealed highest transcription levels at 6-12h of sporulation with a considerable downregulation thereafter. Alignment of IMP1 coding sequence from Houghton, Weybridge, and APU-1 strains of E. maxima revealed single nucleotide polymorphisms that in some instances led to amino acid changes in the encoded protein sequence. The E. maxima (APU-1) IMP1 cDNA sequence was cloned and expressed in 2 different polyHis Escherichia coli expression vectors. Regardless of expression vector, recombinant E. maxima IMP1 (rEmaxIMP1) was fairly unstable in non-denaturing buffer, which is consistent with stability analysis of the primary amino acid sequence. Antisera specific for rEmaxIMP1 identified a single 72 kDa protein or a 61 kDa protein by non-reducing or reducing SDS-PAGE/immunoblotting. Immunofluorescence staining with anti-rEmaxIMP1, revealed intense surface staining of E. maxima sporozoites, with negligible staining of merozoite stages. Immuno-histochemical staining of E. maxima-infected chicken intestinal tissue revealed staining of E. maxima developmental stages in the lamnia propia and crypts at both 24 and 48 h post-infection, and negligible staining thereafter. The expression of IMP1 during early stages of in vivo development and its location on the sporozoite surface may explain in part the immunoprotective effect of this protein against E. maxima infection. Published by Elsevier B.V.

Genome-wide organization and expression profiling of the NAC transcription factor family in potato (Solanum tuberosum L.).

PubMed

Singh, Anil Kumar; Sharma, Vishal; Pal, Awadhesh Kumar; Acharya, Vishal; Ahuja, Paramvir Singh

2013-08-01

NAC [no apical meristem (NAM), Arabidopsis thaliana transcription activation factor [ATAF1/2] and cup-shaped cotyledon (CUC2)] proteins belong to one of the largest plant-specific transcription factor (TF) families and play important roles in plant development processes, response to biotic and abiotic cues and hormone signalling. Our genome-wide analysis identified 110 StNAC genes in potato encoding for 136 proteins, including 14 membrane-bound TFs. The physical map positions of StNAC genes on 12 potato chromosomes were non-random, and 40 genes were found to be distributed in 16 clusters. The StNAC proteins were phylogenetically clustered into 12 subgroups. Phylogenetic analysis of StNACs along with their Arabidopsis and rice counterparts divided these proteins into 18 subgroups. Our comparative analysis has also identified 36 putative TNAC proteins, which appear to be restricted to Solanaceae family. In silico expression analysis, using Illumina RNA-seq transcriptome data, revealed tissue-specific, biotic, abiotic stress and hormone-responsive expression profile of StNAC genes. Several StNAC genes, including StNAC072 and StNAC101that are orthologs of known stress-responsive Arabidopsis RESPONSIVE TO DEHYDRATION 26 (RD26) were identified as highly abiotic stress responsive. Quantitative real-time polymerase chain reaction analysis largely corroborated the expression profile of StNAC genes as revealed by the RNA-seq data. Taken together, this analysis indicates towards putative functions of several StNAC TFs, which will provide blue-print for their functional characterization and utilization in potato improvement.

RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

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

Smialowska, Agata, E-mail: smialowskaa@gmail.com; School of Life Sciences, Södertörn Högskola, Huddinge 141-89; Djupedal, Ingela

Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its rolemore » in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.« less

Dynamic gene and protein expression patterns of the autism-associated met receptor tyrosine kinase in the developing mouse forebrain.

PubMed

Judson, Matthew C; Bergman, Mica Y; Campbell, Daniel B; Eagleson, Kathie L; Levitt, Pat

2009-04-10

The establishment of appropriate neural circuitry depends on the coordination of multiple developmental events across space and time. These events include proliferation, migration, differentiation, and survival-all of which can be mediated by hepatocyte growth factor (HGF) signaling through the Met receptor tyrosine kinase. We previously found a functional promoter variant of the MET gene to be associated with autism spectrum disorder, suggesting that forebrain circuits governing social and emotional function may be especially vulnerable to developmental disruptions in HGF/Met signaling. However, little is known about the spatiotemporal distribution of Met expression in the forebrain during the development of such circuits. To advance our understanding of the neurodevelopmental influences of Met activation, we employed complementary Western blotting, in situ hybridization, and immunohistochemistry to comprehensively map Met transcript and protein expression throughout perinatal and postnatal development of the mouse forebrain. Our studies reveal complex and dynamic spatiotemporal patterns of expression during this period. Spatially, Met transcript is localized primarily to specific populations of projection neurons within the neocortex and in structures of the limbic system, including the amygdala, hippocampus, and septum. Met protein appears to be principally located in axon tracts. Temporally, peak expression of transcript and protein occurs during the second postnatal week. This period is characterized by extensive neurite outgrowth and synaptogenesis, supporting a role for the receptor in these processes. Collectively, these data suggest that Met signaling may be necessary for the appropriate wiring of forebrain circuits, with particular relevance to the social and emotional dimensions of behavior. (c) 2009 Wiley-Liss, Inc.

Two rat brain staufen isoforms differentially bind RNA.

PubMed

Monshausen, M; Putz, U; Rehbein, M; Schweizer, M; DesGroseillers, L; Kuhl, D; Richter, D; Kindler, S

2001-01-01

In neurones, a limited number of mRNAs is found in dendrites, including transcripts encoding the microtubule-associated protein 2 (MAP2). Recently, we identified a cis-acting dendritic targeting element (DTE) in MAP2 mRNAs. Here we used the yeast tri-hybrid system to identify potential trans-acting RNA-binding factors of the DTE. A cDNA clone was isolated that encodes a member of a mammalian protein family that is highly homologous to the Drosophila RNA-binding protein Staufen. Mammalian Staufen appears to be expressed in most tissues and brain areas. Two distinct rat brain Staufen isoforms, rStau+I6 and rStau-I6, are encoded by alternatively spliced mRNAs. Both isoforms contain four double-stranded RNA-binding domains (dsRBD). In the larger rStau+I6 isoform, six additional amino acids are inserted in the second dsRBD. Although both isoforms interacted with the MAP2-DTE and various additional RNA fragments in an in vitro north-western assay, rStau-I6 exhibited a stronger signal of bound radioactively labelled RNAs as compared with rStau+I6. Using an antibody directed against mammalian Staufen, the protein was detected in somata and dendrites of neurones of the adult rat hippocampus and cerebral cortex. Ultrastructural studies revealed that in dendrites, rat Staufen accumulates along microtubules. Thus in neurones, rat Staufen may serve to link RNAs to the dendritic microtubular cytoskeleton and may thereby regulate their subcellular localization.

