Interaction of Low Molecular Weight Hyaluronan (LMW-HA) with CD44 and Toll-Like Receptors Promotes the Actin Filament-Associated Protein (AFAP-110)-Actin Binding and MyD88-NFκB Signaling Leading to Pro-inflammatory Cytokine/Chemokine Production and Breast Tumor Invasion

PubMed Central

Bourguignon, Lilly Y.W.; Wong, Gabriel; Earle, Christine A.; Xia, Weiliang

2011-01-01

Both high and low molecular weight hyaluronan (HMW-HA vs. LMW-HA) exist in various tissues and cells. In this study we investigated LMW-HA-mediated CD44 interaction with Toll-like receptors (TLRs), the actin filament-associated protein (AFAP-110) and a myeloid differentiation factor (MyD88) in breast tumor cells (MDA-MB-231 cells). Our data indicate that LMW-HA (but not HMW-HA) preferentially stimulates a physical association between CD44 and TLRs followed by a concomitant recruitment of AFAP-110 and MyD88 into receptor-containing complexes in breast tumor cells. LMW-HA-activated AFAP-110 then binds to F-actin resulting in MyD88/NF-κB nuclear translocation, NF-κB-specific transcription and target gene (IL-1β and IL-8) expression. These signaling events lead to pro-inflammatory cytokine/chemokine production in the breast tumor cells. AFAP-110-F-actin (activated by LMW-HA) also promotes tumor cell invasion. Downregulation of AFAP-110 or MyD88 by transfecting breast tumor cells with AFAP-110 siRNA or MyD88 siRNA, respectively not only blocks the ability of LMW-HA to stimulate AFAP-110-actin function, but also impairs MyD88-NF-κB nuclear translocation and NF-κB transcriptional activation. Consequently, both IL-1β/IL-8 production and tumor cell invasion are impaired. Taken together, these findings suggest that LMW-HA plays an important role in CD44-TLR-associated AFAP-110-actin interaction and MyD88-NF-κB signaling required for tumor cell behaviors which may contribute to the progression of breast cancer. PMID:22031535

Ectopic Expression of GsPPCK3 and SCMRP in Medicago sativa Enhances Plant Alkaline Stress Tolerance and Methionine Content

PubMed Central

Zhao, Yang; Zhao, Chaoyue; DuanMu, Huizi; Yu, Yang; Ji, Wei; Zhu, Yanming

2014-01-01

So far, it has been suggested that phosphoenolpyruvate carboxylases (PEPCs) and PEPC kinases (PPCKs) fulfill several important non-photosynthetic functions. However, the biological functions of soybean PPCKs, especially in alkali stress response, are not yet well known. In previous studies, we constructed a Glycine soja transcriptional profile, and identified three PPCK genes (GsPPCK1, GsPPCK2 and GsPPCK3) as potential alkali stress responsive genes. In this study, we confirmed the induced expression of GsPPCK3 under alkali stress and investigated its tissue expression specificity by using quantitative real-time PCR analysis. Then we ectopically expressed GsPPCK3 in Medicago sativa and found that GsPPCK3 overexpression improved plant alkali tolerance, as evidenced by lower levels of relative ion leakage and MDA content and higher levels of chlorophyll content and root activity. In this respect, we further co-transformed the GsPPCK3 and SCMRP genes into alfalfa, and demonstrated the increased alkali tolerance of GsPPCK3-SCMRP transgenic lines. Further investigation revealed that GsPPCK3-SCMRP co-overexpression promoted the PEPC activity, net photosynthetic rate and citric acid content of transgenic alfalfa under alkali stress. Moreover, we also observed the up-regulated expression of PEPC, CS (citrate synthase), H+-ATPase and NADP-ME genes in GsPPCK3-SCMRP transgenic alfalfa under alkali stress. As expected, we demonstrated that GsPPCK3-SCMRP transgenic lines displayed higher methionine content than wild type alfalfa. Taken together, results presented in this study supported the positive role of GsPPCK3 in plant response to alkali stress, and provided an effective way to simultaneously improve plant alkaline tolerance and methionine content, at least in legume crops. PMID:24586886

Low molecular weight (LMW) heparin inhibits injury-induced femoral artery remodeling in mouse via upregulating CD44 expression.

PubMed

Zhao, Gaofeng; Shaik, Rahamthulla S; Zhao, Hang; Beagle, John; Kuo, Shuennwen; Hales, Charles A

2011-05-01

The mechanism of postangioplasty restenosis remains poorly understood. Low molecular weight (LMW) heparin has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), which is the principal characteristic of restenosis. Studies have shown that LMW heparin could bind to CD44. We hypothesized that LMW heparin might modulate CD44 expression thereby decreasing vascular remodeling. Vascular remodeling was induced in CD44(+/+) and CD44(-/-) mice and treated with LMW heparin. The arteries were harvested for histologic assessment and determination of CD44 expression. Bone marrow transplantation was introduced to further explore the role and functional sites of CD44. Effects of LMW heparin on growth capacity, CD44 expression were further studied using the cultured mouse VSMCs. Transluminal injury induced remarkable remodeling in mouse femoral artery (sham wall thickness percentage [WT%]: 3.4 ± 1.2% vs injury WT%: 31.8 ± 4.7%; P < .001). LMW heparin reduced the remodeling significantly (WT%: 17.8 ± 3.5%, P < .005). CD44(-/-) mice demonstrated considerably thicker arterial wall remodeling (WT%: 46.2 ± 7.6%, P = .0035), and CD44-chimeric mice exhibited equal contributions of the local and circulating CD44 signal to the neointima formation. LMW heparin markedly upregulated CD44 expression in the injured femoral arteries. In vitro, LMW heparin decreased mouse VSMC growth capacity and upregulated its CD44 expression simultaneously in a dose-dependent and time-dependent manner, which could be partially blocked by CD44 inhibitor. LMW heparin inhibits injury-induced femoral artery remodeling, at least partially, by upregulating CD44 expression. Copyright © 2011. Published by Mosby, Inc.

The Glycine soja NAC transcription factor GsNAC019 mediates the regulation of plant alkaline tolerance and ABA sensitivity.

PubMed

Cao, Lei; Yu, Yang; Ding, Xiaodong; Zhu, Dan; Yang, Fan; Liu, Beidong; Sun, Xiaoli; Duan, Xiangbo; Yin, Kuide; Zhu, Yanming

2017-10-01

Overexpression of Gshdz4 or GsNAC019 enhanced alkaline tolerance in transgenic Arabidopsis. We proved that Gshdz4 up-regulated both GsNAC019 and GsRD29B but GsNAC019 may repress the GsRD29B expression under alkaline stress. Wild soybean (Glycine soja) has a high tolerance to environmental challenges. It is a model species for dissecting the molecular mechanisms of salt-alkaline stresses. Although many NAC transcription factors play important roles in response to multiple abiotic stresses, such as salt, osmotic and cold, their mode of action in alkaline stress resistance is largely unknown. In our study, we identified a G. soja NAC gene, GsNAC019, which is a homolog of the Arabidopsis AtNAC019 gene. GsNAC019 was highly up-regulated by 50 mM NaHCO 3 treatment in the roots of wild soybean. Further investigation showed that a well-characterized transcription factor, Gshdz4 protein, bound the cis-acting element sequences (CAATA/TA), which are located in the promoter of the AtNAC019/GsNAC019 genes. Overexpression of Gshdz4 positively regulated AtNAC019 expression in transgenic Arabidopsis, implying that AtNAC019/GsNAC019 may be the target genes of Gshdz4. GsNAC019 was demonstrated to be a nuclear-localized protein in onion epidermal cells and possessed transactivation activity in yeast cells. Moreover, overexpression of GsNAC019 in Arabidopsis resulted in enhanced tolerance to alkaline stress at the seedling and mature stages, but reduced ABA sensitivity. The closest Arabidopsis homolog mutant plants of Gshdz4, GsNAC019 and GsRD29B containing athb40, atnac019 and atrd29b were sensitive to alkaline stress. Overexpression or the closest Arabidopsis homolog mutant plants of the GsNAC019 gene in Arabidopsis positively or negatively regulated the expression of stress-related genes, such as AHA2, RD29A/B and KIN1. Moreover, this mutation could phenotypically promoted or compromised plant growth under alkaline stress, implying that GsNAC019 may contribute to alkaline stress

Isolation and characterization of a GS2 gene in melon (Cucumis melo L.) and its expression patterns under the fertilization of different forms of N.

PubMed

Deng, Yang-Wu; Zhang, Yi-Dong; Chen, Yi; Wang, Shu; Tang, Dong-Mei; Huang, Dan-Feng

2010-01-01

We isolated a novel glutamine synthetase (GS, EC 6.3.1.2) gene M-GS2 (accession: AY773090) by the RACE approach from melon. The full-length cDNA of M-GS2 is 1807 bp and contains a 1296 bp open reading frame (ORF) encoding 432 amino acids. The deduced protein contains conserved structural domains among plant GS2 proteins and shares extensive sequence homology with GS2 enzymes from other higher plants. M-GS2 expresses with specificity in leaf, and identification of a chloroplast transit peptide (cTP) in M-GS2 suggests that it localizes to the chloroplast. As shown by real-time quantitative PCR, distinct forms of nitrogen (N) found in fertilizers transcriptionally regulated M-GS2 differently. Ammonium and nitrate feeding only significantly regulated M-GS2 transcripts in leaf; starving (0.75 mM) or moderate (3.75 mM) N levels dramatically increased M-GS2 transcripts for 1 day, decreasing to a constant low level after 2-3 days, while sufficient N level (7.5 mM) had a minor effect throughout 3 days compared to controls. Glutamate feeding, however, not only significantly regulated M-GS2 transcripts in leaf (decreased initially then increased to higher levels than controls), but also in root, where it was up-regulated continuously. Our results suggested that M-GS2 is the first GS gene cloned and characterized in melon and melon responds to the variations in N fertilization by differentially expressing M-GS2.

Identification of Low Molecular Weight Glutenin Alleles by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) in Common Wheat (Triticum aestivum L.).

PubMed

Wang, Aili; Liu, Li; Peng, Yanchun; Islam, Shahidul; Applebee, Marie; Appels, Rudi; Yan, Yueming; Ma, Wujun

2015-01-01

Low molecular weight glutenin subunits (LMW-GS) play an important role in determining dough properties and breadmaking quality. However, resolution of the currently used methodologies for analyzing LMW-GS is rather low which prevents an efficient use of genetic variations associated with these alleles in wheat breeding. The aim of the current study is to evaluate and develop a rapid, simple, and accurate method to differentiate LMW-GS alleles using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A set of standard single LMW-GS allele lines as well as a suite of well documented wheat cultivars were collected from France, CIMMYT, and Canada. Method development and optimization were focused on protein extraction procedures and MALDI-TOF instrument settings to generate reproducible diagnostic spectrum peak profiles for each of the known wheat LMW-GS allele. Results revealed a total of 48 unique allele combinations among the studied genotypes. Characteristic MALDI-TOF peak patterns were obtained for 17 common LMW-GS alleles, including 5 (b, a or c, d, e, f), 7 (a, b, c, d or i, f, g, h) and 5 (a, b, c, d, f) patterns or alleles for the Glu-A3, Glu-B3, and Glu-D3 loci, respectively. In addition, some reproducible MALDI-TOF peak patterns were also obtained that did not match with any known alleles. The results demonstrated a high resolution and throughput nature of MALDI-TOF technology in analyzing LMW-GS alleles, which is suitable for application in wheat breeding programs in processing a large number of wheat lines with high accuracy in limited time. It also suggested that the variation of LMW-GS alleles is more abundant than what has been defined by the current nomenclature system that is mainly based on SDS-PAGE system. The MALDI-TOF technology is useful to differentiate these variations. An international joint effort may be needed to assign allele symbols to these newly identified alleles and determine their effects on end

Identification of Low Molecular Weight Glutenin Alleles by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) in Common Wheat (Triticum aestivum L.)

PubMed Central

Islam, Shahidul; Applebee, Marie; Appels, Rudi; Yan, Yueming; Ma, Wujun

2015-01-01

Low molecular weight glutenin subunits (LMW-GS) play an important role in determining dough properties and breadmaking quality. However, resolution of the currently used methodologies for analyzing LMW-GS is rather low which prevents an efficient use of genetic variations associated with these alleles in wheat breeding. The aim of the current study is to evaluate and develop a rapid, simple, and accurate method to differentiate LMW-GS alleles using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A set of standard single LMW-GS allele lines as well as a suite of well documented wheat cultivars were collected from France, CIMMYT, and Canada. Method development and optimization were focused on protein extraction procedures and MALDI-TOF instrument settings to generate reproducible diagnostic spectrum peak profiles for each of the known wheat LMW-GS allele. Results revealed a total of 48 unique allele combinations among the studied genotypes. Characteristic MALDI-TOF peak patterns were obtained for 17 common LMW-GS alleles, including 5 (b, a or c, d, e, f), 7 (a, b, c, d or i, f, g, h) and 5 (a, b, c, d, f) patterns or alleles for the Glu-A3, Glu-B3, and Glu-D3 loci, respectively. In addition, some reproducible MALDI-TOF peak patterns were also obtained that did not match with any known alleles. The results demonstrated a high resolution and throughput nature of MALDI-TOF technology in analyzing LMW-GS alleles, which is suitable for application in wheat breeding programs in processing a large number of wheat lines with high accuracy in limited time. It also suggested that the variation of LMW-GS alleles is more abundant than what has been defined by the current nomenclature system that is mainly based on SDS-PAGE system. The MALDI-TOF technology is useful to differentiate these variations. An international joint effort may be needed to assign allele symbols to these newly identified alleles and determine their effects on end

GsCBRLK, a calcium/calmodulin-binding receptor-like kinase, is a positive regulator of plant tolerance to salt and ABA stress.

PubMed

Yang, Liang; Ji, Wei; Zhu, Yanming; Gao, Peng; Li, Yong; Cai, Hua; Bai, Xi; Guo, Dianjing

2010-05-01

Calcium/calmodulin-dependent kinases play vital roles in protein phosphorylation in eukaryotes, yet little is known about the phosphorylation process of calcium/calmodulin-dependent protein kinase and its role in stress signal transduction in plants. A novel plant-specific calcium-dependent calmodulin-binding receptor-like kinase (GsCBRLK) has been isolated from Glycine soja. A subcellular localization study using GFP fusion protein indicated that GsCBRLK is localized in the plasma membrane. Binding assays demonstrated that calmodulin binds to GsCBRLK with an affinity of 25.9 nM in a calcium-dependent manner and the binding motif lies between amino acids 147 to169 within subdomain II of the kinase domain. GsCBRLK undergoes autophosphorylation and Myelin Basis Protein phosphorylation in the presence of calcium. It was also found that calcium/calmodulin positively regulates GsCBRLK kinase activity through direct interaction between the calmodulin-binding domain and calmodulin. So, it is likely that GsCBRLK responds to an environmental stimulus in two ways: by increasing the protein expression level and by regulating its kinase activity through the calcium/calmodulin complex. Furthermore, cold, salinity, drought, and ABA stress induce GsCBRLK gene transcripts. Over-expression of GsCBRLK in transgenic Arabidopsis resulted in enhanced plant tolerance to high salinity and ABA and increased the expression pattern of a number of stress gene markers in response to ABA and high salt. These results identify GsCBRLK as a molecular link between the stress- and ABA-induced calcium/calmodulin signal and gene expression in plant cells.

Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis.

PubMed

Feary, Marc; Racher, Andrew J; Young, Robert J; Smales, C Mark

2017-01-01

In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for

Low-molecular-weight hyaluronan (LMW-HA) accelerates lymph node metastasis of melanoma cells by inducing disruption of lymphatic intercellular adhesion.

PubMed

Du, Yan; Cao, Manlin; Liu, Yiwen; He, Yiqing; Yang, Cuixia; Wu, Man; Zhang, Guoliang; Gao, Feng

2016-01-01

Endothelial integrity defects initiate lymphatic metastasis of tumor cells. Low-molecular-weight hyaluronan (LMW-HA) derived from plasma and interstitial fluid was reported to be associated with tumor lymphatic metastasis. In addition, LMW-HA was proved to disrupt lymphatic vessel endothelium integrity, thus promoting lymphatic metastasis of tumor cells. Until now, there are few reports on how LMW-HA modulates lymphatic endothelial cells adhesion junctions and affects cancer cells metastasizing into lymph vessels. The aim of our study is to unravel the novel mechanism of LMW-HA in mediating tumor lymphatic metastasis. Here, we employed a melanoma metastasis model to investigate whether LMW-HA facilitates tumor cells transferring from foci to remote lymph nodes by disrupting the lymphatic endothelial integrity. Our data indicate that LMW-HA significantly induces metastasis of melanoma cells to lymph nodes and accelerates interstitial-lymphatic flow in vivo . Further experiments show that increased migration of melanoma cells across human dermal lymphatic endothelial cell (HDLEC) monolayers is accompanied by impaired lymphatic endothelial barrier function and increased permeability. The mechanism study reveals that VE-cadherin-β-catenin pathway and relevant signals are involved in modulating the interactions between endothelial cells and that a significant inhibition of lymphatic endothelium disruption is observed when antibodies to the LMW-HA receptor (LYVE-1) are present. Thus, our findings demonstrate a disruptive effect of LMW-HA on lymphatic endothelium continuity which leads to a promotion on melanoma lymphatic metastasis and also suggest a cellular signaling mechanism associated with VE-cadherin-mediated lymphatic intercellular junctions.

Follicular variant of papillary thyroid carcinoma presenting as toxic nodule in an adolescent: coexistent polymorphism of the TSHR and Gsα genes.

PubMed

Ruggeri, Rosaria Maddalena; Campennì, Alfredo; Giovinazzo, Salvatore; Saraceno, Giovanna; Vicchio, Teresa Manuela; Carlotta, Dario; Cucinotta, Maria Paola; Micali, Carmelo; Trimarchi, Francesco; Tuccari, Giovanni; Baldari, Sergio; Benvenga, Salvatore

2013-02-01

Autonomously functioning, "hot", thyroid nodules are not common in children and adolescents. Such nodules are not considered alarming because they are assumed to be benign adenomas. Herein, we present a 15-year-old girl with a papillary thyroid carcinoma of 3.5 cm in diameter, which was functionally autonomous and scintigraphically hot. The patient, initially referred to our Endocrine Unit because of a thyroid nodule, returned 6 months later for symptoms of hyperthyroidism. Hyperthyroidism was confirmed biochemically. Radioactive iodine ((131)I) thyroid scintigraphy was consistent with an autonomous thyroid nodule. As per guidelines, the patient underwent surgery and a pathological examination revealed papillary carcinoma, follicular variant. The excised nodule was examined for activating mutations of the thyrotropin receptor (TSHR), Gsα (GNAS1), H-RAS, N-RAS, K-RAS, and BRAF genes by direct sequencing. No mutations were found. Nevertheless, two combined nonfunctioning mutations were detected: a single-nucleotide polymorphism (SNP) of the TSHR gene, in exon 7, at codon 187 (AAT→AAC, both encoding asparagine), and a SNP within exon 8 of the Gsα gene at codon 185 (ATC→ATT, both encoding isoleucine). Both SNPs were also identified in the germline DNA of the patient. The same SNPs were sought in the parents and brother of our patient. Her father was heterozygous for the TSHR SNP, her mother heterozygous for the Gsα SNP, and her brother was wild type. This case demonstrates that the presence of hyperfunctioning thyroid nodule(s) does not rule out cancer and warrants careful evaluation, especially in childhood and adolescence to overlook malignancy.

Ligand-Mediated Activation of an Engineered Gs G Protein-Coupled Receptor in Osteoblasts Increases Trabecular Bone Formation

PubMed Central

Hsiao, Edward C.; Millard, Susan M.; Louie, Alyssa; Huang, Yong; Conklin, Bruce R.; Nissenson, Robert A.

2010-01-01

Age-dependent changes in skeletal growth play important roles in regulating skeletal expansion and in the course of many diseases affecting bone. How G protein-coupled receptor (GPCR) signaling affects these changes is poorly understood. Previously, we described a mouse model expressing Rs1, an engineered receptor with constitutive Gs activity. Rs1 expression in osteoblasts from gestation induced a dramatic age-dependent increase in trabecular bone with features resembling fibrous dysplasia; however, these changes were greatly minimized if Rs1 expression was delayed until after puberty. To further investigate whether ligand-induced activation of the Gs-GPCR pathway affects bone formation in adult mice, we activated Rs1 in adult mice with the synthetic ligand RS67333 delivered continuously via an osmotic pump or intermittently by daily injections. We found that osteoblasts from adult animals can be stimulated to form large amounts of bone, indicating that adult mice are sensitive to the dramatic bone- forming actions of Gs signaling in osteoblasts. In addition, our results show that intermittent and continuous activation of Rs1 led to structurally similar but quantitatively different degrees of trabecular bone formation. These results indicate that activation of a Gs-coupled receptor in osteoblasts of adult animals by either intermittent or continuous ligand administration can increase trabecular bone formation. In addition, osteoblasts located at the bone epiphyses may be more responsive to Gs signaling than osteoblasts at the bone diaphysis. This model provides a powerful tool for investigating the effects of ligand-activated Gs-GPCR signaling on dynamic bone growth and remodeling. PMID:20150184

GS-2, a pyrazolo[1,5-a]indole derivative with inhibitory activity of topoisomerases, exerts its potent cytotoxic activity by ROS generation.

PubMed

Ji, Yuan Yuan; Zhu, Yong Ming; Wang, Jian Wen

2013-11-01

Pyrazolo[1,5-a]indole derivatives, a new type of topoisomerase (topo) inhibitor, demonstrate a broad spectrum of antitumor activities. However, the mechanism underlying the induced cytotoxicity remains unclear. In this study, we investigated whether GS-2, one of the derivatives, altered the levels of ROS in breast cancer MDA-231 cells and whether these ROS contributed to the observed antitumoral activity. Our data revealed that GS-2 caused a time- and dose-dependent elevation of intracellular ROS level in MDA-231 cells. GS-2 subsequently elicited notable inhibition on the expression of topos, DNA damage, activation of caspase-3, -9. The loss of mitochondrial membrane potential (MMP) was observed during the induction. The addition of N-acetyl cysteine (NAC, a well-known antioxidant) could effectively attenuate the GS-2-induced ROS enhancement and subsequent apoptosis. NAC attenuated the induced inhibition on expression of topos, indicating that topos might be the target of GS-2-induced ROS. The finding of the induced ROS provides new evidence for the molecular mechanisms of antitumor activity of pyrazolo[1,5-a]indole derivatives. Copyright © 2013 Elsevier B.V. All rights reserved.

Juxta-articular myxoma and intramuscular myxoma are two distinct entities. Activating Gs alpha mutation at Arg 201 codon does not occur in juxta-articular myxoma.

PubMed

Okamoto, Sumika; Hisaoka, Masanori; Meis-Kindblom, Jeanne M; Kindblom, Lars-Gunnar; Hashimoto, Hiroshi

2002-01-01

Juxta-articular myxoma is a rare myxoid tumor of soft tissue that bears a close histologic resemblance to intramuscular myxoma but is distinguished from the latter by its clinical setting and behavior. Activating missense mutations at the Arg 201 codon of the Gs alpha gene ultimately leading to increased levels of cyclic adenosine monophosphate have been implicated in McCune-Albright syndrome and sporadic fibrous dysplasia of bone. Recently, we have demonstrated that the same Gs alpha mutations occur in intramuscular myxomas associated with fibrous dysplasia of bone (Mazabraud's syndrome) as well as in sporadic intramuscular myxoma. The overlapping histologic appearances of juxta-articular myxoma and intramuscular myxoma prompted us to investigate whether there is a relationship between the two entities. We studied this possibility by looking for Gs alpha mutations in juxta-articular myxoma using polymerase chain reaction (PCR) to amplify appropriate genomic DNA fragments extracted from formalin-fixed, paraffin-embedded specimens of five juxta-articular myxomas, followed by single-strand conformation polymorphism analysis. Using these techniques, no aberrant bands were detected in any of the five juxta-articular myxomas, indicating that they lack Gs alpha mutations. Moreover, DNA sequencing of the PCR products of two JAMs showed no abnormalities. We conclude that juxta-articular myxomas, in contrast to intramuscular myxomas, do not involve Arg 201 mutations of the Gs alpha gene, indicating that they represent distinct entities with different underlying molecular mechanisms.

LMW Heparin Prevents Increased Kidney Expression of Proinflammatory Mediators in (NZBxNZW)F1 Mice

PubMed Central

Kanapathippillai, Premasany; Rekvig, Ole Petter; Fenton, Kristin Andreassen

2013-01-01

We have previously demonstrated that continuous infusion of low molecular weight (LMW) heparin delays autoantibody production and development of lupus nephritis in (NZBxNZW)F1 (B/W) mice. In this study we investigated the effect of LMW heparin on renal cytokine and chemokine expression and on nucleosome-mediated activation of nucleosome-specific splenocytes. Total mRNA extracted from kidneys of heparin-treated or -untreated B/W mice was analysed by qPCR for the expression of several cytokines, chemokines, and Toll-like receptors. Splenocytes taken from B/W mice were stimulated with nucleosomes with or without the presence of heparin. Splenocyte cell proliferation as thymidine incorporation and the expression of costimulatory molecules and cell activation markers were measured. Heparin treatment of B/W mice reduced the in vivo expression of CCR2, IL1β, and TLR7 compared to untreated B/W mice. Nucleosome-induced cell proliferation of splenocytes was not influenced by heparin. The expression of CD80, CD86, CD69, CD25, CTLA-4, and TLR 2, 7, 8, and 9 was upregulated upon stimulation by nucleosomes, irrespective of whether heparin was added to the cell culture or not. In conclusion, treatment with heparin lowers the kidney expression of proinflammatory mediators in B/W mice but does not affect nucleosomal activation of splenocytes. PMID:24151519

Impact of concurrent overexpression of cytosolic glutamine synthetase (GS1) and sucrose phosphate synthase (SPS) on growth and development in transgenic tobacco.

PubMed

Seger, Mark; Gebril, Sayed; Tabilona, Jules; Peel, Amanda; Sengupta-Gopalan, Champa

2015-01-01

The outcome of simultaneously increasing SPS and GS activities in transgenic tobacco, suggests that sucrose is the major determinant of growth and development, and is not affected by changes in N assimilation. Carbon (C) and nitrogen (N) are the major components required for plant growth and the metabolic pathways for C and N assimilation are very closely interlinked. Maintaining an appropriate balance or ratio of sugar to nitrogen metabolites in the cell, is important for the regulation of plant growth and development. To understand how C and N metabolism interact, we manipulated the expression of key genes in C and N metabolism individually and concurrently and checked for the repercussions. Transgenic tobacco plants with a cytosolic soybean glutamine synthetase (GS1) gene and a sucrose phosphate synthase (SPS) gene from maize, both driven by the CaMV 35S promoter were produced. Co-transformants, with both the transgenes were produced by sexual crosses. While GS is the key enzyme in N assimilation, involved in the synthesis of glutamine, SPS plays a key role in C metabolism by catalyzing the synthesis of sucrose. Moreover, to check if nitrate has any role in this interaction, the plants were grown under both low and high nitrogen. The SPS enzyme activity in the SPS and SPS/GS1 co-transformants were the same under both nitrogen regimens. However, the GS activity was lower in the co-transformants compared to the GS1 transformants, specifically under low nitrogen conditions. The GS1/SPS transformants showed a phenotype similar to the SPS transformants, suggesting that sucrose is the major determinant of growth and development in tobacco, and its effect is only marginally affected by increased N assimilation. Sucrose may be functioning in a metabolic capacity or as a signaling molecule.

Growth hormone-releasing hormone resistance in pseudohypoparathyroidism type ia: new evidence for imprinting of the Gs alpha gene.

PubMed

Mantovani, Giovanna; Maghnie, Mohamad; Weber, Giovanna; De Menis, Ernesto; Brunelli, Valeria; Cappa, Marco; Loli, Paola; Beck-Peccoz, Paolo; Spada, Anna

2003-09-01

Heterozygous inactivating mutations in the Gs alpha gene cause Albright's hereditary osteodystrophy. Consistent with the observation that only maternally inherited mutations lead to resistance to hormone action [pseudohypoparathyroidism type Ia (PHP Ia)], recent studies provided evidence for a predominant maternal origin of Gs alpha transcripts in endocrine organs, such as thyroid, gonad, and pituitary. The aim of this study was to investigate the presence of pituitary resistance to hypothalamic hormones acting via Gs alpha-coupled receptors in patients with PHP Ia. Six of nine patients showed an impaired GH responsiveness to GHRH plus arginine, consistent with a complete GH deficiency (GH peak from 2.6-8.6 microg/liter, normal > 16.5), and partial (GH peak 13.9 and 13.6 microg/liter) and normal responses were found in two and one patient, respectively. Accordingly, IGF-I levels were below and in the low-normal range in seven and two patients. All patients had a normal cortisol response to 1 microg ACTH test, suggesting a normal corticotroph function that was confirmed by a normal ACTH and cortisol response to CRH test in three patients. In conclusion, we report that in addition to PTH and TSH resistance, patients with PHP Ia display variable degrees of GHRH resistance, consistent with Gs alpha imprinting in human pituitary.

Assessing the osteoblast transcriptome in a model of enhanced bone formation due to constitutive Gs-G protein signaling in osteoblasts

PubMed Central

Wattanachanya, Lalita; Wang, Liping; Millard, Susan M.; Lu, Wei-Dar; O’Carroll, Dylan; Hsiao, Edward C.; Conklin, Bruce R.; Nissenson, Robert A.

2015-01-01

G protein–coupled receptor (GPCR) signaling in osteoblasts (OBs) is an important regulator of bone formation. We previously described a mouse model expressing Rs1, an engineered constitutively active Gs-coupled GPCR, under the control of the 2.3-kb Col I promoter. These mice showed a dramatic age-dependent increase in trabecular bone of femurs. Here, we further evaluated the effects of enhanced Gs signaling in OBs on intramembranous bone formation by examining calvariae of 1- and 9-week-old Col1(2.3)/Rs1 mice and characterized the in vivo gene expression specifically occurring in osteoblasts with activated Gs G protein–coupled receptor signaling, at the cellular level rather than in a whole bone. Rs1 calvariae displayed a dramatic increase in bone volume with partial loss of cortical structure. By immunohistochemistry, Osterix was detected in cells throughout the inter-trabecular space while Osteocalcin was expressed predominantly in cells along bone surfaces, suggesting the role of paracrine mediators secreted from OBs driven by 2.3-kb Col I promoter could influence early OB commitment, differentiation, and/or proliferation. Gene expression analysis of calvarial OBs revealed that genes affected by Rs1 signaling include those encoding proteins important for cell differentiation, cytokines and growth factors, angiogenesis, coagulation, and energy metabolism. The set of Gs-GPCRs and other GPCRs that may contribute to the observed skeletal phenotype and candidate paracrine mediators of the effect of Gs signaling in OBs were also determined. Our results identify novel detailed in vivo cellular changes of the anabolic response of the skeleton to Gs signaling in mature OBs. PMID:25704759

Enhancement of heterogeneous alkaline xylanase production in Pichia pastoris GS115

NASA Astrophysics Data System (ADS)

Zheng, Wei

2017-08-01

A series of strategies were applied to improve expression level of the recombinant alkaline xylanase from Bacillus pumilus G1-3 in Pichia pastoris GS115. Codon optimization of xylanase gene xynG1-3 from B. pumilus G1-3 were carried out for its heterogeneous expression in P. pastoris. The activity of xylanase encoded by optimized gene (xynG1-3-opt) was up to 33641 U/mL, which was 37% higher than that by wild-type (xynG1-3) gene. The results will greatly contribute to increasing the production of recombinant proteins in P. pastoris and improving the industrial production of the alkaline xylanase.

GsERF6, an ethylene-responsive factor from Glycine soja, mediates the regulation of plant bicarbonate tolerance in Arabidopsis.

PubMed

Yu, Yang; Liu, Ailin; Duan, Xiangbo; Wang, Sunting; Sun, Xiaoli; Duanmu, Huizi; Zhu, Dan; Chen, Chao; Cao, Lei; Xiao, Jialei; Li, Qiang; Nisa, Zaib Un; Zhu, Yanming; Ding, Xiaodong

2016-09-01

This is an original study focus on ERF gene response to alkaline stress. GsERF6 functions as transcription factor and significantly enhanced plant tolerance to bicarbonate (HCO 3 (-) ) in transgenic Arabidopsis . Alkaline stress is one of the most harmful, but little studied environmental factors, which negatively affects plant growth, development and yield. The cause of alkaline stress is mainly due to the damaging consequence of high concentration of the bicarbonate ion, high-pH, and osmotic shock to plants. The AP2/ERF family genes encode plant-specific transcription factors involved in diverse environmental stresses. However, little is known about their physiological functions, especially in alkaline stress responses. In this study, we functionally characterized a novel ERF subfamily gene, GsERF6 from alkaline-tolerant wild soybean (Glycine soja). In wild soybean, GsERF6 was rapidly induced by NaHCO3 treatment, and its overexpression in Arabidopsis enhanced transgenic plant tolerance to NaHCO3 challenge. Interestingly, GsERF6 transgenic lines also displayed increased tolerance to KHCO3 treatment, but not to high pH stress, implicating that GsERF6 may participate specifically in bicarbonate stress responses. We also found that GsERF6 overexpression up-regulated the transcription levels of bicarbonate-stress-inducible genes such as NADP-ME, H (+)-Ppase and H (+)-ATPase, as well as downstream stress-tolerant genes such as RD29A, COR47 and KINI. GsERF6 overexpression and NaHCO3 stress also altered the expression patterns of plant hormone synthesis and hormone-responsive genes. Conjointly, our results suggested that GsERF6 is a positive regulator of plant alkaline stress by increasing bicarbonate ionic resistance specifically, providing a new insight into the regulation of gene expression under alkaline conditions.

Guggulsterone (GS) inhibits smokeless tobacco and nicotine-induced NF-κB and STAT3 pathways in head and neck cancer cells.

PubMed

Macha, Muzafar A; Matta, Ajay; Chauhan, S S; Siu, K W Michael; Ralhan, Ranju

2011-03-01

Understanding the molecular pathways perturbed in smokeless tobacco- (ST) associated head and neck squamous cell carcinoma (HNSCC) is critical for identifying novel complementary agents for effective disease management. Activation of nuclear factor-kappaB (NF-κB) and cyclooxygenase-2 (COX-2) was reported in ST-associated HNSCC by us [Sawhney,M. et al. (2007) Expression of NF-kappaB parallels COX-2 expression in oral precancer and cancer: association with smokeless tobacco. Int. J. Cancer, 120, 2545-2556]. In search of novel agents for treatment of HNSCC, we investigated the potential of guggulsterone (GS), (4,17(20)-pregnadiene-3,16-dione), a biosafe nutraceutical, in inhibiting ST- and nicotine-induced activation of NF-κB and signal transducer and activator of transcription (STAT) 3 pathways in HNSCC cells. GS inhibited the activation of NF-κB and STAT3 proteins in head and neck cancer cells. This inhibition of NF-κB by GS resulted from decreased phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha the inhibitory subunit of NF-κB. Importantly, treatment of HNSCC cells with GS abrogated both ST- and nicotine-induced nuclear activation of NF-κB and pSTAT3 proteins and their downstream targets COX-2 and vascular endothelial growth factor. Furthermore, GS treatment decreased the levels of ST- and nicotine-induced secreted interleukin-6 in culture media of HNSCC cells. In conclusion, our findings demonstrated that GS treatment abrogates the effects of ST and nicotine on activation of NF-κB and STAT3 pathways in HNSCC cells that contribute to inflammatory and angiogenic responses as well as its progression and metastasis. These findings provide a biologic rationale for further clinical investigation of GS as an effective complementary agent for inhibiting ST-induced head and neck cancer.

Ectopic Expression of GsSRK in Medicago sativa Reveals Its Involvement in Plant Architecture and Salt Stress Responses.

PubMed

Sun, Mingzhe; Qian, Xue; Chen, Chao; Cheng, Shufei; Jia, Bowei; Zhu, Yanming; Sun, Xiaoli

2018-01-01

Receptor-like kinases (RLK) play fundamental roles in plant growth and stress responses. Compared with other RLKs, little information is provided concerning the S-locus LecRLK subfamily, which is characterized by an extracellular G-type lectin domain and an S-locus-glycop domain. Until now, the function of the G-type lectin domain is still unknown. In a previous research, we identified a Glycine soja S-locus LecRLK gene GsSRK , which conferred increased salt stress tolerance in transgenic Arabidopsis . In this study, to investigate the role of the G-type lectin domain and to breed transgenic alfalfa with superior salt stress tolerance, we transformed the full-length GsSRK ( GsSRK-f ) and a truncated version of GsSRK ( GsSRK-t ) deleting the G-type lectin domain into alfalfa. Our results showed that overexpression of GsSRK-t , but not GsSRK-f , resulted in changes of plant architecture, as evidenced by more branches but shorter shoots of GsSRK-t transgenic alfalfa, indicating a potential role of the extracellular G-type lectin domain in regulating plant architecture. Furthermore, we also found that transgenic alfalfa overexpressing either GsSRK-f or GsSRK-t showed increased salt stress tolerance, and GsSRK-t transgenic alfalfa displayed better growth (more branches and higher fresh weight) than GsSRK-f lines under salt stress. In addition, our results suggested that both GsSRK-f and GsSRK-t were involved in ion homeostasis, ROS scavenging, and osmotic regulation. Under salt stress, the Na + content in the transgenic lines was significantly lower, while the K + content was slightly higher than that in WT. Moreover, the transgenic lines displayed reduced ion leakage and MDA content, but increased SOD activity and proline content than WT. Notably, no obvious difference in these physiological indices was observed between GsSRK-f and GsSRK-t transgenic lines, implying that deletion of the GsSRK G-type lectin domain does not affect its physiological function in salt

Bioreducible Zinc(II)-Coordinative Polyethylenimine with Low Molecular Weight for Robust Gene Delivery of Primary and Stem Cells.

PubMed

Liu, Shuai; Zhou, Dezhong; Yang, Jixiang; Zhou, Hao; Chen, Jiatong; Guo, Tianying

2017-03-30

To transform common low-molecular-weight (LMW) cationic polymers, such as polyethylenimine (PEI), to highly efficient gene vectors would be of great significance but remains challenging. Because LMW cationic polymers perform far less efficiently than their high-molecular-weight counterparts, mainly due to weaker nucleic acid encapsulation, herein we report the design and synthesis of a dipicolylamine-based disulfide-containing zinc(II) coordinative module (Zn-DDAC), which is used to functionalize LMW PEI (M w ≈ 1800 Da) to give a non-viral vector (Zn-PD) with high efficiency and safety in primary and stem cells. Given its high phosphate binding affinity, Zn-DDAC can significantly promote the DNA packaging functionality of PEI 1.8k and improve the cellular uptake of formulated polyplexes, which is particularly critical for hard-to-transfect cell types. Furthermore, Zn-PD polymer can be cleaved by glutathione in cytoplasm to facilitate DNA release post internalization and diminish the cytotoxicity. Consequently, the optimal Zn-PD mediates 1-2 orders of magnitude higher gluciferase activity than commercial transfection reagents, Xfect and PEI 25k , across diverse cell types, including primary and stem cells. Our findings provide a valuable insight into the exploitation of LMW cationic polymers for gene delivery and demonstrate great promise for the development of next-generation non-viral vectors for clinically viable gene therapy.

BcGs1, a glycoprotein from Botrytis cinerea, elicits defence response and improves disease resistance in host plants.

PubMed

Zhang, Yi; Zhang, Yunhua; Qiu, Dewen; Zeng, Hongmei; Guo, Lihua; Yang, Xiufen

2015-02-20

In this study, a necrosis-inducing protein was purified from the culture filtrate of the necrotrophic fungus Botrytis cinerea BC-98 strain. Secreted proteins were collected and fractionated by liquid chromatography. The fraction with the highest necrosis-inducing activity was further purified. A glycoprotein named BcGs1 was identified by 2D electrophoresis and mass spectrometry. The BcGs1 protein consisted of 672 amino acids with a theoretical molecular weight of 70.487 kDa. Functional domain analysis indicated that BcGs1 was a glucan 1,4-alpha-glucosidase, a cell wall-degrading enzyme, with a Glyco_hydro_15 domain and a CBM20_glucoamylase domain. The BcGs1 protein caused necrotic lesions that mimicked a typical hypersensitive response and H2O2 production in tomato and tobacco leaves. BcGs1-treated plants exhibited resistance to B. cinerea, Pseudomonas syringae pv. tomato DC3000 and tobacco mosaic virus in systemic leaves. In addition, BcGs1 triggered elevation of the transcript levels of the defence-related genes PR-1a, TPK1b and Prosystemin. This is the first report of a Botrytis glucan 1,4-alpha-glucosidase triggering host plant immunity as an elicitor. These results lay a foundation for further study of the comprehensive interaction between plants and necrotrophic fungi. Copyright © 2015 Elsevier Inc. All rights reserved.

Investigation of structure and function of mitochondrial alcohol dehydrogenase isozyme III from Komagataella phaffii GS115.

PubMed

Zhang, Huaidong; Li, Qin; Wang, Lina; Chen, Yan

2018-05-01

Alcohol dehydrogenases (ADHs) catalyze the reversible oxidation of alcohol using NAD + or NADP + as cofactor. Three ADH homologues have been identified in Komagataella phaffii GS115 (also named Pichia pastoris GS115), ADH1, ADH2 and ADH3, among which adh3 is the only gene responsible for consumption of ethanol in Komagataella phaffii GS115. However, the relationship between structure and function of mitochondrial alcohol dehydrogenase isozyme III from Komagataella phaffii GS115 (KpADH3) is still not clear yet. KpADH3 was purified, identified and characterized by multiple biophysical techniques (Nano LC-MS/MS, Enzymatic activity assay, X-ray crystallography). The crystal structure of KpADH3, which was the first ADH structure from Komagataella phaffii GS115, was solved at 1.745 Å resolution. Structural analysis indicated that KpADH3 was the sole dimeric ADH structure with face-to-face orientation quaternary structure from yeast. The major structural different conformations located on residues 100-114 (the structural zinc binding loop) and residues 337-344 (the loop between α12 and β15 which covered the catalytic domain). In addition, three channels were observed in KpADH3 crystal structure, channel 2 and channel 3 may be essential for substrate specific recognition, ingress and egress, channel 1 may be the pass-through for cofactor. KpADH3 plays an important role in the metabolism of alcohols in Komagataella phaffii GS115, and its crystal structure is the only dimeric medium-chain ADH from yeast described so far. Knowledge of the relationship between structure and function of KpADH3 is crucial for understanding the role of KpADH3 in Komagataella phaffii GS115 mitochondrial metabolism. Copyright © 2018 Elsevier B.V. All rights reserved.

Overexpression of GsZFP1 enhances salt and drought tolerance in transgenic alfalfa (Medicago sativa L.).

PubMed

Tang, Lili; Cai, Hua; Ji, Wei; Luo, Xiao; Wang, Zhenyu; Wu, Jing; Wang, Xuedong; Cui, Lin; Wang, Yang; Zhu, Yanming; Bai, Xi

2013-10-01

GsZFP1 encodes a Cys2/His2-type zinc-finger protein. In our previous study, when GsZFP1 was heterologously expressed in Arabidopsis, the transgenic Arabidopsis plants exhibited enhanced drought and cold tolerance. However, it is still unknown whether GsZFP1 is also involved in salt stress. GsZFP1 is from the wild legume Glycine soja. Therefore, the aims of this study were to further elucidate the functions of the GsZFP1 gene under salt and drought stress in the forage legume alfalfa and to investigate its biochemical and physiological functions under these stress conditions. Our data showed that overexpression of GsZFP1 in alfalfa resulted in enhanced salt tolerance. Under high salinity stress, greater relative membrane permeability and malondialdehyde (MDA) content were observed and more free proline and soluble sugars accumulated in transgenic alfalfa than in the wild-type (WT) plants; in addition, the transgenic lines accumulated less Na(+) and more K(+) in both the shoots and roots. Overexpression of GsZFP1 also enhanced the drought tolerance of alfalfa. The fold-inductions of stress-responsive marker gene expression, including MtCOR47, MtRAB18, MtP5CS, and MtRD2, were greater in transgenic alfalfa than those of WT under drought stress conditions. In conclusion, the transgenic alfalfa plants generated in this study could be used for farming in salt-affected as well as arid and semi-arid areas. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Influence of low-molecular-weight glutenin subunit haplotypes on dough rheology in elite common wheat varieties

USDA-ARS?s Scientific Manuscript database

The low molecular weight glutenin subunits (LMW-GSs) are a class of wheat seed storage proteins. They are encoded by a multigene family located at the Glu-3 loci, and their allelic variation strongly influences wheat end-use quality. Due to ambiguities in the LMW-GS allele nomenclature and to the co...