Enhanced aggressive behaviour in a mouse model of depression.

PubMed

Yang, C R; Bai, Y Y; Ruan, C S; Zhou, H F; Liu, D; Wang, X F; Shen, L J; Zheng, H Y; Zhou, X F

2015-02-01

Depression is one of the most common chronic mental disorders, which is a leading cause of morbidity and mortality in patients. Depression often leads to offensive and defensive behaviours but the underlying mechanisms are not known. We propose that the aggressive behaviours in depression can be modelled in animal experiments. In this study, we successfully established a mouse model of depression using the chronic unpredictable mild stress (CUMS) paradigm and detected aggressive and social dominance behaviours in rodents by resident/intruder test and social dominance tube test (SDTT), respectively. The CUMS-exposed mice showed increased defensive, offensive and aggressive behaviours in the resident-intruder test. In the SDTT, these mice showed enhanced social dominance. These alterations were associated with reduced MAP-2 expression in the hippocampus while no difference in β-tubulin expression was detected. In addition, the treatment of anti-depressant fluoxetine reversed the aggressive behaviours without reducing the social dominance behaviour induced by CUMS. However, fluoxetine did effectively reverted the changes in MAP-2 expression in the hippocampus. In addition, the nonspecific tricyclic antipsychotic drug, clozapine, reversed all symptoms of CUMS-exposed mice including aggressive tendencies, impulsive violence, social dominance behaviour and MAP-2 expression in the hippocampus. The results suggests that social maladjustment such as competition and social dominance are likely related to the dopaminergic system rather than the serotonergic system and the hippocampal dendritic structure protein MAP-2. Thus, dominance can be separated from aggression. This study shows that aggression/hostility and social hierarchy/dominance are increased in the CUMS-exposed mice and thus provide an excellent model for further study in the diagnosis and the treatment of depression-associated aggression.

Downregulation of the glucocorticoid-induced leucine zipper (GILZ) promotes vascular inflammation.

PubMed

Hahn, Rebecca T; Hoppstädter, Jessica; Hirschfelder, Kerstin; Hachenthal, Nina; Diesel, Britta; Kessler, Sonja M; Huwer, Hanno; Kiemer, Alexandra K

2014-06-01

Glucocorticoid-induced leucine zipper (GILZ) represents an anti-inflammatory mediator, whose downregulation has been described in various inflammatory processes. Aim of our study was to decipher the regulation of GILZ in vascular inflammation. Degenerated aortocoronary saphenous vein bypass grafts (n = 15), which exhibited inflammatory cell activation as determined by enhanced monocyte chemoattractrant protein 1 (MCP-1, CCL2) and Toll-like receptor 2 (TLR2) expression, showed significantly diminished GILZ protein and mRNA levels compared to healthy veins (n = 23). GILZ was also downregulated in human umbilical vein endothelial cells (HUVEC) and macrophages upon treatment with the inflammatory cytokine TNF-α in a tristetraprolin (ZFP36, TTP)- and p38 MAPK-dependent manner. To assess the functional implications of decreased GILZ expression, we determined NF-κB activation after GILZ knockdown by siRNA and found that NF-κB activity and inflammatory gene expression were significantly enhanced. Importantly, ZFP36 is induced in TNF-α-activated HUVEC as well as in degenerated vein bypasses. When atheroprotective laminar shear stress was employed, GILZ levels in HUVEC increased on mRNA and protein level. Laminar flow also counteracted TNF-α-induced ZFP36 expression and GILZ downregulation. MAP kinase phosphatase 1 (MKP-1, DUSP1), a negative regulator of ZFP36 expression, was distinctly upregulated under laminar shear stress conditions and downregulated in degenerated vein bypasses. Our data show a diminished expression of the anti-inflammatory mediator GILZ in the inflamed vasculature and indicate that GILZ downregulation requires the mRNA binding protein ZFP36. We suggest that reduced GILZ levels play a role in cardiovascular disease. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Neogenin mediates the action of repulsive guidance molecule.

PubMed

Rajagopalan, Srikanth; Deitinghoff, Lutz; Davis, Denise; Conrad, Sabine; Skutella, Thomas; Chedotal, Alain; Mueller, Bernhard K; Strittmatter, Stephen M

2004-08-01

Repulsive guidance molecule (RGM) is a recently identified protein implicated in both axonal guidance and neural tube closure. The avoidance of chick RGM in the posterior optic tectum by growing temporal, but not nasal, retinal ganglion cell axons is thought to contribute to visual map formation. In contrast to ephrins, semaphorins, netrins and slits, no receptor mechanism for RGM action has been defined. Here, an expression cloning strategy identified neogenin as a binding site for RGM, with a sub-nanomolar affinity. Consistent with selective axonal responsiveness to RGM, neogenin is expressed in a gradient across the chick retina. Neogenin is known to be one of several netrin-binding proteins but only neogenin interacts with RGM. The avoidance of RGM by temporal retinal axons is blocked by the anti-neogenin antibody and the soluble neogenin ectodomain. Dorsal root ganglion axons are unresponsive to RGM but are converted to a responsive state by neogenin expression. Thus, neogenin functions as an RGM receptor.

The helix-loop-helix protein id1 controls stem cell proliferation during regenerative neurogenesis in the adult zebrafish telencephalon.

PubMed

Rodriguez Viales, Rebecca; Diotel, Nicolas; Ferg, Marco; Armant, Olivier; Eich, Julia; Alunni, Alessandro; März, Martin; Bally-Cuif, Laure; Rastegar, Sepand; Strähle, Uwe

2015-03-01

The teleost brain has the remarkable ability to generate new neurons and to repair injuries during adult life stages. Maintaining life-long neurogenesis requires careful management of neural stem cell pools. In a genome-wide expression screen for transcription regulators, the id1 gene, encoding a negative regulator of E-proteins, was found to be upregulated in response to injury. id1 expression was mapped to quiescent type I neural stem cells in the adult telencephalic stem cell niche. Gain and loss of id1 function in vivo demonstrated that Id1 promotes stem cell quiescence. The increased id1 expression observed in neural stem cells in response to injury appeared independent of inflammatory signals, suggesting multiple antagonistic pathways in the regulation of reactive neurogenesis. Together, we propose that Id1 acts to maintain the neural stem cell pool by counteracting neurogenesis-promoting signals. © 2014 AlphaMed Press.