Variations in yield and gluten proteins in durum wheat varieties under late-season foliar versus soil application of nitrogen fertilizer in a northern Mediterranean environment.

PubMed

Visioli, Giovanna; Bonas, Urbana; Dal Cortivo, Cristian; Pasini, Gabriella; Marmiroli, Nelson; Mosca, Giuliano; Vamerali, Teofilo

2018-04-01

With the increasing demand for high-quality foodstuffs and concern for environmental sustainability, late-season nitrogen (N) foliar fertilization of common wheat is now an important and widespread practice. This study investigated the effects of late-season foliar versus soil N fertilization on yield and protein content of four varieties of durum wheat, Aureo, Ariosto, Biensur and Liberdur, in a three-year field trial in northern Italy. Variations in low-molecular-weight glutenins (LMW-GS), high-molecular-weight glutenins (HMW-GS) and gliadins were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It was found that N applied to the canopy did not improve protein rate compared with N application to the soil (general mean 138 mg g -1 ), but moderately increased productivity in the high-yielding varieties Liberdur and Biensur (three-year means 7.23 vs 7.13 and 7.53 vs 7.09 t ha -1 respectively). Technological quality was mainly related to variety choice, Aureo and Ariosto having higher protein rates and glutenin/gliadin ratios. Also found was a strong 'variety × N application method' interaction in the proportions of protein subunits within each class, particularly LMW-GS and gliadins. A promising result was the higher N uptake efficiency, although as apparent balance, combined with higher HMW/LMW-GS ratio in var. Biensur. Late-season foliar N fertilization allows N fertilizer saving, potentially providing environmental benefits in the rainy climate of the northern Mediterranean area, and also leads to variety-dependent up-regulation of essential LMW-GS and gliadins. Variety choice is a key factor in obtaining high technological quality, although it is currently associated with modest grain yield. This study provides evidence of high quality in the specific high-yielding variety Biensur, suggesting its potential as a mono-varietal semolina for pasta production. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Docosahexaenoic acid alters Gsα localization in lipid raft and potentiates adenylate cyclase.

PubMed

Zhu, Zhuoran; Tan, Zhoubin; Li, Yan; Luo, Hongyan; Hu, Xinwu; Tang, Ming; Hescheler, Jürgen; Mu, Yangling; Zhang, Lanqiu

2015-01-01

Supplementation with docosahexaenoic acid (DHA), an ω-3 polyunsaturated fatty acid (PUFA), recently has become popular for the amelioration of depression; however the molecular mechanism of DHA action remains unclear. The aim of this study was to investigate the mechanism underlying the antidepressant effect of DHA by evaluating Gsα localization in lipid raft and the activity of adenylate cyclase in an in vitro glioma cell model. Lipid raft fractions from C6 glioma cells treated chronically with DHA were isolated by sucrose gradient ultracentrifugation. The content of Gsα in lipid raft was analyzed by immunoblotting and colocalization of Gsα with lipid raft was subjected to confocal microscopic analysis. The intracellular cyclic adenosine monophosphate (cAMP) level was determined by cAMP immunoassay kit. DHA decreased the amount of Gsα in lipid raft, whereas whole cell lysate Gsα was not changed. Confocal microscopic analysis demonstrated that colocalization of Gsα with lipid raft was decreased, whereas DHA increased intracellular cAMP accumulation in a dose-dependent manner. Interestingly, we found that DHA increased the lipid raft level, instead of disrupting it. The results of this study suggest that DHA may exert its antidepressant effect by translocating Gsα from lipid raft and potentiating the activity of adenylate cyclase. Importantly, the reduced Gsα in lipid raft by DHA is independent of disruption of lipid raft. Overall, the study provides partial preclinical evidence supporting a safe and effective therapy using DHA for depression. Copyright © 2015 Elsevier Inc. All rights reserved.

Effector gene birth in plant parasitic nematodes: Neofunctionalization of a housekeeping glutathione synthetase gene

PubMed Central

Lilley, Catherine J.; Maqbool, Abbas; Wu, Duqing; Yusup, Hazijah B.; Jones, Laura M.; Birch, Paul R. J.; Urwin, Peter E.

2018-01-01

Plant pathogens and parasites are a major threat to global food security. Plant parasitism has arisen four times independently within the phylum Nematoda, resulting in at least one parasite of every major food crop in the world. Some species within the most economically important order (Tylenchida) secrete proteins termed effectors into their host during infection to re-programme host development and immunity. The precise detail of how nematodes evolve new effectors is not clear. Here we reconstruct the evolutionary history of a novel effector gene family. We show that during the evolution of plant parasitism in the Tylenchida, the housekeeping glutathione synthetase (GS) gene was extensively replicated. New GS paralogues acquired multiple dorsal gland promoter elements, altered spatial expression to the secretory dorsal gland, altered temporal expression to primarily parasitic stages, and gained a signal peptide for secretion. The gene products are delivered into the host plant cell during infection, giving rise to “GS-like effectors”. Remarkably, by solving the structure of GS-like effectors we show that during this process they have also diversified in biochemical activity, and likely represent the founding members of a novel class of GS-like enzyme. Our results demonstrate the re-purposing of an endogenous housekeeping gene to form a family of effectors with modified functions. We anticipate that our discovery will be a blueprint to understand the evolution of other plant-parasitic nematode effectors, and the foundation to uncover a novel enzymatic function. PMID:29641602

Chimeric microbial rhodopsins for optical activation of Gs-proteins

PubMed Central

Yoshida, Kazuho; Yamashita, Takahiro; Sasaki, Kengo; Inoue, Keiichi; Shichida, Yoshinori; Kandori, Hideki

2017-01-01

We previously showed that the chimeric proteins of microbial rhodopsins, such as light-driven proton pump bacteriorhodopsin (BR) and Gloeobacter rhodopsin (GR) that contain cytoplasmic loops of bovine rhodopsin, are able to activate Gt protein upon light absorption. These facts suggest similar protein structural changes in both the light-driven proton pump and animal rhodopsin. Here we report two trials to engineer chimeric rhodopsins, one for the inserted loop, and another for the microbial rhodopsin template. For the former, we successfully activated Gs protein by light through the incorporation of the cytoplasmic loop of β2-adrenergic receptor (β2AR). For the latter, we did not observe any G-protein activation for the light-driven sodium pump from Indibacter alkaliphilus (IndiR2) or a light-driven chloride pump halorhodopsin from Natronomonas pharaonis (NpHR), whereas the light-driven proton pump GR showed light-dependent G-protein activation. This fact suggests that a helix opening motion is common to G protein coupled receptor (GPCR) and GR, but not to IndiR2 and NpHR. Light-induced difference FTIR spectroscopy revealed similar structural changes between WT and the third loop chimera for each light-driven pump. A helical structural perturbation, which was largest for GR, was further enhanced in the chimera. We conclude that similar structural dynamics that occur on the cytoplasmic side of GPCR are needed to design chimeric microbial rhodopsins. PMID:29362703

Limitations to the development of recombinant human embryonic kidney 293E cells using glutamine synthetase-mediated gene amplification: Methionine sulfoximine resistance.

PubMed

Yu, Da Young; Noh, Soo Min; Lee, Gyun Min

2016-08-10

To investigate the feasibility of glutamine synthetase (GS)-mediated gene amplification in HEK293 cells for the high-level stable production of therapeutic proteins, HEK293E cells were transfected by the GS expression vector containing antibody genes and were selected at various methionine sulfoximine (MSX) concentrations in 96-well plates. For a comparison, CHOK1 cells were transfected by the same GS expression vector and selected at various MSX concentrations. Unlike CHOK1 cells, HEK293E cells producing high levels of antibodies were not selected at all. For HEK293E cells, the number of wells with the cell pool did not decrease with an increase in the concentration of MSX up to 500μM MSX. A q-RT-PCR analysis confirmed that the antibody genes in the HEK293E cells, unlike the CHOK1 cells, were not amplified after increasing the MSX concentration. It was found that the GS activity in HEK293E cells was much higher than that in CHOK1 cells (P<0.05). In a glutamine-free medium, the GS activity of HEK293E cells was approximately 4.8 times higher than that in CHOK1 cells. Accordingly, it is inferred that high GS activity of HEK293E cells results in elevated resistance to MSX and therefore hampers GS-mediated gene amplification by MSX. Thus, in order to apply the GS-mediated gene amplification system to HEK293 cells, the endogenous GS expression level in HEK293 cells needs to be minimized by knock-out or down-regulation methods. Copyright © 2016 Elsevier B.V. All rights reserved.

The 2015 hard-state only outburst of GS 1354-64

NASA Astrophysics Data System (ADS)

Stiele, H.; Kong, A. K. H.

2016-07-01

Since its outburst in 1997, GS 1354-64 stayed in quiescence. In 2015 June, renewed activity of GS 1354-64 was observed. Based on our analysis of energy spectra and timing properties obtained from Swift/X-ray telescope monitoring data, we found that GS 1354-64 stayed in the hard state during the entire outburst. Such a hard state only (or `failed' outburst) has also been observed in 1997. In addition, we analysed an XMM-Newton observation taken on August 6th. We compared variability on long and short time-scales using covariance ratio and found that the ratio showed a decrease towards lower energies instead of the increase that has been found in other black hole X-ray binaries. There are now two sources (H1743-322 and GS 1354-64) that do not show an increase towards lower energies in their covariance ratio. Both sources have been observed during `failed' outbursts and showed photon indices much harder than what is usually observed in black hole X-ray binaries.

Attenuation of Quorum Sensing Regulated Virulence of Pectobacterium carotovorum subsp. carotovorum through an AHL Lactonase Produced by Lysinibacillus sp. Gs50

PubMed Central

Garge, Sneha S.; Nerurkar, Anuradha S.

2016-01-01

Quorum sensing (QS) is a mechanism in which Gram negative bacterial pathogens sense their population density through acyl homoserine lactones (AHLs) and regulate the expression of virulence factors. Enzymatic degradation of AHLs by lactonases, known as quorum quenching (QQ), is thus a potential strategy for attenuating QS regulated bacterial infections. We characterised the QQ activity of soil isolate Lysinibacillus sp. Gs50 and explored its potential for controlling bacterial soft rot of crop plants. Lysinibacillus sp. Gs50 inactivated AHL, which could be restored upon acidification, suggested that inactivation was due to the lactone ring hydrolysis of AHL. Heterologous expression of cloned gene for putative hydrolase (792 bp) designated adeH from Lysinibacillus sp. Gs50 produced a ~29 kDa protein which degraded AHLs of varying chain length. Mass spectrometry analysis of AdeH enzymatic reaction product revealed that AdeH hydrolyses the lactone ring of AHL and hence is an AHL lactonase. Multiple sequence alignment of the amino acid sequence of AdeH showed that it belongs to the metallo- β- lactamase superfamily, has a conserved “HXHXDH” motif typical of AHL lactonases. KM for AdeH for C6HSL was found to be 3.089 μM and the specific activity was 0.8 picomol min-1μg-1. AdeH has not so far been reported from any Lysinibacillus sp. and has less than 40% identity with known AHL lactonases. Finally we found that Lysinibacillus sp. Gs50 can degrade AHL produced by Pectobacterium carotovorum subsp. carotovorum (Pcc), a common cause of soft rot. This QQ activity causes a decrease in production of plant cell wall degrading enzymes of Pcc and attenuates symptoms of soft rot in experimental infection of potato, carrot and cucumber. Our results demonstrate the potential of Lysinibacillus sp. Gs50 as a preventive and curative biocontrol agent. PMID:27911925

Randomised clinical trial: a phase 1, dose-ranging study of the anti-matrix metalloproteinase-9 monoclonal antibody GS-5745 versus placebo for ulcerative colitis.

PubMed

Sandborn, W J; Bhandari, B R; Fogel, R; Onken, J; Yen, E; Zhao, X; Jiang, Z; Ge, D; Xin, Y; Ye, Z; French, D; Silverman, J A; Kanwar, B; Subramanian, G M; McHutchison, J G; Lee, S D; Shackelton, L M; Pai, R K; Levesque, B G; Feagan, B G

2016-07-01

.008) significant improvements in histopathology scores (confirmed with three separate histological disease activity indices), as well as changes in colonic gene expression that were consistent with reduced inflammation. This phase 1 trial provides preliminary evidence for the safety and therapeutic potential of GS-5745 in the treatment of ulcerative colitis. © 2016 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd.

Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys.

PubMed

Warren, Travis K; Jordan, Robert; Lo, Michael K; Ray, Adrian S; Mackman, Richard L; Soloveva, Veronica; Siegel, Dustin; Perron, Michel; Bannister, Roy; Hui, Hon C; Larson, Nate; Strickley, Robert; Wells, Jay; Stuthman, Kelly S; Van Tongeren, Sean A; Garza, Nicole L; Donnelly, Ginger; Shurtleff, Amy C; Retterer, Cary J; Gharaibeh, Dima; Zamani, Rouzbeh; Kenny, Tara; Eaton, Brett P; Grimes, Elizabeth; Welch, Lisa S; Gomba, Laura; Wilhelmsen, Catherine L; Nichols, Donald K; Nuss, Jonathan E; Nagle, Elyse R; Kugelman, Jeffrey R; Palacios, Gustavo; Doerffler, Edward; Neville, Sean; Carra, Ernest; Clarke, Michael O; Zhang, Lijun; Lew, Willard; Ross, Bruce; Wang, Queenie; Chun, Kwon; Wolfe, Lydia; Babusis, Darius; Park, Yeojin; Stray, Kirsten M; Trancheva, Iva; Feng, Joy Y; Barauskas, Ona; Xu, Yili; Wong, Pamela; Braun, Molly R; Flint, Mike; McMullan, Laura K; Chen, Shan-Shan; Fearns, Rachel; Swaminathan, Swami; Mayers, Douglas L; Spiropoulou, Christina F; Lee, William A; Nichol, Stuart T; Cihlar, Tomas; Bavari, Sina

2016-03-17

The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the

Chromatin modification contributes to the expression divergence of three TaGS2 homoeologs in hexaploid wheat

PubMed Central

Zhang, Wei; Fan, Xiaoli; Gao, Yingjie; Liu, Lei; Sun, Lijing; Su, Qiannan; Han, Jie; Zhang, Na; Cui, Fa; Ji, Jun; Tong, Yiping; Li, Junming

2017-01-01

Plastic glutamine synthetase (GS2) is responsible for ammonium assimilation. The reason that TaGS2 homoeologs in hexaploid wheat experience different selection pressures in the breeding process remains unclear. TaGS2 were minimally expressed in roots but predominantly expressed in leaves, and TaGS2-B had higher expression than TaGS2-A and TaGS2-D. ChIP assays revealed that the activation of TaGS2-B expression in leaves was correlated with increased H3K4 trimethylation. The transcriptional silencing of TaGS2 in roots was correlated with greater cytosine methylation and less H3K4 trimethylation. Micrococcal nuclease and DNase I accessibility experiments indicated that the promoter region was more resistant to digestion in roots than leaves, which indicated that the closed nucleosome conformation of the promoter region was important to the transcription initiation for the spatial-temporal expression of TaGS2. In contrast, the transcribed regions possess different nuclease accessibilities of three TaGS2 homoeologs in the same tissue, suggesting that nucleosome conformation of the transcribed region was part of the fine adjustment of TaGS2 homoeologs. This study provides evidence that histone modification, DNA methylation and nuclease accessibility coordinated the control of the transcription of TaGS2 homoeologs. Our results provided important evidence that TaGS2-B experienced the strongest selection pressures during the breeding process. PMID:28300215

Variation in ultrafiltered and LMW organic matter fluorescence properties under simulated estuarine mixing transects: 1. Mixing alone

NASA Astrophysics Data System (ADS)

Boyd, Thomas J.; Barham, Bethany P.; Hall, Gregory J.; Osburn, Christopher L.

2010-09-01

Ultrafiltered and low molecular weight dissolved organic matter (UDOM and LMW-DOM, respectively) fluorescence was studied under simulated estuarine mixing using samples collected from Delaware, Chesapeake, and San Francisco Bays (USA) transects. UDOM was concentrated by tangential flow ultrafiltration (TFF) from the marine (>33 PSU), mid-estuarine (˜16 PSU), and freshwater (<1 PSU) members. TFF permeates (<1 kDa) from the three members were used to create artificial salinity transects ranging from ˜0 to ˜36, with 4 PSU increments. UDOM from the end- or mid-members was added in equal amounts to each salinity-mix. Three-dimensional fluorescence excitation-emission matrix (EEMs) spectra were generated for each end-member permeate and UDOM through the full estuarine mixing transect. Fluorescence components such as proteinaceous, terrigenous, and marine derived humic peaks, and certain fluorescent ratios were noticeably altered by simulated estuarine mixing, suggesting that LMW DOM and UDOM undergo physicochemical alteration as they move to or from the freshwater, mid-estuarine, or coastal ocean members. LMW fluorescence components fit a decreasing linear mixing model from mid salinities to the ocean end-member, but were more highly fluorescent than mixing alone would predict in lower salinities (<8). Significant shifts were also seen in UDOM peak emission wavelengths with blue-shifting toward the ocean end-member. Humic-type components in UDOM generally showed lower fluorescent intensities at low salinities, higher at mid-salinities, and lower again toward the ocean end-member. T (believed to be proteinaceous) and N (labile organic matter) peaks behaved similarly to each other, but not to B peak fluorescence, which showed virtually no variation in permeate or UDOM mixes with salinity. PCA and PARAFAC models showed similar results suggesting trends could be modeled for DOM end- and mid-member sources. Changes in fluorescence properties due to estuarine mixing may be

Changes of Ammonia-Metabolizing Enzyme Activity and Gene Expression of Two Strains in Shrimp Litopenaeus vannamei Under Ammonia Stress

PubMed Central

Qiu, Liguo; Shi, Xiang; Yu, Simeng; Han, Qian; Diao, Xiaoping; Zhou, Hailong

2018-01-01

Ammonia stress can inhibit the survival and growth, and even cause mortality of shrimp. In this study, ammonia-metabolizing enzyme activities and gene expression were compared between two strains of L. vannamei under different ammonia-N (NH4+) concentrations (3.4, 13.8, and 24.6 mg/L). The results showed that elevated ammonia concentrations mainly increased glutamine synthetase (GSase) activities while inhibiting transglutaminase (TGase) activities in the muscle of both strains. Thus, we concluded that L. vannamei could accelerate the synthesis of glutamine from glutamate and NH4+ to alleviate ammonia stress. Compared with the muscle, the hepatopancreas plays a major role in ammonia stress and might be a target tissue to respond to the ammonia stress. Compared to the control group, the treatment of high ammonia concentrations reduced the hepatopancreas TGase (TG) gene expression and increased the gene expression rates of glutamate dehydrogenase-β (GDH-β) and GSase (GS) in both the muscle and the hepatopancreas of the two strains (p < 0.05). These genes (GDH-β and GS) in strain B were not only expressed earlier but also at levels higher than the expression range of strain A. At the gene level, strain B showed a more rapid and positive response than strain A. These data might help reveal the physiological responses mechanisms of shrimp adapt to ammonia stress and speed up the selective breeding process in L. vannamei. PMID:29628893

Glutamine synthetase gene knockout-human embryonic kidney 293E cells for stable production of monoclonal antibodies.

PubMed

Yu, Da Young; Lee, Sang Yoon; Lee, Gyun Min

2018-05-01

Previously, it was inferred that a high glutamine synthetase (GS) activity in human embryonic kidney (HEK) 293E cells results in elevated resistance to methionine sulfoximine (MSX) and consequently hampers GS-mediated gene amplification and selection by MSX. To overcome this MSX resistance in HEK293E cells, a GS-knockout HEK293E cell line was generated using the CRISPR/Cas9 system to target the endogenous human GS gene. The GS-knockout in the HEK293E cell line (RK8) was confirmed by Western blot analysis of GS and by observation of glutamine-dependent growth. Unlike the wild type HEK293E cells, the RK8 cells were successfully used as host cells to generate a recombinant HEK293E cell line (rHEK293E) producing a monoclonal antibody (mAb). When the RK8 cells were transfected with the GS expression vector containing the mAb gene, rHEK293E cells producing the mAb could be selected in the absence as well as in the presence of MSX. The gene copies and mRNA expression levels of the mAb in rHEK293E cells were also quantified using qRT-PCR. Taken together, the GS-knockout HEK293E cell line can be used as host cells to generate stable rHEK293E cells producing a mAb through GS-mediated gene selection in the absence as well as in the presence of MSX. © 2018 Wiley Periodicals, Inc.

Relationship between the dough quality and content of specific glutenin proteins in wheat mill streams, and its application to making flour suitable for instant Chinese noodles.

PubMed

Yahata, Eriko; Maruyama-Funatsuki, Wakako; Nishio, Zenta; Yamamoto, Yoshihiko; Hanaoka, Akihiro; Sugiyama, Hisashi; Tanida, Masatoshi; Saruyama, Haruo

2006-04-01

The content of specific proteins such as high-molecular-weight glutenin subunits HMW-GS 5+10 and low-molecular-weight glutenin subunits LMW-GS KS2 in wheat mill streams of extra-strong Kachikei 33 wheat was quantified by SDS-PAGE and 2D-PAGE. The mill streams showed varied quantities of HMW-GS 5+10 (0.077 to 2.007 mg/g of mill stream), LMW-GS KS2 (0.018 to 0.586 mg/g of mill stream) and total protein (9.42% to 18.98%). The contents of these specific proteins in the mill streams were significantly correlated with the SDS sedimentation volume and the mixing properties, which are respective indices of specific loaf volume and dough strength. The contents of these specific glutenin proteins in the mill streams were therefore found to be significantly important for improving the dough quality suitable for bread and Chinese noodles. Accordingly, we present here the application of this information to the development of an effective method for producing mill streams with high quality and yield that are suitable for instant Chinese noodles.

A Rare Allele of GS2 Enhances Grain Size and Grain Yield in Rice.

PubMed

Hu, Jiang; Wang, Yuexing; Fang, Yunxia; Zeng, Longjun; Xu, Jie; Yu, Haiping; Shi, Zhenyuan; Pan, Jiangjie; Zhang, Dong; Kang, Shujing; Zhu, Li; Dong, Guojun; Guo, Longbiao; Zeng, Dali; Zhang, Guangheng; Xie, Lihong; Xiong, Guosheng; Li, Jiayang; Qian, Qian

2015-10-05

Grain size determines grain weight and affects grain quality. Several major quantitative trait loci (QTLs) regulating grain size have been cloned; however, our understanding of the underlying mechanism that regulates the size of rice grains remains fragmentary. Here, we report the cloning and characterization of a dominant QTL, grain size on chromosome 2 (GS2), which encodes Growth-Regulating Factor 4 (OsGRF4), a transcriptional regulator. GS2 localizes to the nucleus and may act as a transcription activator. A rare mutation of GS2 affecting the binding site of a microRNA, OsmiR396c, causes elevated expression of GS2/OsGRF4. The increase in GS2 expression leads to larger cells and increased numbers of cells, which thus enhances grain weight and yield. The introduction of this rare allele of GS2/OsGRF4 into rice cultivars could significantly enhance grain weight and increase grain yield, with possible applications in breeding high-yield rice varieties. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

Laboratory testing of the (Japan Storage Battery) traction batteries GS E75A and GS E150H

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

Not Available

This report describes the testing of the GS E75A and GS E150H flooded lead-acid 12-volt traction batteries and compares the selected batteries to U.S.-made electric vehicle batteries. The results and conclusions of the testing are presented.

Metal-dependent hydrolysis of myelin basic protein by IgGs from the sera of patients with multiple sclerosis.

PubMed

Polosukhina, Dar'ya I; Kanyshkova, Tat'yana G; Doronin, Boris M; Tyshkevich, Olga B; Buneva, Valentina N; Boiko, Alexey N; Gusev, Evgenii I; Nevinsky, Georgy A; Favorova, Olga O

2006-02-28

Homogeneous IgG fractions were obtained by chromatography of the sera of patients with multiple sclerosis (MS) on Protein G-Sepharose under conditions that remove non-specifically bound proteins. These IgGs contained several chelated metals, the relative amount of which decreases in the order: Fe>or=Ca>Cu>or=Zn>or=Mg>or=Mn>or=Pb>or=Co>or=Ni. In contrast to homogeneous IgGs of healthy individuals, Abs of MS patients effectively hydrolyzed human myelin basic protein (MBP). A minor metal-dependent fraction was obtained by chromatography of highly purified IgGs from MS patient on Chelex-100. This IgG fraction did not hydrolyze human MBP in the absence of Me(2+) ions but was activated after addition of Me(2+) ions: Mg(2+)>Mn(2+)>Cu(2+)>Ca(2+). Proteolytic activities of IgGs from other MS patients were also activated by other metal ions (Ni(2+), Fe(2+), Co(2+), Zn(2+), Pb(2+), and Co(2+)) and especially Ni(2+). Ni(2+)-activated IgGs were separated into distinct MBP-hydrolyzing fractions by chromatography on HiTraptrade mark Chelating Sepharose charged with Ni(2+). Detection of Mg(2+)-dependent proteolytic activity in the SDS-PAGE area corresponding only to IgG provided direct evidence that IgG from sera of MS patients possesses metal-dependent human MBP-hydrolyzing activity. Observed properties of MS abzymes distinguish them from other known mammalian metalloproteases and demonstrate their pronounced catalytic diversity. Metal-dependent IgGs from MS patients represent the first example of abzymes with metal-dependent proteolytic activity.

Fluorophore-assisted carbohydrate electrophoresis for the determination of molecular mass of heparins and low-molecular-weight (LMW) heparins.

PubMed

Buzzega, Dania; Maccari, Francesca; Volpi, Nicola

2008-11-01

We report the use of fluorophore-assisted carbohydrate electrophoresis (FACE) to determine the molecular mass (M) values of heparins (Heps) and low-molecular-weight (LMW)-Hep derivatives. Hep are labeled with 8-aminonaphthalene-1,3,6-trisulfonic acid and FACE is able to resolve each fraction as a discrete band depending on their M. After densitometric acquisition, the migration distance of each Hep standard is acquired and the third-grade polynomial calibration standard curve is determined by plotting the logarithms of the M values as a function of migration ratio. Purified Hep samples having different properties, pharmaceutical Heps and various LMW-Heps were analyzed by both FACE and conventional high-performance size-exclusion liquid chromatography (HPSEC) methods. The molecular weight value on the top of the chromatographic peak (Mp), the number-average Mn, weight-average Mw and polydispersity (Mw/Mn) were examined by both techniques and found to be similar. This approach offers certain advantages over the HPSEC method. The derivatization process with 8-aminonaphthalene-1,3,6-trisulfonic acid is complete after 4 h so that many samples may be analyzed in a day also considering that multiple samples can be run simultaneously and in parallel and that a single FACE analysis requires approx. 15 min. Furthermore, FACE is a very sensitive method as it requires approx. 5-10 microg of Heps, about 10-100-fold lower than samples and standards used in HPSEC evaluation. Finally, the utilization of mini-gels allows the use of very low amounts of reagents with neither expensive equipment nor any complicated procedures having to be applied. This study demonstrates that FACE analysis is a sensitive method for the determination of the M values of Heps and LMW-Heps with possible utilization in virtually any kind of research and development such as quality control laboratories due to its rapid, parallel analysis of multiple samples by means of common and simple largely used

Studies on Antiviral and Immuno-Regulation Activity of Low Molecular Weight Fucoidan from Laminaria japonica

NASA Astrophysics Data System (ADS)

Sun, Taohua; Zhang, Xinhui; Miao, Ying; Zhou, Yang; Shi, Jie; Yan, Meixing; Chen, Anjin

2018-06-01

The antiviral activity in vitro and in vivo and the effect of the immune system of two fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica (LMW fucoidans) were investigated in order to examine the possible mechanism. In vitro, I-type influenza virus, adenovirus and Parainfluenza virus I were used to infect Hep-2, Hela and MDCK cells, respectively. And 50% tissue culture infective dose was calculated to detect the antiviral activity of two LMW fucoidans. The results indicated that compared with the control group, 2 kinds of LMW fucoidans had remarkable antiviral activity in vitro in middle and high doses, while at low doses, the antiviral activity of 2 kinds of LMW fucoidans was not statistically different from that in the blank control group. And there was no statistically difference between two LMW fucoidans in antiviral activity. In vivo, LMW fucoidans could prolong the survival time of virus-infected mice, and could improve the lung index of virus-infected mice significantly, which have statistical differences with the control group significantly ( p < 0.01). However, the survival time of the two LMW fucoidans was not statistically significant ( p > 0.05). In this study, it was shown that both of two LMW fucoidans (LF1, LF2) could increase the thymus index, spleen index, phagocytic index, phagocytosis coefficient and half hemolysin value in middle and high doses, which suggested that LMW fucoidans could play an antiviral role by improving the quality of immune organs, improving immune cell phagocytosis and humoral immunity.

High profile backing for GS1 drive.

PubMed

Hodgson, Glen

2016-09-01

At the 2016 GS1 UK Healthcare Conference in London, delegates heard from speakers including Pat Mills, the Department of Health's commercial director, on the ongoing work to embed GS1 standards throughout the NHS in England in line with the DH's eProcurement Strategy, published in April 2014. This mandated that any service or product procured by an English NHS acute Trust comply with the standards--one of the most obvious representations of which is on barcodes--'to enable Trusts to manage their non-pay spending by adopting master procurement data, automating the exchange of such data, and benchmarking their procurement against other Trusts and healthcare providers'. One of six 'demonstrator site' Trusts to provide a speaker at the 2016 GS1 UK national conference to report on their progress to date was Leeds Teaching Hospitals NHS Trust. Shortly after, HEJ editor, Jonathan Baillie, spoke to the Trust's associate director, Commercial and Procurement, Chris Slater, and to head of Healthcare at GS1 UK, Glen Hodgson.

Differential expression of multiple glutamine synthetase genes in air-breathing magur catfish, Clarias magur and their induction under hyper-ammonia stress.

PubMed

Banerjee, Bodhisattwa; Koner, Debaprasad; Bhuyan, Gitalee; Saha, Nirmalendu

2018-06-01

The present study demonstrates the unique presence of three different gs genes (cmgs01, cmgs02, and cmgs03) in air-breathing ureogenic magur catfish (Clarias magur), which is otherwise reported to be encoded by a single gene in higher vertebrates. Of these three genes, two (cmgs01and cmgs03) were identified as 'liver' form, predominantly expressed in liver cells, and the third one as 'brain' form (cmgs02), expressed chiefly in brain cells. Molecular characterization studies have revealed conservation of homologous active site residues in all the three gs genes. In silico analysis, accompanied by GS enzyme assay and Western blot analysis of different GS isoforms in different subcellular fractions indicated the mitochondrial localization of cmGS01 and cmGS03 in liver and kidney cells and cytosolic localization of cmGS02 in brain cells. Further, exposure of magur catfish to high external ammonia (HEA; 25 mM NH 4 Cl) led to a significant induction of multiple gs genes as evidenced by higher expression of different gs mRNAs at variable levels in different tissues. The cmgs01 and cmgs03 mRNA levels elevated significantly in liver, kidney, muscle, and gills, whereas the cmgs02 mRNA level increased considerably in the brain after 14 days of exposure to HEA. These increases in mRNA levels were associated with a significant rise in cmGS01 and cmGS03 proteins in liver, kidney, muscle, and gills, and the cmGS02 protein in the brain after 14 days of exposure to HEA. Therefore, it can be concluded that the unique differential expression of three gs genes and their induction under high ammonia level probably helps in detoxification of ammonia to glutamine and further to urea via the ornithine-urea cycle in ureogenic as well as non-ureogenic tissues of these magur catfish. Copyright © 2017. Published by Elsevier B.V.

Hydrophobic modification of low molecular weight polyethylenimine for improved gene transfection.

PubMed

Teo, Pei Yun; Yang, Chuan; Hedrick, James L; Engler, Amanda C; Coady, Daniel J; Ghaem-Maghami, Sadaf; George, Andrew J T; Yang, Yi Yan

2013-10-01

Hydrophobic modification of low molecular weight (LMW) polyethylenimine (PEI) is known to increase gene transfection efficiency of LMW PEI. However, few studies have explored how the conjugated hydrophobic groups influence the properties of the modified LMW PEI mainly due to difficulties in obtaining well defined final product compositions and limitations in current chemical synthesis routes. The aim of this study was to modify LMW PEI (Mn 1.8 kDa, PEI-1.8) judiciously with different hydrophobic functional groups and to investigate how hydrophobicity, molecular structure and inclusion of hydrogen bonding properties in the conjugated side groups as well as the conjugation degree (number of primary amine groups of PEI-1.8 modified with hydrophobic groups) influence PEI-1.8 gene transfection efficiency. The modified polymers were characterized for DNA binding ability, particle size, zeta potential, in vitro gene transfection efficiency and cytotoxicity in SKOV-3 human ovarian cancer and HepG2 human liver carcinoma cell lines. The study shows that modified PEI-1.8 polymers are able to condense plasmid DNA into cationic nanoparticles, of sizes ~100 nm, whereas unmodified polymer/DNA complexes display larger particle sizes of 2 μm. Hydrophobic modification also increases the zeta potential of polymer/DNA complexes. Importantly, modified PEI-1.8 shows enhanced transfection efficiency over the unmodified counterpart. Higher transfection efficiency is obtained when PEI-1.8 is modified with shorter hydrophobic groups (MTC-ethyl) as opposed to longer ones (MTC-octyl and MTC-deodecyl). An aromatic structured functional group (MTC-benzyl) also enhances transfection efficiency more than an alkyl functional group (MTC-octyl). An added hydrogen-bonding urea group in the conjugated functional group (MTC-urea) does not enhance transfection efficiency over one without urea (MTC-benzyl). The study also demonstrates that modification degree greatly influences gene transfection, and

Proteomics of Durum Wheat Grain during Transition to Conservation Agriculture

PubMed Central

Galieni, Angelica; Stagnari, Fabio; Bonas, Urbana; Speca, Stefano; Faccini, Andrea; Pisante, Michele; Marmiroli, Nelson

2016-01-01

Nitrogen management in combination with sustainable agronomic techniques can have a great impact on the wheat grain proteome influencing its technological quality. In this study, proteomic analyses were used to document changes in the proportion of prolamins in mature grains of the newly released Italian durum wheat cv Achille. Such an approach was applied to wheat fertilized with urea (UREA) and calcium nitrate (NITRATE), during the transition to no-till Conservation Agriculture (CA) practice in a Mediterranean environment. Results obtained in a two-years field experiment study suggest low molecular weight glutenins (LMW-GS) as the fraction particularly inducible regardless of the N-form. Quantitative analyses of LMW-GS by 2D-GE followed by protein identification by LC-ESI-MS/MS showed that the stable increase was principally due to C-type LMW-GS. The highest accumulation resulted from a physiologically healthier state of plants treated with UREA and NITRATE. Proteomic analysis on the total protein fraction during the active phase of grain filling was also performed. For both N treatments, but at different extent, an up-regulation of different classes of proteins was observed: i) enzymes involved in glycolysis and citric acid cycles which contribute to an enhanced source of energy and carbohydrates, ii) stress proteins like heat shock proteins (HSPs) and antioxidant enzymes, such as peroxidases and superoxide dismutase which protect the grain from abiotic stress during starch and storage protein synthesis. In conclusion N inputs, which combined rate with N form gave high yield and improved quality traits in the selected durum wheat cultivar. The specific up-regulation of some HSPs, antioxidant enzymes and defense proteins in the early stages of grain development and physiological indicators related to fitness traits, could be useful bio-indicators, for wheat genotype screening under more sustainable agronomic conditions, like transition phase to no-till CA in

Predictors of Gleason Score (GS) upgrading on subsequent prostatectomy: a single Institution study in a cohort of patients with GS 6

PubMed Central

Mehta, Vikas; Rycyna, Kevin; Baesens, Bart MM; Barkan, Güliz A; Paner, Gladell P; Flanigan, Robert C; Wojcik, Eva M; Venkataraman, Girish

2012-01-01

Background Biopsy Gleason score (bGS) remains an important prognostic indicator for adverse outcomes in Prostate Cancer (PCA). In the light of recent studies purporting difference in prognostic outcomes for the subgroups of GS7 group (primary Gleason pattern 4 vs. 3), upgrading of a bGS of 6 to a GS≥7 has serious implications. We sought to identify pre-operative factors associated with upgrading in a cohort of GS6 patients who underwent prostatectomy. Design We identified 281 cases of GS6 PCA on biopsy with subsequent prostatectomies. Using data on pre-operative variables (age, PSA, biopsy pathology parameters), logistic regression models (LRM) were developed to identify factors that could be used to predict upgrading to GS≥7 on subsequent prostatectomy. A decision tree (DT) was constructed. Results 92 of 281 cases (32.7%) were upgraded on subsequent prostatectomy. LRM identified a model with two variables with statistically significant ability to predict upgrading, including pre-biopsy PSA (Odds Ratio 8.66; 2.03-37.49, 95% CI) and highest percentage of cancer at any single biopsy site (Odds Ratio 1.03, 1.01-1.05, 95% CI). This two-parameter model yielded an area under curve of 0.67. The decision tree was constructed using only 3 leave nodes; with a test set classification accuracy of 70%. Conclusions A simplistic model using clinical and biopsy data is able to predict the likelihood of upgrading of GS with an acceptable level of certainty. External validation of these findings along with development of a nomogram will aid in better stratifying the cohort of low risk patients as based on the GS. PMID:22949931

Mechanistic Characterization of GS-9190 (Tegobuvir), a Novel Nonnucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase▿

PubMed Central

Shih, I-hung; Vliegen, Inge; Peng, Betty; Yang, Huiling; Hebner, Christy; Paeshuyse, Jan; Pürstinger, Gerhard; Fenaux, Martijn; Tian, Yang; Mabery, Eric; Qi, Xiaoping; Bahador, Gina; Paulson, Matthew; Lehman, Laura S.; Bondy, Steven; Tse, Winston; Reiser, Hans; Lee, William A.; Schmitz, Uli; Neyts, Johan; Zhong, Weidong

2011-01-01

GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitro and has demonstrated potent antiviral activity in patients chronically infected with genotype 1 (GT1) HCV. GS-9190 exhibits reduced activity against GT2a (JFH1) subgenomic replicons and GT2a (J6/JFH1) infectious virus, suggesting that the compound's mechanism of action involves a genotype-specific viral component. To further investigate the GS-9190 mechanism of action, we utilized the susceptibility differences between GT1b and GT2a by constructing a series of replicon chimeras where combinations of 1b and 2a nonstructural proteins were encoded within the same replicon. The antiviral activities of GS-9190 against the chimeric replicons were reduced to levels comparable to that of the wild-type GT2a replicon in chimeras expressing GT2a NS5B. GT1b replicons in which the β-hairpin region (amino acids 435 to 455) was replaced by the corresponding sequence of GT2a were markedly less susceptible to GS-9190, indicating the importance of the thumb subdomain of the polymerase in this effect. Resistance selection in GT1b replicon cells identified several mutations in NS5B (C316Y, Y448H, Y452H, and C445F) that contributed to the drug resistance phenotype. Reintroduction of these mutations into wild-type replicons conferred resistance to GS-9190, with the number of NS5B mutations correlating with the degree of resistance. Analysis of GS-9190 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of GS-9190 is different from other nonnucleoside inhibitors. Collectively, these data demonstrate that GS-9190 represents a novel class of nonnucleoside polymerase inhibitors that interact with NS5B likely through involvement of the β-hairpin in the thumb subdomain. PMID:21746939

Genome-Wide Analysis of Mycoplasma bovirhinis GS01 Reveals Potential Virulence Factors and Phylogenetic Relationships.

PubMed

Chen, Shengli; Hao, Huafang; Zhao, Ping; Liu, Yongsheng; Chu, Yuefeng

2018-05-04

Mycoplasma bovirhinis is a significant etiology in bovine pneumonia and mastitis, but our knowledge about the genetic and pathogenic mechanisms of M. bovirhinis is very limited. In this study, we sequenced the complete genome of M. bovirhinis strain GS01 isolated from the nasal swab of pneumonic calves in Gansu, China, and we found that its genome forms a 847,985 bp single circular chromosome with a GC content of 27.57% and with 707 protein-coding genes. The putative virulence determinants of M. bovirhinis were then analyzed. Results showed that three genomic islands and 16 putative virulence genes, including one adhesion gene enolase, seven surface lipoproteins, proteins involved in glycerol metabolism, and cation transporters, might be potential virulence factors. Glycerol and pyruvate metabolic pathways were defective. Comparative analysis revealed remarkable genome variations between GS01 and a recently reported HAZ141_2 strain, and extremely low homology with others mycoplasma species. Phylogenetic analysis demonstrated that M. bovirhinis was most genetically close to M. canis , distant from other bovine Mycoplasma species. Genomic dissection may provide useful information on the pathogenic mechanisms and genetics of M. bovirhinis . Copyright © 2018 Chen et al.

Cytosolic Glutamine Synthetase Gln1;2 Is the Main Isozyme Contributing to GS1 Activity and Can Be Up-Regulated to Relieve Ammonium Toxicity1[OPEN

PubMed Central

Pedersen, Carsten

2016-01-01

Cytosolic GS1 (Gln synthetase) is central for ammonium assimilation in plants. High ammonium treatment enhanced the expression of the GS1 isogene Gln-1;2 encoding a low-affinity high-capacity GS1 protein in Arabidopsis (Arabidopsis thaliana) shoots. Under the same conditions, the expression of the high-affinity low-capacity isoform Gln-1;1 was reduced. The expression of Gln-1;3 did not respond to ammonium treatment while Gln-1;4 and Gln-1;5 isogenes in all cases were expressed at a very low level. Gln-2 was highly expressed in shoots but only at a very low level in roots. To investigate the specific functions of the two isogenes Gln-1;1 and Gln-1;2 in shoots for ammonium detoxification, single and double knock-out mutants were grown under standard N supply or with high ammonium provision. Phenotypes of the single mutant gln1;1 were similar to the wild type, while growth of the gln1;2 single mutant and the gln1;1:gln1;2 double mutant was significantly impaired irrespective of N regime. GS1 activity was significantly reduced in both gln1;2 and gln1;1:gln1;2. Along with this, the ammonium content increased while that of Gln decreased, showing that Gln-1;2 was essential for ammonium assimilation and amino acid synthesis. We conclude that Gln-1;2 is the main isozyme contributing to shoot GS1 activity in vegetative growth stages and can be up-regulated to relieve ammonium toxicity. This reveals, to our knowledge, a novel shoot function of Gln-1;2 in Arabidopsis shoots. PMID:27231101

Gs-coupled GPCR signalling in AgRP neurons triggers sustained increase in food intake

PubMed Central

Nakajima, Ken-ichiro; Cui, Zhenzhong; Li, Chia; Meister, Jaroslawna; Cui, Yinghong; Fu, Ou; Smith, Adam S.; Jain, Shalini; Lowell, Bradford B.; Krashes, Michael J.; Wess, Jürgen

2016-01-01

Agouti-related peptide (AgRP) neurons of the hypothalamus play a key role in regulating food intake and body weight, by releasing three different orexigenic molecules: AgRP; GABA; and neuropeptide Y. AgRP neurons express various G protein-coupled receptors (GPCRs) with different coupling properties, including Gs-linked GPCRs. At present, the potential role of Gs-coupled GPCRs in regulating the activity of AgRP neurons remains unknown. Here we show that the activation of Gs-coupled receptors expressed by AgRP neurons leads to a robust and sustained increase in food intake. We also provide detailed mechanistic data linking the stimulation of this class of receptors to the observed feeding phenotype. Moreover, we show that this pathway is clearly distinct from other GPCR signalling cascades that are operative in AgRP neurons. Our data suggest that drugs able to inhibit this signalling pathway may become useful for the treatment of obesity. PMID:26743492

Gs-coupled GPCR signalling in AgRP neurons triggers sustained increase in food intake.

PubMed

Nakajima, Ken-ichiro; Cui, Zhenzhong; Li, Chia; Meister, Jaroslawna; Cui, Yinghong; Fu, Ou; Smith, Adam S; Jain, Shalini; Lowell, Bradford B; Krashes, Michael J; Wess, Jürgen

2016-01-08

Agouti-related peptide (AgRP) neurons of the hypothalamus play a key role in regulating food intake and body weight, by releasing three different orexigenic molecules: AgRP; GABA; and neuropeptide Y. AgRP neurons express various G protein-coupled receptors (GPCRs) with different coupling properties, including Gs-linked GPCRs. At present, the potential role of Gs-coupled GPCRs in regulating the activity of AgRP neurons remains unknown. Here we show that the activation of Gs-coupled receptors expressed by AgRP neurons leads to a robust and sustained increase in food intake. We also provide detailed mechanistic data linking the stimulation of this class of receptors to the observed feeding phenotype. Moreover, we show that this pathway is clearly distinct from other GPCR signalling cascades that are operative in AgRP neurons. Our data suggest that drugs able to inhibit this signalling pathway may become useful for the treatment of obesity.

Study on antithrombotic and antiplatelet activities of low molecular weight fucoidan from Laminaria japonica

NASA Astrophysics Data System (ADS)

Chen, Anjin; Zhang, Fang; Shi, Jie; Zhao, Xue

2012-06-01

The antithrombotic and antiplatelet effects of two fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica were compared in order to examine the influence of chemical character on their antithrombotic activity and the possible mechanism. Both LMW fucoidan fractions exhibited favorable antithrombotic activity in an Fecl3-induced arterial thrombosis. The antithrombotic activity of LMW fucoidan was related with decrease of TXB2 and whole blood viscosity and hematocrit. LMW fucoidan showed a correlation between anticoagulant, antiaggregant and antithrombotic effects in vivo. For LMW fucoidan, antithrombotic activity required high dose of 5-10 nmol kg-1, concomitantly with increase in anticoagulant activity and inhibition of platelet aggregation. Administration of LMW fucoidan significantly promoted the 6-keto-PGF1α content and decreased the TXB2 content, indicating its inhibition of tissue factor pathway and regulation of metabolism of arachidonic acid. By comparison, highly sulfated fucoidan LF2 with Mw 3900 seemed to be a more suitable choice for antithrombotic drug for its antithrombotic activity accompanied with specific inhibitory activity on platelet aggregation, low anticoagulant activity and low hemorrhagic risk in vivo.

Antioxidation activities of low-molecular-weight gelatin hydrolysate isolated from the sea cucumber Stichopus japonicus

NASA Astrophysics Data System (ADS)

Wang, Jingfeng; Wang, Yuming; Tang, Qingjuan; Wang, Yi; Chang, Yaoguang; Zhao, Qin; Xue, Changhu

2010-03-01

Gelatin extracted from the body wall of the sea cucumber ( Stichopus japonicus) was hydrolyzed with flavourzyme. Low-molecular-weight gelatin hydrolysate (LMW-GH) of 700-1700 Da was produced using an ultrafiltration membrane bioreactor system. Chemiluminescence analysis revealed that LMW-GH scavenges high free radicals in a concentration-dependent manner; IC50 value for superoxide and hydroxyl radicals was 442 and 285 μg mL-1, respectively. LMW-GH exhibited excellent inhibitory characteristics against melanin synthesis and tyrosinase activity in B16 cells. Furthermore, LMW-GH notably increased intracellular glutathione (GSH), which in turn suppressed melanogenesis. LMW-GH performs antioxidation activity, holding the potential of being used as a valuable ingredient in function foods, cosmetics and pharmaceuticals or nutriceuticals.

51g-s-219

NASA Image and Video Library

2011-04-25

51G-S-219 (June 1985) --- Public Affairs Office (PAO) commentator Janet K. Ross is pictured at her console in the Mission Operations Control Room (MOCR) in the Mission Control Center at NASA's Johnson Space Center during the STS-51G mission. Photo credit: NASA

51g-s-213

NASA Image and Video Library

2011-04-25

51G-S-213 (June 1985) --- Public Affairs Office (PAO) commentator Janet K. Ross is pictured at her console in the Mission Operations Control Room (MOCR) in the Mission Control Center at NASA's Johnson Space Center during the STS-51G mission. Photo credit: NASA

Overexpression of a glutamine synthetase gene affects growth and development in sorghum.

PubMed

Urriola, Jazmina; Rathore, Keerti S

2015-06-01

Nitrogen is a primary macronutrient in plants, and nitrogen fertilizers play a critical role in crop production and yield. In this study, we investigated the effects of overexpressing a glutamine synthetase (GS) gene on nitrogen metabolism, and plant growth and development in sorghum (Sorghum bicolor L., Moench). GS catalyzes the ATP dependent reaction between ammonia and glutamate to produce glutamine. A 1,071 bp long coding sequence of a sorghum cytosolic GS gene (Gln1) under the control of the maize ubiquitin (Ubq) promoter was introduced into sorghum immature embryos by Agrobacterium-mediated transformation. Progeny of the transformants exhibited higher accumulation of the Gln1 transcripts and up to 2.2-fold higher GS activity compared to the non-transgenic controls. When grown under optimal nitrogen conditions, these Gln1 transgenic lines showed greater tillering and up to 2.1-fold increase in shoot vegetative biomass. Interestingly, even under greenhouse conditions, we observed a seasonal component to both these parameters and the grain yield. Our results, showing that the growth and development of sorghum Gln1 transformants are also affected by N availability and other environmental factors, suggest complexity of the relationship between GS activity and plant growth and development. A better understanding of other control points and the ability to manipulate these will be needed to utilize the transgenic technology to improve nitrogen use efficiency of crop plants.

GS143, an I{kappa}B ubiquitination inhibitor, inhibits allergic airway inflammation in mice

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

Hirose, Koichi; Wakashin, Hidefumi; Oki, Mie

2008-09-26

Asthma is characterized by airway inflammation with intense eosinophil infiltration and mucus hyper-production, in which antigen-specific Th2 cells play critical roles. Nuclear factor-{kappa}B (NF-{kappa}B) pathway has been demonstrated to be essential for the production of Th2 cytokines and chemokines in the airways in murine asthma models. In the present study, we examined the effect of GS143, a novel small-molecule inhibitor of I{kappa}B ubiquitination, on antigen-induced airway inflammation and Th2 cytokine production in mice. Intranasal administration of GS143 prior to antigen challenge suppressed antigen-induced NF-{kappa}B activation in the lung of sensitized mice. Intranasal administration of GS143 also inhibited antigen-induced eosinophil andmore » lymphocyte recruitment into the airways as well as the expression of Th2 cytokines and eotaxin in the airways. Moreover, GS143 inhibited antigen-induced differentiation of Th2 cells but not of Th1 cells in vitro. Taken together, these results suggest that I{kappa}B ubiquitination inhibitor may have therapeutic potential against asthma.« less

Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses

PubMed Central

Sheahan, Timothy P.; Sims, Amy C.; Graham, Rachel L.; Menachery, Vineet D.; Gralinski, Lisa E.; Case, James B.; Leist, Sarah R.; Pyrc, Krzysztof; Feng, Joy Y.; Trantcheva, Iva; Bannister, Roy; Park, Yeojin; Babusis, Darius; Clarke, Michael O.; Mackman, Richard L.; Spahn, Jamie E.; Palmiotti, Christopher A.; Siegel, Dustin; Ray, Adrian S.; Cihlar, Tomas; Jordan, Robert; Denison, Mark R.; Baric, Ralph S.

2017-01-01

Emerging viral infections are difficult to control as heterogeneous members periodically cycle in and out of humans and zoonotic hosts, complicating the development of specific antiviral therapies and vaccines. Coronaviruses (CoVs) have a proclivity to spread rapidly into new host species causing severe disease. SARS-CoV and MERS-CoV successively emerged causing severe epidemic respiratory disease in immunologically naïve human populations throughout the globe. Broad-spectrum therapies capable of inhibiting CoV infections would address an immediate unmet medical need and could be invaluable in the treatment of emerging and endemic CoV infections. Here we show that a nucleotide prodrug GS-5734, currently in clinical development for treatment of Ebola virus disease, can inhibit SARS-CoV and MERS-CoV replication in multiple in vitro systems including primary human airway epithelial cell cultures with submicromolar IC50 values. GS-5734 was also effective against bat-CoVs, prepandemic bat-CoVs and circulating contemporary human CoV in primary human lung cells, thus demonstrating broad-spectrum anti-CoV activity. In a mouse model of SARS-CoV pathogenesis, prophylactic and early therapeutic administration of GS-5734 significantly reduced lung viral load and improved clinical signs of disease as well as respiratory functions. These data provide substantive evidence that GS-5734 may prove effective against endemic MERS-CoV in the Middle East, circulating human CoV, and possibly most importantly, emerging CoV of the future. PMID:28659436

Toll-Like Receptor 7 Agonist GS-9620 Induces HIV Expression and HIV-Specific Immunity in Cells from HIV-Infected Individuals on Suppressive Antiretroviral Therapy.

PubMed

Tsai, Angela; Irrinki, Alivelu; Kaur, Jasmine; Cihlar, Tomas; Kukolj, George; Sloan, Derek D; Murry, Jeffrey P

2017-04-15

Antiretroviral therapy can suppress HIV replication to undetectable levels but does not eliminate latent HIV, thus necessitating lifelong therapy. Recent efforts to target this persistent reservoir have focused on inducing the expression of latent HIV so that infected cells may be recognized and eliminated by the immune system. Toll-like receptor (TLR) activation stimulates antiviral immunity and has been shown to induce HIV from latently infected cells. Activation of TLR7 leads to the production of several stimulatory cytokines, including type I interferons (IFNs). In this study, we show that the selective TLR7 agonist GS-9620 induced HIV in peripheral blood mononuclear cells (PBMCs) from HIV-infected individuals on suppressive antiretroviral therapy. GS-9620 increased extracellular HIV RNA 1.5- to 2-fold through a mechanism that required type I IFN signaling. GS-9620 also activated HIV-specific T cells and enhanced antibody-mediated clearance of HIV-infected cells. Activation by GS-9620 in combination with HIV peptide stimulation increased CD8 T cell degranulation, production of intracellular cytokines, and cytolytic activity. T cell activation was again dependent on type I IFNs produced by plasmacytoid dendritic cells. GS-9620 induced phagocytic cell maturation and improved effector-mediated killing of HIV-infected CD4 T cells by the HIV envelope-specific broadly neutralizing antibody PGT121. Collectively, these data show that GS-9620 can activate HIV production and improve the effector functions that target latently infected cells. GS-9620 may effectively complement orthogonal therapies designed to stimulate antiviral immunity, such as therapeutic vaccines or broadly neutralizing antibodies. Clinical studies are under way to determine if GS-9620 can target HIV reservoirs. IMPORTANCE Though antiretroviral therapies effectively suppress viral replication, they do not eliminate integrated proviral DNA. This stable intermediate of viral infection is persistently

Toll-Like Receptor 7 Agonist GS-9620 Induces HIV Expression and HIV-Specific Immunity in Cells from HIV-Infected Individuals on Suppressive Antiretroviral Therapy

PubMed Central

Tsai, Angela; Irrinki, Alivelu; Kaur, Jasmine; Cihlar, Tomas; Kukolj, George

2017-01-01

ABSTRACT Antiretroviral therapy can suppress HIV replication to undetectable levels but does not eliminate latent HIV, thus necessitating lifelong therapy. Recent efforts to target this persistent reservoir have focused on inducing the expression of latent HIV so that infected cells may be recognized and eliminated by the immune system. Toll-like receptor (TLR) activation stimulates antiviral immunity and has been shown to induce HIV from latently infected cells. Activation of TLR7 leads to the production of several stimulatory cytokines, including type I interferons (IFNs). In this study, we show that the selective TLR7 agonist GS-9620 induced HIV in peripheral blood mononuclear cells (PBMCs) from HIV-infected individuals on suppressive antiretroviral therapy. GS-9620 increased extracellular HIV RNA 1.5- to 2-fold through a mechanism that required type I IFN signaling. GS-9620 also activated HIV-specific T cells and enhanced antibody-mediated clearance of HIV-infected cells. Activation by GS-9620 in combination with HIV peptide stimulation increased CD8 T cell degranulation, production of intracellular cytokines, and cytolytic activity. T cell activation was again dependent on type I IFNs produced by plasmacytoid dendritic cells. GS-9620 induced phagocytic cell maturation and improved effector-mediated killing of HIV-infected CD4 T cells by the HIV envelope-specific broadly neutralizing antibody PGT121. Collectively, these data show that GS-9620 can activate HIV production and improve the effector functions that target latently infected cells. GS-9620 may effectively complement orthogonal therapies designed to stimulate antiviral immunity, such as therapeutic vaccines or broadly neutralizing antibodies. Clinical studies are under way to determine if GS-9620 can target HIV reservoirs. IMPORTANCE Though antiretroviral therapies effectively suppress viral replication, they do not eliminate integrated proviral DNA. This stable intermediate of viral infection is

Gsα deficiency in the paraventricular nucleus of the hypothalamus partially contributes to obesity associated with Gsα mutations.

PubMed

Chen, Min; Berger, Alta; Kablan, Ahmed; Zhang, Jiandi; Gavrilova, Oksana; Weinstein, Lee S

2012-09-01

The G protein α-subunit G(s)α mediates receptor-stimulated cAMP generation. Heterozygous inactivating G(s)α mutations on the maternal allele result in obesity primarily due to reduced energy expenditure in Albright hereditary osteodystrophy patients and in mice. We previously showed that mice with central nervous system (CNS)-specific G(s)α deletion on the maternal allele (mBrGs KO) also develop severe obesity with reduced energy expenditure and that G(s)α is primarily expressed from the maternal allele in the paraventricular nucleus (PVN) of the hypothalamus, an important site of energy balance regulation. We now generated mice with PVN-specific G(s)α deficiency by mating Single-minded 1-cre and G(s)α-floxed mice. Homozygous G(s)α deletion produced early lethality. Heterozygotes with maternal G(s)α deletion (mPVNGsKO) also developed obesity and had small reductions in energy expenditure. However, this effect was much milder than that found in mBrGsKO mice and was more prominent in males. We previously showed mBrGsKO mice to have significant reductions in melanocortin receptor agonist-stimulated energy expenditure and now show that mBrGsKO mice have impaired cold-induced brown adipose tissue stimulation. In contrast, these effects were absent in mPVNGsKO mice. mPVNGsKO mice also had minimal effects on glucose metabolism as compared with mBrGsKO mice. Consistent with the presence of G(s)α imprinting, paternal heterozygotes showed no changes in energy or glucose metabolism. These results indicate that although G(s)α deficiency in PVN partially contributes to the metabolic phenotype resulting from maternal G(s)α mutations, G(s)α imprinting in other CNS regions is also important in mediating the CNS effects of G(s)α mutations on energy and glucose metabolism.

Polyelectrolyte Complexes of Low Molecular Weight PEI and Citric Acid as Efficient and Nontoxic Vectors for in Vitro and in Vivo Gene Delivery.

PubMed

Giron-Gonzalez, M Dolores; Salto-Gonzalez, Rafael; Lopez-Jaramillo, F Javier; Salinas-Castillo, Alfonso; Jodar-Reyes, Ana Belen; Ortega-Muñoz, Mariano; Hernandez-Mateo, Fernando; Santoyo-Gonzalez, Francisco

2016-03-16

Gene transfection mediated by the cationic polymer polyethylenimine (PEI) is considered a standard methodology. However, while highly branched PEIs form smaller polyplexes with DNA that exhibit high transfection efficiencies, they have significant cell toxicity. Conversely, low molecular weight PEIs (LMW-PEIs) with favorable cytotoxicity profiles display minimum transfection activities as a result of inadequate DNA complexation and protection. To solve this paradox, a novel polyelectrolyte complex was prepared by the ionic cross-linking of branched 1.8 kDa PEI with citric acid (CA). This system synergistically exploits the good cytotoxicity profile exhibited by LMW-PEI with the high transfection efficiencies shown by highly branched and high molecular weight PEIs. The polyectrolyte complex (1.8 kDa-PEI@CA) was obtained by a simple synthetic protocol based on the microwave irradiation of a solution of 1.8 kDa PEI and CA. Upon complexation with DNA, intrinsic properties of the resulting particles (size and surface charge) were measured and their ability to form stable polyplexes was determined. Compared with unmodified PEIs the new complexes behave as efficient gene vectors and showed enhanced DNA binding capability associated with facilitated intracellular DNA release and enhanced DNA protection from endonuclease degradation. In addition, while transfection values for LMW-PEIs are almost null, transfection efficiencies of the new reagent range from 2.5- to 3.8-fold to those of Lipofectamine 2000 and 25 kDa PEI in several cell lines in culture such as CHO-k1, FTO2B hepatomas, L6 myoblasts, or NRK cells, simultaneously showing a negligible toxicity. Furthermore, the 1.8 kDa-PEI@CA polyelectrolyte complexes retained the capability to transfect eukaryotic cells in the presence of serum and exhibited the capability to promote in vivo transfection in mouse (as an animal model) with an enhanced efficiency compared to 25 kDa PEI. Results support the polyelectrolyte complex

An integrated -omics analysis of the epigenetic landscape of gene expression in human blood cells.

PubMed

Kennedy, Elizabeth M; Goehring, George N; Nichols, Michael H; Robins, Chloe; Mehta, Divya; Klengel, Torsten; Eskin, Eleazar; Smith, Alicia K; Conneely, Karen N

2018-06-19

Gene expression can be influenced by DNA methylation 1) distally, at regulatory elements such as enhancers, as well as 2) proximally, at promoters. Our current understanding of the influence of distal DNA methylation changes on gene expression patterns is incomplete. Here, we characterize genome-wide methylation and expression patterns for ~ 13 k genes to explore how DNA methylation interacts with gene expression, throughout the genome. We used a linear mixed model framework to assess the correlation of DNA methylation at ~ 400 k CpGs with gene expression changes at ~ 13 k transcripts in two independent datasets from human blood cells. Among CpGs at which methylation significantly associates with transcription (eCpGs), > 50% are distal (> 50 kb) or trans (different chromosome) to the correlated gene. Many eCpG-transcript pairs are consistent between studies and ~ 90% of neighboring eCpGs associate with the same gene, within studies. We find that enhancers (P < 5e-18) and microRNA genes (P = 9e-3) are overrepresented among trans eCpGs, and insulators and long intergenic non-coding RNAs are enriched among cis and distal eCpGs. Intragenic-eCpG-transcript correlations are negative in 60-70% of occurrences and are enriched for annotated gene promoters and enhancers (P < 0.002), highlighting the importance of intragenic regulation. Gene Ontology analysis indicates that trans eCpGs are enriched for transcription factor genes and chromatin modifiers, suggesting that some trans eCpGs represent the influence of gene networks and higher-order transcriptional control. This work sheds new light on the interplay between epigenetic changes and gene expression, and provides useful data for mining biologically-relevant results from epigenome-wide association studies.

Mechanism study of endothelial protection and inhibits platelet activation of low molecular weight fucoidan from Laminaria japonica

NASA Astrophysics Data System (ADS)

Chen, Anjin; Zhang, Fang; Shi, Jie; Zhao, Xue; Yan, Meixing

2016-10-01

Several studies have indicated that fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica could inhibit the activation of platelets directly by reducing the platelet aggregation. To explore the direct effect of LMW fucoidan on the platelet system furthermore and examine the possible mechanism, the endothelial protection and inhibits platelet activation effects of two LMW fucoidan were investigated. In the present study, Endothelial injury model of rats was made by injection of adrenaline (0.4 mg kg-1) and human umbilical vein endothelial cells were cultured. vWF level was be investigated in vivo and in vitro as an important index of endothelial injury. LMW fucoidan could significantly reduce vWF level in vascular endothelial injury rats and also significantly reduce vWF level in vitro. The number of EMPs was be detected as another important index of endothelial injury. The results showed that LMW fucoidan reduced EMPs stimulated by tumor necrosis factor. In this study, it was found that by inhibiting platelet adhesion, LMW fucoidan played a role in anti-thrombosis and the specific mechanism of action is to inhibit the flow of extracellular Ca2+. All in a word, LMW fucoidan could inhibit the activation of platelets indirectly by reducing the concentration of EMPs and vWF, at the same time; LMW fucoidan inhibited the activation of platelets directly by inhibiting the flow of extracellular Ca2+.

Expression of Genes Encoding the Enzymes for Glycogen and Trehalose Metabolism in L3 and L4 Larvae of Anisakis simplex.

PubMed

Łopieńska-Biernat, E; Zaobidna, E A; Dmitryjuk, M

2015-01-01

Trehalose and glycogen metabolism plays an important role in supporting life processes in many nematodes, including Anisakis simplex. Nematodes, cosmopolitan helminths parasitizing sea mammals and humans, cause a disease known as anisakiasis. The aim of this study was to investigate the expression of genes encoding the enzymes involved in the metabolism of trehalose and glycogen-trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), glycogen synthase (GS), and glycogen phosphorylase (GP)-in stage L3 and stage L4 larvae of A. simplex. The expression of mRNA all four genes, tps, tpp, gs, and gp, was examined by real-time polymerase chain reaction. The A. simplex ribosomal gene (18S) was used as a reference gene. Enzymatic activity was determined. The expression of trehalose enzyme genes was higher in L3 than in L4 larvae, but an inverse relationship was noted for the expression of gs and gp genes.

Expression of Genes Encoding the Enzymes for Glycogen and Trehalose Metabolism in L3 and L4 Larvae of Anisakis simplex

PubMed Central

Łopieńska-Biernat, E.; Zaobidna, E. A.; Dmitryjuk, M.

2015-01-01

Trehalose and glycogen metabolism plays an important role in supporting life processes in many nematodes, including Anisakis simplex. Nematodes, cosmopolitan helminths parasitizing sea mammals and humans, cause a disease known as anisakiasis. The aim of this study was to investigate the expression of genes encoding the enzymes involved in the metabolism of trehalose and glycogen—trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), glycogen synthase (GS), and glycogen phosphorylase (GP)—in stage L3 and stage L4 larvae of A. simplex. The expression of mRNA all four genes, tps, tpp, gs, and gp, was examined by real-time polymerase chain reaction. The A. simplex ribosomal gene (18S) was used as a reference gene. Enzymatic activity was determined. The expression of trehalose enzyme genes was higher in L3 than in L4 larvae, but an inverse relationship was noted for the expression of gs and gp genes. PMID:26783451

The novel sodium channel modulator GS-458967 (GS967) is an effective treatment in a mouse model of SCN8A encephalopathy.

PubMed

Baker, Erin M; Thompson, Christopher H; Hawkins, Nicole A; Wagnon, Jacy L; Wengert, Eric R; Patel, Manoj K; George, Alfred L; Meisler, Miriam H; Kearney, Jennifer A

2018-06-01

De novo mutations of SCN8A, encoding the voltage-gated sodium channel Na V 1.6, have been associated with a severe infant onset epileptic encephalopathy. Individuals with SCN8A encephalopathy have a mean age of seizure onset of 4-5 months, with multiple seizure types that are often refractory to treatment with available drugs. Anecdotal reports suggest that high-dose phenytoin is effective for some patients, but there are associated adverse effects and potential for toxicity. Functional characterization of several SCN8A encephalopathy variants has shown that elevated persistent sodium current is one of several common biophysical defects. Therefore, specifically targeting elevated persistent current may be a useful therapeutic strategy in some cases. The novel sodium channel modulator GS967 has greater preference for persistent as opposed to peak current and nearly 10-fold greater potency than phenytoin. We evaluated the therapeutic effect of GS967 in the Scn8a N1768D/+ mouse model carrying an SCN8A patient mutation that results in elevated persistent sodium current. We also performed patch clamp recordings to assess the effect of GS967 on peak and persistent sodium current and excitability in hippocampal neurons from Scn8a N1768D/+ mice. GS967 potently blocked persistent sodium current without affecting peak current, normalized action potential morphology, and attenuated excitability in neurons from heterozygous Scn8a N1768D/+ mice. Acute treatment with GS967 provided dose-dependent protection against maximal electroshock-induced seizures in Scn8a N1768D/+ and wild-type mice. Chronic treatment of Scn8a N1768D/+ mice with GS967 resulted in lower seizure burden and complete protection from seizure-associated lethality observed in untreated Scn8a N1768D/+ mice. Protection was achieved at a chronic dose that did not cause overt behavioral toxicity or sedation. Persistent sodium current modulators like GS967 may be an effective precision targeting strategy for SCN8A

The PA-Gene-Mediated Lethal Dissemination and Excessive Innate Immune Response Contribute to the High Virulence of H5N1 Avian Influenza Virus in Mice

PubMed Central

Hu, Jiao; Hu, Zenglei; Song, Qingqing; Gu, Min; Liu, Xiaowen; Wang, Xiaoquan; Hu, Shunlin; Chen, Chaoyang; Liu, Huimou; Liu, Wenbo; Chen, Sujuan; Peng, Daxin

2013-01-01

Highly pathogenic H5N1 influenza A virus remains a substantial threat to public health. To understand the molecular basis and host mechanism for the high virulence of H5N1 viruses in mammals, we compared two H5N1 isolates which have similar genetic backgrounds but greatly differ in their virulence in mice. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is nonpathogenic. We first showed that CK10 elicited a more potent innate immune response than did GS10 in mouse lungs by increasing the number and expression levels of activated genes. We then generated a series of reassortants between the two viruses and evaluated their virulence in mice. Inclusion of the CK10 PA gene in the GS10 background resulted in a dramatic increase in virulence. Conversely, expression of the GS10 PA gene in the CK10 background significantly attenuated the virulence. These results demonstrated that the PA gene mainly determines the pathogenicity discrepancy between CK10 and GS10 in mice. We further determined that arginine (R) at position 353 of the PA gene contributes to the high virulence of CK10 in mice. The reciprocal substitution at position 353 in PA or the exchange of the entire PA gene largely caused the transfer of viral phenotypes, including virus replication, polymerase activity, and manipulation of the innate response, between CK10 and GS10. We therefore defined a novel molecular marker associated with the high virulence of H5N1 influenza viruses, providing further insights into the pathogenesis of H5N1 viruses in mammals. PMID:23255810

Anti-tumor efficacy study of the Bruton's tyrosine kinase (BTK) inhibitor, ONO/GS-4059, in combination with the glycoengineered type II anti-CD20 monoclonal antibody obinutuzumab (GA101) demonstrates superior in vivo efficacy compared to ONO/GS-4059 in combination with rituximab.

PubMed

Yasuhiro, Tomoko; Sawada, Wako; Klein, Christian; Kozaki, Ryohei; Hotta, Shingo; Yoshizawa, Toshio

2017-03-01

The activated B-cell diffuse large B-cell-like lymphoma (ABC-DLBCL) correlates with poor prognosis. The B-cell receptor signaling pathway is known to be dysregulated in NHL/CLL and given BTK is a downstream mediator of BCR signaling, BTK constitutes an interesting and obvious therapeutic target. Given the high potency and selectivity of the BTK inhibitor, ONO/GS-4059, it was hypothesized that, the anti-tumor activity of ONO/GS-4059 could be further enhanced by combining it with the anti-CD20 Abs, rituximab (RTX) or obinutuzumab (GA101). ONO/GS-4059 combined with GA101 or RTX was significantly better than the respective monotherapy with tumor growth inhibition (TGI) of 90% for the GA101 combination and 86% for the RTX combination. In contrast, ibrutinib (PCI-32765) combined with RTX did not result in improved efficacy compared with respective monotherapy. Taken together these data indicate that the combination of ONO/GS-4059 with rituximab and particularly obinutuzumab may be an effective treatment for ABC-DLBCL.

Evaluation of Two Guralp Preamplifiers for GS13 Seismometer Application.

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

Merchant, Bion J.

Sandia National Laboratories has tested and evaluated a new preamplifier, the Guralp Preamplifier for GS13, manufactured by Guralp. These preamplifiers are used to interface between Guralp digitizers and Geotech GS13 Seismometers. The purpose of the preamplifier evaluation was to measure the performance characteristics in such areas as power consumption, input impedance, sensitivity, full scale, self-noise, dynamic range, system noise, response, passband, and timing. The Guralp GS13 Preamplifiers are being evaluated for potential use in the International Monitoring System (IMS) of the Comprehensive Nuclear Test-Ban-Treaty Organization (CTBTO).

5 CFR 531.217 - Special conversion rules for certain non-GS employees.

Code of Federal Regulations, 2012 CFR

2012-01-01

... be converted to GS-equivalent rates immediately before leaving the non-GS system, the employee is... 5 Administrative Personnel 1 2012-01-01 2012-01-01 false Special conversion rules for certain non... Appointment Or Position Changes § 531.217 Special conversion rules for certain non-GS employees. When an...

5 CFR 531.217 - Special conversion rules for certain non-GS employees.

Code of Federal Regulations, 2014 CFR

2014-01-01

... be converted to GS-equivalent rates immediately before leaving the non-GS system, the employee is... 5 Administrative Personnel 1 2014-01-01 2014-01-01 false Special conversion rules for certain non... Appointment Or Position Changes § 531.217 Special conversion rules for certain non-GS employees. When an...

5 CFR 531.217 - Special conversion rules for certain non-GS employees.

Code of Federal Regulations, 2011 CFR

2011-01-01

... be converted to GS-equivalent rates immediately before leaving the non-GS system, the employee is... 5 Administrative Personnel 1 2011-01-01 2011-01-01 false Special conversion rules for certain non... Appointment Or Position Changes § 531.217 Special conversion rules for certain non-GS employees. When an...

5 CFR 531.217 - Special conversion rules for certain non-GS employees.

Code of Federal Regulations, 2013 CFR

2013-01-01

... be converted to GS-equivalent rates immediately before leaving the non-GS system, the employee is... 5 Administrative Personnel 1 2013-01-01 2013-01-01 false Special conversion rules for certain non... Appointment Or Position Changes § 531.217 Special conversion rules for certain non-GS employees. When an...

5 CFR 531.217 - Special conversion rules for certain non-GS employees.

Code of Federal Regulations, 2010 CFR

2010-01-01

... be converted to GS-equivalent rates immediately before leaving the non-GS system, the employee is... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Special conversion rules for certain non... Appointment Or Position Changes § 531.217 Special conversion rules for certain non-GS employees. When an...

Influence of temperature during grain filling on gluten viscoelastic properties and gluten protein composition.

PubMed

Koga, Shiori; Böcker, Ulrike; Moldestad, Anette; Tosi, Paola; Shewry, Peter R; Mosleth, Ellen F; Uhlen, Anne Kjersti

2016-01-15

The aim of this study was to investigate the effects of low to moderate temperatures on gluten functionality and gluten protein composition. Four spring wheat cultivars were grown in climate chambers with three temperature regimes (day/night temperatures of 13/10, 18/15 and 23/20 °C) during grain filling. The temperature strongly influenced grain weight and protein content. Gluten quality measured by maximum resistance to extension (Rmax ) was highest in three cultivars grown at 13 °C. Rmax was positively correlated with the proportion of sodium dodecyl sulfate-unextractable polymeric proteins (%UPP). The proportions of ω-gliadins and D-type low-molecular-weight glutenin subunits (LMW-GS) increased and the proportions of α- and γ-gliadins and B-type LMW-GS decreased with higher temperature, while the proportion of high-molecular-weight glutenin subunits (HMW-GS) was constant between temperatures. The cultivar Berserk had strong and constant Rmax between the different temperatures. Constant low temperature, even as low as 13 °C, had no negative effects on gluten quality. The observed variation in Rmax related to temperature could be explained more by %UPP than by changes in the proportions of HMW-GS or other gluten proteins. The four cultivars responded differently to temperature, as gluten from Berserk was stronger and more stable over a wide range of temperatures. © 2015 Society of Chemical Industry.

The transcriptional activator NtrC controls the expression and activity of glutamine synthetase in Herbaspirillum seropedicae.

PubMed

Persuhn, D C; Souza, E M; Steffens, M B; Pedrosa, F O; Yates, M G; Rigo, L U

2000-11-15

The role of the Ntr system in Herbaspirillum seropedicae was determined via ntrB and ntrC mutants. Three phenotypes were identified in these mutants: Nif(-), deficiency in growth using nitrate, and low glutamine synthetase (GS) activity. All phenotypes were restored by the plasmid pKRT1 containing the intact glnA, ntrB and ntrC genes of H. seropedicae. The promoter region of glnA was subcloned into a beta-galactosidase fusion vector and the results suggested that NtrC positively regulates the glnA promoter in response to low nitrogen. The H. seropedicae ntrC and ntrB mutant strains showed a deficiency of adenylylation/deadenylylation of GS, indicating that NtrC and NtrB are involved in both transcription and activity control of GS in this organism.

Spatiotemporal distribution and source apportionment of low molecular weight organic acids in wet precipitation at a coastal city, China.

PubMed

Du, Wenjiao; Hong, Zhenyu; Chen, Yanting; Deng, Junjun; Chen, Jinsheng; Xu, Lingling; Hong, Youwei; Xiao, Hang

2017-03-01

To investigate the characteristics and sources of low molecular weight (LMW) organic acids in wet precipitation at a coastal city, Xiamen, a total of 313 rainwater samples were collected at seven different functional areas from September 2012 to August 2013. Spatiotemporal characteristics of LMW organic acids as well as pH and electrical conductivity were analyzed. Meanwhile, air mass clusters in different seasons and the positive matrix factorization (PMF) source apportion model were comprehensively used to identify the sources of organic acids. In conclusion, the volume-weighted mean (VWM) concentration of formic (3.20 μmol/L), acetic (1.84 μmol/L), lactic (0.44 μmol/L), and oxalic acid (0.53 μmol/L) were obtained, which jointly contributed to 4.33% of the total free acidity (TFA). At the same time, the highest wet deposition flux of LMW organic acids and contribution of that to TFA were achieved at the forest protection area during growing season in Xiamen. In addition, biogenic emissions (77.12%), sea salts (13.77%), regional agriculture activities (3.92%), soil emissions (2.56%), biomass burning (1.47%), and secondary aerosols (1.15%) were determined as the source of LMW organic acids. Besides, the dominancy of biomass burning via long-range transport in non-growing season (NGS) and the contribution of biogenic emission in growing season (GS) were recognized. Finally, the considerable influence of sea salts on the LMW organic acids (13.77%) in Xiamen was quantified, especially for oxalic acid.

Identification of novel interactors and potential phosphorylation substrates of GsSnRK1 from wild soybean (Glycine soja).

PubMed

Song, Yu; Zhang, Hang; You, Hongguang; Liu, Yuanming; Chen, Chao; Feng, Xu; Yu, Xingyu; Wu, Shengyang; Wang, Libo; Zhong, Shihua; Li, Qiang; Zhu, Yanming; Ding, Xiaodong

2018-04-17

The plant sucrose nonfermenting kinase 1 (SnRK1) kinases play the central roles in the processes of energy balance, hormone perception, stress resistance, metabolism, growth, and development. However, the functions of these kinases are still elusive. In this study, we used GsSnRK1 of wild soybean as bait to perform library-scale screens by the means of yeast two-hybrid to identify its interacting proteins. The putative interactions were verified by yeast retransformation and β-galactosidase assays, and the selected interactions were further confirmed in planta by bimolecular fluorescence complementation and biochemical Co-IP assays. Protein phosphorylation analyses were carried out by phos-tag assay and anti-phospho-(Ser/Thr) substrate antibodies. Finally, we obtained 24 GsSnRK1 interactors and several putative substrates that can be categorized into SnRK1 regulatory β subunit, protein modification, biotic and abiotic stress-related, hormone perception and signalling, gene expression regulation, water and nitrogen transport, metabolism, and unknown proteins. Intriguingly, we first discovered that GsSnRK1 interacted with and phosphorylated the components of soybean nodulation and symbiotic nitrogen fixation. The interactions and potential functions of GsSnRK1 and its associated proteins were extensively discussed and analysed. This work provides plausible clues to elucidate the novel functions of SnRK1 in response to variable environmental, metabolic, and physiological requirements. © 2018 John Wiley & Sons Ltd.

Gsα Deficiency in the Ventromedial Hypothalamus Enhances Leptin Sensitivity and Improves Glucose Homeostasis in Mice on a High-Fat Diet.

PubMed

Berger, Alta; Kablan, Ahmed; Yao, Catherine; Ho, Thuy; Podyma, Brandon; Weinstein, Lee S; Chen, Min

2016-02-01

In both mice and patients with Albright hereditary osteodystrophy, heterozygous inactivating mutations of Gsα, a ubiquitously expressed G protein that mediates receptor-stimulated intracellular cAMP production, lead to obesity and insulin resistance but only when the mutation is present on the maternal allele. This parent-of-origin effect in mice was shown to be due to Gsα imprinting in one or more brain regions. The ventromedial hypothalamus (VMH) is involved in the regulation of energy and glucose homeostasis, but the role of Gsα in VMH on metabolic regulation is unknown. To examine this, we created VMH-specific Gsα-deficient mice by mating Gsα-floxed mice with SF1-cre mice. Heterozygotes with Gsα mutation on either the maternal or paternal allele had a normal metabolic phenotype, and there was no molecular evidence of Gsα imprinting, indicating that the parent-of-origin metabolic effects associated with Gsα mutations is not due to Gsα deficiency in VMH SF1 neurons. Homozygous VMH Gsα knockout mice (VMHGsKO) showed no changes in body weight on either a regular or high-fat diet. However, glucose metabolism (fasting glucose, glucose tolerance, insulin sensitivity) was significantly improved in male VMHGsKO mice, with the difference more dramatic on the high-fat diet. In addition, male VMHGsKO mice on the high-fat diet showed a greater anorexigenic effect and increased VMH signal transducer and activator of transcription-3 phosphorylation in response to leptin. These results indicate that VMH Gsα/cyclic AMP signaling regulates glucose homeostasis and alters leptin sensitivity in mice, particularly in the setting of excess caloric intake.

Time to take action to meet GS1 mandate.

PubMed

Doyle, Chris

2014-10-01

A recent paper, NHS eProcurement Strategy,(1) published earlier this year by the Department of Health, mandated the use of GS1 standards throughout the NHS in England. The Strategy requires all NHS Trusts in England to produce a Board-approved adoption plan. Chris Doyle, head of Healthcare at independent global supply chain standards organization, GS1 UK, explains the next steps to becoming compliant.

Construction of a multiplex mutation hot spot PCR panel: the first step towards colorectal cancer genotyping on the GS Junior platform.

PubMed

Péterfia, Bálint; Kalmár, Alexandra; Patai, Árpád V; Csabai, István; Bodor, András; Micsik, Tamás; Wichmann, Barnabás; Egedi, Krisztina; Hollósi, Péter; Kovalszky, Ilona; Tulassay, Zsolt; Molnár, Béla

2017-01-01

Background: To support cancer therapy, development of low cost library preparation techniques for targeted next generation sequencing (NGS) is needed. In this study we designed and tested a PCR-based library preparation panel with limited target area for sequencing the top 12 somatic mutation hot spots in colorectal cancer on the GS Junior instrument. Materials and Methods: A multiplex PCR panel was designed to amplify regions of mutation hot spots in 12 selected genes ( APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53 ). Amplicons were sequenced on a GS Junior instrument using ligated and barcoded adaptors. Eight samples were sequenced in a single run. Colonic DNA samples (8 normal mucosa; 33 adenomas; 17 adenocarcinomas) as well as HT-29 and Caco-2 cell lines with known mutation profiles were analyzed. Variants found by the panel on APC, BRAF, KRAS and NRAS genes were validated by conventional sequencing. Results: In total, 34 kinds of mutations were detected including two novel mutations ( FBXW7 c.1740:C>G and SMAD4 c.413C>G) that have not been recorded in mutation databases, and one potential germline mutation ( APC ). The most frequently mutated genes were APC, TP53 and KRAS with 30%, 15% and 21% frequencies in adenomas and 29%, 53% and 29% frequencies in carcinomas, respectively. In cell lines, all the expected mutations were detected except for one located in a homopolymer region. According to re-sequencing results sensitivity and specificity was 100% and 92% respectively. Conclusions: Our NGS-based screening panel denotes a promising step towards low cost colorectal cancer genotyping on the GS Junior instrument. Despite the relatively low coverage, we discovered two novel mutations and obtained mutation frequencies comparable to literature data. Additionally, as an advantage, this panel requires less template DNA than sequence capture colon cancer panels currently available for the GS Junior instrument.

Nuclear Expression of GS28 Protein: A Novel Biomarker that Predicts Prognosis in Colorectal Cancers

PubMed Central

Lee, Sung Hak; Yoo, Hyung Jae; Rim, Do Eun; Cui, Yinji; Lee, Ahwon; Jung, Eun Sun; Oh, Seung Taek; Kim, Jun Gi; Kwon, Oh-Joo; Kim, Su Young; Jeong, Seong-Whan

2017-01-01

Aims: GS28 (Golgi SNARE protein, 28 kDa), a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) protein family, plays a critical role in mammalian endoplasmic reticulum (ER)-Golgi or intra-Golgi vesicle transport. To date, few researches on the GS28 protein in human cancer tissues have been reported. In this study, we assessed the prognostic value of GS28 in patients with colorectal cancer (CRC). Methods and results: We screened for GS28 expression using immunohistochemistry in 230 surgical CRC specimens. The CRCs were right-sided and left-sided in 28.3% (65/230) and 71.3% (164/230) of patients, respectively. GS28 staining results were available in 214 cases. Among these, there were 26 nuclear predominant cases and 188 non-nuclear predominant cases. Stromal GS28 expression was noted in 152 cases of CRC. GS28 nuclear predominant immunoreactivity was significantly associated with advanced tumour stage (p = 0.045) and marginally associated with perineural invasion (p = 0.064). Decreased GS28 expression in the stromal cells was significantly associated with lymph node metastasis (N stage; p = 0.036). GS28 expression was not associated with epidermal growth factor receptor (EGFR) immunohistochemical positivity or KRAS mutation status. Investigation of the prognostic value of GS28 with Kaplan-Meier analysis revealed a correlation with overall survival (p = 0.004). Cases with GS28 nuclear predominant expression had significantly poorer overall survival than those with a non-nuclear predominant pattern. Conclusions: Taken together, these results indicate that GS28 nuclear predominant expression could serve as a prognostic marker for CRC and may help in identifying aggressive forms of CRC. PMID:28638266

Functional analysis of ars gene cluster of Pannonibacter indicus strain HT23(T) (DSM 23407(T)) and identification of a proline residue essential for arsenate reductase activity.

PubMed

Bandyopadhyay, Saumya; Das, Subrata K

2016-04-01

Arsenic is a naturally occurring ubiquitous highly toxic metalloid. In this study, we have identified ars gene cluster in Pannonibacter indicus strain HT23(T) (DSM 23407(T)), responsible for reduction of toxic pentavalent arsenate. The ars gene cluster is comprised of four non-overlapping open reading frames (ORFs) encoding a transcriptional regulator (ArsR), a low molecular weight protein tyrosine phosphatases (LMW-PTPase) with hypothetical function, an arsenite efflux pump (Acr3), and an arsenate reductase (ArsC). Heterologous expression of arsenic inducible ars gene cluster conferred arsenic resistance to Escherichia coli ∆ars mutant strain AW3110. The recombinant ArsC was purified and assayed. Site-directed mutagenesis was employed to ascertain the role of specific amino acids in ArsC catalysis. Pro94X (X = Ala, Arg, Cys, and His) amino acid substitutions led to enzyme inactivation. Circular dichroism spectra analysis suggested Pro94 as an essential amino acid for enzyme catalytic activity as it is indispensable for optimum protein folding in P. indicus Grx-coupled ArsC.

The PA and HA Gene-Mediated High Viral Load and Intense Innate Immune Response in the Brain Contribute to the High Pathogenicity of H5N1 Avian Influenza Virus in Mallard Ducks

PubMed Central

Hu, Jiao; Hu, Zenglei; Mo, Yiqun; Wu, Qiwen; Cui, Zhu; Duan, Zhiqiang; Huang, Junqing; Chen, Hongzhi; Chen, Yuxin; Gu, Min; Wang, Xiaoquan; Hu, Shunlin; Liu, Huimou; Liu, Wenbo; Liu, Xiaowen

2013-01-01

Most highly pathogenic avian influenza A viruses cause only mild clinical signs in ducks, serving as an important natural reservoir of influenza A viruses. However, we isolated two H5N1 viruses that are genetically similar but differ greatly in virulence in ducks. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is low pathogenic. To determine the genetic basis for the high virulence of CK10 in ducks, we generated a series of single-gene reassortants between CK10 and GS10 and tested their virulence in ducks. Expression of the CK10 PA or hemagglutinin (HA) gene in the GS10 context resulted in increased virulence and virus replication. Conversely, inclusion of the GS10 PA or HA gene in the CK10 background attenuated the virulence and virus replication. Moreover, the PA gene had a greater contribution. We further determined that residues 101G and 237E in the PA gene contribute to the high virulence of CK10. Mutations at these two positions produced changes in virulence, virus replication, and polymerase activity of CK10 or GS10. Position 237 plays a greater role in determining these phenotypes. Moreover, the K237E mutation in the GS10 PA gene increased PA nuclear accumulation. Mutant GS10 viruses carrying the CK10 HA gene or the PA101G or PA237E mutation induced an enhanced innate immune response. A sustained innate response was detected in the brain rather than in the lung and spleen. Our results suggest that the PA and HA gene-mediated high virus replication and the intense innate immune response in the brain contribute to the high virulence of H5N1 virus in ducks. PMID:23926340

Allelic analysis of low molecular weight glutenin subunits using 2-DGE in Korean wheat cultivars

USDA-ARS?s Scientific Manuscript database

Two-dimensional gel electrophoresis (2-DGE) was used to determine the allelic compositions of LMW-GS in 32 Korean wheat cultivars. Protein patterns generated by 2-DGE from each cultivar were compared to patterns from standard wheat cultivars for each allele. At the Glu-A3 locus, thirteen c, twelve ...