From Allergen Back to Antigen:. a Rational Approach to New Forms of Immunotherapy

NASA Astrophysics Data System (ADS)

Colombo, Paolo; Trapani, Antonino; Geraci, Domenico; Golino, Massimiliano; Gianguzza, Fabrizio; Bonura, Angela

2007-12-01

Mapping an epitope on a protein by gene fragmentation and/or point mutations is often expensive and time consuming. Analysis of a 3D model can be utilized to detect the amino acids residues which are exposed to the solvent surface and thus represent potential epitope residues. Parj1 and Parj2 are the two major allergens of the Parietaria judaica pollen belonging to the Lipid Transfer Protein family. Using their three-dimensional structures as a guide, a head to tail dimer expressing disulphide bond variants of the major allergens was generated by means of DNA recombinant technology. The hybrid was expressed in E.coli and its immunological activity studied in vivo and in vitro. Our results demonstrate that a hybrid polypeptide expressing disulphide bond variants of the major allergens of the Parietaria pollen displayed reduced allergenicity and enhanced T cell reactivity for induction of protective antibodies able to block human IgE induced during the natural course of sensitization against the Parietaria pollen.

Negative regulatory loci coupling flagellin synthesis to flagellar assembly in Salmonella typhimurium.

PubMed Central

Gillen, K L; Hughes, K T

1991-01-01

The complex regulation of flagellin gene expression in Salmonella typhimurium was characterized in vivo by using lac transcriptional fusions to the two flagellin structural genes (fliC [H1] and fljB [H2]). Phase variation was measured as the rate of switching of flagellin gene expression. Switching frequencies varied from 1/500 per cell per generation to 1/10,000 per cell per generation depending on the particular insertion and the direction of switching. There is a 4- to 20-fold bias in favor of switching from the fljB(On) to the fljB(Off) orientation. Random Tn10dTc insertions were isolated which failed to express flagellin. While most of these insertions mapped to loci known to be required for flagellin expression, several new loci were identified. The presence of functional copies of all of the genes responsible for complete flagellar assembly, except the hook-associated proteins (flgK, flgL, and fliD gene products), were required for expression of the fliC or fljB flagellin genes. Two novel loci involved in negative regulation of fliC and fljB in fla mutant backgrounds were identified. One of these loci, designated the flgR locus, mapped to the flg operon at 23 min on the Salmonella linkage map. An flgR insertion mutation resulted in relief of repression of the fliC and fljB genes in all fla mutant backgrounds except for mutants in the positive regulatory loci (flhC, flhD, and fliA genes). PMID:1848842

Towards a complete map of the human long non-coding RNA transcriptome.

PubMed

Uszczynska-Ratajczak, Barbara; Lagarde, Julien; Frankish, Adam; Guigó, Roderic; Johnson, Rory

2018-05-23

Gene maps, or annotations, enable us to navigate the functional landscape of our genome. They are a resource upon which virtually all studies depend, from single-gene to genome-wide scales and from basic molecular biology to medical genetics. Yet present-day annotations suffer from trade-offs between quality and size, with serious but often unappreciated consequences for downstream studies. This is particularly true for long non-coding RNAs (lncRNAs), which are poorly characterized compared to protein-coding genes. Long-read sequencing technologies promise to improve current annotations, paving the way towards a complete annotation of lncRNAs expressed throughout a human lifetime.

DAPK1 as an independent prognostic marker in liver cancer.

PubMed

Li, Ling; Guo, Libin; Wang, Qingshui; Liu, Xiaolong; Zeng, Yongyi; Wen, Qing; Zhang, Shudong; Kwok, Hang Fai; Lin, Yao; Liu, Jingfeng

2017-01-01

The death-associated protein kinase 1 (DAPK1) can act as an oncogene or a tumor suppressor gene depending on the cellular context as well as external stimuli. Our study aims to investigate the prognostic significance of DAPK1 in liver cancer in both mRNA and protein levels. The mRNA expression of DAPK1 was extracted from the Gene Expression Omnibus database in three independent liver cancer datasets while protein expression of DAPK1 was detected by immunohistochemistry in our Chinese liver cancer patient cohort. The associations between DAPK1 expression and clinical characteristics were tested. DAPK1 mRNA expression was down-regulated in liver cancer. Low levels of DAPK1 mRNA were associated with shorter survival in a liver cancer patient cohort ( n  = 115;  p  = 0.041), while negative staining of DAPK1 protein was significantly correlated with shorter time to progression ( p  = 0.002) and overall survival ( p  = 0.02). DAPK1 was an independent prognostic marker for both time to progression and overall survival by multivariate analysis. Liver cancer with the b-catenin mutation has a lower DAPK1 expression, suggesting that DAPK1 may be regulated under the b-catenin pathway. In addition, we also identified genes that are co-regulated with DAPK1. DAPK1 expression was positively correlated with IRF2, IL7R, PCOLCE and ZBTB16, and negatively correlated with SLC16A3 in both liver cancer datasets. Among these genes, PCOLCE and ZBTB16 were significantly down-regulated, while SLC16A3 was significantly upregulated in liver cancer. By using connectivity mapping of these co-regulated genes, we have identified amcinonide and sulpiride as potential small molecules that could potentially reverse DAPK1/PCOLCE/ZBTB16/SLC16A3 expression. Our study demonstrated for the first time that both DAPK1 mRNA and protein expression levels are important prognostic markers in liver cancer, and have identified genes that may contribute to DAPK1-mediated liver carcinogenesis.

Separation and identification of Musa acuminate Colla (banana) leaf proteins by two-dimensional gel electrophoresis and mass spectrometry.

PubMed

Lu, Y; Qi, Y X; Zhang, H; Zhang, H Q; Pu, J J; Xie, Y X

2013-12-19

To establish a proteomic reference map of Musa acuminate Colla (banana) leaf, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 44 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Three spots that were not identified by MALDI-TOF MS analysis were identified by searching against the NCBInr, SwissProt, and expressed sequence tag (EST) databases. We identified 41 unique proteins. The majority of the identified leaf proteins were found to be involved in energy metabolism. The results indicate that 2D-PAGE is a sensitive and powerful technique for the separation and identification of Musa leaf proteins. A summary of the identified proteins and their putative functions is discussed.

Identification and characterization of a novel serine-threonine kinase gene from the Xp22 region.