Comparison of Two Methods for Detecting Alternative Splice Variants Using GeneChip® Exon Arrays

PubMed Central

Fan, Wenhong; Stirewalt, Derek L.; Radich, Jerald P.; Zhao, Lueping

2011-01-01

The Affymetrix GeneChip Exon Array can be used to detect alternative splice variants. Microarray Detection of Alternative Splicing (MIDAS) and Partek® Genomics Suite (Partek® GS) are among the most popular analytical methods used to analyze exon array data. While both methods utilize statistical significance for testing, MIDAS and Partek® GS could produce somewhat different results due to different underlying assumptions. Comparing MIDAS and Partek® GS is quite difficult due to their substantially different mathematical formulations and assumptions regarding alternative splice variants. For meaningful comparison, we have used the previously published generalized probe model (GPM) which encompasses both MIDAS and Partek® GS under different assumptions. We analyzed a colon cancer exon array data set using MIDAS, Partek® GS and GPM. MIDAS and Partek® GS produced quite different sets of genes that are considered to have alternative splice variants. Further, we found that GPM produced results similar to MIDAS as well as to Partek® GS under their respective assumptions. Within the GPM, we show how discoveries relating to alternative variants can be quite different due to different assumptions. MIDAS focuses on relative changes in expression values across different exons within genes and tends to be robust but less efficient. Partek® GS, however, uses absolute expression values of individual exons within genes and tends to be more efficient but more sensitive to the presence of outliers. From our observations, we conclude that MIDAS and Partek® GS produce complementary results, and discoveries from both analyses should be considered. PMID:23675234

Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds.

PubMed

Seabra, Ana R; Vieira, Cristina P; Cullimore, Julie V; Carvalho, Helena G

2010-08-19

Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS), occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in Medicago truncatula. This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2) in M. truncatula. The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene. This study shows that Medicago truncatula contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate that this gene duplication represents a functional

Characterization of a wheat mutant missing low-molecular-weight glutenin subunits encoded by the B-genome

USDA-ARS?s Scientific Manuscript database

DH20, a new wheat mutant missing low-molecular weight glutenin subunits encoded by the Glu-B3 locus, was discovered among double haploid lines obtained from a cross between the Korean wheat cultivars Keumkang and Olgeuru. Absence of the Glu-B3 LMW-GS proteins was determined by one-dimensional gel e...

Molecular cloning of the perilipin gene and its association with carcass and fat traits in Chinese ducks.

PubMed

Zhang, H L; Fan, H J; Liu, X L; Wu, Y; Hou, S S

2013-05-13

The perilipin (PLIN) gene is a candidate gene of carcass and fat traits in ducks. In order to study the molecular character of the PLIN gene and its function in different breeds of Chinese ducks, samples were obtained from the Chinese Academy of Agricultural Sciences Research Center for Birds, including 95 Peking ducks of the Z2 series, 91 Peking ducks of the Z4 series, 82 hybrid systems (Z2 x Z4), and 93 Cherry Valley ducks. We used RT-PCR and 3'-RACE to clone the duck PLIN gene, detect SNPs and analyze their associations with carcass and fat traits. A 2212-bp sequence was cloned with the complete coding region and a 3'-untranslated region. We found a nucleotide mutation (C → T) in exon 2 of the PLIN gene. There were no significant correlations between the 3 genotypes (CC, CT, TT) in breast muscle weight (BMW), leg muscle weight (LMW), subcutaneous fat weight (SFW), and intramuscular fat (IMF) in the Cherry Valley duck. The CC and CT genotypes had significant differences in carcass weight (CW), carcass net weight (CNW), and percentage of abdominal fat weight (AFW); there were significant differences in AFW and percentage of SFW. In Z4, there were no significant correlations between the 3 genotypes (TT, CC, and CT) in CW, BMW, LMW, SFW, AFW, the percentage of SFW and AFW, and IMF. CNW was significantly different between TT, CC, and CT genotypes. In Z2 x Z4, there were no significant correlations between the 3 genotypes in CW, BMW, LMW, SFW, AFW, the percentage of SFW and AFW, and IMF, while the CC and CT genotypes had significant differences in CNW. In Z2, there were no significant differences between the 3 genotypes in all traits. We deduced that the PLIN gene is a potential major gene. It is linked to a major gene affecting meat quality traits. This SNP has potential as a molecular marker for marker-assisted selection.

The GS (genetic selection) Principle.

PubMed

Abel, David L

2009-01-01

The GS (Genetic Selection) Principle states that biological selection must occur at the nucleotide-sequencing molecular-genetic level of 3'5' phosphodiester bond formation. After-the-fact differential survival and reproduction of already-living phenotypic organisms (ordinary natural selection) does not explain polynucleotide prescription and coding. All life depends upon literal genetic algorithms. Even epigenetic and "genomic" factors such as regulation by DNA methylation, histone proteins and microRNAs are ultimately instructed by prior linear digital programming. Biological control requires selection of particular configurable switch-settings to achieve potential function. This occurs largely at the level of nucleotide selection, prior to the realization of any integrated biofunction. Each selection of a nucleotide corresponds to the setting of two formal binary logic gates. The setting of these switches only later determines folding and binding function through minimum-free-energy sinks. These sinks are determined by the primary structure of both the protein itself and the independently prescribed sequencing of chaperones. The GS Principle distinguishes selection of existing function (natural selection) from selection for potential function (formal selection at decision nodes, logic gates and configurable switch-settings).

GOES-R GS Product Generation Infrastructure Operations

NASA Astrophysics Data System (ADS)

Blanton, M.; Gundy, J.

2012-12-01

GOES-R GS Product Generation Infrastructure Operations: The GOES-R Ground System (GS) will produce a much larger set of products with higher data density than previous GOES systems. This requires considerably greater compute and memory resources to achieve the necessary latency and availability for these products. Over time, new algorithms could be added and existing ones removed or updated, but the GOES-R GS cannot go down during this time. To meet these GOES-R GS processing needs, the Harris Corporation will implement a Product Generation (PG) infrastructure that is scalable, extensible, extendable, modular and reliable. The primary parts of the PG infrastructure are the Service Based Architecture (SBA), which includes the Distributed Data Fabric (DDF). The SBA is the middleware that encapsulates and manages science algorithms that generate products. The SBA is divided into three parts, the Executive, which manages and configures the algorithm as a service, the Dispatcher, which provides data to the algorithm, and the Strategy, which determines when the algorithm can execute with the available data. The SBA is a distributed architecture, with services connected to each other over a compute grid and is highly scalable. This plug-and-play architecture allows algorithms to be added, removed, or updated without affecting any other services or software currently running and producing data. Algorithms require product data from other algorithms, so a scalable and reliable messaging is necessary. The SBA uses the DDF to provide this data communication layer between algorithms. The DDF provides an abstract interface over a distributed and persistent multi-layered storage system (memory based caching above disk-based storage) and an event system that allows algorithm services to know when data is available and to get the data that they need to begin processing when they need it. Together, the SBA and the DDF provide a flexible, high performance architecture that can meet

Glutaredoxin modulates platelet-derived growth factor-dependent cell signaling by regulating the redox status of low molecular weight protein-tyrosine phosphatase.

PubMed

Kanda, Munetake; Ihara, Yoshito; Murata, Hiroaki; Urata, Yoshishige; Kono, Takaaki; Yodoi, Junji; Seto, Shinji; Yano, Katsusuke; Kondo, Takahito

2006-09-29

Glutaredoxin (GRX) is a glutathione-disulfide oxidoreductase involved in various cellular functions, including the redox-dependent regulation of certain integral proteins. Here we demonstrated that overexpression of GRX suppressed the proliferation of myocardiac H9c2 cells treated with platelet-derived growth factor (PDGF)-BB. After stimulation with PDGF-BB, the phosphorylation of PDGF receptor (PDGFR) beta was suppressed in GRX gene-transfected cells, compared with controls. Conversely, the phosphorylation was enhanced by depletion of GRX by RNA interference. In this study we focused on the role of low molecular weight protein-tyrosine phosphatase (LMW-PTP) in the dephosphorylation of PDGFRbeta via a redox-dependent mechanism. We found that depletion of LMW-PTP using RNA interference enhanced the PDGF-BB-induced phosphorylation of PDGFRbeta, indicating that LMW-PTP works for PDGFRbeta. The enhancement of the phosphorylation of PDGFRbeta was well correlated with inactivation of LMW-PTP by cellular peroxide generated in the cells stimulated with PDGF-BB. In vitro, with hydrogen peroxide treatment, LMW-PTP showed decreased activity with the concomitant formation of dithiothreitol-reducible oligomers. GRX protected LMW-PTP from hydrogen peroxide-induced oxidation and inactivation in concert with glutathione, NADPH, and glutathione disulfide reductase. This strongly suggests that retention of activity of LMW-PTP by enhanced GRX expression suppresses the proliferation of cells treated with PDGF-BB via enhanced dephosphorylation of PDGFRbeta. Thus, GRX plays an important role in PDGF-BB-dependent cell proliferation by regulating the redox state of LMW-PTP.

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

PubMed

Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

2012-04-01

Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was

The Vigna Genome Server, 'VigGS': A Genomic Knowledge Base of the Genus Vigna Based on High-Quality, Annotated Genome Sequence of the Azuki Bean, Vigna angularis (Willd.) Ohwi & Ohashi.

PubMed

Sakai, Hiroaki; Naito, Ken; Takahashi, Yu; Sato, Toshiyuki; Yamamoto, Toshiya; Muto, Isamu; Itoh, Takeshi; Tomooka, Norihiko

2016-01-01

The genus Vigna includes legume crops such as cowpea, mungbean and azuki bean, as well as >100 wild species. A number of the wild species are highly tolerant to severe environmental conditions including high-salinity, acid or alkaline soil; drought; flooding; and pests and diseases. These features of the genus Vigna make it a good target for investigation of genetic diversity in adaptation to stressful environments; however, a lack of genomic information has hindered such research in this genus. Here, we present a genome database of the genus Vigna, Vigna Genome Server ('VigGS', http://viggs.dna.affrc.go.jp), based on the recently sequenced azuki bean genome, which incorporates annotated exon-intron structures, along with evidence for transcripts and proteins, visualized in GBrowse. VigGS also facilitates user construction of multiple alignments between azuki bean genes and those of six related dicot species. In addition, the database displays sequence polymorphisms between azuki bean and its wild relatives and enables users to design primer sequences targeting any variant site. VigGS offers a simple keyword search in addition to sequence similarity searches using BLAST and BLAT. To incorporate up to date genomic information, VigGS automatically receives newly deposited mRNA sequences of pre-set species from the public database once a week. Users can refer to not only gene structures mapped on the azuki bean genome on GBrowse but also relevant literature of the genes. VigGS will contribute to genomic research into plant biotic and abiotic stresses and to the future development of new stress-tolerant crops. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR-based GS-FLX sequencing.

PubMed

Goossens, Dirk; Moens, Lotte N; Nelis, Eva; Lenaerts, An-Sofie; Glassee, Wim; Kalbe, Andreas; Frey, Bruno; Kopal, Guido; De Jonghe, Peter; De Rijk, Peter; Del-Favero, Jurgen

2009-03-01

We evaluated multiplex PCR amplification as a front-end for high-throughput sequencing, to widen the applicability of massive parallel sequencers for the detailed analysis of complex genomes. Using multiplex PCR reactions, we sequenced the complete coding regions of seven genes implicated in peripheral neuropathies in 40 individuals on a GS-FLX genome sequencer (Roche). The resulting dataset showed highly specific and uniform amplification. Comparison of the GS-FLX sequencing data with the dataset generated by Sanger sequencing confirmed the detection of all variants present and proved the sensitivity of the method for mutation detection. In addition, we showed that we could exploit the multiplexed PCR amplicons to determine individual copy number variation (CNV), increasing the spectrum of detected variations to both genetic and genomic variants. We conclude that our straightforward procedure substantially expands the applicability of the massive parallel sequencers for sequencing projects of a moderate number of amplicons (50-500) with typical applications in resequencing exons in positional or functional candidate regions and molecular genetic diagnostics. 2008 Wiley-Liss, Inc.

Ovarian cancer therapeutic potential of glutamine depletion based on GS expression.

PubMed

Furusawa, Akiko; Miyamoto, Morikazu; Takano, Masashi; Tsuda, Hitoshi; Song, Yong Sang; Aoki, Daisuke; Miyasaka, Naoyuki; Inazawa, Johji; Inoue, Jun

2018-05-28

Amino acids (AAs) are biologically important nutrient compounds necessary for the survival of any cell. Of the 20 AAs, cancer cells depend on the uptake of several extracellular AAs for survival. However, which extracellular AA is indispensable for the survival of cancer cells and the molecular mechanism involved have not been fully defined. In this study, we found that the reduction of cell survival caused by glutamine (Gln) depletion is inversely correlated with the expression level of glutamine synthetase (GS) in ovarian cancer (OVC) cells. GS expression was downregulated in 45 of 316 OVC cases (14.2%). The depletion of extracellular Gln by treatment with l-asparaginase, in addition to inhibiting Gln uptake via the knockdown of a Gln transporter, led to the inhibition of cell growth in OVC cells with low expression of GS (GSlow-OVC cells). Furthermore, the re-expression of GS in GSlow-OVC cells induced the inhibition of tumor growth in vitro and in vivo. Thus, these findings provide novel insight into the development of an OVC therapy based on the requirement of Gln.

Improved stress tolerance and productivity in transgenic rice plants constitutively expressing the Oryza sativa glutathione synthetase OsGS under paddy field conditions.

PubMed

Park, Seong-Im; Kim, Young-Saeng; Kim, Jin-Ju; Mok, Ji-Eun; Kim, Yul-Ho; Park, Hyang-Mi; Kim, Il-Sup; Yoon, Ho-Sung

2017-08-01

Reactive oxygen species, which increase under various environmental stresses, have deleterious effects on plants. An important antioxidant, glutathione, is used to detoxify reactive oxygen species in plant cells and is mainly produced by two enzymes: gamma-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GS). To evaluate the functional roles of the glutathione synthetase gene (OsGS) in rice, we generated four independent transgenic rice plants (TG1-TG4) that overexpressed OsGS under the control of the constitutively expressed OsCc1 promoter. When grown under natural paddy field conditions, the TG rice plants exhibited greater growth development, higher chlorophyll content, and higher GSH/GSSH ratios than control wild-type (WT) rice plants. Subsequently, the TG rice plants enhanced redox homeostasis by preventing hydroperoxide-mediated membrane damage, which improved their adaptation to environmental stresses. As a result, TG rice plants improved rice grain yield and total biomass following increases in panicle number and number of spikelets per panicle, despite differences in climate during the cultivation periods of 2014 and 2015. Overall, our results indicate that OsGS overexpression improved redox homeostasis by enhancing the glutathione pool, which resulted in greater tolerance to environmental stresses in the paddy fields. Copyright © 2017. Published by Elsevier GmbH.

Deletion of the low-molecular-weight glutenin subunit allele Glu-A3a of wheat (Triticum aestivum L.) significantly reduces dough strength and breadmaking quality.

PubMed

Zhen, Shoumin; Han, Caixia; Ma, Chaoying; Gu, Aiqin; Zhang, Ming; Shen, Xixi; Li, Xiaohui; Yan, Yueming

2014-12-19

Low-molecular-weight glutenin subunits (LMW-GS), encoded by Glu-3 complex loci in hexaploid wheat, play important roles in the processing quality of wheat flour. To date, the molecular characteristics and effects on dough quality of individual Glu-3 alleles and their encoding proteins have been poorly studied. We used a Glu-A3 deletion line of the Chinese Spring (CS-n) wheat variety to conduct the first comprehensive study on the molecular characteristics and functional properties of the LMW-GS allele Glu-A3a. The Glu-A3a allele at the Glu-A3 locus in CS and its deletion in CS-n were identified and characterized by proteome and molecular marker methods. The deletion of Glu-A3a had no significant influence on plant morphological and yield traits, but significantly reduced the dough strength and breadmaking quality compared to CS. The complete sequence of the Glu-A3a allele was cloned and characterized, which was found to encode a B-subunit with longer repetitive domains and an increased number of α-helices. The Glu-A3a-encoded B-subunit showed a higher expression level and accumulation rate during grain development. These characteristics of the Glu-A3a allele could contribute to achieving superior gluten quality and demonstrate its potential application to wheat quality improvement. Furthermore, an allele-specific polymerase chain reaction (AS-PCR) marker for the Glu-A3a allele was developed and validated using different bread wheat cultivars, including near-isogenic lines (NILs) and recombinant inbred lines (RILs), which could be used as an effective molecular marker for gluten quality improvement through marker-assisted selection. This work demonstrated that the LMW-GS allele Glu-A3a encodes a specific LMW-i type B-subunit that significantly affects wheat dough strength and breadmaking quality. The Glu-A3a-encoded B-subunit has a long repetitive domain and more α-helix structures as well as a higher expression level and accumulation rate during grain development

Regulating unfolded protein response activator HAC1p for production of thermostable raw-starch hydrolyzing α-amylase in Pichia pastoris.

PubMed

Huang, Mengmeng; Gao, Yanyun; Zhou, Xiangshan; Zhang, Yuanxing; Cai, Menghao

2017-03-01

Unfolded protein response (UPR) usually happens when expressing heterologous proteins in high level, which may help cells to facilitate protein processing. Here, we evaluated the effects of the UPR activator HAC1p on a raw-starch hydrolyzing α-amylase (Gs4j-amyA), so as to improve heterologous production of the enzyme in Pichia pastoris. The gene (amyA) encoding Gs4j-amyA was first codon-optimized and expressed in P. pastoris under the control of the AOX1 promoter. A high gene dosage (12 copies) of amyA facilitated amylase expression which produced an enzyme activity of 305 U/ml. A spliced HAC1 encoding an UPR activator HAC1p was then co-expressed and the dosage effects of HAC1 on amylase expression was investigated. Six copies of HAC1 driven by AOX1 promoter produced a high amylase activity of 2200 U/ml, further increasing by 621%. However, excessive gene dosages driven by the same promoter led to a titration effect of its transcription factors and decreased the amount of amyA transcripts. Thus, constitutive expression of HAC1 by GAP promotor was further involved and Gs4j-amyA activity reached 3700 U/ml finally, which was further increased by 68.2%. Moreover, Gs4j-amyA was glycosylated in P. pastoris which generated higher enzyme activity than that in E. coli. Generally, regulating HAC1p expression by different strategies enhanced amylase production by 11.1 folds, indicating a reference for expression of other proteins in P. pastoris.

Antiangiogenic activity of semisynthetic biotechnological heparins: low-molecular-weight-sulfated Escherichia coli K5 polysaccharide derivatives as fibroblast growth factor antagonists.

PubMed

Presta, Marco; Oreste, Pasqua; Zoppetti, Giorgio; Belleri, Mirella; Tanghetti, Elena; Leali, Daria; Urbinati, Chiara; Bugatti, Antonella; Ronca, Roberto; Nicoli, Stefania; Moroni, Emanuela; Stabile, Helena; Camozzi, Maura; Hernandez, German Andrés; Mitola, Stefania; Dell'Era, Patrizia; Rusnati, Marco; Ribatti, Domenico

2005-01-01

Low-molecular-weight heparin (LMWH) exerts antitumor activity in clinical trials. The K5 polysaccharide from Escherichia coli has the same structure as the heparin precursor. Chemical and enzymatic modifications of K5 polysaccharide lead to the production of biotechnological heparin-like compounds. We investigated the fibroblast growth factor-2 (FGF2) antagonist and antiangiogenic activity of a series of LMW N,O-sulfated K5 derivatives. Surface plasmon resonance analysis showed that LMW-K5 derivatives bind FGF2, thus inhibiting its interaction with heparin immobilized to a BIAcore sensor chip. Interaction of FGF2 with tyrosine-kinase receptors (FGFRs), heparan sulfate proteoglycans (HSPGs), and alpha(v)beta3 integrin is required for biological response in endothelial cells. Similar to LMWH, LMW-K5 derivatives abrogate the formation of HSPG/FGF2/FGFR ternary complexes by preventing FGF2-mediated attachment of FGFR1-overexpressing cells to HSPG-bearing cells and inhibit FGF2-mediated endothelial cell proliferation. However, LMW-K5 derivatives, but not LMWH, also inhibit FGF2/alpha(v)beta3 integrin interaction and consequent FGF2-mediated endothelial cell sprouting in vitro and angiogenesis in vivo in the chick embryo chorioallantoic membrane. LMW N,O-sulfated K5 derivatives affect both HSPG/FGF2/FGFR and FGF2/alpha(v)beta3 interactions and are endowed with FGF2 antagonist and antiangiogenic activity. These compounds may provide the basis for the design of novel LMW heparin-like angiostatic compounds.

Piezo channels and GsMTx4: Two milestones in our understanding of excitatory mechanosensitive channels and their role in pathology.

PubMed

Suchyna, Thomas M

2017-11-01

Discovery of Piezo channels and the reporting of their sensitivity to the inhibitor GsMTx4 were important milestones in the study of non-selective cationic mechanosensitive channels (MSCs) in normal physiology and pathogenesis. GsMTx4 had been used for years to investigate the functional role of cationic MSCs, especially in muscle tissue, but with little understanding of its target or inhibitory mechanism. The sensitivity of Piezo channels to bilayer stress and its robust mechanosensitivity when expressed in heterologous systems were keys to determining GsMTx4's mechanism of action. However, questions remain regarding Piezo's role in muscle function due to the non-selective nature of GsMTx4 inhibition toward membrane mechanoenzymes and the implication of MCS channel types by genetic knockdown. Evidence supporting Piezo like activity, at least in the developmental stages of muscle, is presented. While the MSC targets of GsMTx4 in muscle pathology are unclear, its muscle protective effects are clearly demonstrated in two recent in situ studies on normal cardiomyocytes and dystrophic skeletal muscle. The muscle protective function may be due to the combined effect of GsMTx4's inhibitory action on cationic MSCs like Piezo and TRP, and its potentiation of repolarizing K + selective MSCs like K2P and SAKCa. Paradoxically, the potent in vitro action of GsMTx4 on many physiological functions seems to conflict with its lack of in situ side-effects on normal animal physiology. Future investigations into cytoskeletal control of sarcolemma mechanics and the suspected inclusion of MSCs in membrane micro/nano sized domains with distinct mechanical properties will aide our understanding of this dichotomy. Published by Elsevier Ltd.

Differences in gluten protein composition between old and modern durum wheat genotypes in relation to 20th century breeding in Italy.

PubMed

De Santis, Michele A; Giuliani, Marcella M; Giuzio, Luigia; De Vita, Pasquale; Lovegrove, Alison; Shewry, Peter R; Flagella, Zina

2017-07-01

The impact of breeding on grain yields of wheat varieties released during the 20th century has been extensively studied, whereas less information is available on the changes in gluten quality associated with effects on the amount and composition of glutenins and gliadins. In order to explore the effects of breeding during the 20th century on gluten quality of durum wheat for processing and health we have compared a set of old and modern Italian genotypes grown under Mediterranean conditions. The better technological performance observed for the modern varieties was found to be due not only to the introgression of superior alleles of high (HMW-GS) and low molecular weight (LMW-GS) glutenin subunits encoded at Glu-B1 and Glu-B3 loci , but also to differential expression of specific storage proteins. In particular, the higher gluten index observed in modern genotypes was correlated with an increased glutenin/gliadin ratio and the expression of B-type LMW-GS which was, on average, two times higher in the modern than in the old group of durum wheat genotypes. By contrast, no significant differences were found between old and modern durum wheat genotypes in relation to the expression of α-type and γ-type gliadins which are major fractions that trigger coeliac disease (CD) in susceptible individuals. Furthermore, a drastic decrease was observed in the expression of ω-type gliadins in the modern genotypes, mainly ω-5 gliadin (also known as Tri a 19) which is a major allergen in wheat dependent exercise induced anaphylaxis (WDEIA). Immunological and 2DE SDS-PAGE analyses indicated that these differences could be related either to a general down-regulation or to differences in numbers of isoforms. Lower rainfall during grain filling period was related to overall higher expression of HMW-GS and ω-gliadins. In conclusion, breeding activity carried out in Italy during the 20th century appears to have improved durum wheat gluten quality, both in relation to technological

Comparison of low molecular weight glutenin subunits identified by SDS-PAGE, 2-DE, MALDI-TOF-MS and PCR in common wheat.

PubMed

Liu, Li; Ikeda, Tatsuya M; Branlard, Gerard; Peña, Roberto J; Rogers, William J; Lerner, Silvia E; Kolman, María A; Xia, Xianchun; Wang, Linhai; Ma, Wujun; Appels, Rudi; Yoshida, Hisashi; Wang, Aili; Yan, Yueming; He, Zhonghu

2010-06-24

Low-molecular-weight glutenin subunits (LMW-GS) play a crucial role in determining end-use quality of common wheat by influencing the viscoelastic properties of dough. Four different methods - sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE, IEF x SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and polymerase chain reaction (PCR), were used to characterize the LMW-GS composition in 103 cultivars from 12 countries. At the Glu-A3 locus, all seven alleles could be reliably identified by 2-DE and PCR. However, the alleles Glu-A3e and Glu-A3d could not be routinely distinguished from Glu-A3f and Glu-A3g, respectively, based on SDS-PAGE, and the allele Glu-A3a could not be differentiated from Glu-A3c by MALDI-TOF-MS. At the Glu-B3 locus, alleles Glu-B3a, Glu-B3b, Glu-B3c, Glu-B3g, Glu-B3h and Glu-B3j could be clearly identified by all four methods, whereas Glu-B3ab, Glu-B3ac, Glu-B3ad could only be identified by the 2-DE method. At the Glu-D3 locus, allelic identification was problematic for the electrophoresis based methods and PCR. MALDI-TOF-MS has the potential to reliably identify the Glu-D3 alleles. PCR is the simplest, most accurate, lowest cost, and therefore recommended method for identification of Glu-A3 and Glu-B3 alleles in breeding programs. A combination of methods was required to identify certain alleles, and would be especially useful when characterizing new alleles. A standard set of 30 cultivars for use in future studies was chosen to represent all LMW-GS allelic variants in the collection. Among them, Chinese Spring, Opata 85, Seri 82 and Pavon 76 were recommended as a core set for use in SDS-PAGE gels. Glu-D3c and Glu-D3e are the same allele. Two new alleles, namely, Glu-D3m in cultivar Darius, and Glu-D3n in Fengmai 27, were identified by 2-DE. Utilization of the suggested standard cultivar set, seed of which is available from the

Association of 70-Gene Signature Assay Findings With Physicians' Treatment Guidance for Patients With Early Breast Cancer Classified as Intermediate Risk by the 21-Gene Assay.

PubMed

Tsai, Michaela; Lo, Shelly; Audeh, William; Qamar, Rubina; Budway, Raye; Levine, Ellis; Whitworth, Pat; Mavromatis, Blanche; Zon, Robin; Oldham, Dwight; Untch, Sarah; Treece, Tina; Blumencranz, Lisa; Soliman, Hatem

2018-01-11

Among patients who undergo the 21-gene assay (21-GA), 39% to 67% receive an intermediate risk result and may receive ambiguous treatment guidance. The 70-gene signature assay (70-GS) may be associated with physicians' treatment decisions in this population with early breast cancer. To determine whether 70-GS findings are associated with physicians' decisions about adjuvant treatment and confidence in their recommendations and to evaluate the dichotomous (high- vs low-risk) and continuous distribution of 70-GS indices among this group of patients with intermediate risk. The Prospective Study of MammaPrint in Breast Cancer Patients With an Intermediate Recurrence Score (PROMIS trial) was an impact study conducted from May 20, 2012, through December 31, 2015, that enrolled 840 patients with early-stage breast cancer and a 21-gene assay recurrence score of 18 to 30. Patients were treated in 58 US institutions. The 70-GS result was given to physicians before adjuvant treatment. Change in physician treatment decision before vs after receiving the 70-GS result. With a treatment change of greater than 20%, the odds ratio (OR) was applied. Among the 840 patients who underwent 70-GS classification (mean age, 59 years; range, 27-93 years), 374 (44.5%) had a low-risk and 466 (55.5%) had a high-risk result. The distribution of 70-GS indices did not correlate with recurrence score within the 21-GA intermediate range, with 70-GS low- and high-risk patients observed at every recurrence score. A significant change in adjuvant treatment was associated with receiving the 70-GS classifications with an OR of 0.64 (95% CI, 0.50-0.82; McNemar test, P < .001) for all patients. Among the low-risk patients, 108 of 374 (28.9%) had chemotherapy removed from their treatment recommendation; among the high-risk patients, 171 of 466 (36.7%) had chemotherapy added. Results of the 70-GS were associated with the physician's adjuvant treatment recommendation; 409 high-risk patients (87.8%) were

Anti-HBV response to toll-like receptor 7 agonist GS-9620 is associated with intrahepatic aggregates of T cells and B cells.

PubMed

Li, Li; Barry, Vivian; Daffis, Stephane; Niu, Congrong; Huntzicker, Erik; French, Dorothy M; Mikaelian, Igor; Lanford, Robert E; Delaney, William E; Fletcher, Simon P

2018-05-01

GS-9620, an oral agonist of toll-like receptor 7, is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the chimpanzee and woodchuck models of CHB. Herein, we investigated the immunomodulatory mechanisms underlying these antiviral effects. Archived liver biopsies and paired peripheral blood mononuclear cell samples from a previous chimpanzee study were analyzed by RNA sequencing, quantitative reverse transcription PCR, immunohistochemistry (IHC) and in situ hybridization (ISH). GS-9620 treatment of CHB chimpanzees induced an intrahepatic transcriptional profile significantly enriched with genes associated with hepatitis B virus (HBV) clearance in acutely infected chimpanzees. Type I and II interferon, CD8 + T cell and B cell transcriptional signatures were associated with treatment response, together with evidence of hepatocyte death and liver regeneration. IHC and ISH confirmed an increase in intrahepatic CD8 + T cell and B cell numbers during treatment, and revealed that GS-9620 transiently induced aggregates predominantly comprised of CD8 + T cells and B cells in portal regions. There were no follicular dendritic cells or IgG-positive cells in these lymphoid aggregates and very few CD11b + myeloid cells. There was no change in intrahepatic natural killer cell number during GS-9620 treatment. The antiviral response to GS-9620 treatment in CHB chimpanzees was associated with an intrahepatic interferon response and formation of lymphoid aggregates in the liver. Our data indicate these intrahepatic structures are not fully differentiated follicles containing germinal center reactions. However, the temporal correlation between development of these T and B cell aggregates and the antiviral response to treatment suggests they play a role in promoting an effective immune response against HBV. New therapies to treat chronic hepatitis B (CHB) are urgently

Rhizobia from Lanzarote, the Canary Islands, that nodulate Phaseolus vulgaris have characteristics in common with LMW RNA group II Sinorhizobium meliloti of Medicago, Melilotus and Trigonella from soils of mainland Spain

USDA-ARS?s Scientific Manuscript database

Several isolates from nodules of Phaseolus vulgaris grown in soil of Lanzarote, an island of the Canaries, had electrophoretic LMW RNA patterns identical with a less common pattern within S. meliloti (assigned as group II) obtained from nodules of alfalfa and alfalfa-related legumes grown in northe...

The SERTS-97 Rocket Experiment on Study Activity on the Sun: Flight 36.167-GS on 1997 November 18

NASA Technical Reports Server (NTRS)

Swartz, Marvin; Condor, Charles E.; Davila, Joseph M.; Haas, J. Patrick; Jordan, Stuart D.; Linard, David L.; Miko, Joseph J.; Nash, I. Carol; Novello, Joseph; Payne, Leslie J.;

Connections between Transcription Downstream of Genes and cis-SAGe Chimeric RNA.

PubMed

Chwalenia, Katarzyna; Qin, Fujun; Singh, Sandeep; Tangtrongstittikul, Panjapon; Li, Hui

2017-11-22

cis-Splicing between adjacent genes (cis-SAGe) is being recognized as one way to produce chimeric fusion RNAs. However, its detail mechanism is not clear. Recent study revealed induction of transcriptions downstream of genes (DoGs) under osmotic stress. Here, we investigated the influence of osmotic stress on cis-SAGe chimeric RNAs and their connection to DoGs. We found,the absence of induction of at least some cis-SAGe fusions and/or their corresponding DoGs at early time point(s). In fact, these DoGs and their cis-SAGe fusions are inversely correlated. This negative correlation was changed to positive at a later time point. These results suggest a direct competition between the two categories of transcripts when total pool of readthrough transcripts is limited at an early time point. At a later time point, DoGs and corresponding cis-SAGe fusions are both induced, indicating that total readthrough transcripts become more abundant. Finally, we observed overall enhancement of cis-SAGe chimeric RNAs in KCl-treated samples by RNA-Seq analysis.

Molecular characterization of the celiac disease epitope domains in α-gliadin genes in Aegilops tauschii and hexaploid wheats (Triticum aestivum L.).

PubMed

Xie, Zhenze; Wang, Congyan; Wang, Ke; Wang, Shunli; Li, Xiaohui; Zhang, Zhao; Ma, Wujun; Yan, Yueming

2010-11-01

Nineteen novel full-ORF α-gliadin genes and 32 pseudogenes containing at least one stop codon were cloned and sequenced from three Aegilops tauschii accessions (T15, T43 and T26) and two bread wheat cultivars (Gaocheng 8901 and Zhongyou 9507). Analysis of three typical α-gliadin genes (Gli-At4, Gli-G1 and Gli-Z4) revealed some InDels and a considerable number of SNPs among them. Most of the pseudogenes were resulted from C to T change, leading to the generation of TAG or TAA in-frame stop codon. The putative proteins of both Gli-At3 and Gli-Z7 genes contained an extra cysteine residue in the unique domain II. Analysis of toxic epitodes among 19 deduced α-gliadins demonstrated that 14 of these contained 1-5 T cell stimulatory toxic epitopes while the other 5 did not contain any toxic epitopes. The glutamine residues in two specific ployglutamine domains ranged from 7 to 27, indicating a high variation in length. According to the numbers of 4 T cell stimulatory toxic epitopes and glutamine residues in the two ployglutamine domains among the 19 α-gliadin genes, 2 were assigned to chromosome 6A, 5 to chromosome 6B and 12 to chromosome 6D. These results were consistent with those from wheat cv. Chinese Spring nulli-tetrasomic and phylogenetic analysis. Secondary structure prediction showed that all α-gliadins had high content of β-strands and most of the α-helixes and β-strands were present in two unique domains. Phylogenetic analysis demonstrated that α-gliadin genes had a high homology with γ-gliadin, B-hordein, and LMW-GS genes and they diverged at approximate 39 MYA. Finally, the five α-gliadin genes were successfully expressed in E. coli, and their expression amount reached to the maximum after 4 h induced by IPTG, indicating that the α-gliadin genes can express in a high level under the control of T(7) promoter.

Sequential treatment of icotinib after first-line pemetrexed in advanced lung adenocarcinoma with unknown EGFR gene status.

PubMed

Zheng, Yulong; Fang, Weijia; Deng, Jing; Zhao, Peng; Xu, Nong; Zhou, Jianying

2014-07-01

In non-small cell lung cancer (NSCLC), the well-developed epidermal growth factor receptor (EGFR) is an important therapeutic target. EGFR activating gene mutations have been proved strongly predictive of response to EGFR-tyrosine kinase inhibitors (TKI) in NSCLC. However, both in daily clinical practice and clinical trials, patients with unknown EGFR gene status (UN-EGFR-GS) are very common. In this study, we assessed efficacy and tolerability of sequential treatment of first-line pemetrexed followed by icotinib in Chinese advanced lung adenocarcinoma with UN-EGFR-GS. We analyzed 38 patients with advanced lung adenocarcinoma with UN-EGFR-GS treated with first-line pemetrexed-based chemotherapy followed by icotinib as maintenance or second-line therapy. The response rates to pemetrexed and icotinib were 21.1% and 42.1%, respectively. The median overall survival was 27.0 months (95% CI, 19.7-34.2 months). The 12-month overall survival probability was 68.4%. The most common toxicities observed in icotinib phase were rashes, diarrheas, and elevated aminotransferase. Subgroup analysis indicated that the overall survival is correlated with response to icotinib. The sequence of first-line pemetrexed-based chemotherapy followed by icotinib treatment is a promising option for advanced lung adenocarcinoma with UN-EGFR-GS in China.

Highly efficient antibody immobilization with multimeric protein Gs coupled magnetic silica nanoparticles

NASA Astrophysics Data System (ADS)

Lee, J. H.; Choi, H. K.; Chang, J. H.

2011-10-01

This work reports the immobilization of monomeric, dimeric and trimer protein Gs onto silica magnetic nanoparticles for self-oriented antibody immobilization. To achieve this, we initially prepared the silica-coated magnetic nanoparticle having about 170 nm diameters. The surface of the silica coated magnetic nanoparticles was modified with 3- aminopropyl-trimethoxysilane (APTMS) to chemically link to multimeric protein Gs. The conjugation of amino groups on the SiO2-MNPs to cysteine tagged in multimeric protein Gs was performed using a sulfo-SMCC coupling procedure. The binding efficiencies of monomer, dimer and trimer were 77 %, 67 % and 55 % respectively. However, the efficiencies of antibody immobilization were 70 %, 83 % and 95 % for monomeric, dimeric and trimeric protein G, respectively. To prove the enhancement of accessibility by using multimeric protein G, FITC labeled goat-anti-mouse IgG was treated to mouse IgG immobilized magnetic silica nanoparticles through multimeric protein G. FITC labeled goat anti-mouse IgGs were more easily bound to mouse IgG immobilized by trimeric protein G than others. Finally protein G bound silica magnetic nanoparticles were utilized to develop highly sensitive immunoassay to detect hepatitis B antigen.

Glycogen Phosphorylase and Glycogen Synthase: Gene Cloning and Expression Analysis Reveal Their Role in Trehalose Metabolism in the Brown Planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae)

PubMed Central

Zhang, Lu; Wang, Huijuan; Chen, Jianyi; Shen, Qida; Wang, Shigui; Xu, Hongxing

2017-01-01

RNA interference has been used to study insects’ gene function and regulation. Glycogen synthase (GS) and glycogen phosphorylase (GP) are two key enzymes in carbohydrates’ conversion in insects. Glycogen content and GP and GS gene expression in several tissues and developmental stages of the Brown planthopper Nilaparvata lugens Stål (Hemiptera: Delphacidae) were analyzed in the present study, using quantitative reverse-transcription polymerase chain reaction to determine their response to double-stranded trehalases (dsTREs), trehalose-6-phosphate synthases (dsTPSs), and validamycin injection. The highest expression of both genes was detected in the wing bud, followed by leg and head tissues, and different expression patterns were shown across the developmental stages analyzed. Glycogen content significantly decreased 48 and 72 h after dsTPSs injection and 48 h after dsTREs injection. GP expression increased 48 h after dsTREs and dsTPSs injection and significantly decreased 72 h after dsTPSs, dsTRE1-1, and dsTRE1-2 injection. GS expression significantly decreased 48 h after dsTPS2 and dsTRE2 injection and 72 h after dsTRE1-1 and dsTRE1-2 injection. GP and GS expression and glycogen content significantly decreased 48 h after validamycin injection. The GP activity significantly decreased 48 h after validamycin injection, while GS activities of dsTPS1 and dsTRE2 injection groups were significantly higher than that of double-stranded GFP (dsGFP) 48 h after injection, respectively. Thus, glycogen is synthesized, released, and degraded across several insect tissues according to the need to maintain stable trehalose levels. PMID:28365765

FLiGS Score: A New Method of Outcome Assessment for Lip Carcinoma–Treated Patients

PubMed Central

Grassi, Rita; Toia, Francesca; Di Rosa, Luigi; Cordova, Adriana

2015-01-01

Background: Lip cancer and its treatment have considerable functional and cosmetic effects with resultant nutritional and physical detriments. As we continue to investigate new treatment regimens, we are simultaneously required to assess postoperative outcomes to design interventions that lessen the adverse impact of this disease process. We wish to introduce Functional Lip Glasgow Scale (FLiGS) score as a new method of outcome assessment to measure the effect of lip cancer and its treatment on patients’ daily functioning. Methods: Fifty patients affected by lip squamous cell carcinoma were recruited between 2009 and 2013. Patients were asked to fill the FLiGS questionnaire before surgery, 1 month, 6 months, and 1 year after surgery. The subscores were used to calculate a total FLiGS score of global oral disability. Statistical analysis was performed to test validity and reliability. Results: FLiGS scores improved significantly from preoperative to 12 months postoperative values (P = 0.000). Statistical evidence of validity was provided through rs (Spearman correlation coefficient) that resulted >0.30 for all surveys and for which P < 0.001. FLiGS score reliability was shown through examination of internal consistency and test-retest reliability. Conclusions: FLiGS score is a simple way of assessing functional impairment related to lip cancer before and after surgery; it is sensitive, valid, reliable, and clinically relevant: it provides useful information to orient the physician in the postoperative management and in the rehabilitation program. PMID:26034652

Functional identification of genes responsible for the biosynthesis of 1-methoxy-indol-3-ylmethyl-glucosinolate in Brassica rapa ssp. chinensis

PubMed Central

2014-01-01

Background Brassica vegetables contain a class of secondary metabolites, the glucosinolates (GS), whose specific degradation products determine the characteristic flavor and smell. While some of the respective degradation products of particular GS are recognized as health promoting substances for humans, recent studies also show evidence that namely the 1-methoxy-indol-3-ylmethyl GS might be deleterious by forming characteristic DNA adducts. Therefore, a deeper knowledge of aspects involved in the biosynthesis of indole GS is crucial to design vegetables with an improved secondary metabolite profile. Results Initially the leafy Brassica vegetable pak choi (Brassica rapa ssp. chinensis) was established as suitable tool to elicit very high concentrations of 1-methoxy-indol-3-ylmethyl GS by application of methyl jasmonate. Differentially expressed candidate genes were discovered in a comparative microarray analysis using the 2 × 104 K format Brassica Array and compared to available gene expression data from the Arabidopsis AtGenExpress effort. Arabidopsis knock out mutants of the respective candidate gene homologs were subjected to a comprehensive examination of their GS profiles and confirmed the exclusive involvement of polypeptide 4 of the cytochrome P450 monooxygenase subfamily CYP81F in 1-methoxy-indol-3-ylmethyl GS biosynthesis. Functional characterization of the two identified isoforms coding for CYP81F4 in the Brassica rapa genome was performed using expression analysis and heterologous complementation of the respective Arabidopsis mutant. Conclusions Specific differences discovered in a comparative microarray and glucosinolate profiling analysis enables the functional attribution of Brassica rapa ssp. chinensis genes coding for polypeptide 4 of the cytochrome P450 monooxygenase subfamily CYP81F to their metabolic role in indole glucosinolate biosynthesis. These new identified Brassica genes will enable the development of genetic tools for breeding

Dietary vitamin A impacts DNA methylation patterns of adipogenesis-related genes in suckling rats.

PubMed

Arreguín, A; Ribot, J; Mušinović, H; von Lintig, J; Palou, A; Bonet, M L

2018-05-11

We previously showed that vitamin A supplementation in early life impacts white adipose tissue (WAT) biology. We here studied the vitamin's effects on DNA methylation of genes crucial for WAT cell development, determination and metabolism. CpG promoter methylation and mRNA expression of Pparg, Zfp423, Pcna, and Rbp4 was compared in inguinal WAT of 21-day-old rats supplemented during the suckling period with vehicle (controls) or an emulsion of vitamin A as retinyl ester (RE) or β-carotene (BC). The methylation profile of promoters was affected by vitamin A supplementation with pronounced differences between the RE and BC groups. In the RE group, hypermethylation of the Rbp4 (at multiple CpGs) and the Pparg2 (at a specific CpG) promoters and hypomethylation of the Pcna promoter (at multiple CpGs) was observed, together with inverse changes in gene expression levels. In the BC group, hypomethylation of the Rbp4 and hypermethylation of the Pcna promoter at distinct CpGs was observed, with no effects on gene expression. In both supplemention groups, hypomethylation and increased expression was found for Zfp423. Thus, modest vitamin A supplementation in early postnatal life impacts methylation marks in developing WAT. Differential epigenetic effects of RE and BC in early life may affect adipose tissue programming activity. Copyright © 2018 Elsevier Inc. All rights reserved.

Hvr-Gs at MT. Graham:. Optical Turbulence Vertical Distribution with Standard and High Resolution

NASA Astrophysics Data System (ADS)

Masciadri, E.; Stoesz, J.; Lascaux, F.; Hagelin, S.

2009-09-01

Since a few years measurements of the optical turbulence vertical distribution have been done at Mt. Graham with a Generalized Scidar (GS) located at the focus of the 1.75 m Vatican Advance Technological Telescope (VATT). Such a telescope is placed on the summit of Mt. Graham (Arizona) at around 250 m far away from the Large Binocular Telescope (LBT). Thanks to a new technique1 that we recently proposed based on the GS observations of wide-binaries (30-35 arcsec), measurements of CN2 profiles characterized by a vertical resolution as high as 20-30 m in the first 600 m have been also collected. The statistic sample of measurements consists, at present, of 43 nights distributed in different periods of the year. In this contribution we present the main scientific motivations as well as the analysis of this extended survey and new insights into the turbulence characterization achieved so far by this on-going activity.

RoMo: An efficient strategy for functional mosaic analysis via stochastic Cre recombination and gene targeting in the ROSA26 locus.

PubMed

Movahedi, Kiavash; Wiegmann, Robert; De Vlaminck, Karen; Van Ginderachter, Jo A; Nikolaev, Viacheslav O

2018-07-01

Functional mosaic analysis allows for the direct comparison of mutant cells with differentially marked control cells in the same organism. While this offers a powerful approach for elucidating the role of specific genes or signalling pathways in cell populations of interest, genetic strategies for generating functional mosaicism remain challenging. We describe a novel and streamlined approach for functional mosaic analysis, which combines stochastic Cre/lox recombination with gene targeting in the ROSA26 locus. With the RoMo strategy a cell population of interest is randomly split into a cyan fluorescent and red fluorescent subset, of which the latter overexpresses a chosen transgene. To integrate this approach into high-throughput gene targeting initiatives, we developed a procedure that utilizes Gateway cloning for the generation of new targeting vectors. RoMo can be used for gain-of-function experiments or for altering signaling pathways in a mosaic fashion. To demonstrate this, we developed RoMo-dnGs mice, in which Cre-recombined red fluorescent cells co-express a dominant-negative Gs protein. RoMo-dnGs mice allowed us to inhibit G protein-coupled receptor activation in a fraction of cells, which could then be directly compared to differentially marked control cells in the same animal. We demonstrate how RoMo-dnGs mice can be used to obtain mosaicism in the brain and in peripheral organs for various cell types. RoMo offers an efficient new approach for functional mosaic analysis that extends the current toolbox and may reveal important new insights into in vivo gene function. © 2018 Wiley Periodicals, Inc.

Regulation of glutamine synthetase isoforms in two differentially drought-tolerant rice (Oryza sativa L.) cultivars under water deficit conditions.

PubMed

Singh, Kamal Krishna; Ghosh, Shilpi

2013-02-01

KEY MESSAGE : The regulation of GS isoforms by WD was organ specific. Two GS isoforms i.e. OsGS1;1 and OsGS2 were differentially regulated in IR-64 (drought-sensitive) and Khitish (drought-tolerant) cultivars of rice. Water deficit (WD) has adverse effect on rice (Oryza sativa L.) and acclimation requires essential reactions of primary metabolism to continue. Rice plants utilize ammonium as major nitrogen source, which is assimilated into glutamine by the reaction of Glutamine synthetase (GS, EC 6.3.1.2). Rice plants possess one gene (OsGS2) for chloroplastic GS2 and three genes (OsGS1;1, OsGS1;2 and OsGS1;3) for cytosolic GS1. Here, we report the effect of WD on regulation of GS isoforms in drought-sensitive (cv. IR-64) and drought-tolerant (cv. Khitish) rice cultivars. Under WD, total GS activity in root and leaf decreased significantly in IR-64 seedlings in comparison to Khitish seedlings. The reduced GS activity in IR-64 leaf was mainly due to decrease in GS2 activity, which correlated with decrease in corresponding transcript and polypeptide contents. GS1 transcript and polypeptide accumulated in leaf during WD, however, GS1 activity was maintained at a constant level. Total GS activity in stem of both the varieties was insensitive to WD. Among GS1 genes, OsGS1;1 expression was differently regulated by WD in the two rice varieties. Its transcript accumulated more abundantly in IR-64 leaf than in Khitish leaf. Following WD, OsGS1;1 mRNA level in stem and root tissues declined in IR-64 and enhanced in Khitish. A steady OsGS1;2 expression patterns were noted in leaf, stem and root of both the cultivars. Results suggest that OsGS2 and OsGS1;1 expression may contribute to drought tolerance of Khitish cultivar under WD conditions.

Glycogen Phosphorylase and Glycogen Synthase: Gene Cloning and Expression Analysis Reveal Their Role in Trehalose Metabolism in the Brown Planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae).

PubMed

Zhang, Lu; Wang, Huijuan; Chen, Jianyi; Shen, Qida; Wang, Shigui; Xu, Hongxing; Tang, Bin

2017-01-01

RNA interference has been used to study insects' gene function and regulation. Glycogen synthase (GS) and glycogen phosphorylase (GP) are two key enzymes in carbohydrates' conversion in insects. Glycogen content and GP and GS gene expression in several tissues and developmental stages of the Brown planthopper Nilaparvata lugens Stål (Hemiptera: Delphacidae) were analyzed in the present study, using quantitative reverse-transcription polymerase chain reaction to determine their response to double-stranded trehalases (dsTREs), trehalose-6-phosphate synthases (dsTPSs), and validamycin injection. The highest expression of both genes was detected in the wing bud, followed by leg and head tissues, and different expression patterns were shown across the developmental stages analyzed. Glycogen content significantly decreased 48 and 72 h after dsTPSs injection and 48 h after dsTREs injection. GP expression increased 48 h after dsTREs and dsTPSs injection and significantly decreased 72 h after dsTPSs, dsTRE1-1, and dsTRE1-2 injection. GS expression significantly decreased 48 h after dsTPS2 and dsTRE2 injection and 72 h after dsTRE1-1 and dsTRE1-2 injection. GP and GS expression and glycogen content significantly decreased 48 h after validamycin injection. The GP activity significantly decreased 48 h after validamycin injection, while GS activities of dsTPS1 and dsTRE2 injection groups were significantly higher than that of double-stranded GFP (dsGFP) 48 h after injection, respectively. Thus, glycogen is synthesized, released, and degraded across several insect tissues according to the need to maintain stable trehalose levels. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.

Transgenic approach to improve wheat (Triticum aestivum L.) nutritional quality.

PubMed

Tamás, Cecília; Kisgyörgy, Boglárka N; Rakszegi, Mariann; Wilkinson, Mark D; Yang, Moon-Sik; Láng, László; Tamás, László; Bedo, Zoltán

2009-07-01

An amaranth (Amaranthus hypochondriacus) albumin gene, encoding the 35-kDa AmA1 protein of the seed, with a high content of essential amino acids, was used in the biolistic transformation of bread wheat (Triticum aestivum L.) variety Cadenza. The transformation cassette carried the ama1 gene under the control of a powerful wheat endosperm-specific promoter (1Bx17 HMW-GS). Southern-blot analysis of T(1) lines confirmed the integration of the foreign gene, while RT-PCR and Western-blot analyses of the samples confirmed the transcription and translation of the transgene. The effects of the extra albumin protein on the properties of flour, produced from bulked T(2) seeds, were calculated using total protein and essential amino acid content analysis, polymeric/monomeric protein and HMW/LMW glutenin subunit ratio measurements. The results indicated that not only can essential amino acid content be increased, but some parameters associated with functional quality may also be improved because of the expression of the AmA1 protein.

Enhanced delignification of lignocellulosic substrates by Pichia GS115 expressed recombinant laccase.

PubMed

Kumar, Vidya Pradeep; Kolte, Atul P; Dhali, Arindam; Naik, Chandrashekar; Sridhar, Manpal

2018-04-25

Utilization of energy-rich crop residues by ruminants is restricted by the presence of lignin, which is recalcitrant to digestion. Application of lignin degrading enzymes on the lignocellulosic biomass exposes the cellulose for easy digestion by ruminants. Laccases have been found to be considerably effective in improving the digestibility by way of delignification. However, laccase yields from natural hosts are not sufficient for industrial scale applications, which restricts their use. A viable option would be to express the laccase gene in compatible hosts to achieve higher production yields. A codon-optimized synthetic variant of Schizophyllum commune laccase gene was cloned into a pPIC9K vector and expressed in P. pastoris GS115 (his4) under the control of an alcohol oxidase promoter. Colonies were screened for G418 resistance and the methanol utilization phenotype was established. The transformant yielded a laccase activity of 344 U·mL -1 after 5 days of growth at 30°C (0.019 g·mL -1 wet cell weight). The laccase protein produced by the recombinant Pichia clone was detected as two bands with apparent molecular weights of 55 kDa and 70 kDa on SDS-PAGE. Activity staining on native PAGE confirmed the presence of bioactive laccase. Treatment of five common crop residues with recombinant laccase recorded a lignin loss ranging between 1.64% in sorghum stover, to 4.83% in finger millet, with an enhancement in digestibility ranging between 8.71% in maize straw to 24.61% in finger millet straw. Treatment with recombinant laccase was effective in enhancing the digestibility of lignocellulosic biomass for ruminant feeding through delignification. To date, a number of hosts have been adventured to produce laccase in large quantities, but, to our knowledge, there are no reports of the expression of laccase protein from Schizophyllum commune in Pichia pastoris, and also on the treatment of crop residues using recombinant laccase for ruminant feeding.

Oxycodone Plus Ultra-Low-Dose Naltrexone Attenuates Neuropathic Pain and Associated μ-Opioid Receptor–Gs Coupling

PubMed Central

Largent-Milnes, Tally M.; Guo, Wenhong; Wang, Hoau-Yan; Burns, Lindsay H.; Vanderah, Todd W.

2017-01-01

Both peripheral nerve injury and chronic opioid treatment can result in hyperalgesia associated with enhanced excitatory neurotransmission at the level of the spinal cord. Chronic opioid administration leads to a shift in μ-opioid receptor (MOR)–G protein coupling from Gi/o to Gs that can be prevented by cotreatment with an ultra-low-dose opioid antagonist. In this study, using lumbar spinal cord tissue from rats with L5/L6 spinal nerve ligation (SNL), we demonstrated that SNL injury induces MOR linkage to Gs in the damaged (ipsilateral) spinal dorsal horn. This MOR-Gs coupling occurred without changing Gi/o coupling levels and without changing the expression of MOR or Gα proteins. Repeated administration of oxycodone alone or in combination with ultra-low-dose naltrexone (NTX) was assessed on the SNL-induced MOR-Gs coupling as well as on neuropathic pain behavior. Repeated spinal oxycodone exacerbated the SNL-induced MOR-Gs coupling, whereas ultra-low-dose NTX cotreatment slightly but significantly attenuated this Gs coupling. Either spinal or oral administration of oxycodone plus ultra-low-dose NTX markedly enhanced the reductions in allodynia and thermal hyperalgesia produced by oxycodone alone and minimized tolerance to these effects. The MOR-Gs coupling observed in response to SNL may in part contribute to the excitatory neurotransmission in spinal dorsal horn in neuropathic pain states. The antihyperalgesic and antiallodynic effects of oxycodone plus ultra-low-dose NTX (Oxytrex, Pain Therapeutics, Inc., San Mateo, CA) suggest a promising new treatment for neuropathic pain. PMID:18468954

Role of the C-terminal extension peptide of plastid located glutamine synthetase from Medicago truncatula: Crucial for enzyme activity and needless for protein import into the plastids.

PubMed

Ferreira, Maria João; Vale, Diogo; Cunha, Luis; Melo, Paula

2017-02-01

Glutamine synthetase (GS), a key enzyme in plant nitrogen metabolism, is encoded by a small family of highly homologous nuclear genes that produce cytosolic (GS1) and plastidic (GS2) isoforms. Compared to GS1, GS2 proteins have two extension peptides, one at the N- and the other at the C-terminus, which show a high degree of conservation among plant species. It has long been known that the N-terminal peptide acts as a transit peptide, targeting the protein to the plastids however, the function of the C-terminal extension is still unknown. To investigate whether the C-terminal extension influences the activity of the enzyme, we produced a C-terminal truncated version of Medicago truncatula GS2a in Escherechia coli and studied its catalytic properties. The activity of the truncated protein was found to be lower than that of MtGS2a and with less affinity for glutamate. The importance of the C-terminal extension for the protein import into the chloroplast was also assessed by transient expression of fluorescently-tagged MtGS2a truncated at the C-terminus, which was correctly detected in the chloroplast. The results obtained in this work demonstrate that the C-terminal extension of M. truncatula GS2a is important for the activity of the enzyme and does not contain crucial information for the import process. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

solGS: a web-based tool for genomic selection

USDA-ARS?s Scientific Manuscript database

Genomic selection (GS) promises to improve accuracy in estimating breeding values and genetic gain for quantitative traits compared to traditional breeding methods. Its reliance on high-throughput genome-wide markers and statistical complexity, however, is a serious challenge in data management, ana...

A Japanese family with nonautoimmune hyperthyroidism caused by a novel heterozygous thyrotropin receptor gene mutation.

PubMed

Nakamura, Akie; Morikawa, Shuntaro; Aoyagi, Hayato; Ishizu, Katsura; Tajima, Toshihiro

2014-06-01

Hyperthyroidism caused by activating mutations of the thyrotropin receptor gene (TSHR) is rare in the pediatric population. We found a Japanese family with hyperthyroidism without autoantibody. DNA sequence analysis of TSHR was undertaken in this family. The functional consequences for the Gs-adenylyl cyclase and Gq/11-phospholipase C signaling pathways and cell surface expression of receptors were determined in vitro using transiently transfected human embryonic kidney 293 cells. We identified a heterozygous mutation (M453R) in exon 10 of TSHR. In this family, this mutation was found in all individuals who exhibited hyperthyroidism. The results showed that this mutation resulted in constitutive activation of the Gs-adenylyl cyclase system. However, this mutation also caused a reduction in the activation capacity of the Gq/11-phospholipase C pathway, compared with the wild type. We demonstrate that the M453R mutation is the cause of nonautoimmune hyperthyroidism.

A Narrative Approach to Exploring TGfU-GS

ERIC Educational Resources Information Center

Stolz, Steven A.; Pill, Shane

2016-01-01

This paper uses a narrative approach in the form of a fictional dialogue between a physical education teacher educator (PETE) and an enquiring physical education teacher (EPET) in order to both contextualise the problem posed by Almond, that Teaching Games for Understanding-Game Sense (TGfU-GS) has been better accepted in academia than in the…

A novel Fe(III) dependent bioflocculant from Klebsiella oxytoca GS-4-08: culture conditions optimization and flocculation mechanism

PubMed Central

Yu, Lei; Tang, Qing-wen; Zhang, Yu-jia; Chen, Rong-ping; Liu, Xin; Qiao, Wei-chuan; Li, Wen-wei; Ruan, Hong-hua; Song, Xin

2016-01-01

In this work, the effect of cultivation factors on the flocculation efficiency (FE) of bioflocculant P-GS408 from Klebsiella oxytoca was optimized by the response surface methodology. The most significant factor, i.e. culture time, was determined by gray relational analysis. A total of 240 mg of purified P-GS408 was prepared from 1 liter of culture solution under the optimal conditions. GC-MS analysis results indicated that the polysaccharide of P-GS408 mainly contains Rhamnose and Galactose, and the existence of abundant hydroxyl, carboxyl and amino groups was evidenced by FTIR and XPS analyses. With the aid of Fe3+, the FE of kaolin solution by P-GS408 could achieve 99.48% in ten minutes. Functional groups of polysaccharide were involved in the first adsorption step and the zeta potential of kaolin solution changed from −39.0 mV to 43.4 mV in the presence of Fe3+ and P-GS408. Three-dimensional excitation-emission (EEM) fluorescence spectra demonstrates that the trivalent Fe3+ and Al3+ can bind efficiently with P-GS408, while those univalent and divalent cations cannot. With the help of SEM images, FTIR, zeta potential and EEM spectra, we proposed the P-GS408 flocculation mechanism, which consists of coordination bond combination, charge neutrality, adsorption and bridging, and net catching. PMID:27713559

A novel Fe(III) dependent bioflocculant from Klebsiella oxytoca GS-4-08: culture conditions optimization and flocculation mechanism.

PubMed

Yu, Lei; Tang, Qing-Wen; Zhang, Yu-Jia; Chen, Rong-Ping; Liu, Xin; Qiao, Wei-Chuan; Li, Wen-Wei; Ruan, Hong-Hua; Song, Xin

2016-10-07

In this work, the effect of cultivation factors on the flocculation efficiency (FE) of bioflocculant P-GS408 from Klebsiella oxytoca was optimized by the response surface methodology. The most significant factor, i.e. culture time, was determined by gray relational analysis. A total of 240 mg of purified P-GS408 was prepared from 1 liter of culture solution under the optimal conditions. GC-MS analysis results indicated that the polysaccharide of P-GS408 mainly contains Rhamnose and Galactose, and the existence of abundant hydroxyl, carboxyl and amino groups was evidenced by FTIR and XPS analyses. With the aid of Fe 3+ , the FE of kaolin solution by P-GS408 could achieve 99.48% in ten minutes. Functional groups of polysaccharide were involved in the first adsorption step and the zeta potential of kaolin solution changed from -39.0 mV to 43.4 mV in the presence of Fe 3+ and P-GS408. Three-dimensional excitation-emission (EEM) fluorescence spectra demonstrates that the trivalent Fe 3+ and Al 3+ can bind efficiently with P-GS408, while those univalent and divalent cations cannot. With the help of SEM images, FTIR, zeta potential and EEM spectra, we proposed the P-GS408 flocculation mechanism, which consists of coordination bond combination, charge neutrality, adsorption and bridging, and net catching.

Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris.

PubMed

Yang, Xiaoxue; Cong, Hua; Song, Jinzhu; Zhang, Junzheng

2013-11-01

Trichoderma asperellum parasitizes a large variety of phytopathogenic fungi. The mycoparasitic activity of T. asperellum depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall and proteases which are a group of enzymes capable of degrading proteins from host. In this study, a full-length cDNA clone of aspartic protease gene, TaAsp, from T. asperellum was obtained and sequenced. The 1,185 bp long cDNA sequence was predicted to encode a 395 amino acid polypeptide with molecular mass of 42.3 kDa. The cDNA of TaAsp was inserted into the pPIC9K vector and transformed into yeast Pichia pastoris GS115 for heterologous expression. A clearly visible band with molecular mass about 42 kDa in the SDS-PAGE gel indicated that the transformant harboring the gene TaAsp had been successfully translated in P. pastoris and produced a recombinant protein. Enzyme characterization test showed that the optimum fermentation time for P. pastoris GS115 transformant was 72 h. Enzyme activity of the recombinant aspartic proteinase remained relatively stable at 25-60 °C and pH 3.0-9.0, which indicated its good prospect of application in biocontrol. The optimal pH value and temperature of the enzyme activity were pH 4.0 and 40 °C, and under this condition, with casein as the substrate, the recombinant protease activity was 18.5 U mL(-1). In order to evaluate antagonistic activity of the recombinant protease against pathogenic fungi, five pathogenic fungi, Fusarium oxysporum, Alternaria alternata, Cytospora chrysosperma, Sclerotinia sclerotiorum and Rhizoctonia solani, were applied to the test of in vitro inhibition of their mycelial growth by culture supernatant of P. pastoris GS115 transformant.

PI3Kδ inhibitor idelalisib in combination with BTK inhibitor ONO/GS-4059 in diffuse large B cell lymphoma with acquired resistance to PI3Kδ and BTK inhibitors.

PubMed

Yahiaoui, Anella; Meadows, Sarah A; Sorensen, Rick A; Cui, Zhi-Hua; Keegan, Kathleen S; Brockett, Robert; Chen, Guang; Quéva, Christophe; Li, Li; Tannheimer, Stacey L

2017-01-01

Activated B-cell-like diffuse large B-cell lymphoma relies on B-cell receptor signaling to drive proliferation and survival. Downstream of the B-cell receptor, the key signaling kinases Bruton's tyrosine kinase and phosphoinositide 3-kinase δ offer opportunities for therapeutic intervention by agents such as ibrutinib, ONO/GS-4059, and idelalisib. Combination therapy with such targeted agents could provide enhanced efficacy due to complimentary mechanisms of action. In this study, we describe both the additive interaction of and resistance mechanisms to idelalisib and ONO/GS-4059 in a model of activated B-cell-like diffuse large B-cell lymphoma. Significant tumor regression was observed with a combination of PI3Kδ and Bruton's tyrosine kinase inhibitors in the mouse TMD8 xenograft. Acquired resistance to idelalisib in the TMD8 cell line occurred by loss of phosphatase and tensin homolog and phosphoinositide 3-kinase pathway upregulation, but not by mutation of PIK3CD. Sensitivity to idelalisib could be restored by combining idelalisib and ONO/GS-4059. Further evaluation of targeted inhibitors revealed that the combination of idelalisib and the phosphoinositide-dependent kinase-1 inhibitor GSK2334470 or the AKT inhibitor MK-2206 could partially overcome resistance. Characterization of acquired Bruton's tyrosine kinase inhibitor resistance revealed a novel tumor necrosis factor alpha induced protein 3 mutation (TNFAIP3 Q143*), which led to a loss of A20 protein, and increased p-IκBα. The combination of idelalisib and ONO/GS-4059 partially restored sensitivity in this resistant line. Additionally, a mutation in Bruton's tyrosine kinase at C481F was identified as a mechanism of resistance. The combination activity observed with idelalisib and ONO/GS-4059, taken together with the ability to overcome resistance, could lead to a new therapeutic option in activated B-cell-like diffuse large B-cell lymphoma. A clinical trial is currently underway to evaluate the

Evaluation of Two Guralp Preamplifiers for GS21 Seismometer Application.

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

Merchant, Bion J.; Slad, George William

2015-08-01

Sandia National Laboratories has tested and evaluated two Guralp preamplifiers for use with a GS21 seismometer application. The two preamplifiers have a gain factor of 61.39. The purpose of the preamplifier evaluation was to determine a measured gain factor, transfer function, total harmonic distortion, self-noise, application passband, dynamic range, seismometer calibration pass-through, and to comment on any issues encountered during the evaluation. The test results included in this report were in response to static, tonal, and dynamic input signals. The Guralp GS21 preamplifiers are being evaluated for potential use in the International Monitoring System (IMS) of the Comprehensive Nuclear Test-Ban-Treatymore » Organization (CTBTO). Test methodologies used were based on IEEE Standards 1057 for Digitizing Waveform Recorders and 1241 for Analog to Digital Converters« less

Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR-DNA complexes.

PubMed

Fisher, Susan H; Wray, Lewis V

2008-01-22

The Bacillus subtilis GlnR repressor controls gene expression in response to nitrogen availability. Because all GlnR-regulated genes are expressed constitutively in mutants lacking glutamine synthetase (GS), GS is required for repression by GlnR. Feedback-inhibited GS (FBI-GS) was shown to activate GlnR DNA binding with an in vitro electophoretic mobility shift assay (EMSA). The activation of GlnR DNA binding by GS in these experiments depended on the feedback inhibitor glutamine and did not occur with mutant GS proteins defective in regulating GlnR activity in vivo. Although stable GS-GlnR-DNA ternary complexes were not observed in the EMSA experiments, cross-linking experiments showed that a protein-protein interaction occurs between GlnR and FBI-GS. This interaction was reduced in the absence of the feedback inhibitor glutamine and with mutant GS proteins. Because FBI-GS significantly reduced the dissociation rate of the GlnR-DNA complexes, the stability of these complexes is enhanced by FBI-GS. These results argue that FBI-GS acts as a chaperone that activates GlnR DNA binding through a transient protein-protein interaction that stabilizes GlnR-DNA complexes. GS was shown to control the activity of the B. subtilis nitrogen transcription factor TnrA by forming a stable complex between FBI-GS and TnrA that inhibits TnrA DNA binding. Thus, B. subtilis GS is an enzyme with dual catalytic and regulatory functions that uses distinct mechanisms to control the activity of two different transcription factors.

Safety and immunogenicity of MAGE-A3 cancer immunotherapeutic with dacarbazine in patients with MAGE-A3-positive metastatic cutaneous melanoma: an open phase I/II study with a first assessment of a predictive gene signature.

PubMed

Grob, Jean-Jacques; Mortier, Laurent; D'Hondt, Lionel; Grange, Florent; Baurain, Jean Francois; Dréno, Brigitte; Lebbe, Céleste; Robert, Caroline; Dompmartin, Anne; Neyns, Bart; Gillet, Marc; Louahed, Jamila; Jarnjak, Silvija; Lehmann, Frédéric F

2017-01-01

We assessed safety, immunogenicity and clinical activity of recombinant MAGE-A3 antigen combined with AS15 immunostimulant (MAGE-A3 immunotherapeutic) in association with dacarbazine in patients with metastatic melanoma. In this open-label, phase I/II, uncontrolled multicentre trial conducted in Belgium and France, patients with MAGE-A3-positive melanoma received up to 24 doses of MAGE-A3 immunotherapeutic (four cycles) coadministered with eight doses of dacarbazine. Adverse events (AE) were recorded until 31 days postvaccination, and serious AEs (SAE), until 30 days following the last dose. MAGE-A3-specific antibodies were measured by ELISA. Clinical activity of MAGE-A3 immunotherapeutic was assessed in patients positive/negative for previously identified gene signature (GS) associated with clinical outcome. Forty-eight patients were enrolled and treated (32 GS+, 15 GS-, 1 unknown GS status); two patients completed the study. All patients reported AEs, the most common were 'general disorders and administration site conditions' (94%). Treatment-related AEs were reported by 85% of patients; the most common was pain at injection site (38%). Sixteen SAEs were reported by 21% of patients; two were considered as treatment related (neutropenia and thrombocytopenia; grade 4). Postdose 4, all patients were seropositive for MAGE-A3-specific antibodies, with a geometric mean titre of 2778.7 ELISA units (EU)/mL (95% CI 1638.3 to 4712.8). One complete and three partial responses were reported (only in GS+ patients). Median overall survival was 11.4 months for GS+ and 5.3 months for GS- patients. Although this trial shows poor results compared with the new results with checkpoint inhibitors, it gives an interesting insight in rapidly developing fields like combinations of immunotherapy and chemotherapy, new generation vaccines and the use of gene profile as a predictive marker. NCT00849875.

Granular activated carbon adsorption of organic micro-pollutants in drinking water and treated wastewater--Aligning breakthrough curves and capacities.

PubMed

Zietzschmann, Frederik; Stützer, Christian; Jekel, Martin

2016-04-01

Small-scale granular activated carbon (GAC) tests for the adsorption of organic micro-pollutants (OMP) were conducted with drinking water and wastewater treatment plant (WWTP) effluent. In both waters, three influent OMP concentration levels were tested. As long as the influent OMP concentrations are below certain thresholds, the relative breakthrough behavior is not impacted in the respective water. Accordingly, the GAC capacity for OMP is directly proportional to the influent OMP concentration in the corresponding water. The differences between the OMP breakthrough curves in drinking water and WWTP effluent can be attributed to the concentrations of the low molecular weight acid and neutral (LMW) organics of the waters. Presenting the relative OMP concentrations (c/c0) over the specific throughput of the LMW organics (mg LMW organics/g GAC), the OMP breakthrough curves in drinking water and WWTP effluent superimpose each other. This superimposition can be further increased if the UV absorbance at 254 nm (UV254) of the LMW organics is considered. In contrast, using the specific throughput of the dissolved organic carbon (DOC) did not suffice to obtain superimposed breakthrough curves. Thus, the LMW organics are the major water constituent impacting OMP adsorption onto GAC. The results demonstrate that knowing the influent OMP and LMW organics concentrations (and UV254) of different waters, the OMP breakthroughs and GAC capacities corresponding to any water can be applied to all other waters. Copyright © 2016 Elsevier Ltd. All rights reserved.

Anaerobic oxidation of toluene, phenol, and p-cresol by the dissimilatory iron-reducing organism, GS-15

USGS Publications Warehouse

Lovley, D.R.; Lonergan, D.J.

1990-01-01

The dissimilatory Fe(III) reducer, GS-15, is the first microorganism known to couple the oxidation of aromatic compounds to the reduction of Fe(III) and the first example of a pure culture of any kind known to anaerobically oxidize an aromatic hydrocarbon, toluene. In this study, the metabolism of toluene, phenol, and p-cresol by GS-15 was investigated in more detail. GS-15 grew in an anaerobic medium with toluene as the sole electron donor and Fe(III) oxide as the electron acceptor. Growth coincided with Fe(III) reduction. [ring-14C]toluene was oxidized to 14CO2, and the stoichiometry of 14CO2 production and Fe(III) reduction indicated that GS-15 completely oxidized toluene to carbon dioxide with Fe(III) as the electron acceptor. Magnetite was the primary iron end product during toluene oxidation. Phenol and p-cresol were also completely oxidized to carbon dioxide with Fe(III) as the sole electron acceptor, and GS-15 could obtain energy to support growth by oxidizing either of these compounds as the sole electron donor. p-Hydroxybenzoate was a transitory extracellular intermediate of phenol and p-cresol metabolism but not of toluene metabolism. GS-15 oxidized potential aromatic intermediates in the oxidation of toluene (benzylalcohol and benzaldehyde) and p-cresol (p-hydroxybenzylalcohol and p-hydroxybenzaldehyde). The metabolism described here provides a model for how aromatic hydrocarbons and phenols may be oxidized with the reduction of Fe(III) in contaminated aquifers and petroleum-containing sediments.

Prospective assessment of a gene signature potentially predictive of clinical benefit in metastatic melanoma patients following MAGE-A3 immunotherapeutic (PREDICT).

PubMed

Saiag, P; Gutzmer, R; Ascierto, P A; Maio, M; Grob, J-J; Murawa, P; Dreno, B; Ross, M; Weber, J; Hauschild, A; Rutkowski, P; Testori, A; Levchenko, E; Enk, A; Misery, L; Vanden Abeele, C; Vojtek, I; Peeters, O; Brichard, V G; Therasse, P

2016-10-01

Genomic profiling of tumor tissue may aid in identifying predictive or prognostic gene signatures (GS) in some cancers. Retrospective gene expression profiling of melanoma and non-small-cell lung cancer led to the characterization of a GS associated with clinical benefit, including improved overall survival (OS), following immunization with the MAGE-A3 immunotherapeutic. The goal of the present study was to prospectively evaluate the predictive value of the previously characterized GS. An open-label prospective phase II trial ('PREDICT') in patients with MAGE-A3-positive unresectable stage IIIB-C/IV-M1a melanoma. Of 123 subjects who received the MAGE-A3 immunotherapeutic, 71 (58.7%) displayed the predictive GS (GS+). The 1-year OS rate was 83.1%/83.3% in the GS+/GS- populations. The rate of progression-free survival at 12 months was 5.8%/4.1% in GS+/GS- patients. The median time-to-treatment failure was 2.7/2.4 months (GS+/GS-). There was one complete response (GS-) and two partial responses (GS+). The MAGE-A3 immunotherapeutic was similarly immunogenic in both populations and had a clinically acceptable safety profile. Treatment of patients with MAGE-A3-positive unresectable stage IIIB-C/IV-M1a melanoma with the MAGE-A3 immunotherapeutic demonstrated an overall 1-year OS rate of 83.5%. GS- and GS+ patients had similar 1-year OS rates, indicating that in this study, GS was not predictive of outcome. Unexpectedly, the objective response rate was lower in this study than in other studies carried out in the same setting with the MAGE-A3 immunotherapeutic. Investigation of a GS to predict clinical benefit to adjuvant MAGE-A3 immunotherapeutic treatment is ongoing in another melanoma study.This study is registered at www.clinicatrials.gov NCT00942162. © The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

Comparative Transcriptome Analyses Reveal a Special Glucosinolate Metabolism Mechanism in Brassica alboglabra Sprouts

PubMed Central

Guo, Rongfang; Huang, Zhongkai; Deng, Yanping; Chen, Xiaodong; XuHan, Xu; Lai, Zhongxiong

2016-01-01

Brassica sprouts contain abundant phytochemicals, especially glucosinolates (GSs). Various methods have been used to enhance GS content in sprouts. However, the molecular basis of GS metabolism in sprouts remains an open question. Here we employed RNA-seq analysis to compare the transcriptomes of high-GS (JL-08) and low-GS (JL-09) Brassica alboglabra sprouts. Paired-end Illumina RNA-seq reads were generated and mapped to the Brassica oleracea reference genome. The differentially expressed genes were analyzed between JL-08 and JL-09. Among these, 1477 genes were up-regulated and 1239 down-regulated in JL-09 compared with JL-08. Enrichment analysis of these differentially expressed genes showed that the GS biosynthesis had the smallest enrichment factor and the highest Q-value of all metabolic pathways in Kyoto Encyclopedia of Genes and Genomes database, indicating the main metabolic difference between JL-08 and JL-09 is the GS biosynthetic pathway. Thirty-seven genes of the sequenced data were annotated as putatively involved in GS biosynthesis, degradation, and regulation, of which 11 were differentially expressed in JL-08 and JL-09. The expression level of GS degradation enzyme myrosinase in high-GS JL-08 was lower compared with low-GS JL-09. Surprisingly, in high-GS JL-08, the expression levels of GS biosynthesis genes were also lower than those in low-GS JL-09. As the GS contents in sprouts are determined by dynamic equilibrium of seed stored GS mobilization, de novo synthesis, degradation, and extra transport, the result of this study leads us to suggest that efforts to increase GS content should focus on either raising GS content in seeds or decreasing myrosinase activity, rather than improving the expression level of GS biosynthesis genes in sprouts. PMID:27757119

Computational Study on the Different Ligands Induced Conformation Change of β2 Adrenergic Receptor-Gs Protein Complex

PubMed Central

Bai, Qifeng; Zhang, Yang; Ban, Yihe; Liu, Huanxiang; Yao, Xiaojun

2013-01-01

β2 adrenergic receptor (β2AR) regulated many key physiological processes by activation of a heterotrimeric GTP binding protein (Gs protein). This process could be modulated by different types of ligands. But the details about this modulation process were still not depicted. Here, we performed molecular dynamics (MD) simulations on the structures of β2AR-Gs protein in complex with different types of ligands. The simulation results demonstrated that the agonist BI-167107 could form hydrogen bonds with Ser2035.42, Ser2075.46 and Asn2936.55 more than the inverse agonist ICI 118,551. The different binding modes of ligands further affected the conformation of β2AR. The energy landscape profiled the energy contour map of the stable and dissociated conformation of Gαs and Gβγ when different types of ligands bound to β2AR. It also showed the minimum energy pathway about the conformational change of Gαs and Gβγ along the reaction coordinates. By using interactive essential dynamics analysis, we found that Gαs and Gβγ domain of Gs protein had the tendency to separate when the inverse agonist ICI 118,551 bound to β2AR. The α5-helix had a relatively quick movement with respect to transmembrane segments of β2AR when the inverse agonist ICI 118,551 bound to β2AR. Besides, the analysis of the centroid distance of Gαs and Gβγ showed that the Gαs was separated from Gβγ during the MD simulations. Our results not only could provide details about the different types of ligands that induced conformational change of β2AR and Gs protein, but also supplied more information for different efficacies of drug design of β2AR. PMID:23922653

Skeletal muscle glycogen synthase subcellular localization: effects of insulin and PPAR-alpha agonist (K-111) administration in rhesus monkeys.

PubMed

Ortmeyer, Heidi K; Adall, Yohannes; Marciani, Karina R; Katsiaras, Andreas; Ryan, Alice S; Bodkin, Noni L; Hansen, Barbara C

2005-06-01

Insulin covalently and allosterically regulates glycogen synthase (GS) and may also cause the translocation of GS from glycogen-poor to glycogen-rich locations. We examined the possible role of subcellular localization of GS and glycogen in insulin activation of GS in skeletal muscle of six obese monkeys and determined whether 1) insulin stimulation during a hyperinsulinemic euglycemic clamp and/or peroxisome proliferator-activated receptor (PPAR)-alpha agonist treatment (K-111, 3 mg.kg(-1).day(-1); Kowa) induced translocation of GS and 2) translocation of GS was associated with insulin activation of GS. GS and glycogen were present in all fractions obtained by differential centrifugation, except for the cytosolic fraction, under both basal and insulin-stimulated conditions. We found no evidence for translocation of GS by insulin. GS total (GST) activity was strongly associated with glycogen content (r = 0.70, P < 0.001). Six weeks of treatment with K-111 increased GST activity in all fractions, except the cytosolic fraction, and mean GST activity, GS independent activity, and glycogen content were significantly higher in the insulin-stimulated samples compared with basal samples, effects not seen with vehicle. The increase in GST activity was strongly related to the increase in glycogen content during the hyperinsulinemic euglycemic clamp after K-111 administration (r = 0.74, P < 0.001). Neither GS protein expression nor GS gene expression was affected by insulin or by K-111 treatment. We conclude that 1) in vivo insulin does not cause translocation of GS from a glycogen-poor to a glycogen-rich location in primate skeletal muscle and 2) the mechanism of action of K-111 to improve insulin sensitivity includes an increase in GST activity without an increase in GS gene or protein expression.

Genes Interacting with Occupational Exposures to Low Molecular Weight Agents and Irritants on Adult-Onset Asthma in Three European Studies

PubMed Central

Rava, Marta; Ahmed, Ismail; Kogevinas, Manolis; Le Moual, Nicole; Bouzigon, Emmanuelle; Curjuric, Ivan; Dizier, Marie-Hélène; Dumas, Orianne; Gonzalez, Juan R.; Imboden, Medea; Mehta, Amar J.; Tubert-Bitter, Pascale; Zock, Jan-Paul; Jarvis, Deborah; Probst-Hensch, Nicole M.; Demenais, Florence; Nadif, Rachel

2016-01-01

Background: The biological mechanisms by which cleaning products and disinfectants—an emerging risk factor—affect respiratory health remain incompletely evaluated. Studying genes by environment interactions (G × E) may help identify new genes related to adult-onset asthma. Objectives: We identified interactions between genetic polymorphisms of a large set of genes involved in the response to oxidative stress and occupational exposures to low molecular weight (LMW) agents or irritants on adult-onset asthma. Methods: Our data came from three large European cohorts: Epidemiological Family-based Study of the Genetics and Environment of Asthma (EGEA), Swiss Cohort Study on Air Pollution and Lung and Heart Disease in Adults (SAPALDIA), and European Community Respiratory Health Survey in Adults (ECRHS). A candidate pathway–based strategy identified 163 genes involved in the response to oxidative stress and potentially related to exposures to LMW agents/irritants. Occupational exposures were evaluated using an asthma job-exposure matrix and job-specific questionnaires for cleaners and healthcare workers. Logistic regression models were used to detect G × E interactions, adjusted for age, sex, and population ancestry, in 2,599 adults (mean age, 47 years; 60% women, 36% exposed, 18% asthmatics). p-Values were corrected for multiple comparisons. Results: Ever exposure to LMW agents/irritants was associated with current adult-onset asthma [OR = 1.28 (95% CI: 1.04, 1.58)]. Eight single nucleotide polymorphism (SNP) by exposure interactions at five loci were found at p < 0.005: PLA2G4A (rs932476, chromosome 1), near PLA2R1 (rs2667026, chromosome 2), near RELA (rs931127, rs7949980, chromosome 11), PRKD1 (rs1958980, rs11847351, rs1958987, chromosome 14), and PRKCA (rs6504453, chromosome 17). Results were consistent across the three studies and after accounting for smoking. Conclusions: Using a pathway-based selection process, we identified novel genes potentially involved

Membrane and Integrative Nuclear Fibroblastic Growth Factor Receptor (FGFR) Regulation of FGF-23*

PubMed Central

Han, Xiaobin; Xiao, Zhousheng; Quarles, L. Darryl

2015-01-01

Fibroblastic growth factor receptor 1 (FGFR1) signaling pathways are implicated in the regulation of FGF-23 gene transcription, but the molecular pathways remain poorly defined. We used low molecular weight (LMW, 18 kDa) FGF-2 and high molecular weight (HMW) FGF-2 isoforms, which, respectively, activate cell surface FGF receptors and intranuclear FGFR1, to determine the roles of membrane FGFRs and integrative nuclear FGFR1 signaling (INFS) in the regulation of FGF-23 gene transcription in osteoblasts. We found that LMW-FGF-2 induced NFAT and Ets1 binding to conserved cis-elements in the proximal FGF-23 promoter and stimulated FGF-23 promoter activity through PLCγ/calcineurin/NFAT and MAPK pathways in SaOS-2 and MC3T3-E1 osteoblasts. In contrast, HMW-FGF-2 stimulated FGF-23 promoter activity in osteoblasts through a cAMP-dependent binding of FGFR1 and cAMP-response element-binding protein (CREB) to a conserved cAMP response element (CRE) contiguous with the NFAT binding site in the FGF-23 promoter. Mutagenesis of the NFAT and CRE binding sites, respectively, inhibited the effects of LMW-FGF-2 and HMW-FGF-23 to stimulate FGF-23 promoter activity. FGF-2 activation of both membrane FGFRs and INFS-dependent FGFR1 pathways may provide a means to integrate systemic and local regulation of FGF-23 transcription under diverse physiological and pathological conditions. PMID:25752607

ICO amplicon NGS data analysis: a Web tool for variant detection in common high-risk hereditary cancer genes analyzed by amplicon GS Junior next-generation sequencing.

PubMed

Lopez-Doriga, Adriana; Feliubadaló, Lídia; Menéndez, Mireia; Lopez-Doriga, Sergio; Morón-Duran, Francisco D; del Valle, Jesús; Tornero, Eva; Montes, Eva; Cuesta, Raquel; Campos, Olga; Gómez, Carolina; Pineda, Marta; González, Sara; Moreno, Victor; Capellá, Gabriel; Lázaro, Conxi

2014-03-01

Next-generation sequencing (NGS) has revolutionized genomic research and is set to have a major impact on genetic diagnostics thanks to the advent of benchtop sequencers and flexible kits for targeted libraries. Among the main hurdles in NGS are the difficulty of performing bioinformatic analysis of the huge volume of data generated and the high number of false positive calls that could be obtained, depending on the NGS technology and the analysis pipeline. Here, we present the development of a free and user-friendly Web data analysis tool that detects and filters sequence variants, provides coverage information, and allows the user to customize some basic parameters. The tool has been developed to provide accurate genetic analysis of targeted sequencing of common high-risk hereditary cancer genes using amplicon libraries run in a GS Junior System. The Web resource is linked to our own mutation database, to assist in the clinical classification of identified variants. We believe that this tool will greatly facilitate the use of the NGS approach in routine laboratories.

Amino- and carboxy-terminal deletion mutants of Gs alpha are localized to the particulate fraction of transfected COS cells

PubMed Central

1992-01-01

To elucidate the structural basis for membrane attachment of the alpha subunit of the stimulatory G protein (Gs alpha), mutant Gs alpha cDNAs with deletions of amino acid residues in the amino and/or carboxy termini were transiently expressed in COS-7 cells. The particulate and soluble fractions prepared from these cells were analyzed by immunoblot using peptide specific antibodies to monitor distribution of the expressed proteins. Transfection of mutant forms of Gs alpha with either 26 amino terminal residues deleted (delta 3-28) or with 59 amino terminal residues deleted (delta 1-59) resulted in immunoreactive proteins which localized primarily to the particulate fraction. Similarly, mutants with 10 (delta 385-394), 32 (delta 353-384), or 42 (delta 353-394) amino acid residues deleted from the carboxy terminus also localized to the particulate fraction, as did a mutant form of Gs alpha lacking amino acid residues at both the amino and carboxy termini (delta 3-28)/(delta 353-384). Mutant and wild type forms of Gs alpha demonstrated a similar degree of tightness in their binding to membranes as demonstrated by treatment with 2.5 M NaCl or 6 M urea, but some mutant forms were relatively resistant compared with wild type Gs alpha to solubilization by 15 mM NaOH or 1% sodium cholate. We conclude that: (a) deletion of significant portions of the amino and/or carboxyl terminus of Gs alpha is still compatible with protein expression; (b) deletion of these regions is insufficient to cause cytosolic localization of the expressed protein. The basis of Gs alpha membrane targeting remains to be elucidated. PMID:1400589

Heterologous expression of type I antifreeze peptide GS-5 in baker's yeast increases freeze tolerance and provides enhanced gas production in frozen dough.