PubMed

Montini, E; Andolfi, G; Caruso, A; Buchner, G; Walpole, S M; Mariani, M; Consalez, G; Trump, D; Ballabio, A; Franco, B

1998-08-01

Eukaryotic protein kinases are part of a large and expanding family of proteins. Through our transcriptional mapping effort in the Xp22 region, we have isolated and sequenced the full-length transcript of STK9, a novel cDNA highly homologous to serine-threonine kinases. A number of human genetic disorders have been mapped to the region where STK9 has been localized including Nance-Horan (NH) syndrome, oral-facial-digital syndrome type 1 (OFD1), and a novel locus for nonsyndromic sensorineural deafness (DFN6). To evaluate the possible involvement of STK9 in any of the above-mentioned disorders, a 2416-bp full-length cDNA was assembled. The entire genomic structure of the gene, which is composed of 20 coding exons, was determined. Northern analysis revealed a transcript larger than 9.5 kb in several tissues including brain, lung, and kidney. The mouse homologue (Stk9) was identified and mapped in the mouse in the region syntenic to human Xp. This location is compatible with the location of the Xcat mutant, which shows congenital cataracts very similar to those observed in NH patients. Sequence homologies, expression pattern, and mapping information in both human and mouse make STK9 a candidate gene for the above-mentioned disorders. Copyright 1998 Academic Press.

Mutations in a Novel Isoform of TRIOBP That Encodes a Filamentous-Actin Binding Protein Are Responsible for DFNB28 Recessive Nonsyndromic Hearing Loss

PubMed Central

Shahin, Hashem; Walsh, Tom; Sobe, Tama; Abu Sa’ed, Judeh; Abu Rayan, Amal; Lynch, Eric D.; Lee, Ming K.; Avraham, Karen B.; King, Mary-Claire; Kanaan, Moein

2006-01-01

In a large consanguineous Palestinian kindred, we previously mapped DFNB28—a locus associated with recessively inherited, prelingual, profound sensorineural hearing impairment—to chromosome 22q13.1. We report here that mutations in a novel 218-kDa isoform of TRIOBP (TRIO and filamentous actin [F-actin] binding protein) are associated with DFNB28 hearing loss in a total of nine Palestinian families. Two nonsense mutations (R347X and Q581X) truncate the protein, and a potentially deleterious missense mutation (G1019R) occurs in a conserved motif in a putative SH3-binding domain. In seven families, 27 deaf individuals are homozygous for one of the nonsense mutations; in two other families, 3 deaf individuals are compound heterozygous for the two nonsense mutations or for Q581X and G1019R. The novel long isoform of TRIOBP has a restricted expression profile, including cochlea, retina, and fetal brain, whereas the original short isoform is widely expressed. Antibodies to TRIOBP reveal expression in sensory cells of the inner ear and colocalization with F-actin along the length of the stereocilia. PMID:16385458

Painting of fourth and chromosome-wide regulation of the 4th chromosome in Drosophila melanogaster.

PubMed

Johansson, Anna-Mia; Stenberg, Per; Bernhardsson, Carolina; Larsson, Jan

2007-05-02

Drosophila melanogaster exhibits two expression-regulating systems that target whole, specific chromosomes: the dosage compensation system whereby the male-specific lethal complex doubles transcription of genes on the male X-chromosome and the chromosome 4-specific protein Painting of fourth, POF. POF is the first example of an autosome-specific protein and its presence raises the question of the universality of chromosome-specific regulation. Here we show that POF and heterochromatin protein 1 (HP1) are involved in the global regulation of the 4th chromosome. Contrary to previous conclusions, Pof is not essential for survival of diplo-4th karyotype flies. However, Pof is essential for survival of haplo-4th individuals and expression of chromosome 4 genes in diplo-4th individuals is decreased in the absence of Pof. Mapping of POF using chromatin immunoprecipitation suggested that it binds within genes. Furthermore, we show that POF binding is dependent on heterochromatin and that POF and HP1 bind interdependently to the 4th chromosome. We propose a balancing mechanism involving POF and HP1 that provides a feedback system for fine-tuning expression status of genes on the 4th chromosome.

Painting of fourth and chromosome-wide regulation of the 4th chromosome in Drosophila melanogaster

PubMed Central

Johansson, Anna-Mia; Stenberg, Per; Bernhardsson, Carolina; Larsson, Jan

2007-01-01

Drosophila melanogaster exhibits two expression-regulating systems that target whole, specific chromosomes: the dosage compensation system whereby the male-specific lethal complex doubles transcription of genes on the male X-chromosome and the chromosome 4-specific protein Painting of fourth, POF. POF is the first example of an autosome-specific protein and its presence raises the question of the universality of chromosome-specific regulation. Here we show that POF and heterochromatin protein 1 (HP1) are involved in the global regulation of the 4th chromosome. Contrary to previous conclusions, Pof is not essential for survival of diplo-4th karyotype flies. However, Pof is essential for survival of haplo-4th individuals and expression of chromosome 4 genes in diplo-4th individuals is decreased in the absence of Pof. Mapping of POF using chromatin immunoprecipitation suggested that it binds within genes. Furthermore, we show that POF binding is dependent on heterochromatin and that POF and HP1 bind interdependently to the 4th chromosome. We propose a balancing mechanism involving POF and HP1 that provides a feedback system for fine-tuning expression status of genes on the 4th chromosome. PMID:17318176

Human Hrs, a tyrosine kinase substrate in growth factor-stimulated cells: cDNA cloning and mapping of the gene to chromosome 17.

PubMed

Lu, L; Komada, M; Kitamura, N

1998-06-15

Hrs is a 115kDa zinc finger protein which is rapidly tyrosine phosphorylated in cells stimulated with various growth factors. We previously purified the protein from a mouse cell line and cloned its cDNA. In the present study, we cloned a human Hrs cDNA from a human placenta cDNA library by cross-hybridization, using the mouse cDNA as a probe, and determined its nucleotide sequence. The human Hrs cDNA encoded a 777-amino-acid protein whose sequence was 93% identical to that of mouse Hrs. Northern blot analysis showed that the Hrs mRNA was about 3.0kb long and was expressed in all the human adult and fetal tissues tested. In addition, we showed by genomic Southern blot analysis that the human Hrs gene was a single-copy gene with a size of about 20kb. Furthermore, the human Hrs gene was mapped to chromosome 17 by Southern blotting of genomic DNAs from human/rodent somatic cell hybrids. Copyright 1998 Elsevier Science B.V. All rights reserved.

Looking for Cancer Clues in Publicly Accessible Databases

PubMed Central

Lemkin, Peter F.; Smythers, Gary W.; Munroe, David J.

2004-01-01

What started out as a mere attempt to tentatively identify proteins in experimental cancer-related 2D-PAGE maps developed into VIRTUAL2D, a web-accessible repository for theoretical pI/MW charts for 92 organisms. Using publicly available expression data, we developed a collection of tissue-specific plots based on differential gene expression between normal and diseased states. We use this comparative cancer proteomics knowledge base, known as the tissue molecular anatomy project (TMAP), to uncover threads of cancer markers common to several types of cancer and to relate this information to established biological pathways. PMID:18629065

Looking for cancer clues in publicly accessible databases.