PubMed

Panadero, Joaquin; Randez-Gil, Francisca; Prieto, Jose Antonio

2005-12-28

The demand for frozen-dough products has increased notably in the baking industry. Nowadays, no appropriate industrial baker's yeast with optimal gassing capacity in frozen dough is, however, available, and it is unlikely that classical breeding programs could provide significant improvements of this trait. Antifreeze proteins, found in diverse organisms, display the ability to inhibit the growth of ice, allowing them to survive at temperatures below 0 degrees C. In this study a recombinant antifreeze peptide GS-5 was expressed from the polar fish grubby sculpin (Myoxocephalus aenaeus) in laboratory and industrial baker's yeast strains of Saccharomyces cerevisiae. Production of the recombinant protein increased freezing tolerance in both strains tested. Furthermore, expression of the GS-5 encoding gene enhanced notably the gassing rate and total gas production in frozen and frozen sweet doughs. These effects are unlikely to be due to reduced osmotic damage during freezing/thawing, because recombinant cells showed growth behavior similar to that of the parent under hypermosmotic stress conditions.

How human IgGs against myelin basic protein (MBP) recognize oligopeptides and MBP.

PubMed

Belov, Sergey; Buneva, Valentina N; Nevinsky, Georgy A

2017-10-01

Myelin basic protein (MBP) is a major protein of myelin-proteolipid shell of axons, and it plays an important role in pathogenesis of multiple sclerosis. In the literature, there are no data on how antibodies recognize different protein antigens including MBP. A stepwise increase in ligand complexity was used to estimate the relative contributions of virtually every amino acid residue (AA) of a specific 12-mer LSRFSWGAEGQK oligopeptide corresponding to immunodominant sequence of MBP to the light chains and to intact anti-MBP IgGs from sera of patients with multiple sclerosis. It was shown that the minimal ligands of the light chains of IgGs are many different free AAs (K d  = 0.51-0.016 M), and each free AA interacts with the specific subsite of the light chain intended for recognition of this AA in specific LSRFSW oligopeptide. A gradual transition from Leu to LSRFSWGAEGQK leads to an increase in the affinity from 10 -1 to 2.3 × 10 -4  M because of additive interactions of the light chain with 6 AAs of this oligopeptide and then the affinity reaches plateau. The contributions of 6 various AAs to the affinity of the oligopeptide are different (K d , M): 0.71 (S), 0.44 (R), 0.14 (F), 0.17 (S), and 0.62 (W). Affinity of nonspecific oligopeptides to the light chains of IgGs is significantly lower. Intact MBP interacts with both light and heavy chains of IgGs demonstrating 192-fold higher affinity than the specific oligopeptide. It is a first quantitative analysis of the mechanism of proteins recognition by antibodies. The thermodynamic model was constructed to describe the interactions of IgGs with MBP. The data obtained can be very useful for understanding how antibodies against many different proteins can recognize these proteins. Copyright © 2017 John Wiley & Sons, Ltd.

MDM Redshift of the Host of ASASSN-17gs

NASA Astrophysics Data System (ADS)

Chornock, R.; Margutti, R.

2017-06-01

The optical transient ASASSN-17gs = AT2017egv (http://www.astronomy.ohio-state.edu/ assassin/transients.html, https://wis-tns.weizmann.ac.il/object/2017egv) was associated with the new Fermi LAT source J1544-0649 and a new, bright X-ray source Swift 154419.7-064915 by Ciprini et al. (ATel #10482).

A phase 1 clinical trial of the selective BTK inhibitor ONO/GS-4059 in relapsed and refractory mature B-cell malignancies.

PubMed

Walter, Harriet S; Rule, Simon A; Dyer, Martin J S; Karlin, Lionel; Jones, Ceri; Cazin, Bruno; Quittet, Philippe; Shah, Nimish; Hutchinson, Claire V; Honda, Hideyuki; Duffy, Kevin; Birkett, Joseph; Jamieson, Virginia; Courtenay-Luck, Nigel; Yoshizawa, Toshio; Sharpe, John; Ohno, Tomoya; Abe, Shinichiro; Nishimura, Akihisa; Cartron, Guillaume; Morschhauser, Franck; Fegan, Christopher; Salles, Gilles

2016-01-28

We report the results of a multicenter phase 1 dose-escalation study of the selective Bruton tyrosine kinase (BTK) inhibitor ONO/GS-4059 in 90 patients with relapsed/refractory B-cell malignancies. There were 9 dose-escalation cohorts ranging from 20 mg to 600 mg once daily with twice-daily regimens of 240 mg and 300 mg. Twenty-four of 25 evaluable chronic lymphocytic leukemia (CLL) patients (96%) responded to ONO/GS-4059, with a median treatment duration of 80 weeks; 21 CLL patients remain on treatment. Lymph node responses were rapid and associated with a concurrent lymphocytosis. Eleven of 12 evaluable patients with mantle cell lymphoma (92%) responded (median treatment duration, 40 weeks). Eleven of 31 non-germinal center B-cell diffuse large B-cell lymphoma patients (35%) responded but median treatment duration was 12 weeks due to development of progressive disease. ONO/GS-4059 was very well tolerated with 75% of adverse events (AEs) being Common Toxicity Criteria for Adverse Events version 4.0 grade 1 or grade 2. Grade 3/4 AEs were mainly hematologic and recovered spontaneously during therapy. One CLL patient experienced a grade 3 treatment-related bleeding event (spontaneous muscle hematoma) but no clinically significant diarrhea, cardiac dysrhythmias, or arthralgia were observed. No maximal tolerated dose (MTD) was reached in the CLL cohort. In the non-Hodgkin lymphoma cohort, 4 patients developed a dose-limiting toxicity, yielding an MTD of 480 mg once daily. ONO/GS-4059 has significant activity in relapsed/refractory B-cell malignancies without major drug-related toxicity. The selectivity of ONO/GS-4059 should confer advantages in combination therapies. This trial was registered at www.clinicaltrials.gov as #NCT01659255. © 2016 by The American Society of Hematology.

Synthesis of silver nanoparticles on the basis of low and high molar mass exopolysaccharides of Bradyrhizobium japonicum 36 and its antimicrobial activity against some pathogens.

PubMed

Rasulov, Bakhtiyor; Rustamova, Nigora; Yili, Abulimiti; Zhao, Hai-Qing; Aisa, Haji A

2016-07-01

Silver nanoparticles (SNPs) were synthesized on the basis of exopolysaccharides (low and high molar mass) of diazotrophic Bradyrhizobium japonicum 36 strain. The synthesis of SNPs was carried out by direct reduction of silver nitrate with ethanol-insoluble (high molar mass, HMW) and ethanol-soluble (low molar mass, LMW) fractions of exopolysaccharides (EPS), produced by diazotrophic strain B. japonicum 36. SNPs were characterized using UV-vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). SNPs synthesized on the basis of LMW EPS absorbed radiation in the visible regions of 420 nm, whereas SNPs based on the HMW EPS have a wavelength maximum at 450 nm because of the strong SPR transition. Moreover, the antibacterial and antifungal activities of the SNPs were examined in vitro against Escherichia coli, Staphylococcus aureus, and Candida albicans. SNPs synthesized on the basis of LMW EPS were active than those synthesized on the basis of HMW EPS. Besides, UV-visible spectroscopic evaluation confirmed that SNPs synthesized on the basis of LMW EPS were far more stable than those obtained on the basis of HMW EPS.

Improved method for reliable HMW-GS identification by RP-HPLC and SDS-PAGE in common wheat cultivars

USDA-ARS?s Scientific Manuscript database

The accurate identification of alleles for high-molecular weight glutenins (HMW-GS) is critical for wheat breeding programs targeting end-use quality. RP-HPLC methods were optimized for separation of HMW-GS, resulting in enhanced resolution of 1By and 1Dx subunits. Statistically significant differe...

AJS1669, a novel small-molecule muscle glycogen synthase activator, improves glucose metabolism and reduces body fat mass in mice

PubMed Central

Nakano, Kazuhiro; Takeshita, Sen; Kawasaki, Noriko; Miyanaga, Wataru; Okamatsu, Yoriko; Dohi, Mizuki; Nakagawa, Tadakiyo

2017-01-01

Impaired glycogen synthesis and turnover are common in insulin resistance and type 2 diabetes. As glycogen synthase (GS) is a key enzyme involved in the synthetic process, it presents a promising therapeutic target for the treatment of type 2 diabetes. In the present study, we identified a novel, potent and orally available GS activator AJS1669 {sodium 2-[[5-[[4-(4,5-difluoro-2-methylsulfanyl-phenyl) phenoxy] methyl]furan-2-carbonyl]-(2-furylmethyl)amino] acetate}. In vitro, we performed a glycogen synthase 1 (GYS1) activation assay for screening GS activators and identified that the activity of AJS1669 was further potentiated in the presence of glucose-6-phosphate (G6P). In vivo, we used ob/ob mice to evaluate the novel anti-diabetic effects of AJS1669 by measuring basal blood glucose levels, glucose tolerance and body fat mass index. Repeated administration of AJS1669 over 4 weeks reduced blood glucose and hemoglobin A1c (HbA1c) levels in ob/ob mice. AJS1669 also improved glucose tolerance in a dose-dependent manner, and decreased body fat mass. The mRNA levels of genes involved in mitochondrial fatty acid oxidation and mitochondrial biogenesis were elevated in skeletal muscle tissue following AJS1669 treatment. Hepatic tissue of treated mice also exhibited elevated expression of genes associated with fatty acid oxidation. In contrast to ob/ob mice, in C57Bl/6 mice AJS1669 administration did not alter body weight or reduce glucose levels. These results demonstrate that pharmacological agents that activate GYS1, the main GS subtype found in skeletal muscle, have potential for use as novel treatments for diabetes that improve glucose metabolism in skeletal muscle. PMID:28290602

AJS1669, a novel small-molecule muscle glycogen synthase activator, improves glucose metabolism and reduces body fat mass in mice.

PubMed

Nakano, Kazuhiro; Takeshita, Sen; Kawasaki, Noriko; Miyanaga, Wataru; Okamatsu, Yoriko; Dohi, Mizuki; Nakagawa, Tadakiyo

2017-04-01

Impaired glycogen synthesis and turnover are common in insulin resistance and type 2 diabetes. As glycogen synthase (GS) is a key enzyme involved in the synthetic process, it presents a promising therapeutic target for the treatment of type 2 diabetes. In the present study, we identified a novel, potent and orally available GS activator AJS1669 {sodium 2-[[5-[[4-(4,5-difluoro-2-methylsulfanyl-phenyl)phenoxy] methyl]furan-2-carbonyl]-(2-furylmethyl)amino] acetate}. In vitro, we performed a glycogen synthase 1 (GYS1) activation assay for screening GS activators and identified that the activity of AJS1669 was further potentiated in the presence of glucose-6-phosphate (G6P). In vivo, we used ob/ob mice to evaluate the novel anti-diabetic effects of AJS1669 by measuring basal blood glucose levels, glucose tolerance and body fat mass index. Repeated administration of AJS1669 over 4 weeks reduced blood glucose and hemoglobin A1c (HbA1c) levels in ob/ob mice. AJS1669 also improved glucose tolerance in a dose-dependent manner, and decreased body fat mass. The mRNA levels of genes involved in mitochondrial fatty acid oxidation and mitochondrial biogenesis were elevated in skeletal muscle tissue following AJS1669 treatment. Hepatic tissue of treated mice also exhibited elevated expression of genes associated with fatty acid oxidation. In contrast to ob/ob mice, in C57Bl/6 mice AJS1669 administration did not alter body weight or reduce glucose levels. These results demonstrate that pharmacological agents that activate GYS1, the main GS subtype found in skeletal muscle, have potential for use as novel treatments for diabetes that improve glucose metabolism in skeletal muscle.

Power line identification of millimeter wave radar based on PCA-GS-SVM

NASA Astrophysics Data System (ADS)

Fang, Fang; Zhang, Guifeng; Cheng, Yansheng

2017-12-01

Aiming at the problem that the existing detection method can not effectively solve the security of UAV's ultra low altitude flight caused by power line, a power line recognition method based on grid search (GS) and the principal component analysis and support vector machine (PCA-SVM) is proposed. Firstly, the candidate line of Hough transform is reduced by PCA, and the main feature of candidate line is extracted. Then, upport vector machine (SVM is) optimized by grid search method (GS). Finally, using support vector machine classifier optimized parameters to classify the candidate line. MATLAB simulation results show that this method can effectively identify the power line and noise, and has high recognition accuracy and algorithm efficiency.

Effects of heat acclimation on photosynthesis, antioxidant enzyme activities, and gene expression in orchardgrass under heat stress.

PubMed

Zhao, Xin Xin; Huang, Lin Kai; Zhang, Xin Quan; Li, Zhou; Peng, Yan

2014-09-01

The present study was designed to examine the effects of heat acclimation on enzymatic activity, transcription levels, the photosynthesis processes associated with thermostability in orchardgrass (Dactylis glomerata L.).The stomatal conductance (Gs), net photosynthetic rate (Pn), and transpiration rates (Tr) of both heat-acclimated (HA) and non-acclimated (NA) plants were drastically reduced during heat treatment [using a 5-day heat stress treatment (38/30 °C ‒ day/night) followed by a 3-day recovery under control conditions (25/20 °C ‒ day/night), in order to consolidate the second cycle was permitted]. Water use efficiency increased more steeply in the HA (4.9 times) versus the NA (1.8 times) plants, and the intercellular CO2 concentration decreased gently in NA (10.9%) and HA (25.3%) plants after 20 d of treatments compared to 0 days'. Furthermore, heat-acclimated plants were able to maintain significant activity levels of superoxide disumutase (SOD), catalase (CAT), guaiacol peroxidase (POD), and transcription levels of genes encoding these enzymes; in addition, HA plants displayed lower malondialdehyde content and lower electrolyte leakage than NA plants. These results suggest that maintenance of activity and transcription levels of antioxidant enzymes as well as photosynthesis are associated with variable thermostability in HA and NA plants. This likely occurs through cellular membrane stabilization and improvements in water use efficiency in the photosynthetic process during heat stress. The association between antioxidant enzyme activity and gene expression, both of which may vary with genetic variation in heat tolerance, is important to further understand the molecular mechanisms that contribute to heat tolerance.

Methyl Jasmonate and 1-Methylcyclopropene Treatment Effects on Quinone Reductase Inducing Activity and Post-Harvest Quality of Broccoli

PubMed Central

Ku, Kang Mo; Choi, Jeong Hee; Kim, Hyoung Seok; Kushad, Mosbah M.; Jeffery, Elizabeth H.; Juvik, John A.

2013-01-01

Effect of pre-harvest methyl jasmonate (MeJA) and post-harvest 1-methylcyclopropene (1-MCP) treatments on broccoli floret glucosinolate (GS) concentrations and quinone reductase (QR, an in vitro anti-cancer biomarker) inducing activity were evaluated two days prior to harvest, at harvest and at 10, 20, and 30 days of post-harvest storage at 4 °C. MeJA treatments four days prior to harvest of broccoli heads was observed to significantly increase floret ethylene biosynthesis resulting in chlorophyll catabolism during post-harvest storage and reduced product quality. Post-harvest treatment with 1-methylcyclopropene (1-MCP), which competitively binds to protein ethylene receptors, maintained post-harvest floret chlorophyll concentrations and product visual quality in both control and MeJA-treated broccoli. Transcript abundance of BoPPH, a gene which is responsible for the synthesis of pheophytinase, the primary enzyme associated with chlorophyll catabolism in broccoli, was reduced by 1-MCP treatment and showed a significant, negative correlation with floret chlorophyll concentrations. The GS, glucobrassicin, neoglucobrassicin, and gluconasturtiin were significantly increased by MeJA treatments. The products of some of the GS from endogenous myrosinase hydrolysis [sulforaphane (SF), neoascorbigen (NeoASG), N-methoxyindole-3-carbinol (NI3C), and phenethyl isothiocyanate (PEITC)] were also quantified and found to be significantly correlated with QR. Sulforaphane, the isothiocyanate hydrolysis product of the GS glucoraphanin, was found to be the most potent QR induction agent. Increased sulforaphane formation from the hydrolysis of glucoraphanin was associated with up-regulated gene expression of myrosinase (BoMyo) and the myrosinase enzyme co-factor gene, epithiospecifier modifier1 (BoESM1). This study demonstrates the combined treatment of MeJA and 1-MCP increased QR activity without post-harvest quality loss. PMID:24146962

Swift observations of GS 1826-238

NASA Astrophysics Data System (ADS)

Ji, L.; Santangelo, A.; Zhang, S.; Ducci, L.; Suleimanov, V.

2018-02-01

GS 1826-238 is a well-studied low-mass X-ray binary neutron star. This source was in a persistent hard state since its discovery in 1988 and until 2014 June. After that, the source exhibited several softer periods of enhanced intensity in the energy range 2-20 keV. We studied the long-term light curves of MAXI (Monitor of All Sky X-ray Image) and Swift/BAT, and found clearly two branches in the MAXI-BAT and hardness-intensity diagrams, which correspond to the persistent state and softer periods, respectively. We analysed 21 Swift/XRT observations, of which four were located in the persistent state while the others were in softer periods or in a state between them. The XRT spectra could be generally fitted by using an absorbed Comptonization model with no other components required. We found a peculiar relationship between the luminosity and the hardness in the energy range of 0.6-10 keV: when the luminosity is larger (smaller) than 4 per cent-6 per cent Ledd, the hardness is anti-correlated (correlated) with luminosity. We also estimated the variability for each observation by using the fractional rms in the 0.1-10 Hz range. We found that the observations in the persistent state had a large fractional rms of ˜25 per cent, similar to other low-mass X-ray binaries. However, the variability is mainly found in the range of 5 per cent-20 per cent during softer periods. We suggest that GS 1826-238 did not evolve into the soft state of atoll sources, and all the observed XRT observations during the softer periods resemble a peculiar intermediate state of atoll sources.

Construction of a star-shaped copolymer as a vector for FGF receptor-mediated gene delivery in vitro and in vivo.

PubMed

Li, Da; Ping, Yuan; Xu, Fujian; Yu, Hai; Pan, Hongming; Huang, Hongliang; Wang, Qingqing; Tang, Guping; Li, Jun

2010-09-13

The success of cancer gene therapy highly relies on the gene delivery vector with high transfection activity and low toxicity. In the present study, eight-armed polyethylene glycol (EAP) and low molecular weight (LMW) polyethylenimine (PEI) were used as basic units to construct the architecture of a new star-shaped EAP-PEI copolymer (EAPP). MC11, a peptide capable of selectively binding fibroblast growth factor receptor (FGFR) on tumor cell membranes, was further conjugated to EAPP to produce the vector EAPP-MC11 (EAPPM) to enhance tumor targetability. This tumor-targeting vector EAPPM was observed to retard the plasmids mobility at a nitrogen/phosphorus (N/P) ratio of 3. The vector could efficiently condense plasmids within 300 nm nanoparticles with a positive zeta potential at the N/P ratio of 20 or above. While the cytotoxicity of EAPPM polyplexes was similar to that of LMW PEI, it was significantly lower than that of PEI (25 kDa) in HepG2 and PC3 cell lines. In vitro gene transfection with pDNA mediated by EAPPM showed that the transfection efficiency increased 15 times in HepG2 cells but remained at a similar level in PC3 cells in comparison with that of EAPP. By systemic injection of EAPPM/pDNA complexes into a HepG2-bearing mice model, luciferase expression detected in lung, liver, and tumor tissues demonstrated EAPPM could deliver in a targeted manner a reporter gene into tumor tissues, where the luciferase expression of EAPPM was 4 times higher than that of EAPP and even 23 times higher than that of PEI (25 kDa). Furthermore, it was found that the systemic delivery of EAPPM/pCSK-α-interferon complexes in vivo were much more effective in inhibiting tumor growth than EAPP or PEI (25 kDa). These results clearly show that EAPPM is an efficient and safe vector for FGFR-mediated targeted gene delivery both in vitro and in vivo. With low cytotoxicity and high targetability, EAPPM may have great potential as a delivery vector for future cancer gene therapy

Feather hydrolysate from Streptomyces sampsonii GS 1322: A potential low cost soil amendment.

PubMed

Jain, Richa; Jain, Aakanchha; Rawat, Neha; Nair, Meera; Gumashta, Raghvendra

2016-06-01

Process parameters for obtaining hydrolysate from hen feathers, i.e., initial pH (5.0-9.0) and incubation period (1-6 day), were set and studied, using Streptomyces sampsonii GS 1322 in submerged and solid state conditions. Under submerged conditions, complete hydrolysis of feathers was observed on fifth day [initial pH 8.0, 28 ± 2°C, shaking (150 rpm)] with release of soluble protein (2985 μg ml(-1)) and amino acids (2407 μg ml(-1)). Cell free hydrolysate showed hydrolysis of casein (18 mm), gelatin (26 mm), collagen (31 mm), hemoglobin (23 mm) and Tween 80 (35 mm) while 445 U keratinase activity. Total soluble protein reached 5 mg ml(-1) in solid state conditions. During Pot experimentation using barren agriculture soil the effect of feather hydrolysate on wheat crop were recorded. Significant increase (p<0.01) in wheat seed germination was observed in treated soils as compared to untreated. Treatment significantly increased plant height from 30 to 60 days and 30-90 days (p<0.001). Treated soil showed an increase in total microbial count, proteolytic activity, phosphate solubilizing bacteria and ammonifying bacteria, whereas pathogenic fungi load was reduced. S. sampsonii GS 1322 can be used for bio-processing of poultry wastes yielding feather hydrolysate rich in proteins and amino acids for development of low-cost organic amendment to accelerate wheat crop growth in barren agricultural land. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

In Vivo Bio-distribution and Efficient Tumor Targeting of Gelatin/Silica Nanoparticles for Gene Delivery

NASA Astrophysics Data System (ADS)

Zhao, Xueqin; Wang, Jun; Tao, SiJie; Ye, Ting; Kong, Xiangdong; Ren, Lei

2016-04-01

The non-viral gene delivery system is an attractive alternative to cancer therapy. The clinical success of non-viral gene delivery is hampered by transfection efficiency and tumor targeting, which can be individually overcome by addition of functional modules such as cell penetration or targeting. Here, we first engineered the multifunctional gelatin/silica (GS) nanovectors with separately controllable modules, including tumor-targeting aptamer AGRO100, membrane-destabilizing peptide HA2, and polyethylene glycol (PEG), and then studied their bio-distribution and in vivo transfection efficiencies by contrast resonance imaging (CRI). The results suggest that the sizes and zeta potentials of multifunctional gelatin/silica nanovectors were 203-217 nm and 2-8 mV, respectively. Functional GS-PEG nanoparticles mainly accumulated in the liver and tumor, with the lowest uptake by the heart and brain. Moreover, the synergistic effects of tumor-targeting aptamer AGRO100 and fusogenic peptide HA2 promoted the efficient cellular internalization in the tumor site. More importantly, the combined use of AGRO100 and PEG enhanced tumor gene expression specificity and effectively reduced toxicity in reticuloendothelial system (RES) organs after intravenous injection. Additionally, low accumulation of GS-PEG was observed in the heart tissues with high gene expression levels, which could provide opportunities for non-invasive gene therapy.

Solvothermal-induced self-assembly of Fe2O3/GS aerogels for high Li-storage and excellent stability.

PubMed

Wang, Ronghua; Xu, Chaohe; Du, Meng; Sun, Jing; Gao, Lian; Zhang, Peng; Yao, Heliang; Lin, Chucheng

2014-06-12

A novel solvothermal-induced self-assembly approach, using colloid sol as precursor, is developed to construct monolithic 3D metal oxide/GS (graphene sheets) aerogels. During the solvothermal process, graphene oxide (GO) is highly reduced to GS and self-assembles into 3D macroscopic hydrogels, accompanying with in situ transformation of colloid sol to metal oxides. As a proof of concept, Fe2 O3 /GS aerogels are synthesized based on Fe(OH)3 sol, in which GS self-assemble into an interconnected macroporous framework and Fe2 O3 nanocrystals (20-50 nm) uniformly deposit on GS. Benefitting from the integration of macroporous structures, large surface area, high electrical conductivity, and good electrode homogeneity, the hybrid electrode manifests a superior rate capability (930, 660 and 520 mAh g(-1) at 500, 2000 and 4000 mA g(-1), respectively) and excellent prolonged cycling stability at high rates (733 mAh g(-1) during 1000 charge/discharge cycles at 2000 mA g(-1)), demonstrating its great potential for application in high performance lithium ion batteries. The work described here provides a versatile pathway to construct various graphene-based hybrid aerogels. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Constitutive stimulatory G protein activity in limb mesenchyme impairs bone growth.

PubMed

Karaca, Anara; Malladi, Vijayram Reddy; Zhu, Yan; Tafaj, Olta; Paltrinieri, Elena; Wu, Joy Y; He, Qing; Bastepe, Murat

2018-05-01

GNAS mutations leading to constitutively active stimulatory G protein alpha-subunit (Gsα) cause different tumors, fibrous dysplasia of bone, and McCune-Albright syndrome, which are typically not associated with short stature. Enhanced signaling of the parathyroid hormone/parathyroid hormone-related peptide receptor, which couples to multiple G proteins including Gsα, leads to short bones with delayed endochondral ossification. It has remained unknown whether constitutive Gsα activity also impairs bone growth. Here we generated mice expressing a constitutively active Gsα mutant (Gsα-R201H) conditionally upon Cre recombinase (cGsα R201H mice). Gsα-R201H was expressed in cultured bone marrow stromal cells from cGsα R201H mice upon adenoviral-Cre transduction. When crossed with mice in which Cre is expressed in a tamoxifen-regulatable fashion (CAGGCre-ER™), tamoxifen injection resulted in mosaic expression of the transgene in double mutant offspring. We then crossed the cGsα R201H mice with Prx1-Cre mice, in which Cre is expressed in early limb-bud mesenchyme. The double mutant offspring displayed short limbs at birth, with narrow hypertrophic chondrocyte zones in growth plates and delayed formation of secondary ossification center. Consistent with enhanced Gsα signaling, bone marrow stromal cells from these mice demonstrated increased levels of c-fos mRNA. Our findings indicate that constitutive Gsα activity during limb development disrupts endochondral ossification and bone growth. Given that Gsα haploinsufficiency also leads to short bones, as in patients with Albright's hereditary osteodystrophy, these results suggest that a tight control of Gsα activity is essential for normal growth plate physiology. Copyright © 2018 Elsevier Inc. All rights reserved.

Early- and late-onset preeclampsia and the DNA methylation of circadian clock and clock-controlled genes in placental and newborn tissues.

PubMed

van den Berg, C B; Chaves, I; Herzog, E M; Willemsen, S P; van der Horst, G T J; Steegers-Theunissen, R P M

2017-01-01

The placenta is important in providing a healthy environment for the fetus and plays a central role in the pathophysiology of preeclampsia (PE). Fetal and placental developments are influenced by epigenetic programming. There is some evidence that PE is controlled to an altered circadian homeostasis. In a nested case-control study embedded in the Rotterdam Periconceptional Cohort, we obtained placental tissue, umbilical cord leukocytes (UCL), and human umbilical venous endothelial cells of 13 early-onset PE, 16 late-onset PE and 83 controls comprising 36 uncomplicated and 47 complicated pregnancies, i.e. 27 fetal growth restricted and 20 spontaneous preterm birth. To investigate the associations between PE and the epigenetics of circadian clock and clock-controlled genes in placental and newborn tissues, genome-wide DNA methylation analysis was performed using the Illumina HumanMethylation450K BeadChip and a candidate-gene approach using ANCOVA was applied on 939 CpGs of 39 circadian clock and clock-controlled genes. DNA methylation significantly differed in early-onset PE compared with spontaneous preterm birth at 6 CpGs in placental tissue (3.73 E-5 ≤ p ≤ 0.016) and at 21 CpGs in UCL (1.09 E-5 ≤ p ≤ 0.024). In early-onset PE compared with fetal growth restriction 2 CpGs in placental tissue (p < 0.05) and 8 CpGs in uncomplicated controls (4.78 E-5 ≤ p ≤ 0.049) were significantly different. Moreover, significantly different DNA methylation in early-onset PE compared with uncomplicated controls was shown at 6 CpGs in placental tissue (1.36 E-4 ≤ p ≤ 0.045) and 11 CpGs in uncomplicated controls (2.52 E-6 ≤ p ≤ 0.009). No significant associations were shown with late-onset PE between study groups or tissues. The most differentially methylated CpGs showed hypomethylation in placental tissue and hypermethylation in uncomplicated controls. In conclusion, DNA methylation of circadian clock and clock-controlled genes demonstrated most differences in UCL

GS 455534 selectively suppresses binge eating of palatable food and attenuates dopamine release in the accumbens of sugar-bingeing rats.

PubMed

Bocarsly, Miriam E; Hoebel, Bartley G; Paredes, Daniel; von Loga, Isabell; Murray, Susan M; Wang, Miaoyuan; Arolfo, Maria P; Yao, Lina; Diamond, Ivan; Avena, Nicole M

2014-04-01

Binge eating palatable foods has been shown to have behavioral and neurochemical similarities to drug addiction. GS 455534 is a highly selective reversible aldehyde dehydrogenase 2 inhibitor that has been shown to reduce alcohol and cocaine intake in rats. Given the overlaps between binge eating and drug abuse, we examined the effects of GS 455534 on binge eating and subsequent dopamine release. Sprague-Dawley rats were maintained on a sugar (experiment 1) or fat (experiment 2) binge eating diet. After 25 days, GS 455534 was administered at 7.5 and 15 mg/kg by an intraperitoneal injection, and food intake was monitored. In experiment 3, rats with cannulae aimed at the nucleus accumbens shell were maintained on the binge sugar diet for 25 days. Microdialysis was performed, during which GS 455534 15 mg/kg was administered, and sugar was available. Dialysate samples were analyzed to determine extracellular levels of dopamine. In experiment 1, GS 455534 selectively decreased sugar intake food was made available in the Binge Sugar group but not the Ad libitum Sugar group, with no effect on chow intake. In experiment 2, GS 455534 decreased fat intake in the Binge Fat group, but not the Ad libitum Fat group, however, it also reduced chow intake. In experiment 3, GS 455534 attenuated accumbens dopamine release by almost 50% in binge eating rats compared with the vehicle injection. The findings suggest that selective reversible aldehyde dehydrogenase 2 inhibitors may have the therapeutic potential to reduce binge eating of palatable foods in clinical populations.

Quality-Assurance Plan for Water-Quality Activities of the U.S. Geological Survey in Miami, Florida

DTIC Science & Technology

2003-01-01

Jacqueline Lima GS-12 GS-10 GS-09 GS-09 GS-08 GS-07 GS-06 GS-06 GS-04 GS-04Eleanor Seymore Surface-Water Data Collections & Jon Woolverton Jorge Agis...Thomas J. Smith, III Gordon Anderson Kevin Whelan Don De Angelis Allison Snow GS-13 GS-07 GS-05 GS-12 GS-13 GS-07 GS-12 GS-13 GS-09 GS-07 GS-14 GS-07...fan, active, inactive, inventory only playa , month day year stream channel, digital rec- order, North American Datum of 1927 North American Datum of

Tissue-specific changes of glutamine synthetase activity in oats after rhizosphere infestation by Pseudomonas syringae pv. tabaci. Final report

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

Knight, T.J.; Temple, S.; Sengupta-Gopalan, C.

1996-05-15

Oats (Avena sativa L. lodi) tolerant of rhizosphere infestation by Pseudomonas syringae pv. tabaci when challenged by the pathogen experience tissue-specific alterations of ammonia assimilatory capabilities. Altered ammonia assimilatory potentials between root and leaf tissue result from selective inactivation of glutamine synthetase (GS) by the toxin Tabtoxinine-B-lactam (TBL). Root GS is sensitive and leaf GSs are resistant to TBL inactivation. With prolonged challenge by the pathogen root GS activity decreases but leaf GS specific activity increase. Higher leaf GS activity is due to decreased rates of degradation rather than increased GS synthesis. Higher leaf GS activity and elevated levels ofmore » GS polypeptide appear to result from a limited interaction between GS and TBL leading to the accumulation of a less active but more stable GS holoenzyme. Tolerant challenged oats besides surviving rhizosphere infestation, experience enhanced growth. A strong correlation exists between leaf GS activity and whole plant fresh weight, suggesting that tissue-specific changes in ammonia assimilatory capability provides the plant a more efficient mechanism for uptake and utilization of nitrogen.« less

Electron Transport Controls Glutamine Synthetase Activity in the Facultative Heterotrophic Cyanobacterium Synechocystis sp. PCC 6803.

PubMed Central

Reyes, J. C.; Crespo, J. L.; Garcia-Dominguez, M.; Florencio, F. J.

1995-01-01

Glutamine synthetase (GS) from Synechocystis sp. PCC 6803 was inactivated in vivo by transferring cells from light to darkness or by incubation with the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea but not with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. Addition of glucose prevented both dark and 3-(3,4-dichlorophenyl)-1,1-dimethylurea GS inactivation. In a Synechocystis psbE-psbF mutant (T1297) lacking photosystem II, glucose was required to maintain active GS, even in the light. However, in nitrogen-starved T1297 cells the removal of glucose did not affect GS activity. The fact that dark-inactivated GS was reactivated in vitro by the same treatments that reactivate the ammonium-inactivated GS points out that both nitrogen metabolism and redox state of the cells lead to the same molecular regulatory mechanism in the control of GS activity. Using GS antibodies we detected that dark-inactivated GS displayed a different electrophoretic migration with respect to the active form in nondenaturing polyacrylamide gel electrophoresis but not in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The possible pathway to modulate GS activity by the electron transport flow in Synechocystis cells is discussed. PMID:12228640

General Framework for Animal Food Safety Traceability Using GS1 and RFID

NASA Astrophysics Data System (ADS)

Cao, Weizhu; Zheng, Limin; Zhu, Hong; Wu, Ping

GS1 is global traceability standard, which is composed by the encoding system (EAN/UCC, EPC), the data carriers identified automatically (bar codes, RFID), electronic data interchange standards (EDI, XML). RFID is a non-contact, multi-objective automatic identification technique. Tracing of source food, standardization of RFID tags, sharing of dynamic data are problems to solve urgently for recent traceability systems. The paper designed general framework for animal food safety traceability using GS1 and RFID. This framework uses RFID tags encoding with EPCglobal tag data standards. Each information server has access tier, business tier and resource tier. These servers are heterogeneous and distributed, providing user access interfaces by SOAP or HTTP protocols. For sharing dynamic data, discovery service and object name service are used to locate dynamic distributed information servers.

Gene transfer of heterologous G protein-coupled receptors to cardiomyocytes: differential effects on contractility.

PubMed

Laugwitz, K L; Weig, H J; Moretti, A; Hoffmann, E; Ueblacker, P; Pragst, I; Rosport, K; Schömig, A; Ungerer, M

2001-04-13

In heart failure, reduced cardiac contractility is accompanied by blunted cAMP responses to beta-adrenergic stimulation. Parathyroid hormone (PTH)-related peptide and arginine vasopressin are released from the myocardium in response to increased wall stress but do not stimulate contractility or adenylyl cyclase at physiological concentrations. To bypass the defective beta-adrenergic signaling cascade, recombinant P1 PTH/PTH-related peptide receptors (rPTH1-Rs) and V(2) vasopressin receptors (rV(2)-Rs), which are normally not expressed in the myocardium and which are both strongly coupled to adenylyl cyclase, and recombinant beta(2)-adrenergic receptors (rbeta(2)-ARs) were overexpressed in cardiomyocytes by viral gene transfer. The capacity of endogenous hormones to increase contractility via the heterologous, recombinant receptors was compared. Whereas V(2)-Rs are uniquely coupled to Gs, PTH1-Rs and beta(2)-ARs are also coupled to other G proteins. Gene transfer of rPTH1-Rs or rbeta(2)-ARs to adult cardiomyocytes resulted in maximally increased basal contractility, which could not be further stimulated by adding receptor agonists. Agonists at rPTH1-Rs induced increased cAMP formation and phospholipase C activity. In contrast, healthy or failing rV(2)-R-expressing cardiomyocytes showed unaltered basal contractility. Their contractility and cAMP formation increased only at agonist exposure, which did not activate phospholipase C. In summary, we found that gene transfer of PTH1-Rs to cardiomyocytes results in constitutive activity of the transgene, as does that of beta(2)-ARS: In the absence of receptor agonists, rPTH1-Rs and rbeta(2)-ARs increase basal contractility, coupling to 2 G proteins simultaneously. In contrast, rV(2)-Rs are uniquely coupled to Gs and are not constitutively active, retaining their property to be activated exclusively on agonist stimulation. Therefore, gene transfer of V(2)-Rs might be more suited to test the effects of c

Observations of whitecaps during HiWinGS, their dependence on wave field, and relation to gas transfer velocities

NASA Astrophysics Data System (ADS)

Brumer, S. E.; Zappa, C. J.; Fairall, C. W.; Blomquist, B.; Brooks, I. M.; Tamura, H.; Yang, M.; Huebert, B. J.

2016-02-01

The High Wind Gas exchange Study (HiWinGS) presents the unique opportunity to gain new insights on the poorly understood aspects of air-sea interaction under high winds. The HiWinGS cruise took place in the North Atlantic during October and November 2013. Wind speeds exceeded 15 m s-1 25% of the time, including 48 hrs with U10 > 20 m s-1. Continuous measurements of turbulent fluxes of heat, momentum, and gas were taken from the bow of the R/V Knorr. Visible imagery was acquired from the port and starboard side of the flying bridge during daylight hours at 20Hz and directional wave spectra were obtained when on station from a wave rider buoy. Additional wave field statistics were computed from a laser altimeter as well as from a Wavewatch III hindcast. Taking advantage of the range of physical forcing and wave conditions sampled during HiWinGS, we investigate how the fractional whitecap coverage (W) and gas transfer velocity (K) vary with sea state. We distinguish between windseas and swell based on a separation algorithm applied to directional wave spectra, allowing contrasting pure windseas to swell dominated periods. For mixed seas, system alignment is considered when interpreting results. The four gases sampled during HiWinGS ranged from being mostly waterside controlled to almost entirely airside controlled. While bubble-mediated transfer appears to be small for moderately soluble gases like DMS, the importance of wave breaking turbulence transport has yet to be determined for all gases regardless of their solubility. This will be addressed by correlating measured K to estimates of active whitecap fraction (WA) and turbulent kinetic energy dissipation rate (ɛ). WA and ɛ are estimated from moments of the breaking crest length distribution derived from the imagery, focusing on young seas, when it is likely that large-scale breaking waves (i.e., whitecapping) will dominate the ɛ.

Molecular characterization of a novel algal glutamine synthetase (GS) and an algal glutamate synthase (GOGAT) from the colorful outer mantle of the giant clam, Tridacna squamosa, and the putative GS-GOGAT cycle in its symbiotic zooxanthellae.

PubMed

Fam, Rachel R S; Hiong, Kum C; Choo, Celine Y L; Wong, Wai P; Chew, Shit F; Ip, Yuen K

2018-05-20

Giant clams harbor symbiotic zooxanthellae (Symbiodinium), which are nitrogen-deficient, mainly in the fleshy and colorful outer mantle. This study aimed to sequence and characterize the algal Glutamine Synthetase (GS) and Glutamate Synthase (GLT), which constitute the glutamate synthase cycle (or GS-GOGAT cycle, whereby GOGAT is the protein acronym of GLT) of nitrogen assimilation, from the outer mantle of the fluted giant clam, Tridacna squamosa. We had identified a novel GS-like cDNA coding sequence of 2325 bp, and named it as T. squamosa Symbiodinium GS1 (TSSGS1). The deduced TSSGS1 sequence had 774 amino acids with a molecular mass of 85 kDa, and displayed the characteristics of GS1 and Nucleotide Diphosphate Kinase. The cDNA coding sequence of the algal GLT, named as T. squamosa Symbiodinium GLT (TSSGLT), comprised 6399 bp, encoding a protein of 2133 amino acids and 232.4 kDa. The zooxanthellal origin of TSSGS1 and TSSGOGAT was confirmed by sequence comparison and phylogenetic analyses. Indeed, TSSGS1 and TSSGOGAT were expressed predominately in the outer mantle, which contained the majority of the zooxanthellae. Immunofluorescence microscopy confirmed the expression of TSSGS1 and TSSGOGAT in the cytoplasm and the plastids, respectively, of the zooxanthellae in the outer mantle. It can be concluded that the symbiotic zooxanthellae of T. squamosa possesses a glutamate synthase (TSSGS1-TSSGOGAT) cycle that can assimilate endogenous ammonia produced by the host clam into glutamate, which can act as a substrate for amino acid syntheses. Thus, our results provide insights into why intact giant clam-zooxanthellae associations do not excrete ammonia under normal circumstances. Copyright © 2018 Elsevier B.V. All rights reserved.

Optimisation of the expression of a Trametes versicolor laccase gene in Pichia pastoris.

PubMed

O'Callaghan, J; O'Brien, M M; McClean, K; Dobson, A D W

2002-08-01

A cDNA encoding a laccase enzyme was isolated from a Trametes versicolor cDNA library. The gene was subcloned into the Pichia pastoris expression vector pPIC3.5 and transformed into the P. pastoris strains KM71 and GS115. Laccase-secreting transformants were selected by their ability to oxidise the substrate ABTS. No difference in laccase activity was observed between culture supernatants from GS115 (proteolytic) and KM71 (nonproteolytic) strains. The presence of at least 200 microM copper was necessary for optimal laccase activity in the culture supernatants. During growth of P. pastoris on minimal medium the pH of the medium was reduced to <3.0. If alanine was added to the medium the pH reduction was not as pronounced and at alanine concentrations >0.6% w/v the pH was kept constant for >7 days. Cultures in which the pH was maintained by alanine metabolism produced higher levels of laccase activity than those grown in the absence of alanine. This study describes the development of a medium that allows convenient pH control of P. pastoris without the need for continuous neutralisation.

Cloning and characterization of a novel chitinase gene (chi46) from Chaetomium globosum and identification of its biological activity.

PubMed

Liu, Z H; Yang, Q; Hu, S; Zhang, J D; Ma, J

2008-08-01

Chitinases play a major role in the defensive strategies of plants against fungal pathogens. In the current study, the gene for a 46-kDa endochitinase (chi46) was cloned from Chaetomium globosum, an important biocontrol fungus. The corresponding complementary deoxyribonucleic acid sequence was 1,350 bp in length, encoding 449 amino acid residues. The temporal expression of chi46, in response to the treatments of cell walls of six pathogens and confrontation against two fungal pathogens, was measured in C. globosum using real-time reverse transcription polymerase chain reaction. The expression of chi46 can be highly induced by exposure to the cell walls of plant pathogens and living pathogens, suggesting a role in plant disease resistance. The chi46 gene was inserted into the pPIC9 vector and transferred into the cells of Pichia pastoris GS115 for heterologous expression. The optimal reaction conditions for chitinase CHI46 activity were: 45 degrees C, pH of 5.0, and 5 mmol l(-1) of Cu2+. The maximum enzyme activity was 1.42 U ml(-1) following exposure to the cell wall chitin of Septoria tritici. The CHI46 enzyme can efficiently degrade cell walls of the phytopathogenic Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum, Valsa sordida, S. tritici, and Phytophthora sojae, demonstrating that it may be involved in the biocontrol mechanism of C. globosum.

Gitelman or Bartter type 3 syndrome? A case of distal convoluted tubulopathy caused by CLCNKB gene mutation

PubMed Central

Cruz, António José; Castro, Alexandra

2013-01-01

A 32-year-old woman with no significant medical history was sent to our consultation due to hypokalaemia (<3.0 mmol/l). Her main complaints were longstanding polyuria and nocturia. Physical examination was normal. Basic investigations showed normal renal function, low serum potassium (2.7 mmol/l) and magnesium (0.79 mmol/l), metabolic alkalosis (pH 7.54; bicarbonate 32.5 mmol/l), elevated urinary potassium (185 mmol/24 h) and normal urinary calcium (246 mg/24 h). Thiazide test revealed blunted response. Chronic vomiting and the abuse of diuretics were excluded. Genetic tests for SLC12A3 gene mutation described in Gitelman syndrome (GS) came negative. CLCNKB gene mutation analysis present in both GS and Bartter (BS) type 3 syndromes was positive. The patient is now being treated with potassium and magnesium oral supplements, ramipril and spironolactone with stable near-normal potassium and magnesium levels. This article presents the case of a patient with hypokalaemia caused by CLCNKB gene mutation hard to categorise as GS or BS type 3. PMID:23345488

Gitelman or Bartter type 3 syndrome? A case of distal convoluted tubulopathy caused by CLCNKB gene mutation.

PubMed

Cruz, António José; Castro, Alexandra

2013-01-22

A 32-year-old woman with no significant medical history was sent to our consultation due to hypokalaemia (<3.0 mmol/l). Her main complaints were longstanding polyuria and nocturia. Physical examination was normal. Basic investigations showed normal renal function, low serum potassium (2.7 mmol/l) and magnesium (0.79 mmol/l), metabolic alkalosis (pH 7.54; bicarbonate 32.5 mmol/l), elevated urinary potassium (185 mmol/24 h) and normal urinary calcium (246 mg/24 h). Thiazide test revealed blunted response. Chronic vomiting and the abuse of diuretics were excluded. Genetic tests for SLC12A3 gene mutation described in Gitelman syndrome (GS) came negative. CLCNKB gene mutation analysis present in both GS and Bartter (BS) type 3 syndromes was positive. The patient is now being treated with potassium and magnesium oral supplements, ramipril and spironolactone with stable near-normal potassium and magnesium levels. This article presents the case of a patient with hypokalaemia caused by CLCNKB gene mutation hard to categorise as GS or BS type 3.

GSNFS: Gene subnetwork biomarker identification of lung cancer expression data.

PubMed

Doungpan, Narumol; Engchuan, Worrawat; Chan, Jonathan H; Meechai, Asawin

2016-12-05

Gene expression has been used to identify disease gene biomarkers, but there are ongoing challenges. Single gene or gene-set biomarkers are inadequate to provide sufficient understanding of complex disease mechanisms and the relationship among those genes. Network-based methods have thus been considered for inferring the interaction within a group of genes to further study the disease mechanism. Recently, the Gene-Network-based Feature Set (GNFS), which is capable of handling case-control and multiclass expression for gene biomarker identification, has been proposed, partly taking into account of network topology. However, its performance relies on a greedy search for building subnetworks and thus requires further improvement. In this work, we establish a new approach named Gene Sub-Network-based Feature Selection (GSNFS) by implementing the GNFS framework with two proposed searching and scoring algorithms, namely gene-set-based (GS) search and parent-node-based (PN) search, to identify subnetworks. An additional dataset is used to validate the results. The two proposed searching algorithms of the GSNFS method for subnetwork expansion are concerned with the degree of connectivity and the scoring scheme for building subnetworks and their topology. For each iteration of expansion, the neighbour genes of a current subnetwork, whose expression data improved the overall subnetwork score, is recruited. While the GS search calculated the subnetwork score using an activity score of a current subnetwork and the gene expression values of its neighbours, the PN search uses the expression value of the corresponding parent of each neighbour gene. Four lung cancer expression datasets were used for subnetwork identification. In addition, using pathway data and protein-protein interaction as network data in order to consider the interaction among significant genes were discussed. Classification was performed to compare the performance of the identified gene subnetworks with three

Safety and immunogenicity of MAGE-A3 cancer immunotherapeutic with dacarbazine in patients with MAGE-A3-positive metastatic cutaneous melanoma: an open phase I/II study with a first assessment of a predictive gene signature

PubMed Central

Grob, Jean-Jacques; Mortier, Laurent; D’Hondt, Lionel; Grange, Florent; Baurain, Jean Francois; Dréno, Brigitte; Lebbe, Céleste; Robert, Caroline; Dompmartin, Anne; Neyns, Bart; Gillet, Marc; Louahed, Jamila; Jarnjak, Silvija; Lehmann, Frédéric F

2017-01-01

Background We assessed safety, immunogenicity and clinical activity of recombinant MAGE-A3 antigen combined with AS15 immunostimulant (MAGE-A3 immunotherapeutic) in association with dacarbazine in patients with metastatic melanoma. Methods In this open-label, phase I/II, uncontrolled multicentre trial conducted in Belgium and France, patients with MAGE-A3-positive melanoma received up to 24 doses of MAGE-A3 immunotherapeutic (four cycles) coadministered with eight doses of dacarbazine. Adverse events (AE) were recorded until 31 days postvaccination, and serious AEs (SAE), until 30 days following the last dose. MAGE-A3-specific antibodies were measured by ELISA. Clinical activity of MAGE-A3 immunotherapeutic was assessed in patients positive/negative for previously identified gene signature (GS) associated with clinical outcome. Results Forty-eight patients were enrolled and treated (32 GS+, 15 GS−, 1 unknown GS status); two patients completed the study. All patients reported AEs, the most common were ‘general disorders and administration site conditions’ (94%). Treatment-related AEs were reported by 85% of patients; the most common was pain at injection site (38%). Sixteen SAEs were reported by 21% of patients; two were considered as treatment related (neutropenia and thrombocytopenia; grade 4). Postdose 4, all patients were seropositive for MAGE-A3-specific antibodies, with a geometric mean titre of 2778.7 ELISA units (EU)/mL (95% CI 1638.3 to 4712.8). One complete and three partial responses were reported (only in GS+ patients). Median overall survival was 11.4 months for GS+ and 5.3 months for GS− patients. Conclusion Although this trial shows poor results compared with the new results with checkpoint inhibitors, it gives an interesting insight in rapidly developing fields like combinations of immunotherapy and chemotherapy, new generation vaccines and the use of gene profile as a predictive marker. Trial registration number NCT00849875. PMID:29177094

Structural Characterization and Evolutionary Relationship of High-Molecular-Weight Glutenin Subunit Genes in Roegneria nakaii and Roegneria alashanica.

PubMed

Zhang, Lujun; Li, Zhixin; Fan, Renchun; Wei, Bo; Zhang, Xiangqi

2016-07-19

The Roegneria of Triticeae is a large genus including about 130 allopolyploid species. Little is known about its high-molecular-weight glutenin subunits (HMW-GSs). Here, we reported six novel HMW-GS genes from R. nakaii and R. alashanica. Sequencing indicated that Rny1, Rny3, and Ray1 possessed intact open reading frames (ORFs), whereas Rny2, Rny4, and Ray2 harbored in-frame stop codons. All of the six genes possessed a similar primary structure to known HMW-GS, while showing some unique characteristics. Their coding regions were significantly shorter than Glu-1 genes in wheat. The amino acid sequences revealed that all of the six genes were intermediate towards the y-type. The phylogenetic analysis showed that the HMW-GSs from species with St, StY, or StH genome(s) clustered in an independent clade, varying from the typical x- and y-type clusters. Thus, the Glu-1 locus in R. nakaii and R. alashanica is a very primitive glutenin locus across evolution. The six genes were phylogenetically split into two groups clustered to different clades, respectively, each of the two clades included the HMW-GSs from species with St (diploid and tetraploid species), StY, and StH genomes. Hence, it is concluded that the six Roegneria HMW-GS genes are from two St genomes undergoing slight differentiation.

Gene discovery for enzymes involved in limonene modification or utilization by the mountain pine beetle-associated pathogen Grosmannia clavigera.

PubMed

Wang, Ye; Lim, Lynette; Madilao, Lina; Lah, Ljerka; Bohlmann, Joerg; Breuil, Colette

2014-08-01

To successfully colonize and eventually kill pine trees, Grosmannia clavigera (Gs cryptic species), the main fungal pathogen associated with the mountain pine beetle (Dendroctonus ponderosae), has developed multiple mechanisms to overcome host tree chemical defenses, of which terpenoids are a major component. In addition to a monoterpene efflux system mediated by a recently discovered ABC transporter, Gs has genes that are highly induced by monoterpenes and that encode enzymes that modify or utilize monoterpenes [especially (+)-limonene]. We showed that pine-inhabiting Ophiostomale fungi are tolerant to monoterpenes, but only a few, including Gs, are known to utilize monoterpenes as a carbon source. Gas chromatography-mass spectrometry (GC-MS) revealed that Gs can modify (+)-limonene through various oxygenation pathways, producing carvone, p-mentha-2,8-dienol, perillyl alcohol, and isopiperitenol. It can also degrade (+)-limonene through the C-1-oxygenated pathway, producing limonene-1,2-diol as the most abundant intermediate. Transcriptome sequencing (RNA-seq) data indicated that Gs may utilize limonene 1,2-diol through beta-oxidation and then valine and tricarboxylic acid (TCA) metabolic pathways. The data also suggested that at least two gene clusters, located in genome contigs 108 and 161, were highly induced by monoterpenes and may be involved in monoterpene degradation processes. Further, gene knockouts indicated that limonene degradation required two distinct Baeyer-Villiger monooxygenases (BVMOs), an epoxide hydrolase and an enoyl coenzyme A (enoyl-CoA) hydratase. Our work provides information on enzyme-mediated limonene utilization or modification and a more comprehensive understanding of the interaction between an economically important fungal pathogen and its host's defense chemicals.

Functioning and nonfunctioning thyroid adenomas involve different molecular pathogenetic mechanisms.

PubMed

Tonacchera, M; Vitti, P; Agretti, P; Ceccarini, G; Perri, A; Cavaliere, R; Mazzi, B; Naccarato, A G; Viacava, P; Miccoli, P; Pinchera, A; Chiovato, L

1999-11-01

The molecular biology of follicular cell growth in thyroid nodules is still poorly understood. Because gain-of-function (activating) mutations of the thyroid-stimulating hormone receptor (TShR) and/or Gs alpha genes may confer TSh-independent growth advantage to neoplastic thyroid cells, we searched for somatic mutations of these genes in a series of hyperfunctioning and nonfunctioning follicular thyroid adenomas specifically selected for their homogeneous gross anatomy (single nodule in an otherwise normal thyroid gland). TShR gene mutations were identified by direct sequencing of exons 9 and 10 of the TShR gene in genomic DNA obtained from surgical specimens. Codons 201 and 227 of the Gs alpha gene were also analyzed. At histology, all hyperfunctioning nodules and 13 of 15 nonfunctioning nodules were diagnosed as follicular adenomas. Two nonfunctioning thyroid nodules, although showing a prevalent microfollicular pattern of growth, had histological features indicating malignant transformation (a minimally invasive follicular carcinoma and a focal papillary carcinoma). Activating mutations of the TShR gene were found in 12 of 15 hyperfunctioning follicular thyroid adenomas. In one hyperfunctioning adenoma, which was negative for TShR mutations, a mutation in codon 227 of the Gs alpha gene was identified. At variance with hyperfunctioning thyroid adenomas, no mutation of the TShR or Gs alpha genes was detected in nonfunctioning thyroid nodules. In conclusion, our findings clearly define a different molecular pathogenetic mechanism in hyperfunctioning and nonfunctioning follicular thyroid adenomas. Activation of the cAMP cascade, which leads to proliferation but maintains differentiation of follicular thyroid cells, typically occurs in hyperfunctioning thyroid adenomas. Oncogenes other than the TShR and Gs alpha genes are probably involved in nonfunctioning follicular adenomas.

78 FR 10001 - Proposed Collection; Comment Request for Form 706-GS(D)

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-12

... Form 706-GS(D), Generation-Skipping Transfer Tax Return for Distributions. DATES: Written comments should be received on or before April 15, 2013 to be assured of consideration. ADDRESSES: Direct all... [email protected] . SUPPLEMENTARY INFORMATION: Title: Generation-Skipping Transfer Tax Return for...

Efficacy and tolerance of enzymatic hydrolysed collagen (EHC) vs. glucosamine sulphate (GS) in the treatment of knee osteoarthritis (KOA).

PubMed

Trč, Tomáš; Bohmová, Jana

2011-03-01

This was a 13-week, multicentre, randomised, parallel, double-blind study. One hundred men and women volunteers aged ≥ 40 years with knee osteoarthritis (KOA) were randomised to once daily enzymatic hydrolysed collagen (EHC) 10 g or glucosamine sulphate (GS) 1.5 g for 90 consecutive days. Follow-up took place after two weeks and after one, two and three months. Primary [visual analogue scale (VAS), Western Ontario and McMaster Universities (WOMAC Index)] and secondary outcomes variables, assessed at weeks two, four, eight and 12, were KOA pain intensity measured by quadruple visual analogue scales in the target knee, the WOMAC total score index, patient's and investigator's global assessments of disease activity, joint assessment, use of rescue medication (ibuprofen 400 mg tablets) and assessment of Quality of Life index (SF-36 Questionnaire). Safety and tolerability were also evaluated. Clear improvement was observed in both joint pain and symptoms in patients with KOA treated with EHC (Colatech®) and significant differences were observed. Mean reductions from baseline for EHC 10 g daily and GS 1.5 g, respectively, were KOA pain intensity reduction in the target knee for Colatech® (p < 0.05): WOMAC index decrease ≤ 15 points at the last visit (day 90) for Colatech® in 16 patients (34.04%) (p < 0.05) and for glucosamine in six patients (13.04%); total score index for painful joints: Colatech® 1.6 (p < 0.05) and glucosamine 1.8; total score index for swollen joints: Colatech® 0.5 (p < 0.05) and glucosamine 0.7; patient's global assessment of efficacy as the sum of improvement good + ideal: 80.8% for Colatech® and 46.6% for glucosamine (p < 0.05). EHC (Colatech®) showed superior improvement over GS in the SF-36 Questionnaire in the Physical Health Index (42.0 for Colatech and 40.0 for glucosamine). The incidence of adverse events was similar in both groups. Both EHC and GS were well tolerated.

Adaptive Activation of a Stress Response Pathway Improves Learning and Memory Through Gs and β-Arrestin-1-Regulated Lactate Metabolism.

PubMed

Dong, Jun-Hong; Wang, Yi-Jing; Cui, Min; Wang, Xiao-Jing; Zheng, Wen-Shuai; Ma, Ming-Liang; Yang, Fan; He, Dong-Fang; Hu, Qiao-Xia; Zhang, Dao-Lai; Ning, Shang-Lei; Liu, Chun-Hua; Wang, Chuan; Wang, Yue; Li, Xiang-Yao; Yi, Fan; Lin, Amy; Kahsai, Alem W; Cahill, Thomas Joseph; Chen, Zhe-Yu; Yu, Xiao; Sun, Jin-Peng

2017-04-15

Stress is a conserved physiological response in mammals. Whereas moderate stress strengthens memory to improve reactions to previously experienced difficult situations, too much stress is harmful. We used specific β-adrenergic agonists, as well as β 2 -adrenergic receptor (β2AR) and arrestin knockout models, to study the effects of adaptive β2AR activation on cognitive function using Morris water maze and object recognition experiments. We used molecular and cell biological approaches to elucidate the signaling subnetworks. We observed that the duration of the adaptive β2AR activation determines its consequences on learning and memory. Short-term formoterol treatment, for 3 to 5 days, improved cognitive function; however, prolonged β2AR activation, for more than 6 days, produced harmful effects. We identified the activation of several signaling networks downstream of β2AR, as well as an essential role for arrestin and lactate metabolism in promoting cognitive ability. Whereas Gs-protein kinase A-cyclic adenosine monophosphate response element binding protein signaling modulated monocarboxylate transporter 1 expression, β-arrestin-1 controlled expression levels of monocarboxylate transporter 4 and lactate dehydrogenase A through the formation of a β-arrestin-1/phospho-mitogen-activated protein kinase/hypoxia-inducible factor-1α ternary complex to upregulate lactate metabolism in astrocyte-derived U251 cells. Conversely, long-term treatment with formoterol led to the desensitization of β2ARs, which was responsible for its decreased beneficial effects. Our results not only revealed that β-arrestin-1 regulated lactate metabolism to contribute to β2AR functions in improved memory formation, but also indicated that the appropriate management of one specific stress pathway, such as through the clinical drug formoterol, may exert beneficial effects on cognitive abilities. Copyright © 2016 Society of Biological Psychiatry. All rights reserved.

Improved antimicrobial activity of Pediococcus acidilactici against Salmonella Gallinarum by UV mutagenesis and genome shuffling.

PubMed

Han, Geon Goo; Song, Ahn Ah; Kim, Eun Bae; Yoon, Seong-Hyun; Bok, Jin-Duck; Cho, Chong-Su; Kil, Dong Yong; Kang, Sang-Kee; Choi, Yun-Jaie

2017-07-01

Pediococcus acidilactici is a widely used probiotic, and Salmonella enterica serovar Gallinarum (SG) is a significant pathogen in the poultry industry. In this study, we improved the antimicrobial activity of P. acidilactici against SG using UV mutation and genome shuffling (GS). To improve antimicrobial activity against SG, UV mutagenesis was performed against wild-type P. acidilactici (WT), and five mutants showed improved antimicrobial activity. To further improve antimicrobial activity, GS was performed on five UV mutants. Following GS, four mutants showed improved antimicrobial activity compared with the UV mutants and WT. The antimicrobial activity of GS1 was highest among the mutants; however, the activity was reduced when the culture supernatant was treated with proteinase K, suggesting that the improved antimicrobial activity is due to a proteinous substance such as bacteriocin. To validate the activity of GS1 in vivo, we designed multi-species probiotics and performed broiler feeding experiments. Groups consisted of no treatment (NC), avilamycin-treated (PC), probiotic group 1 containing WT (T1), and probiotic group 2 containing GS1 (T2). In broiler feeding experiments, coliform bacteria were significantly reduced in T2 compared with NC, PC, and T1. The cecal microbiota was modulated and pathogenic bacteria were reduced by GS1 oral administration. In this study, GS1 showed improved antimicrobial activity against SG in vitro and reduced pathogenic bacteria in a broiler feeding experiment. These results suggest that GS1 can serve as an efficient probiotic, as an alternative to antibiotics in the poultry industry.

Corruption of the intra-gene DNA methylation architecture is a hallmark of cancer.

PubMed

Bartlett, Thomas E; Zaikin, Alexey; Olhede, Sofia C; West, James; Teschendorff, Andrew E; Widschwendter, Martin

2013-01-01

Epigenetic processes--including DNA methylation--are increasingly seen as having a fundamental role in chronic diseases like cancer. It is well known that methylation levels at particular genes or loci differ between normal and diseased tissue. Here we investigate whether the intra-gene methylation architecture is corrupted in cancer and whether the variability of levels of methylation of individual CpGs within a defined gene is able to discriminate cancerous from normal tissue, and is associated with heterogeneous tumour phenotype, as defined by gene expression. We analysed 270985 CpGs annotated to 18272 genes, in 3284 cancerous and 681 normal samples, corresponding to 14 different cancer types. In doing so, we found novel differences in intra-gene methylation pattern across phenotypes, particularly in those genes which are crucial for stem cell biology; our measures of intra-gene methylation architecture are a better determinant of phenotype than measures based on mean methylation level alone (K-S test [Formula: see text] in all 14 diseases tested). These per-gene methylation measures also represent a considerable reduction in complexity, compared to conventional per-CpG beta-values. Our findings strongly support the view that intra-gene methylation architecture has great clinical potential for the development of DNA-based cancer biomarkers.

Improved fatigue resistance in Gsα-deficient and aging mouse skeletal muscles due to adaptive increases in slow fibers

PubMed Central

Feng, Han-Zhong; Chen, Min; Weinstein, Lee S.

2011-01-01

Genetically modified mice with deficiency of the G protein α-subunit (Gsα) in skeletal muscle showed metabolic abnormality with reduced glucose tolerance, low muscle mass, and low contractile force, along with a fast-to-slow-fiber-type switch (Chen M, Feng HZ, Gupta D, Kelleher J, Dickerson KE, Wang J, Hunt D, Jou W, Gavrilova O, Jin JP, Weinstein LS. Am J Physiol Cell Physiol 296: C930–C940, 2009). Here we investigated a hypothesis that the switching to more slow fibers is an adaptive response with specific benefit. The results showed that, corresponding to the switch of myosin isoforms, the thin-filament regulatory proteins troponin T and troponin I both switched to their slow isoforms in the atrophic soleus muscle of 3-mo-old Gsα-deficient mice. This fiber-type switch involving coordinated changes of both thick- and thin-myofilament proteins progressed in the Gsα-deficient soleus muscles of 18- to 24-mo-old mice, as reflected by the expression of solely slow isoforms of myosin and troponin. Compared with age-matched controls, Gsα-deficient soleus muscles with higher proportion of slow fibers exhibited slower contractile and relaxation kinetics and lower developed force, but significantly increased resistance to fatigue, followed by a better recovery. Gsα-deficient soleus muscles of neonatal and 3-wk-old mice did not show the increase in slow fibers. Therefore, the fast-to-slow-fiber-type switch in Gsα deficiency at older ages was likely an adaptive response. The benefit of higher fatigue resistance in adaption to metabolic deficiency and aging provides a mechanism to sustain skeletal muscle function in diabetic patients and elderly individuals. PMID:21680879

The Complete Local-Volume Groups Sample (CLoGS): Early results from X-ray and radio observations

NASA Astrophysics Data System (ADS)

Vrtilek, Jan M.; O'Sullivan, Ewan; David, Laurence P.; Giacintucci, Simona; Kolokythas, Konstantinos

2017-08-01

Although the group environment is the dominant locus of galaxy evolution (in contrast to rich clusters, which contain only a few percent of galaxies), there has been a lack of reliable, representative group samples in the local Universe. In particular, X-ray selected samples are strongly biased in favor of the X-ray bright, centrally-concentrated cool-core systems. In response, we have designed the Complete Local-Volume Groups Sample (CLoGS), an optically-selected statistically-complete sample of 53 groups within 80 Mpc which is intended to overcome the limitations of X-ray selected samples and serve as a representative survey of groups in the local Universe. We have supplemented X-ray data from Chandra and XMM (70% complete to date, using both archival and new observations, with a 26-group high richness subsample 100% complete) with GMRT radio continuum observations (at 235 and 610 MHz, complete for the entire sample). CLoGS includes groups with a wide variety of properties in terms of galaxy population, hot gas content, and AGN power. We here describe early results from the survey, including the range of AGN activity observed in the dominant galaxies, the relative fraction of cool-core and non-cool-core groups in our sample, and the degree of disturbance observed in the IGM.

Photometry of the three eclipsing novalike variables EC 21178-5417, GS Pav and V345 Pav

NASA Astrophysics Data System (ADS)

Bruch, Albert

2017-10-01

As part of a project to better characterize comparatively bright, yet little studied cataclysmic variables time resolved photometry of the three eclipsing novalike variables EC 21178-5417, GS Pav und V345 Pav is presented. Previously known orbital periods are significantly improved and long-term ephemeris are derived. Variations of eclipse profiles, occurring on time scales of days to weeks, are analyzed. Out of eclipse the light curves are characterized by low scale flickering superposed on more gradual variations with amplitudes limited to a few tenths of a magnitude and profiles which at least in EC 21178-5417 and GS Pav roughly follow the same pattern in all observed cycles. Additionally, signs for variations on the time scale of some tens of minutes are seen in GS Pav, most clearly in two subsequent nights when in the first of these a signal with a period of 15.7 min was observed over several hours. In the second night variations with twice this period were seen. While no additional insight could be gained on quasi periodic oscillations (QPOs) and dwarf nova oscillations in EC 21178-5417, previously detected by Warner et al. (2003), and while such oscillations could not be found in V345 Pav, stacked power spectra of GS Pav clearly reveal the presence of QPOs over time intervals of several hours with periods varying between 200 s and 500 s in that system.

Glucose dependence of glycogen synthase activity regulation by GSK3 and MEK/ERK inhibitors and angiotensin-(1-7) action on these pathways in cultured human myotubes.

PubMed

Montori-Grau, Marta; Tarrats, Núria; Osorio-Conles, Oscar; Orozco, Anna; Serrano-Marco, Lucía; Vázquez-Carrera, Manuel; Gómez-Foix, Anna M

2013-05-01

Glycogen synthase (GS) is activated by glucose/glycogen depletion in skeletal muscle cells, but the contributing signaling pathways, including the chief GS regulator GSK3, have not been fully defined. The MEK/ERK pathway is known to regulate GSK3 and respond to glucose. The aim of this study was to elucidate the GSK3 and MEK/ERK pathway contribution to GS activation by glucose deprivation in cultured human myotubes. Moreover, we tested the glucose-dependence of GSK3 and MEK/ERK effects on GS and angiotensin (1-7) actions on these pathways. We show that glucose deprivation activated GS, but did not change phospho-GS (Ser640/1), GSK3β activity or activity-activating phosphorylation of ERK1/2. We then treated glucose-replete and -depleted cells with SB415286, U0126, LY294 and rapamycin to inhibit GSK3, MEK1/2, PI3K and mTOR, respectively. SB415286 activated GS and decreased the relative phospho-GS (Ser640/1) level, more in glucose-depleted than -replete cells. U0126 activated GS and reduced the phospho-GS (Ser640/1) content significantly in glucose-depleted cells, while GSK3β activity tended to increase. LY294 inactivated GS in glucose-depleted cells only, without affecting relative phospho-GS (Ser640/1) level. Rapamycin had no effect on GS activation. Angiotensin-(1-7) raised phospho-ERK1/2 but not phospho-GSK3β (Ser9) content, while it inactivated GS and increased GS phosphorylation on Ser640/1, in glucose-replete cells. In glucose-depleted cells, angiotensin-(1-7) effects on ERK1/2 and GS were reverted, while relative phospho-GSK3β (Ser9) content decreased. In conclusion, activation of GS by glucose deprivation is not due to GS Ser640/1 dephosphorylation, GSK3β or ERK1/2 regulation in cultured myotubes. However, glucose depletion enhances GS activation/Ser640/1 dephosphorylation due to both GSK3 and MEK/ERK inhibition. Angiotensin-(1-7) inactivates GS in glucose-replete cells in association with ERK1/2 activation, not with GSK3 regulation, and glucose

Reassimilation of Photorespiratory Ammonium in Lotus japonicus Plants Deficient in Plastidic Glutamine Synthetase

PubMed Central

Pérez-Delgado, Carmen M.; García-Calderón, Margarita; Márquez, Antonio J.; Betti, Marco

2015-01-01

It is well established that the plastidic isoform of glutamine synthetase (GS2) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4 + accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4 + when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH4 +. Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4 + when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity. PMID:26091523

GO-based functional dissimilarity of gene sets.

PubMed

Díaz-Díaz, Norberto; Aguilar-Ruiz, Jesús S

2011-09-01

The Gene Ontology (GO) provides a controlled vocabulary for describing the functions of genes and can be used to evaluate the functional coherence of gene sets. Many functional coherence measures consider each pair of gene functions in a set and produce an output based on all pairwise distances. A single gene can encode multiple proteins that may differ in function. For each functionality, other proteins that exhibit the same activity may also participate. Therefore, an identification of the most common function for all of the genes involved in a biological process is important in evaluating the functional similarity of groups of genes and a quantification of functional coherence can helps to clarify the role of a group of genes working together. To implement this approach to functional assessment, we present GFD (GO-based Functional Dissimilarity), a novel dissimilarity measure for evaluating groups of genes based on the most relevant functions of the whole set. The measure assigns a numerical value to the gene set for each of the three GO sub-ontologies. Results show that GFD performs robustly when applied to gene set of known functionality (extracted from KEGG). It performs particularly well on randomly generated gene sets. An ROC analysis reveals that the performance of GFD in evaluating the functional dissimilarity of gene sets is very satisfactory. A comparative analysis against other functional measures, such as GS2 and those presented by Resnik and Wang, also demonstrates the robustness of GFD.

User-centered design of multi-gene sequencing panel reports for clinicians.

PubMed

Cutting, Elizabeth; Banchero, Meghan; Beitelshees, Amber L; Cimino, James J; Fiol, Guilherme Del; Gurses, Ayse P; Hoffman, Mark A; Jeng, Linda Jo Bone; Kawamoto, Kensaku; Kelemen, Mark; Pincus, Harold Alan; Shuldiner, Alan R; Williams, Marc S; Pollin, Toni I; Overby, Casey Lynnette

2016-10-01

The objective of this study was to develop a high-fidelity prototype for delivering multi-gene sequencing panel (GS) reports to clinicians that simulates the user experience of a final application. The delivery and use of GS reports can occur within complex and high-paced healthcare environments. We employ a user-centered software design approach in a focus group setting in order to facilitate gathering rich contextual information from a diverse group of stakeholders potentially impacted by the delivery of GS reports relevant to two precision medicine programs at the University of Maryland Medical Center. Responses from focus group sessions were transcribed, coded and analyzed by two team members. Notification mechanisms and information resources preferred by participants from our first phase of focus groups were incorporated into scenarios and the design of a software prototype for delivering GS reports. The goal of our second phase of focus group, to gain input on the prototype software design, was accomplished through conducting task walkthroughs with GS reporting scenarios. Preferences for notification, content and consultation from genetics specialists appeared to depend upon familiarity with scenarios for ordering and delivering GS reports. Despite familiarity with some aspects of the scenarios we proposed, many of our participants agreed that they would likely seek consultation from a genetics specialist after viewing the test reports. In addition, participants offered design and content recommendations. Findings illustrated a need to support customized notification approaches, user-specific information, and access to genetics specialists with GS reports. These design principles can be incorporated into software applications that deliver GS reports. Our user-centered approach to conduct this assessment and the specific input we received from clinicians may also be relevant to others working on similar projects. Copyright © 2016 Elsevier Inc. All rights

Three-dimensional structure and ligand interactions of the low molecular weight protein tyrosine phosphatase from Campylobacter jejuni.

PubMed

Tolkatchev, Dmitri; Shaykhutdinov, Rustem; Xu, Ping; Plamondon, Josée; Watson, David C; Young, N Martin; Ni, Feng

2006-10-01

A putative low molecular weight protein tyrosine phosphatase (LMW-PTP) was identified in the genome sequence of the bacterial pathogen, Campylobacter jejuni. This novel gene, cj1258, has sequence homology with a distinctive class of phosphatases widely distributed among prokaryotes and eukaryotes. We report here the solution structure of Cj1258 established by high-resolution NMR spectroscopy using NOE-derived distance restraints, hydrogen bond data, and torsion angle restraints. The three-dimensional structure consists of a central four-stranded parallel beta-sheet flanked by five alpha-helices, revealing an overall structural topology similar to those of the eukaryotic LMW-PTPs, such as human HCPTP-A, bovine BPTP, and Saccharomyces cerevisiae LTP1, and to those of the bacterial LMW-PTPs MPtpA from Mycobacterium tuberculosis and YwlE from Bacillus subtilis. The active site of the enzyme is flexible in solution and readily adapts to the binding of ligands, such as the phosphate ion. An NMR-based screen was carried out against a number of potential inhibitors and activators, including phosphonomethylphenylalanine, derivatives of the cinnamic acid, 2-hydroxy-5-nitrobenzaldehyde, cinnamaldehyde, adenine, and hypoxanthine. Despite its bacterial origin, both the three-dimensional structure and ligand-binding properties of Cj1258 suggest that this novel phosphatase may have functional roles close to those of eukaryotic and mammalian tyrosine phosphatases. The three-dimensional structure along with mapping of small-molecule binding will be discussed in the context of developing high-affinity inhibitors of this novel LMW-PTP.

Catalase activity of IgG antibodies from the sera of healthy donors and patients with schizophrenia

PubMed Central

Ermakov, Evgeny A.; Smirnova, Ludmila P.; Bokhan, Nikolay A.; Semke, Arkadiy V.; Ivanova, Svetlana A.; Buneva, Valentina N.

2017-01-01

We present first evidence showing that some electrophoretically homogeneous IgGs from the sera of patients with schizophrenia (36.4%) and their Fab and F(ab)2 fragments as well as from healthy donors (33.3%) possess catalase activity. The relative catalase activity of IgGs from the sera of individual schizophrenia patients (and healthy donors) significantly varied from patient to patient, but the activity of IgGs from healthy donors is on average 15.8-fold lower than that for schizophrenia patients. After extensive dialysis of purified IgGs against EDTA chelating metal ions, the relative catalase activity of IgGs decreases on average approximately 2.5–3.7-fold; all IgGs possess metal-dependent and independent catalase activity. The addition of external Me2+ ions to dialyzed and non-dialyzed IgGs leads to a significant increase in their activity. The best activator of dialyzed and non-dialyzed IgGs is Co2+, the activation by Cu2+, Mn2+, and Ni2+ ions were rare and always lower than by Co2+. Every IgG preparation demonstrates several individual sets of very well expressed pH optima in the pH range from 4.0 to 9.5. These data speak for the individual repertoire of catalase IgGs in every person and an extreme diversity of abzymes in their pH optima and activation by different metal ions. It is known that antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases represent critical defense mechanisms preventing oxidative modifications of DNA, proteins, and lipids. Catalase activity of human IgGs could probably also play a major role in the protection of organisms from oxidative stress and toxic compounds. PMID:28945759

Catalase activity of IgG antibodies from the sera of healthy donors and patients with schizophrenia.

PubMed

Ermakov, Evgeny A; Smirnova, Ludmila P; Bokhan, Nikolay A; Semke, Arkadiy V; Ivanova, Svetlana A; Buneva, Valentina N; Nevinsky, Georgy A

2017-01-01

We present first evidence showing that some electrophoretically homogeneous IgGs from the sera of patients with schizophrenia (36.4%) and their Fab and F(ab)2 fragments as well as from healthy donors (33.3%) possess catalase activity. The relative catalase activity of IgGs from the sera of individual schizophrenia patients (and healthy donors) significantly varied from patient to patient, but the activity of IgGs from healthy donors is on average 15.8-fold lower than that for schizophrenia patients. After extensive dialysis of purified IgGs against EDTA chelating metal ions, the relative catalase activity of IgGs decreases on average approximately 2.5-3.7-fold; all IgGs possess metal-dependent and independent catalase activity. The addition of external Me2+ ions to dialyzed and non-dialyzed IgGs leads to a significant increase in their activity. The best activator of dialyzed and non-dialyzed IgGs is Co2+, the activation by Cu2+, Mn2+, and Ni2+ ions were rare and always lower than by Co2+. Every IgG preparation demonstrates several individual sets of very well expressed pH optima in the pH range from 4.0 to 9.5. These data speak for the individual repertoire of catalase IgGs in every person and an extreme diversity of abzymes in their pH optima and activation by different metal ions. It is known that antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases represent critical defense mechanisms preventing oxidative modifications of DNA, proteins, and lipids. Catalase activity of human IgGs could probably also play a major role in the protection of organisms from oxidative stress and toxic compounds.

Use of plant trait data in the ISBA-A-gs model

NASA Astrophysics Data System (ADS)

Calvet, Jean-Christophe

2014-05-01

ISBA-A-gs is a CO2-responsive LSM (Calvet et al., 1998; Gibelin et al., 2006), able to simulate the diurnal cycle of carbon and water vapour fluxes, together with LAI and soil moisture evolution. The various components of ISBA-A-gs are based to a large extent on meta-analyses of trait data. (1) Photosynthesis: ISBA-A-gs uses the model of Goudriaan et al. (1985) modified by Jacobs (1994) and Jacobs et al. (1996). The main parameter is mesophyll conductance (gm). Leaf-level photosynthesis observations were used together with canopy level flux observations to derive gm together with other key parameters of the Jacobs model, including in drought conditions. This permitted implementing detailed representations of the soil moisture stress. Two different types of drought responses are distinguished for both herbaceous vegetation (Calvet, 2000) and forests (Calvet et al., 2004), depending on the evolution of the water use efficiency (WUE) under moderate stress: WUE increases in the early soil water stress stages in the case of the drought-avoiding response, whereas WUE decreases or remains stable in the case of the drought-tolerant response. (2) Plant growth: the leaf biomass is provided by a growth model (Calvet et al., 1998; Calvet and Soussana, 2001) driven by photosynthesis. In contrast to other land surface models, no GDD-based phenology model is used in ISBA-A-gs, as the vegetation growth and senescence are entirely driven by photosynthesis. The leaf biomass is supplied with the carbon assimilated by photosynthesis, and decreased by a turnover and a respiration term. Turnover is increased by a deficit in photosynthesis. The leaf onset is triggered by sufficient photosynthesis levels and a minimum LAI value is prescribed. The maximum annual value of LAI is prognostic, i.e. it can be predicted by the model. LAI is derived from leaf biomass using SLA values. The latter are derived from the leaf nitrogen concentration using plasticity parameters. (3) CO2 effect: the

Gene Discovery for Enzymes Involved in Limonene Modification or Utilization by the Mountain Pine Beetle-Associated Pathogen Grosmannia clavigera

PubMed Central

Wang, Ye; Lim, Lynette; Madilao, Lina; Lah, Ljerka; Bohlmann, Joerg

2014-01-01

To successfully colonize and eventually kill pine trees, Grosmannia clavigera (Gs cryptic species), the main fungal pathogen associated with the mountain pine beetle (Dendroctonus ponderosae), has developed multiple mechanisms to overcome host tree chemical defenses, of which terpenoids are a major component. In addition to a monoterpene efflux system mediated by a recently discovered ABC transporter, Gs has genes that are highly induced by monoterpenes and that encode enzymes that modify or utilize monoterpenes [especially (+)-limonene]. We showed that pine-inhabiting Ophiostomale fungi are tolerant to monoterpenes, but only a few, including Gs, are known to utilize monoterpenes as a carbon source. Gas chromatography-mass spectrometry (GC-MS) revealed that Gs can modify (+)-limonene through various oxygenation pathways, producing carvone, p-mentha-2,8-dienol, perillyl alcohol, and isopiperitenol. It can also degrade (+)-limonene through the C-1-oxygenated pathway, producing limonene-1,2-diol as the most abundant intermediate. Transcriptome sequencing (RNA-seq) data indicated that Gs may utilize limonene 1,2-diol through beta-oxidation and then valine and tricarboxylic acid (TCA) metabolic pathways. The data also suggested that at least two gene clusters, located in genome contigs 108 and 161, were highly induced by monoterpenes and may be involved in monoterpene degradation processes. Further, gene knockouts indicated that limonene degradation required two distinct Baeyer-Villiger monooxygenases (BVMOs), an epoxide hydrolase and an enoyl coenzyme A (enoyl-CoA) hydratase. Our work provides information on enzyme-mediated limonene utilization or modification and a more comprehensive understanding of the interaction between an economically important fungal pathogen and its host's defense chemicals. PMID:24837377

A Catalog of Regulatory Sequences for Trait Gene for the Genome Editing of Wheat.

PubMed

Makai, Szabolcs; Tamás, László; Juhász, Angéla

2016-01-01

Wheat has been cultivated for 10000 years and ever since the origin of hexaploid wheat it has been exempt from natural selection. Instead, it was under the constant selective pressure of human agriculture from harvest to sowing during every year, producing a vast array of varieties. Wheat has been adopted globally, accumulating variation for genes involved in yield traits, environmental adaptation and resistance. However, one small but important part of the wheat genome has hardly changed: the regulatory regions of both the x- and y-type high molecular weight glutenin subunit (HMW-GS) genes, which are alone responsible for approximately 12% of the grain protein content. The phylogeny of the HMW-GS regulatory regions of the Triticeae demonstrates that a genetic bottleneck may have led to its decreased diversity during domestication and the subsequent cultivation. It has also highlighted the fact that the wild relatives of wheat may offer an unexploited genetic resource for the regulatory region of these genes. Significant research efforts have been made in the public sector and by international agencies, using wild crosses to exploit the available genetic variation, and as a result synthetic hexaploids are now being utilized by a number of breeding companies. However, a newly emerging tool of genome editing provides significantly improved efficiency in exploiting the natural variation in HMW-GS genes and incorporating this into elite cultivars and breeding lines. Recent advancement in the understanding of the regulation of these genes underlines the needs for an overview of the regulatory elements for genome editing purposes.

DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα)-encoding (GNAS) genomic imprinting domain are associated with performance traits.

PubMed

Sikora, Klaudia M; Magee, David A; Berkowicz, Erik W; Berry, Donagh P; Howard, Dawn J; Mullen, Michael P; Evans, Ross D; Machugh, David E; Spillane, Charles

2011-01-07

Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs) spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS) domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486) were located upstream of the GNAS gene, while one SNP (rs41694646) was located in the second intron of the GNAS gene. The final SNP (rs41694656) was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646) is associated (P ≤ 0.05) with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf) and gestation length. Association (P ≤ 0.01) with direct calving difficulty (i.e. due to calf size) and maternal calving difficulty (i.e. due to the maternal pelvic width size) was also observed at the rs43101491 SNP. Following adjustment for

DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα)-encoding (GNAS) genomic imprinting domain are associated with performance traits

PubMed Central

2011-01-01

Background Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs) spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS) domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486) were located upstream of the GNAS gene, while one SNP (rs41694646) was located in the second intron of the GNAS gene. The final SNP (rs41694656) was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. Results SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646) is associated (P ≤ 0.05) with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf) and gestation length. Association (P ≤ 0.01) with direct calving difficulty (i.e. due to calf size) and maternal calving difficulty (i.e. due to the maternal pelvic width size) was also observed at the rs43101491 SNP. Following

Potent inhibitory effects of D-tagatose on the acid production and water-insoluble glucan synthesis of Streptococcus mutans GS5 in the presence of sucrose.

PubMed

Sawada, Daijo; Ogawa, Takaaki; Miyake, Minoru; Hasui, Yoshinori; Yamaguchi, Fuminori; Izumori, Ken; Tokuda, Masaaki

2015-01-01

We examined and compared the inhibitory effects of D-tagatose on the growth, acid production, and water-insoluble glucan synthesis of GS5, a bacterial strain of Streptococcus mutans, with those of xylitol, D-psicose, L-psicose and L-tagatose. GS5 was cultured for 12h in a medium containing 10% (w/v) of xylitol, D-psicose, L-psicose, D-tagatose or L-tagatose, and the inhibitory effect of GS5 growth was assessed. Each sugar showed different inhibitory effects on GS5. Both D-tagatose and xylitol significantly inhibited the acid production and water-insoluble glucan synthesis of GS5 in the presence of 1% (w/v) sucrose. However, the inhibitory effect of acid production by D-tagatose was significantly stronger than that of xylitol in presence of sucrose.

Gi proteins regulate adenylyl cyclase activity independent of receptor activation.

PubMed

Melsom, Caroline Bull; Ørstavik, Øivind; Osnes, Jan-Bjørn; Skomedal, Tor; Levy, Finn Olav; Krobert, Kurt Allen

2014-01-01

Despite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to G(i), some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. We used the Gs-selective (R,R)- and the Gs- and G(i)-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats. PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response. Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G(i) and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

PubMed Central

Melsom, Caroline Bull; Ørstavik, Øivind; Osnes, Jan-Bjørn; Skomedal, Tor; Levy, Finn Olav; Krobert, Kurt Allen

2014-01-01

Background and purpose Despite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to Gi, some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. Experimental approach We used the Gs-selective (R,R)- and the Gs- and Gi-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats. Key results PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response. Conclusions and implications Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic Gi and Gs activity upon AC towards Gs

Low molecular weight fibroblast growth factor-2 signals via protein kinase C and myofibrillar proteins to protect against postischemic cardiac dysfunction.

PubMed

Manning, Janet R; Perkins, Sarah O; Sinclair, Elizabeth A; Gao, Xiaoqian; Zhang, Yu; Newman, Gilbert; Pyle, W Glen; Schultz, Jo El J

2013-05-15

Among its many biological roles, fibroblast growth factor-2 (FGF2) acutely protects the heart from dysfunction associated with ischemia/reperfusion (I/R) injury. Our laboratory has demonstrated that this is due to the activity of the low molecular weight (LMW) isoform of FGF2 and that FGF2-mediated cardioprotection relies on the activity of protein kinase C (PKC); however, which PKC isoforms are responsible for LMW FGF2-mediated cardioprotection, and their downstream targets, remain to be elucidated. To identify the PKC pathway(s) that contributes to postischemic cardiac recovery by LMW FGF2, mouse hearts expressing only LMW FGF2 (HMWKO) were bred to mouse hearts not expressing PKCα (PKCαKO) or subjected to a selective PKCε inhibitor (εV(1-2)) before and during I/R. Hearts only expressing LMW FGF2 showed significantly improved postischemic recovery of cardiac function following I/R (P < 0.05), which was significantly abrogated in the absence of PKCα (P < 0.05) or presence of PKCε inhibition (P < 0.05). Hearts only expressing LMW FGF2 demonstrated differences in actomyosin ATPase activity as well as increases in the phosphorylation of troponin I and T during I/R compared with wild-type hearts; several of these effects were dependent on PKCα activity. This evidence indicates that both PKCα and PKCε play a role in LMW FGF2-mediated protection from cardiac dysfunction and that PKCα signaling to the contractile apparatus is a key step in the mechanism of LMW FGF2-mediated protection against myocardial dysfunction.