PubMed

Medjahed, Djamel; Lemkin, Peter F; Smythers, Gary W; Munroe, David J

2004-01-01

What started out as a mere attempt to tentatively identify proteins in experimental cancer-related 2D-PAGE maps developed into VIRTUAL2D, a web-accessible repository for theoretical pI/MW charts for 92 organisms. Using publicly available expression data, we developed a collection of tissue-specific plots based on differential gene expression between normal and diseased states. We use this comparative cancer proteomics knowledge base, known as the tissue molecular anatomy project (TMAP), to uncover threads of cancer markers common to several types of cancer and to relate this information to established biological pathways.

ALS3 encodes a phloem-localized ABC transporter-like protein that is required for aluminum tolerance in Arabidopsis.

PubMed

Larsen, Paul B; Geisler, Matt J B; Jones, Carol A; Williams, Kelly M; Cancel, Jesse D

2005-02-01

Aluminum (Al) toxicity in acid soils is a worldwide agricultural problem that severely limits crop productivity through inhibition of root growth. Previously, Arabidopsis mutants with increased Al sensitivity were isolated in order to identify genes important for Al tolerance in plants. One mutant, als3, exhibited extreme root growth inhibition in the presence of Al, suggesting that this mutation negatively impacts a gene required for Al tolerance. Map-based cloning of the als3-1 mutation resulted in the isolation of a novel gene that encodes a previously undescribed ABC transporter-like protein, which is highly homologous to a putative bacterial metal resistance protein, ybbM. Northern analysis for ALS3 expression revealed that it is found in all organs examined, which is consistent with the global nature of Al sensitivity displayed by als3, and that expression increases in roots following Al treatment. Based on GUS fusion and in situ hybridization analyses, ALS3 is primarily expressed in leaf hydathodes and the phloem throughout the plant, along with the root cortex following Al treatment. Immunolocalization indicates that ALS3 predominantly accumulates in the plasma membrane of cells that express ALS3. From our results, it appears that ALS3 encodes an ABC transporter-like protein that is required for Al resistance/tolerance and may function to redistribute accumulated Al away from sensitive tissues in order to protect the growing root from the toxic effects of Al.

The adapter protein, Grb10, is a positive regulator of vascular endothelial growth factor signaling.

PubMed

Giorgetti-Peraldi, S; Murdaca, J; Mas, J C; Van Obberghen, E

2001-07-05

Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis and angiogenesis. Activation of VEGF receptors leads to the recruitment of SH2 containing proteins which link the receptors to the activation of signaling pathways. Here we report that Grb10, an adapter protein of which the biological role remains unknown, is tyrosine phosphorylated in response to VEGF in endothelial cells (HUVEC) and in 293 cells expressing the VEGF receptor KDR. An intact SH2 domain is required for Grb10 tyrosine phosphorylation in response to VEGF, and this phosphorylation is mediated in part through the activation of Src. In HUVEC, VEGF increases Grb10 mRNA level. Expression of Grb10 in HUVEC or in KDR expressing 293 cells results in an increase in the amount and in the tyrosine phosphorylation of KDR. In 293 cells, this is correlated with the activation of signaling molecules, such as MAP kinase. By expressing mutants of Grb10, we found that the positive action of Grb10 is independent of its SH2 domain. Moreover, these Grb10 effects on KDR seem to be specific since Grb10 has no effect on the insulin receptor, and Grb2, another adapter protein, does not mimic the effect of Grb10 on KDR. In conclusion, we propose that VEGF up-regulates Grb10 level, which in turn increases KDR molecules, suggesting that Grb10 could be involved in a positive feedback loop in VEGF signaling.

Characterization of a novel binding partner of the melanocortin-4 receptor: attractin-like protein.

PubMed Central

Haqq, Andrea M; René, Patricia; Kishi, Toshiro; Khong, Kathy; Lee, Charlotte E; Liu, Hongyan; Friedman, Jeffrey M; Elmquist, Joel K; Cone, Roger D

2003-01-01

The gene dosage effect of the MC4-R (melanocortin 4 receptor) on obesity suggests that regulation of MC4-R expression and function is critically important to the central control of energy homoeostasis. In order to identify putative MC4-R regulatory proteins, we performed a yeast two-hybrid screen of a mouse brain cDNA library using the mouse MC4-R intracellular tail (residues 303-332) as bait. We report here on one positive clone that shares 63% amino acid identity with the C-terminal part of the mouse attractin gene product, a single-transmembrane-domain protein characterized as being required for agouti signalling through the melanocortin 1 receptor. We confirmed a direct interaction between this ALP (attractin-like protein) and the C-terminus of the mouse MC4-R by glutathione S-transferase pulldown experiments, and mapped the regions involved in this interaction using N- and C-terminal truncation constructs; residues 303-313 in MC4-R and residues 1280-1317 in ALP are required for binding. ALP is highly expressed in brain, but also in heart, lung, kidney and liver. Furthermore, co-localization analyses in mice showed co-expression of ALP in cells expressing MC4-R in a number of regions known to be important in the regulation of energy homoeostasis by melanocortins, such as the paraventricular nucleus of hypothalamus and the dorsal motor nucleus of the vagus. PMID:14531729

Conserved Regulation of MAP Kinase Expression by PUF RNA-Binding Proteins

PubMed Central

Lee, Myon-Hee; Hook, Brad; Pan, Guangjin; Kershner, Aaron M; Merritt, Christopher; Seydoux, Geraldine; Thomson, James A; Wickens, Marvin; Kimble, Judith

2007-01-01

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression. PMID:18166083

PrP(C) regulates epidermal growth factor receptor function and cell shape dynamics in Neuro2a cells.

PubMed

Llorens, Franc; Carulla, Patricia; Villa, Ana; Torres, Juan M; Fortes, Puri; Ferrer, Isidre; del Río, José A

2013-10-01

The prion protein (PrP) plays a key role in prion disease pathogenesis. Although the misfolded and pathologic variant of this protein (PrP(SC)) has been studied in depth, the physiological role of PrP(C) remains elusive and controversial. PrP(C) is a cell-surface glycoprotein involved in multiple cellular functions at the plasma membrane, where it interacts with a myriad of partners and regulates several intracellular signal transduction cascades. However, little is known about the gene expression changes modulated by PrP(C) in animals and in cellular models. In this article, we present PrP(C)-dependent gene expression signature in N2a cells and its implication in the most overrepresented functions: cell cycle, cell growth and proliferation, and maintenance of cell shape. PrP(C) over-expression enhances cell proliferation and cell cycle re-entrance after serum stimulation, while PrP(C) silencing slows down cell cycle progression. In addition, MAP kinase and protein kinase B (AKT) pathway activation are under the regulation of PrP(C) in asynchronous cells and following mitogenic stimulation. These effects are due in part to the modulation of epidermal growth factor receptor (EGFR) by PrP(C) in the plasma membrane, where the two proteins interact in a multimeric complex. We also describe how PrP(C) over-expression modulates filopodia formation by Rho GTPase regulation mainly in an AKT-Cdc42-N-WASP-dependent pathway. © 2013 International Society for Neurochemistry.

The baculovirus-integrated retrotransposon TED encodes gag and pol proteins that assemble into viruslike particles with reverse transcriptase.

PubMed Central

Lerch, R A; Friesen, P D

1992-01-01

TED is a lepidopteran retrotransposon found inserted within the DNA genome of the Autographa californica nuclear polyhedrosis virus mutant, FP-D. To examine the proteins and functions encoded by this representative of the gypsy family of retrotransposons, the gag- and pol-like open reading frames (ORFs 1 and 2) were expressed in homologous lepidopteran cells by using recombinant baculovirus vectors. Expression of ORF 1 resulted in synthesis of an abundant TED-specific protein (Pr55gag) that assembled into viruslike particles with a diameter of 55 to 60 nm. Expression of ORF 2, requiring a -1 translational frameshift, resulted in synthesis of a protease that mediated cleavage of Pr55gag to generate p37, the major protein component of the resulting particles. Expression of ORF 2 also produced reverse transcriptase that associated with these particles. Both protease and reverse transcriptase activities mapped to domains within ORF 2 that contain sequence similarities with the corresponding functional domains of the pol gene of the vertebrate retroviruses. These results indicated that TED ORFs 1 and 2 functionally resemble the retrovirus gag and pol genes and demonstrated for the first time that an invertebrate member of the gypsy family of elements encodes active forms of the structural and enzymatic functions necessary for transposition via an RNA intermediate. TED integration within the baculovirus genome thus represents one of the first examples of transposon-mediated transfer of host-derived genes to an eukaryotic virus. Images PMID:1371168

Timing Is Everything: Highly Specific and Transient Expression of a MAP Kinase Determines Auxin-Induced Leaf Venation Patterns in Arabidopsis

PubMed Central

Stanko, Vera; Giuliani, Concetta; Retzer, Katarzyna; Djamei, Armin; Wahl, Vanessa; Wurzinger, Bernhard; Wilson, Cathal; Heberle-Bors, Erwin; Teige, Markus; Kragler, Friedrich

2014-01-01

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules present in all eukaryotes. In plants, MAPK cascades were shown to regulate cell division, developmental processes, stress responses, and hormone pathways. The subgroup A of Arabidopsis MAPKs consists of AtMPK3, AtMPK6, and AtMPK10. AtMPK3 and AtMPK6 are activated by their upstream MAP kinase kinases (MKKs) AtMKK4 and AtMKK5 in response to biotic and abiotic stress. In addition, they were identified as key regulators of stomatal development and patterning. AtMPK10 has long been considered as a pseudo-gene, derived from a gene duplication of AtMPK6. Here we show that AtMPK10 is expressed highly but very transiently in seedlings and at sites of local auxin maxima leaves. MPK10 encodes a functional kinase and interacts with the upstream MAP kinase kinase (MAPKK) AtMKK2. mpk10 mutants are delayed in flowering in long-day conditions and in continuous light. Moreover, cotyledons of mpk10 and mkk2 mutants have reduced vein complexity, which can be reversed by inhibiting polar auxin transport (PAT). Auxin does not affect AtMPK10 expression while treatment with the PAT inhibitor HFCA extends the expression in leaves and reverses the mpk10 mutant phenotype. These results suggest that the AtMKK2–AtMPK10 MAPK module regulates venation complexity by altering PAT efficiency. PMID:25064848

Lead-induced changes of cytoskeletal protein is involved in the pathological basis in mice brain.

PubMed

Ge, Yaming; Chen, Lingli; Sun, Xianghe; Yin, Zhihong; Song, Xiaochao; Li, Chong; Liu, Junwei; An, Zhixing; Yang, Xuefeng; Ning, Hongmei

2018-04-01

Lead poisoning is a geochemical disease. On the other hand, lead is highly carcinogenic and exhibits liver and kidney toxicity. This element can also cross the blood-brain barrier, reduce learning and memory ability and damage the structure of the cerebral cortex and hippocampus. To further investigate the mechanism of lead neurotoxicity, 4-week-old Kunming mice were used to explore the effects of different concentrations of Pb 2+ (0, 2.4, 4.8 and 9.6 mM) for 9 days. In this study, pathological and ultrastructural changes in brain cells of the treated group were related to damages to mitochondria, chromatin and the nucleus. Lead content in blood was tested by atomic absorption spectroscopy, which showed high lead concentrations in the blood with increasing doses of lead. Distribution of lead in nerve cells was analysed by transmission electron microscopy with energy dispersive spectroscopy. Data showed the presence of lead in nucleopores, chromatin and nuclear membrane of nerve cells in the treatment groups, whereas lead content increased with increasing doses of lead acetate. Finally, microtubule-associated protein 2 (MAP2) mRNA and protein expression levels were detected by real-time PCR and Western blotting, which showed a reduction in MAP2 expression with increasing lead doses in the mouse brain. These findings suggest that acute lead poisoning can cause significant dose-dependent toxic effects on mouse brain function and can contribute to better understanding of lead-induced toxicity.

MAPs/bFGF-PLGA microsphere composite-coated titanium surfaces promote increased adhesion and proliferation of fibroblasts.

PubMed

Wang, Zhongshan; Wu, Guofeng; Bai, Shizhu; Feng, Zhihong; Dong, Yan; Zhou, Jian; Qin, Haiyan; Zhao, Yimin

2014-06-01

Infection and epithelial downgrowth are two major problems with maxillofacial transcutaneous implants, and both are mainly due to lack of stable closure of soft tissues at transcutaneous sites. Fibroblasts have been shown to play a key role in the formation of biological seals. In this work, titanium (Ti) model surfaces were coated with mussel adhesive proteins (MAPs) utilizing its unique adhesion ability on diverse inorganic and organic surfaces in wet environments. Prepared basic fibroblast growth factor (bFGF)-poly(lactic-co-glycolic acid) (PLGA) microspheres can be easily synthesized and combined onto MAPs-coated Ti surfaces, due to the negative surface charges of microspheres in aqueous solution, which is in contrast to the positive charges of MAPs. Titanium model surfaces were divided into three groups. Group A: MAPs/bFGF-PLGA microspheres composite-coated Ti surfaces. Group B: MAPs-coated Ti surfaces. Group C: uncoated Ti surfaces. The effects of coated Ti surfaces on adhesion of fibroblasts, cytoskeletal organization, proliferation, and extracellular matrix (ECM)-related gene expressions were examined. The results revealed increased adhesion (P < 0.05), enhanced actin cytoskeletal organization, and up-regulated ECM-related gene expressions in groups A and B compared with group C. Increased proliferation of fibroblasts during five days of incubation was observed in group A compared with groups B and C (P < 0.05). Collectively, the results from this in vitro study demonstrated that MAPs/bFGF-PLGA microspheres composite-coated Ti surfaces had the ability to increase fibroblast functionality. In addition, MAPs/bFGF-PLGA microsphere composite-coated Ti surfaces should be studied further as a method of promoting formation of stable biological seals around transcutaneous sites.

A Database of Reaction Monitoring Mass Spectrometry Assays for Elucidating Therapeutic Response in Cancer

PubMed Central

Remily-Wood, Elizabeth R.; Liu, Richard Z.; Xiang, Yun; Chen, Yi; Thomas, C. Eric; Rajyaguru, Neal; Kaufman, Laura M.; Ochoa, Joana E.; Hazlehurst, Lori; Pinilla-Ibarz, Javier; Lancet, Jeffrey; Zhang, Guolin; Haura, Eric; Shibata, David; Yeatman, Timothy; Smalley, Keiran S.M.; Dalton, William S.; Huang, Emina; Scott, Ed; Bloom, Gregory C.; Eschrich, Steven A.; Koomen, John M.

2012-01-01

Purpose The Quantitative Assay Database (QuAD), http://proteome.moffitt.org/QUAD/, facilitates widespread implementation of quantitative mass spectrometry in cancer biology and clinical research through sharing of methods and reagents for monitoring protein expression and modification. Experimental Design Liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM) assays are developed using SDS-PAGE fractionated lysates from cancer cell lines. Pathway maps created using GeneGO Metacore provide the biological relationships between proteins and illustrate concepts for multiplexed analysis; each protein can be selected to examine assay development at the protein and peptide level. Results The coupling of SDS-PAGE and LC-MRM screening has been used to detect 876 peptides from 218 cancer-related proteins in model systems including colon, lung, melanoma, leukemias, and myeloma, which has led to the development of 95 quantitative assays including stable-isotope labeled peptide standards. Methods are published online and peptide standards are made available to the research community. Protein expression measurements for heat shock proteins, including a comparison with ELISA and monitoring response to the HSP90 inhibitor, 17-DMAG, are used to illustrate the components of the QuAD and its potential utility. Conclusions and Clinical Relevance This resource enables quantitative assessment of protein components of signaling pathways and biological processes and holds promise for systematic investigation of treatment responses in cancer. PMID:21656910

Minimum-noise production of translation factor eIF4G maps to a mechanistically determined optimal rate control window for protein synthesis

PubMed Central

Meng, Xiang; Firczuk, Helena; Pietroni, Paola; Westbrook, Richard; Dacheux, Estelle; Mendes, Pedro; McCarthy, John E.G.

2017-01-01

Gene expression noise influences organism evolution and fitness. The mechanisms determining the relationship between stochasticity and the functional role of translation machinery components are critical to viability. eIF4G is an essential translation factor that exerts strong control over protein synthesis. We observe an asymmetric, approximately bell-shaped, relationship between the average intracellular abundance of eIF4G and rates of cell population growth and global mRNA translation, with peak rates occurring at normal physiological abundance. This relationship fits a computational model in which eIF4G is at the core of a multi-component–complex assembly pathway. This model also correctly predicts a plateau-like response of translation to super-physiological increases in abundance of the other cap-complex factors, eIF4E and eIF4A. Engineered changes in eIF4G abundance amplify noise, demonstrating that minimum stochasticity coincides with physiological abundance of this factor. Noise is not increased when eIF4E is overproduced. Plasmid-mediated synthesis of eIF4G imposes increased global gene expression stochasticity and reduced viability because the intrinsic noise for this factor influences total cellular gene noise. The naturally evolved eIF4G gene expression noise minimum maps within the optimal activity zone dictated by eIF4G's mechanistic role. Rate control and noise are therefore interdependent and have co-evolved to share an optimal physiological abundance point. PMID:27928055

Cloning, expression and nuclear localization of human NPM3, a member of the nucleophosmin/nucleoplasmin family of nuclear chaperones

PubMed Central

Shackleford, Gregory M; Ganguly, Amit; MacArthur, Craig A

2001-01-01

Background Studies suggest that the related proteins nucleoplasmin and nucleophosmin (also called B23, NO38 or numatrin) are nuclear chaperones that mediate the assembly of nucleosomes and ribosomes, respectively, and that these activities are accomplished through the binding of basic proteins via their acidic domains. Recently discovered and less well characterized members of this family of acidic phosphoproteins include mouse nucleophosmin/nucleoplasmin 3 (Npm3) and Xenopus NO29. Here we report the cloning and initial characterization of the human ortholog of Npm3. Results Human genomic and cDNA clones of NPM3 were isolated and sequenced. NPM3 lies 5.5 kb upstream of FGF8 and thus maps to chromosome 10q24-26. In addition to amino acid similarities, NPM3 shares many physical characteristics with the nucleophosmin/nucleoplasmin family, including an acidic domain, multiple potential phosphorylation sites and a putative nuclear localization signal. Comparative analyses of 14 members of this family from various metazoans suggest that Xenopus NO29 is a candidate ortholog of human and mouse NPM3, and they further group both proteins closer with the nucleoplasmins than with the nucleophosmins. Northern blot analysis revealed that NPM3 was strongly expressed in all 16 human tissues examined, with especially robust expression in pancreas and testis; lung displayed the lowest level of expression. An analysis of subcellular fractions of NIH3T3 cells expressing epitope-tagged NPM3 revealed that NPM3 protein was localized solely in the nucleus. Conclusions Human NPM3 is an abundant and widely expressed protein with primarily nuclear localization. These biological activities, together with its physical relationship to the chaparones nucleoplasmin and nucleophosmin, are consistent with the proposed function of NPM3 as a molecular chaperone functioning in the nucleus. PMID:11722795

Soluble cysteine-rich tick saliva proteins Salp15 and Iric-1 from E. coli

PubMed Central

Kolb, Philipp; Vorreiter, Jolanta; Habicht, Jüri; Bentrop, Detlef; Wallich, Reinhard; Nassal, Michael

2014-01-01

Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive cysteine-rich secretory glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodesricinus. All tick-borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmission. Hence, Salp15 proteins are attractive targets for anti-tick vaccines that also target borreliae. However, recombinant Salp proteins are not accessible in sufficient quantity for either vaccine manufacturing or for structural characterization. As an alternative to low-yield eukaryotic systems, we investigated cytoplasmic expression in Escherichia coli, even though this would not result in glycosylation. His-tagged Salp15 was efficiently expressed but insoluble. Among the various solubility-enhancing protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric-1 fusions. Easily accessible mutants enabled epitope mapping of two monoclonal antibodies that, importantly, cross-react with glycosylated Salp15, and revealed interaction sites with OspC. Free Salp15 and Iric-1 from protease-cleavable fusions, despite limited solubility, allowed the recording of 1H–15N 2D NMR spectra, suggesting partial folding of the wild-type proteins but not of Cys-free variants. Fusion to the NMR-compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in 13C/15N double-labeled Iric-1. Together, E. coli expression of appropriately fused Salp15 proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant tick proteins. PMID:25628987

Soluble cysteine-rich tick saliva proteins Salp15 and Iric-1 from E. coli.

PubMed

Kolb, Philipp; Vorreiter, Jolanta; Habicht, Jüri; Bentrop, Detlef; Wallich, Reinhard; Nassal, Michael

2015-01-01

Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive cysteine-rich secretory glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodes ricinus. All tick-borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmission. Hence, Salp15 proteins are attractive targets for anti-tick vaccines that also target borreliae. However, recombinant Salp proteins are not accessible in sufficient quantity for either vaccine manufacturing or for structural characterization. As an alternative to low-yield eukaryotic systems, we investigated cytoplasmic expression in Escherichia coli, even though this would not result in glycosylation. His-tagged Salp15 was efficiently expressed but insoluble. Among the various solubility-enhancing protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric-1 fusions. Easily accessible mutants enabled epitope mapping of two monoclonal antibodies that, importantly, cross-react with glycosylated Salp15, and revealed interaction sites with OspC. Free Salp15 and Iric-1 from protease-cleavable fusions, despite limited solubility, allowed the recording of (1)H-(15)N 2D NMR spectra, suggesting partial folding of the wild-type proteins but not of Cys-free variants. Fusion to the NMR-compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in (13)C/(15)N double-labeled Iric-1. Together, E. coli expression of appropriately fused Salp15 proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant tick proteins.

Patterns of gene expression associated with recovery and injury in heat-stressed rats.

PubMed

Stallings, Jonathan D; Ippolito, Danielle L; Rakesh, Vineet; Baer, Christine E; Dennis, William E; Helwig, Bryan G; Jackson, David A; Leon, Lisa R; Lewis, John A; Reifman, Jaques

2014-12-03

The in vivo gene response associated with hyperthermia is poorly understood. Here, we perform a global, multiorgan characterization of the gene response to heat stress using an in vivo conscious rat model. We heated rats until implanted thermal probes indicated a maximal core temperature of 41.8°C (Tc,Max). We then compared transcriptomic profiles of liver, lung, kidney, and heart tissues harvested from groups of experimental animals at Tc,Max, 24 hours, and 48 hours after heat stress to time-matched controls kept at an ambient temperature. Cardiac histopathology at 48 hours supported persistent cardiac injury in three out of six animals. Microarray analysis identified 78 differentially expressed genes common to all four organs at Tc,Max. Self-organizing maps identified gene-specific signatures corresponding to protein-folding disorders in heat-stressed rats with histopathological evidence of cardiac injury at 48 hours. Quantitative proteomics analysis by iTRAQ (isobaric tag for relative and absolute quantitation) demonstrated that differential protein expression most closely matched the transcriptomic profile in heat-injured animals at 48 hours. Calculation of protein supersaturation scores supported an increased propensity of proteins to aggregate for proteins that were found to be changing in abundance at 24 hours and in animals with cardiac injury at 48 hours, suggesting a mechanistic association between protein misfolding and the heat-stress response. Pathway analyses at both the transcript and protein levels supported catastrophic deficits in energetics and cellular metabolism and activation of the unfolded protein response in heat-stressed rats with histopathological evidence of persistent heat injury, providing the basis for a systems-level physiological model of heat illness and recovery.

Investigation of the effects of the level of glycemic control on erectile function and pathophysiological mechanisms in diabetic rats.

PubMed

Cho, Sung Yong; Chai, Ji Sun; Lee, Sun Hee; Park, Kwanjin; Paick, Jae-Seung; Kim, Soo Woong

2012-06-01

Poor glycemic control is associated with erectile dysfunction (ED); however, differences in ED according to the level of glycemic control have been poorly investigated. The aim of this paper is to investigate the change in erectile function according to the level of glycemic control and to clarify the pathophysiological mechanism of diabetes-associated ED. Streptozotocin was injected into 55 male Sprague-Dawley rats classified into four groups: control (group 1), diabetes with multiple insulin injections (group 2), diabetes with a single injection (group 3), and untreated diabetes (group 4). Daily insulin injections in groups 2 and 3 were administered for 4 weeks after 10 weeks of diabetic induction. The main outcome measures are the anova or Kruskal-Wallis tests to evaluate glycosylated hemoglobin (HbA1c), testosterone levels, the ratios of intracavernosal pressure to mean arterial pressure (ICP/MAP), area under the ICP curve to MAP (AUC/MAP), and changes in cavernous tissue and protein expression related to Rho kinase and nitric oxide pathways. HbA1c levels were different between pairs of groups. Group 4 showed the lowest erectile parameters and group 2 showed near normal level. No differences in erectile parameters were found between groups 1 and 2 or between groups 3 and 4, except the ratio of AUC to MAP for group 1 was significantly higher than that of group 2 (20 Hz stimulation). Decrease in erectile function of group 2 was related to decreased expression of nitrergic nitric oxide synthase or decreased testosterone level compared with group 1. Groups 2 and 3 showed significant differences in erectile parameters, which were associated with difference in apoptotic index. Groups 3 and 4 showed no differences in erectile parameters, although these groups had significant differences in apoptotic index, smooth muscle component, and protein expression ratios of phosphorylated to total myosin phosphatase target subunit 1, endothelial nitric oxide synthase, and Akt. Improvement in glycemic control assists recovery from diabetes-associated ED; however, only tight glycemic control can provide recovery from ED to a near normal status. © 2012 International Society for Sexual Medicine.