Facile fabrication of 3D porous MnO@GS/CNT architecture as advanced anode materials for high-performance lithium-ion battery.

PubMed

Wang, Junyong; Deng, Qinglin; Li, Mengjiao; Wu, Cong; Jiang, Kai; Hu, Zhigao; Chu, Junhao

2018-08-03

To overcome inferior rate capability and cycle stability of MnO-based anode materials for lithium-ion batteries (LIBs), we reported a novel 3D porous MnO@GS/CNT composite, consisting of MnO nanoparticles homogeneously distributed on the conductive interconnected framework based on 2D graphene sheets (GS) and 1D carbon nanotubes (CNTs). The distinctive architecture offers highly interpenetrated network along with efficient porous channels for fast electron transfer and ionic diffusion as well as abundant stress buffer space to accommodate the volume expansion of the MnO nanoparticles. The MnO@GS/CNT anode exhibits an ultrahigh capacity of 1115 mAh g -1 at 0.2 A g -1 after 150 cycles and outstanding rate capacity of 306 mAh g -1 at 10.0 A g -1 . Moreover, a stable capacity of 405 mAh g -1 after 3200 cycles can still be achieved, even at a large current density of 5.0 A g -1 . When coupled with LiMn 2 O 4 (LMO) cathode, the LMO [Formula: see text] MnO@GS/CNT full cell characterizes an excellent cycling stability and rate capability, indicating the promising application of MnO@GS/CNT anode in the next-generation LIBs.

Biased Gs versus Gq proteins and β-arrestin signaling in the NK1 receptor determined by interactions in the water hydrogen bond network.

PubMed

Valentin-Hansen, Louise; Frimurer, Thomas M; Mokrosinski, Jacek; Holliday, Nicholas D; Schwartz, Thue W

2015-10-02

X-ray structures, molecular dynamics simulations, and mutational analysis have previously indicated that an extended water hydrogen bond network between trans-membranes I-III, VI, and VII constitutes an allosteric interface essential for stabilizing different active and inactive helical constellations during the seven-trans-membrane receptor activation. The neurokinin-1 receptor signals efficiently through Gq, Gs, and β-arrestin when stimulated by substance P, but it lacks any sign of constitutive activity. In the water hydrogen bond network the neurokinin-1 has a unique Glu residue instead of the highly conserved AspII:10 (2.50). Here, we find that this GluII:10 occupies the space of a putative allosteric modulating Na(+) ion and makes direct inter-helical interactions in particular with SerIII:15 (3.39) and AsnVII:16 (7.49) of the NPXXY motif. Mutational changes in the interface between GluII:10 and AsnVII:16 created receptors that selectively signaled through the following: 1) Gq only; 2) β-arrestin only; and 3) Gq and β-arrestin but not through Gs. Interestingly, increased constitutive Gs but not Gq signaling was observed by Ala substitution of four out of the six core polar residues of the network, in particular SerIII:15. Three residues were essential for all three signaling pathways, i.e. the water-gating micro-switch residues TrpVI:13 (6.48) of the CWXP motif and TyrVII:20 (7.53) of the NPXXY motif plus the totally conserved AsnI:18 (1.50) stabilizing the kink in trans-membrane VII. It is concluded that the interface between position II:10 (2.50), III:15 (3.39), and VII:16 (7.49) in the center of the water hydrogen bond network constitutes a focal point for fine-tuning seven trans-membrane receptor conformations activating different signal transduction pathways. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Multiplexed direct genomic selection (MDiGS): a pooled BAC capture approach for highly accurate CNV and SNP/INDEL detection.

PubMed

Alvarado, David M; Yang, Ping; Druley, Todd E; Lovett, Michael; Gurnett, Christina A

2014-06-01

Despite declining sequencing costs, few methods are available for cost-effective single-nucleotide polymorphism (SNP), insertion/deletion (INDEL) and copy number variation (CNV) discovery in a single assay. Commercially available methods require a high investment to a specific region and are only cost-effective for large samples. Here, we introduce a novel, flexible approach for multiplexed targeted sequencing and CNV analysis of large genomic regions called multiplexed direct genomic selection (MDiGS). MDiGS combines biotinylated bacterial artificial chromosome (BAC) capture and multiplexed pooled capture for SNP/INDEL and CNV detection of 96 multiplexed samples on a single MiSeq run. MDiGS is advantageous over other methods for CNV detection because pooled sample capture and hybridization to large contiguous BAC baits reduces sample and probe hybridization variability inherent in other methods. We performed MDiGS capture for three chromosomal regions consisting of ∼ 550 kb of coding and non-coding sequence with DNA from 253 patients with congenital lower limb disorders. PITX1 nonsense and HOXC11 S191F missense mutations were identified that segregate in clubfoot families. Using a novel pooled-capture reference strategy, we identified recurrent chromosome chr17q23.1q23.2 duplications and small HOXC 5' cluster deletions (51 kb and 12 kb). Given the current interest in coding and non-coding variants in human disease, MDiGS fulfills a niche for comprehensive and low-cost evaluation of CNVs, coding, and non-coding variants across candidate regions of interest. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

The effect of multiple contamination of soil on LMW and MMW PAHs accumulation in the roots of Rubus fruticosus L. naturally growing near The Copper Mining and Smelting Complex Bor (East Serbia).

PubMed

Alagić, Slađana Č; Stankov Jovanović, Vesna P; Mitić, Violeta D; Nikolić, Jelena S; Petrović, Goran M; Tošić, Snežana B; Stojanović, Gordana S

2017-06-01

Samples of roots and spatial soils of native Rubus fruticosus L. were collected from the spots positioned at different distances from the copper smelter and city heating plants in the industrial zone of the town of Bor (Serbia) and subjected to chemical analyses in order to determine the content of several heavy metals, and 15 priority polycyclic aromatic hydrocarbons (PAHs). In this study, the results for 9 low and medium molecular weight PAHs (LMW and MMW PAHs) are represented and processed using the calculation of bio-concentration factors and statistical methods such as hierarchical cluster analysis and Pearson's correlation study with the aim of investigating the plant capabilities for their uptake from the soil and later accumulation into the root tissue, under the hostile circumstances of multiple contamination. The obtained data revealed different accumulation rates for the investigated PAHs and showed that in several cases, the contents of root PAHs were under the strong influence of present contaminants such as soil copper and some soil PAHs, indicating at the same time that R. fruticosus can regulate the processes of LMW and MMW PAHs extraction/accumulation using different mechanisms, depending on the existing environmental circumstances. The used mechanisms could be exploited in phytoremediation methods based not only on the extraction and concentration of PAHs in plant roots but also on PAH degradation or stabilization in the soil. Also, the results of this study confirmed that, except in the case of naphthalene and fluoranthene, there was no PAH pollution, which originated solely from the industrial zone.

Potential effects of a low-molecular-weight fucoidan extracted from brown algae on bone biomaterial osteoconductive properties.

PubMed

Changotade, S Igondjo Tchen; Korb, G; Bassil, J; Barroukh, B; Willig, C; Colliec-Jouault, S; Durand, P; Godeau, G; Senni, K

2008-12-01

In this work, we first tested the influence of low-molecular-weight (LMW) fucoidan extracted from pheophicae cell wall on bidimensional cultured normal human osteoblasts' behaviors. Second, by impregnation procedure with LMW fucoidan of bone biomaterial (Lubboc), we explored in this bone extracellular matrix context its capabilities to support human osteoblastic behavior in 3D culture. In bidimensionnal cultures, we evidenced that LMW fucoidan promotes human osteoblast proliferation and collagen type I expression and favors precocious alkaline phosphatase activity. Furthermore, with LMW fucoidan, von Kossa's staining was positive at 30 days and positive only at 45 days in the absence of LMW fucoidan. In our three-dimensional culture models with the biomaterial pretreated with LMW fucoidan, osteoblasts promptly overgrew the pretreated biomaterial. We also evidenced that osteoblasts increased proliferation with pretreated biomaterial when compared with untreated biomaterial. Osteoblasts secreted osteocalcin and expressed BMP2 receptor on control material as well as with LMW fucoidan impregnated biomaterial. In conclusion, in our experimental conditions, LMW fucoidan stimulated expression of osteoblastic markers differentiation such as alkaline phosphatase activity, collagen type I expression, and mineral deposition; furthermore, cell proliferation was favored. These findings suggest that fucoidan could be clinically useful for bone regeneration and bone substitute design. (c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2008.

Anti-EBOV GP IgGs Lacking α1-3-Galactose and Neu5Gc Prolong Survival and Decrease Blood Viral Load in EBOV-Infected Guinea Pigs

PubMed Central

Reynard, Olivier; Jacquot, Frédéric; Evanno, Gwénaëlle; Mai, Hoa Le; Martinet, Bernard; Duvaux, Odile; Bach, Jean-Marie; Conchon, Sophie; Judor, Jean-Paul; Perota, Andrea; Lagutina, Irina; Duchi, Roberto; Lazzari, Giovanna; Le Berre, Ludmilla; Perreault, Hélène; Lheriteau, Elsa; Raoul, Hervé; Volchkov, Viktor; Galli, Cesare; Soulillou, Jean-Paul

2016-01-01

Polyclonal xenogenic IgGs, although having been used in the prevention and cure of severe infectious diseases, are highly immunogenic, which may restrict their usage in new applications such as Ebola hemorrhagic fever. IgG glycans display powerful xenogeneic antigens in humans, for example α1–3 Galactose and the glycolyl form of neuraminic acid Neu5Gc, and IgGs deprived of these key sugar epitopes may represent an advantage for passive immunotherapy. In this paper, we explored whether low immunogenicity IgGs had a protective effect on a guinea pig model of Ebola virus (EBOV) infection. For this purpose, a double knock-out pig lacking α1–3 Galactose and Neu5Gc was immunized against virus-like particles displaying surface EBOV glycoprotein GP. Following purification from serum, hyper-immune polyclonal IgGs were obtained, exhibiting an anti-EBOV GP titer of 1:100,000 and a virus neutralizing titer of 1:100. Guinea pigs were injected intramuscularly with purified IgGs on day 0 and day 3 post-EBOV infection. Compared to control animals treated with IgGs from non-immunized double KO pigs, the anti-EBOV IgGs-treated animals exhibited a significantly prolonged survival and a decreased virus load in blood on day 3. The data obtained indicated that IgGs lacking α1–3 Galactose and Neu5Gc, two highly immunogenic epitopes in humans, have a protective effect upon EBOV infection. PMID:27280712

A TAD further: exogenous control of gene activation.

PubMed

Mapp, Anna K; Ansari, Aseem Z

2007-01-23

Designer molecules that can be used to impose exogenous control on gene transcription, artificial transcription factors (ATFs), are highly desirable as mechanistic probes of gene regulation, as potential therapeutic agents, and as components of cell-based devices. Recently, several advances have been made in the design of ATFs that activate gene transcription (activator ATFs), including reports of small-molecule-based systems and ATFs that exhibit potent activity. However, the many open mechanistic questions about transcriptional activators, in particular, the structure and function of the transcriptional activation domain (TAD), have hindered rapid development of synthetic ATFs. A compelling need thus exists for chemical tools and insights toward a more detailed portrait of the dynamic process of gene activation.

A verification of the gyrokinetic microstability codes GEM, GYRO, and GS2

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

Bravenec, R. V.; Chen, Y.; Wan, W.

2013-10-15

A previous publication [R. V. Bravenec et al., Phys. Plasmas 18, 122505 (2011)] presented favorable comparisons of linear frequencies and nonlinear fluxes from the Eulerian gyrokinetic codes gyro[J. Candy and R. E. Waltz, J. Comput. Phys. 186, 545 (2003)] and gs2[W. Dorland et al., Phys. Rev. Lett. 85, 5579 (2000)]. The motivation was to verify the codes, i.e., demonstrate that they correctly solve the gyrokinetic-Maxwell equations. The premise was that it is highly unlikely for both codes to yield the same incorrect results. In this work, we add the Lagrangian particle-in-cell code gem[Y. Chen and S. Parker, J. Comput. Phys.more » 220, 839 (2007)] to the comparisons, not simply to add another code, but also to demonstrate that the codes' algorithms do not matter. We find good agreement of gem with gyro and gs2 for the plasma conditions considered earlier, thus establishing confidence that the codes are verified and that ongoing validation efforts for these plasma parameters are warranted.« less

Differential expression of a WRKY gene between wild and cultivated soybeans correlates to seed size.

PubMed

Gu, Yongzhe; Li, Wei; Jiang, Hongwei; Wang, Yan; Gao, Huihui; Liu, Miao; Chen, Qingshan; Lai, Yongcai; He, Chaoying

2017-05-17

Soybean (Glycine max) probably originated from the wild soybean (Glycine soja). Glycine max has a significantly larger seed size, but the underlying genomic changes are largely unknown. Candidate regulatory genes were preliminarily proposed by data co-localizing RNA sequencing with the quantitative loci (QTLs) for seed size. The soybean gene locus SoyWRKY15a and its orthologous genes from G. max (GmWRKY15a) and G. soja (GsWRKY15a) were analyzed in detail. The coding sequences were nearly identical between the two orthologs, but GmWRKY15a was significantly more highly expressed than GsWRKY15a. Four haplotypes (H1-H4) were found and they varied in the size of a CT-core microsatellite locus in the 5'-untranslated region of this gene. H1 (with six CT-repeats) was the only allelic version found in G. max, while H3 (with five CT-repeats) was the dominant G. soja allele. Differential expression of this gene in soybean pods was correlated with CT-repeat variation, and manipulation of the CT copy number altered the reporter gene expression, suggesting a regulatory role for the simple sequence repeats. Seed weight of wild soybeans harboring H1 was significantly greater than that of soybeans having haplotypes H2, H3, or H4, and seed weight was correlated with gene expression, suggesting the influence of GsWRKY15a in controlling seed size. However, the seed size might be refractory to increased SoyWRKY15a expression in cultivated soybeans. The evolutionary significance of SoyWRKY15a variation in soybean seed domestication is discussed. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Diethylstilbestrol (DES)-stimulated hormonal toxicity is mediated by ERα alteration of target gene methylation patterns and epigenetic modifiers (DNMT3A, MBD2, and HDAC2) in the mouse seminal vesicle.

PubMed

Li, Yin; Hamilton, Katherine J; Lai, Anne Y; Burns, Katherine A; Li, Leping; Wade, Paul A; Korach, Kenneth S

2014-03-01

Diethylstilbestrol (DES) is a synthetic estrogen associated with adverse effects on reproductive organs. DES-induced toxicity of the mouse seminal vesicle (SV) is mediated by estrogen receptor α (ERα), which alters expression of seminal vesicle secretory protein IV (Svs4) and lactoferrin (Ltf) genes. We examined a role for nuclear receptor activity in association with DNA methylation and altered gene expression. We used the neonatal DES exposure mouse model to examine DNA methylation patterns via bisulfite conversion sequencing in SVs of wild-type (WT) and ERα-knockout (αERKO) mice. The DNA methylation status at four specific CpGs (-160, -237, -306, and -367) in the Svs4 gene promoter changed during mouse development from methylated to unmethylated, and DES prevented this change at 10 weeks of age in WT SV. At two specific CpGs (-449 and -459) of the Ltf gene promoter, DES altered the methylation status from methylated to unmethylated. Alterations in DNA methylation of Svs4 and Ltf were not observed in αERKO SVs, suggesting that changes of methylation status at these CpGs are ERα dependent. The methylation status was associated with the level of gene expression. In addition, gene expression of three epigenetic modifiers-DNMT3A, MBD2, and HDAC2-increased in the SV of DES-exposed WT mice. DES-induced hormonal toxicity resulted from altered gene expression of Svs4 and Ltf associated with changes in DNA methylation that were mediated by ERα. Alterations in gene expression of DNMT3A, MBD2, and HDAC2 in DES-exposed male mice may be involved in mediating the changes in methylation status in the SV. Li Y, Hamilton KJ, Lai AY, Burns KA, Li L, Wade PA, Korach KS. 2014. Diethylstilbestrol (DES)-stimulated hormonal toxicity is mediated by ERα alteration of target gene methylation patterns and epigenetic modifiers (DNMT3A, MBD2, and HDAC2) in the mouse seminal vesicle. Environ Health Perspect 122:262-268; http://dx.doi.org/10.1289/ehp.1307351.

Changes in DNA Methylation from Age 18 to Pregnancy in Type 1, 2, and 17 T Helper and Regulatory T-Cells Pathway Genes

PubMed Central

Iqbal, Sabrina; Lockett, Gabrielle A.; Arshad, S. Hasan; Zhang, Hongmei; Kaushal, Akhilesh; Tetali, Sabarinath R.; Mukherjee, Nandini

2018-01-01

To succeed, pregnancies need to initiate immune biases towards T helper 2 (Th2) responses, yet little is known about what establishes this bias. Using the Illumina 450 K platform, we explored changes in DNA methylation (DNAm) of Th1, Th2, Th17, and regulatory T cell pathway genes before and during pregnancy. Female participants were recruited at birth (1989), and followed through age 18 years and their pregnancy (2011–2015). Peripheral blood DNAm was measured in 245 girls at 18 years; from among these girls, the DNAm of 54 women was repeatedly measured in the first (weeks 8–21, n = 39) and second (weeks 22–38, n = 35) halves of pregnancy, respectively. M-values (logit-transformed β-values of DNAm) were analyzed: First, with repeated measurement models, cytosine–phosphate–guanine sites (CpGs) of pathway genes in pregnancy and at age 18 (nonpregnant) were compared for changes (p ≤ 0.05). Second, we tested how many of the 348 pathway-related CpGs changed compared to 10 randomly selected subsets of all other CpGs and compared to 10 randomly selected subsets of other CD4+-related CpGs (348 in each subset). Contrasted to the nonpregnant state, 27.7% of Th1-related CpGs changed in the first and 36.1% in the second half of pregnancy. Among the Th2 pathway CpGs, proportions of changes were 35.1% (first) and 33.8% (second half). The methylation changes suggest involvement of both Th1 and Th2 pathway CpGs in the immune bias during pregnancy. Changes in regulatory T cell and Th17 pathways need further exploration. PMID:29415463

DNA methylation analysis of Homeobox genes implicates HOXB7 hypomethylation as risk factor for neural tube defects

PubMed Central

Rochtus, Anne; Izzi, Benedetta; Vangeel, Elise; Louwette, Sophie; Wittevrongel, Christine; Lambrechts, Diether; Moreau, Yves; Winand, Raf; Verpoorten, Carla; Jansen, Katrien; Van Geet, Chris; Freson, Kathleen

2015-01-01

Neural tube defects (NTDs) are common birth defects of complex etiology. Though family- and population-based studies have confirmed a genetic component, the responsible genes for NTDs are still largely unknown. Based on the hypothesis that folic acid prevents NTDs by stimulating methylation reactions, epigenetic factors, such as DNA methylation, are predicted to be involved in NTDs. Homeobox (HOX) genes play a role in spinal cord development and are tightly regulated in a spatiotemporal and collinear manner, partly by epigenetic modifications. We have quantified DNA methylation for the different HOX genes by subtracting values from a genome-wide methylation analysis using leukocyte DNA from 10 myelomeningocele (MMC) patients and 6 healthy controls. From the 1575 CpGs profiled for the 4 HOX clusters, 26 CpGs were differentially methylated (P-value < 0.05; β-difference > 0.05) between MMC patients and controls. Seventy-seven percent of these CpGs were located in the HOXA and HOXB clusters, with the most profound difference for 3 CpGs within the HOXB7 gene body. A validation case-control study including 83 MMC patients and 30 unrelated healthy controls confirmed a significant association between MMC and HOXB7 hypomethylation (-14.4%; 95% CI: 11.9–16.9%; P-value < 0.0001) independent of the MTHFR 667C>T genotype. Significant HOXB7 hypomethylation was also present in 12 unaffected siblings, each related to a MMC patient, suggestive of an epigenetic change induced by the mother. The inclusion of a neural tube formation model using zebrafish showed that Hoxb7a overexpression but not depletion resulted in deformed body axes with dysmorphic neural tube formation. Our results implicate HOXB7 hypomethylation as risk factor for NTDs and highlight the importance for future genome-wide DNA methylation analyses without preselecting candidate pathways. PMID:25565354

Half molecular exchange of IgGs in the blood of healthy humans: chimeric lambda-kappa-immunoglobulins containing HL fragments of antibodies of different subclasses (IgG1-IgG4).

PubMed

Sedykh, Sergey E; Lekchnov, Evgenii A; Prince, Viktor V; Buneva, Valentina N; Nevinsky, Georgy A

2016-10-20

In the classic paradigm, immunoglobulins represent products of clonal B cell populations, each producing antibodies recognizing a single antigen (monospecific). There is a common belief that IgGs in mammalian biological fluids are monospecific molecules having stable structures and two identical antigen-binding sites. But the issue concerning the possibility of exchange by HL-fragments between the antibody molecules in human blood is still unexplored. Different physico-chemical and immunological methods for analysis of half-molecule exchange between human blood IgGs were used. Using eighteen blood samples of healthy humans we have shown unexpected results for the first time: blood antibodies undergo extensive post-transcriptional half-molecule exchange and IgG pools on average consist of 62.4 ± 6.5% IgGs containing kappa light chains (kappa-kappa-IgGs), 29.8.6 ± 5.4% lambda light chains (lambda-lambda-IgGs), and 8.8 ± 2.7% (range 2.6-16.8%) IgGs containing both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained on average (%): IgG1 (36.0 and 32.3), IgG2 (50.9 and 51.4), IgG3 (9.7 and 9.9), and IgG4 (6.5 and 5.7), while chimeric kappa-lambda-IgGs consisted of (%): 25.5 ± 4.2 IgG1, 50.8 ± 3.9 IgG2, 9.1 ± 2.1 IgG3, and 14.5 ± 2.2 IgG4. Our unexpected data are indicative of the possibility of half-molecule exchange between blood IgGs of various subclasses, raised against different antigens. The existence of blood chimeric bifunctional IgGs with different binding sites destroys the classic paradigm. Due to the phenomenon of polyspecificity and cross-reactivity of bifunctional IgGs containing HL-fragments of different types to different antigens, such IgGs may be important in human blood for widening their different biological functions.

Vitamin D receptor gene methylation is associated with ethnicity, tuberculosis and TaqI polymorphism

PubMed Central

Andraos, Charlene; Koorsen, Gerrit; Knight, Julian C; Bornman, Liza

2014-01-01

The Vitamin D Receptor (VDR) gene encodes a transcription factor which, on activation by vitamin D, modulates diverse biological processes including calcium homeostasis and immune function. Genetic variation involving VDR shows striking differences in allele frequency between populations and has been associated with disease susceptibility including tuberculosis and autoimmunity, although results have often been conflicting. We hypothesized that methylation of VDR may be population specific and that the combination of differential methylation and genetic variation may characterise TB predisposition. We use bisulphite conversion and/or pyrosequencing to analyse the methylation status of 17 CpGs of VDR and to genotype 7 SNPs in the 3′ CpG Island (CGI 1060), including the commonly studied SNPs ApaI (rs7975232) and TaqI (rs731236). We show that for lymphoblastoid cell lines from two ethnically diverse populations (Yoruba from HapMap, n=30 and Caucasians, n=30) together with TB cases (n=32) and controls (n=29) from the Venda population of South Africa there are methylation variable positions (MVPs) in the 3′ end that significantly distinguish ethnicity (9/17 CpGs) and TB status (3/17 CpGs). Moreover methylation status shows complex association with TaqI genotype highlighting the need to consider both genetic and epigenetic variants in genetic studies of VDR association with disease. PMID:21168462

Novel DNA variations to characterize low molecular weight glutenin Glu-D3 genes and develop STS markers in common wheat.

PubMed

Zhao, X L; Xia, X C; He, Z H; Lei, Z S; Appels, R; Yang, Y; Sun, Q X; Ma, W

2007-02-01

Low-molecular-weight glutenin subunits (LMW-GS) play an important role in bread and noodle processing quality by influencing the viscoelasticity and extensibility of dough. The objectives of this study were to characterize Glu-D3 subunit coding genes and to develop molecular markers for identifying Glu-D3 gene haplotypes. Gene specific primer sets were designed to amplify eight wheat cultivars containing Glu-D3a, b, c, d and e alleles, defined traditionally by protein electrophoretic mobility. Three novel Glu-D3 DNA sequences, designated as GluD3-4, GluD3-5 and GluD3-6, were amplified from the eight wheat cultivars. GluD3-4 showed three allelic variants or haplotypes at the DNA level in the eight cultivars, which were designated as GluD3-41, GluD3-42 and GluD3-43. Compared with GluD3-42, a single nucleotide polymorphism (SNP) was detected for GluD3-43 in the coding region, resulting in a pseudo-gene with a nonsense mutation at the 119th position of deduced peptide, and a 3-bp insertion was found in the coding region of GluD3-41, leading to a glutamine insertion at the 249th position of its deduced protein. The coding regions for GluD3-5 and GluD3-6 showed no allelic variation in the eight cultivars tested, indicating that they were relatively conservative in common wheat. Based on the 12 allelic variants of three Glu-D3 genes identified in this study and three detected previously, seven STS markers were established to amplify the corresponding gene sequences in wheat cultivars containing five Glu-D3 alleles (a, b, c, d and e). The seven primer sets M2F12/M2R12, M2F2/M2R2, M2F3/M2R3, M3F1/M3R1, M3F2/M3R2, M4F1/M4R1 and M4F3/M4R3 were specific to the allelic variants GluD3-21/22, GluD3-22, GluD3-23, GluD3-31, GluD3-32, GluD3-41 and GluD3-43, respectively, which were validated by amplifying 20 Chinese wheat cultivars containing alleles a, b, c and f based on protein electrophoretic mobility. These markers will be useful to identify the Glu-D3 gene haplotypes in wheat

Mineral nitrogen sources differently affect root glutamine synthetase isoforms and amino acid balance among organs in maize.

PubMed

Prinsi, Bhakti; Espen, Luca

2015-04-03

Glutamine synthetase (GS) catalyzes the first step of nitrogen assimilation in plant cell. The main GS are classified as cytosolic GS1 and plastidial GS2, of which the functionality is variable according to the nitrogen sources, organs and developmental stages. In maize (Zea mays L.) one gene for GS2 and five genes for GS1 subunits are known, but their roles in root metabolism are not yet well defined. In this work, proteomic and biochemical approaches have been used to study root GS enzymes and nitrogen assimilation in maize plants re-supplied with nitrate, ammonium or both. The plant metabolic status highlighted the relevance of root system in maize nitrogen assimilation during both nitrate and ammonium nutrition. The analysis of root proteomes allowed a study to be made of the accumulation and phosphorylation of six GS proteins. Three forms of GS2 were identified, among which only the phosphorylated one showed an accumulation trend consistent with plastidial GS activity. Nitrogen availabilities enabled increments in root total GS synthetase activity, associated with different GS1 isoforms according to the nitrogen sources. Nitrate nutrition induced the specific accumulation of GS1-5 while ammonium led to up-accumulation of both GS1-1 and GS1-5, highlighting co-participation. Moreover, the changes in thermal sensitivity of root GS transferase activity suggested differential rearrangements of the native enzyme. The amino acid accumulation and composition in roots, xylem sap and leaves deeply changed in response to mineral sources. Glutamine showed the prevalent changes in all nitrogen nutritions. Besides, the ammonium nutrition was associated with an accumulation of asparagine and reducing sugars and a drop in glutamic acid level, significantly alleviated by the co-provision with nitrate. This work provides new information about the multifaceted regulation of the GS enzyme in maize roots, indicating the involvement of specific isoenzymes/isoforms, post

Promoter methylation status in genes related with inflammation, nitrosative stress and xenobiotic metabolism in low-level benzene exposure: Searching for biomarkers of oncogenesis.

PubMed

Jiménez-Garza, Octavio; Guo, Liqiong; Byun, Hyang-Min; Carrieri, Mariella; Bartolucci, Giovanni Battista; Zhong, Jia; Baccarelli, Andrea A

2017-11-01

Exposure to low levels of benzene may cause acute myeloid leukemia in humans. Epigenetic effects in benzene exposure have been studied for tumor suppressor genes and oxidative stress-related genes, but other cellular pathways must be explored. Here, we studied promoter DNA methylation of IL6, CYP2E1 and iNOS in blood cells from three groups of workers: a) gas station attendants (GS) exposed to low levels of benzene; b) plastic shoe factory workers (PS) exposed to other solvents different to benzene and c) administrative workers as a reference group with no solvent exposure (C). IL6 promoter methylation was higher in GS workers (p < 0.05). Also in GS, CYP2E1 promoter methylation negatively correlated with benzene levels (r = -0.47, p < 0.05); iNOS promoter methylation positively correlated with CYP2E1 promoter methylation (r = 0.29, p < 0.05), cumulative time of exposure (r = 0.31, p < 0.05) as well as with urinary levels of S- Phenyl mercapturic acid (SPMA), (r = 0.55, p < 0.05). Our results demonstrate alterations in the inflammation pathway at the epigenetic level associated with exposure to benzene. Correlations between iNOS methylation with both CYP2E1 methylation and urinary SPMA levels represent novel evidence about CYP2E1 epigenetic regulation and activity related with nitrosative stress, making promoter methylation status of these genes a potential biomarker in early stages of oncogenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quantification of low molecular weight selenium metabolites in human plasma after treatment with selenite in pharmacological doses by LC-ICP-MS.

PubMed

Flouda, Konstantina; Dersch, Julie Maria; Gabel-Jensen, Charlotte; Stürup, Stefan; Misra, Sougat; Björnstedt, Mikael; Gammelgaard, Bente

2016-03-01

The paper presents an analytical method for quantification of low molecular weight (LMW) selenium compounds in human plasma based on liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS) and post column isotope dilution-based quantification. Prior to analysis, samples were ultrafiltrated using a cut-off value of 3000 Da. The method was validated in aqueous solution as well as plasma using standards of selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), selenite, and the selenosugar Se-methylseleno-N-acetylgalactosamine (SeGal) for linearity, precision, recoveries, and limits of detection and quantitation with satisfactory results. The method was applied for analysis of a set of plasma samples from cancer patients receiving selenite treatment in a clinical trial. Three LMW selenium compounds were observed. The main compounds, SeGal and selenite were tentatively identified by retention time matching with standards in different chromatographic systems, while the third minor compound was not identified. The identity of the selenosugar was verified by ESI-MS-MS product ion scanning, while selenite was identified indirectly as the glutathione (GSH) reaction product, GS-Se-SG.

A high-risk 70-gene signature is not associated with the detection of tumor cell dissemination to the bone marrow.

PubMed

Walter, Vincent P; Taran, Florin-Andrei; Wallwiener, Markus; Walter, Christina; Grischke, Eva-Maria; Wallwiener, Diethelm; Brucker, Sara Y; Hartkopf, Andreas D

2018-06-01

The 70-gene signature (70-GS) is a prognostic tool, grouping patients in risk groups to assess their need for adjuvant chemotherapy. Tumor cell dissemination to the bone marrow is a marker of minimal residual disease and associated with impaired survival. In this study, we aimed to evaluate whether 70-GS is associated with the presence of disseminated tumor cells (DTCs) in the bone marrow of patients with early breast cancer. In patients with hormone receptor-positive HER2-negative early breast cancer, the 70-GS was obtained and the presence of DTCs was immunohistochemically evaluated using cytokeratin staining with the A45-B/B3 antibody. 149 patients were included into the analysis. 40 (27%) had a high-risk 70-GS and 35 (23%) had detectable DTCs in their bone marrow. 9 (22%) of the 40 patients with high-risk 70-GS and 26 (24%) of the 109 patients with a low-risk 70-GS were positive for DTCs (p = 0.863). As both 70-GS and DTC detection are known prognostic factors but do not seem to correlate, a follow-up on a larger cohort is warranted to evaluate if a combination of the two is able to better stratify the relapse risk in early breast cancer patients.

Glutathione Preservation during Storage of Rat Lenses in Optisol-GS and Castor Oil

PubMed Central

Holm, Thomas; Brøgger-Jensen, Martin Rocho; Johnson, Leif; Kessel, Line

2013-01-01

Background Glutathione concentration in the lens decreases in aging and cataractous lenses, providing a marker for tissue condition. Experimental procedures requiring unfrozen lenses from donor banks rely on transportation in storage medium, affecting lens homeostasis and alterations in glutathione levels. The aim of the study was to examine the effects of Optisol-GS and castor oil on lens condition, determined from their ability to maintain glutathione concentrations. Methodology/Principal Findings Rat lenses were stored in the two types of storage media at varying time intervals up to 3 days. Glutathione concentration was afterwards determined in an enzymatic detection assay, specific for both reduced and oxidized forms. Lenses removed immediately after death exhibited a glutathione concentration of 4.70±0.29 mM. In vitro stored lenses in Optisol-GS lost glutathione quickly, ending with a concentration of 0.60±0.34 mM after 3 days while castor oil stored lenses exhibited a slower decline and ended at 3 times the concentration. A group of lenses were additionally stored under post mortem conditions within the host for 6 hours before its removal. Total glutathione after 6 hours was similar to that of lenses removed immediately after death, but with altered GSH and GSSG concentrations. Subsequent storage of these lenses in media showed changes similar to those in the first series of experiments, albeit to a lesser degree. Conclusions/Significance It was determined that storage in Optisol-GS resulted in a higher loss of glutathione than lenses stored in castor oil. Storage for more than 12 hours reduced glutathione to half its original concentration, and was considered unusable after 24 hours. PMID:24260265

Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor.

PubMed

Parameswaran, N; Disa, J; Spielman, W S; Brooks, D P; Nambi, P; Aiyar, N

2000-02-18

Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8-37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11, 11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3, 2-de]indeno[4,5-h]-2

Isolated dorsal root ganglion neurones inhibit receptor-dependent adenylyl cyclase activity in associated glial cells

PubMed Central

Ng, KY; Yeung, BHS; Wong, YH; Wise, H

2013-01-01

Background and Purpose Hyper-nociceptive PGE2 EP4 receptors and prostacyclin (IP) receptors are present in adult rat dorsal root ganglion (DRG) neurones and glial cells in culture. The present study has investigated the cell-specific expression of two other Gs-protein coupled hyper-nociceptive receptor systems: β-adrenoceptors and calcitonin gene-related peptide (CGRP) receptors in isolated DRG cells and has examined the influence of neurone–glial cell interactions in regulating adenylyl cyclase (AC) activity. Experimental Approach Agonist-stimulated AC activity was determined in mixed DRG cell cultures from adult rats and compared with activity in DRG neurone-enriched cell cultures and pure DRG glial cell cultures. Key Results Pharmacological analysis showed the presence of Gs-coupled β2-adrenoceptors and CGRP receptors, but not β1-adrenoceptors, in all three DRG cell preparations. Agonist-stimulated AC activity was weakest in DRG neurone-enriched cell cultures. DRG neurones inhibited IP receptor-stimulated glial cell AC activity by a process dependent on both cell–cell contact and neurone-derived soluble factors, but this is unlikely to involve purine or glutamine receptor activation. Conclusions and Implications Gs-coupled hyper-nociceptive receptors are readily expressed on DRG glial cells in isolated cell cultures and the activity of CGRP, EP4 and IP receptors, but not β2-adrenoceptors, in glial cells is inhibited by DRG neurones. Studies using isolated DRG cells should be aware that hyper-nociceptive ligands may stimulate receptors on glial cells in addition to neurones, and that variable numbers of neurones and glial cells will influence absolute measures of AC activity and affect downstream functional responses. PMID:22924655

Three Cytosolic Glutamine Synthetase Isoforms Located in Different Order Veins Work Together for N Remobilization and Seed Filling in Arabidopsis.

PubMed

Moison, Michael; Marmagne, Anne; Dinant, Sylvie; Soulay, Fabienne; Azzopardi, Marianne; Lothier, Jérémy; Citerne, Sylvie; Morin, Halima; Legay, Nicolas; Chardon, Fabien; Avice, Jean-Christophe; Reisdorf-Cren, Michèle; Masclaux-Daubresse, Céline

2018-06-05

Glutamine synthetase (GS) is central for ammonium assimilation and consists of cytosolic (GS1) and chloroplastic (GS2) isoenzymes. During plant ageing, GS2 protein decreases due to chloroplast degradation, and GS1 activity increases to support glutamine biosynthesis and N remobilization from senescing leaves. The role of the different Arabidopsis GS1 isoforms in nitrogen remobilization was examined using 15N tracing experiments. Only the triple gln1;1-gln1;2-gln1;3 mutation affecting the three GLN1;1, GLN1;2 and GLN1;3 genes reduced significantly N remobilization, seed yield, seed weight, harvest index and vegetative biomass. The triple gln1;1-gln1;2-gln1;3 mutant accumulated large amount of ammonium that cannot be assimilated by GS1. Alternative ammonium assimilation through asparagine biosynthesis was increased and related to higher ASN2 asparagine synthetase transcript levels. The GS2 transcript, protein and activity levels were also increased to compensate for the lack of GS1-related glutamine biosynthesis. Localization of the different GLN1 genes showed that they are all expressed in the phloem companion cells but in different order veins. In conclusion, our study shows that glutamine biosynthesis for N-remobilization occurs in all the order veins (major and minor) in leaves, is mainly catalysed by the three major GS1 isoforms (GLN1;1, GLN1;2 and GLN1;3) and is alternatively supported by AS2 in the veins and GS2 in the mesophyll cells.

Diversity of cultivable β-glycosidase-producing micro-organisms isolated from the soil of a ginseng field and their ginsenosides-hydrolysing activity.

PubMed

Fu, Y; Yin, Z; Wu, L; Yin, C

2014-02-01

This research aimed to explore the diversity of cultivable β-glycosidase-producing micro-organisms in ginseng field soil. Fifty-three strains showing β-glucosidase activity were isolated from a ginseng field, using a newly designed Esculin-R2A agar. All the isolated strains belonged to the genus Agrobacterium, Arthrobacter, Burkholderia, Dyella, Edaphobacter, Luteibacter, Mucilaginibacter, Paenibacillus, Phenylobacterium, Pseudomonas, Sphingomonas and Streptomyces. The main β-glucosidase-producing micro-organisms in the ginseng field soil were Sphingomonas, Burkholderia, Luteibacter and Streptomyces, while concentrations of Agrobacterium, Arthrobacter, Paenibacillus and Pseudomonas were relatively low. Of these micro-organisms, the strain GS 09 could hydrolyse major ginsenosides Rb1, Rb2 and Rc to the active metabolite compound K. The strain GS 09 belonged to the genus Sphingomonas, and its 16S rRNA gene sequence showed 100% similarities with that of Sphingomonas asaccharolytica. This is the first study to provide information of cultivable β-glycosidase-producing micro-organisms in ginseng field soil. The strain GS 09 has potential to be applied on the preparation for minor ginsenoside C-K in pharmaceutical industry. © 2013 The Society for Applied Microbiology.

Clinical Utility of a Coronary Heart Disease Risk Prediction Gene Score in UK Healthy Middle Aged Men and in the Pakistani Population

PubMed Central

Beaney, Katherine E.; Cooper, Jackie A.; Ullah Shahid, Saleem; Ahmed, Waqas; Qamar, Raheel; Drenos, Fotios; Crockard, Martin A.; Humphries, Steve E.

2015-01-01

Background Numerous risk prediction algorithms based on conventional risk factors for Coronary Heart Disease (CHD) are available but provide only modest discrimination. The inclusion of genetic information may improve clinical utility. Methods We tested the use of two gene scores (GS) in the prospective second Northwick Park Heart Study (NPHSII) of 2775 healthy UK men (284 cases), and Pakistani case-control studies from Islamabad/Rawalpindi (321 cases/228 controls) and Lahore (414 cases/219 controls). The 19-SNP GS included SNPs in loci identified by GWAS and candidate gene studies, while the 13-SNP GS only included SNPs in loci identified by the CARDIoGRAMplusC4D consortium. Results In NPHSII, the mean of both gene scores was higher in those who went on to develop CHD over 13.5 years of follow-up (19-SNP p=0.01, 13-SNP p=7x10-3). In combination with the Framingham algorithm the GSs appeared to show improvement in discrimination (increase in area under the ROC curve, 19-SNP p=0.48, 13-SNP p=0.82) and risk classification (net reclassification improvement (NRI), 19-SNP p=0.28, 13-SNP p=0.42) compared to the Framingham algorithm alone, but these were not statistically significant. When considering only individuals who moved up a risk category with inclusion of the GS, the improvement in risk classification was statistically significant (19-SNP p=0.01, 13-SNP p=0.04). In the Pakistani samples, risk allele frequencies were significantly lower compared to NPHSII for 13/19 SNPs. In the Islamabad study, the mean gene score was higher in cases than controls only for the 13-SNP GS (2.24 v 2.34, p=0.04). There was no association with CHD and either score in the Lahore study. Conclusion The performance of both GSs showed potential clinical utility in European men but much less utility in subjects from Pakistan, suggesting that a different set of risk loci or SNPs may be required for risk prediction in the South Asian population. PMID:26133560

Comparisons of 'Identical' Simulations by the Eulerian Gyrokinetic Codes GS2 and GYRO

NASA Astrophysics Data System (ADS)

Bravenec, R. V.; Ross, D. W.; Candy, J.; Dorland, W.; McKee, G. R.

2003-10-01

A major goal of the fusion program is to be able to predict tokamak transport from first-principles theory. To this end, the Eulerian gyrokinetic code GS2 was developed years ago and continues to be improved [1]. Recently, the Eulerian code GYRO was developed [2]. These codes are not subject to the statistical noise inherent to particle-in-cell (PIC) codes, and have been very successful in treating electromagnetic fluctuations. GS2 is fully spectral in the radial coordinate while GYRO uses finite-differences and ``banded" spectral schemes. To gain confidence in nonlinear simulations of experiment with these codes, ``apples-to-apples" comparisons (identical profile inputs, flux-tube geometry, two species, etc.) are first performed. We report on a series of linear and nonlinear comparisons (with overall agreement) including kinetic electrons, collisions, and shaped flux surfaces. We also compare nonlinear simulations of a DIII-D discharge to measurements of not only the fluxes but also the turbulence parameters. [1] F. Jenko, et al., Phys. Plasmas 7, 1904 (2000) and refs. therein. [2] J. Candy, J. Comput. Phys. 186, 545 (2003).

A Low-Molecular-Weight Antagonist for the Human Thyrotropin Receptor with Therapeutic Potential for Hyperthyroidism

PubMed Central

Neumann, Susanne; Kleinau, Gunnar; Costanzi, Stefano; Moore, Susanna; Jiang, Jian-kang; Raaka, Bruce M.; Thomas, Craig J.; Krause, Gerd; Gershengorn, Marvin C.

2008-01-01

Low-molecular-weight (LMW) antagonists for TSH receptor (TSHR) may have therapeutic potential as orally active drugs to block stimulating antibodies (TsAbs) in Graves’ hyperthyroidism. We describe an approach to identify LMW ligands for TSHR based on Org41841, a LMW partial agonist for the LH/choriogonadotropin receptor and TSHR. We used molecular modeling and functional experiments to guide the chemical modification of Org41841. We identified an antagonist (NIDDK/CEB-52) that selectively inhibits activation of TSHR by both TSH and TsAbs. Whereas initially characterized in cultured cells overexpressing TSHRs, the antagonist was also active under more physiologically relevant conditions in primary cultures of human thyrocytes expressing endogenous TSHRs in which it inhibited TSH- and TsAb-induced up-regulation of mRNA transcripts for thyroperoxidase. Our results establish this LMW compound as a lead for the development of higher potency antagonists and serve as proof of principle that LMW ligands that target TSHR could serve as drugs in patients with Graves’ disease. PMID:18669595

Necessary and sufficient liveness condition of GS3PR Petri nets

NASA Astrophysics Data System (ADS)

Liu, GaiYun; Barkaoui, Kamel

2015-05-01

Structural analysis is one of the most important and efficient methods to investigate the behaviour of Petri nets. Liveness is a significant behavioural property of Petri nets. Siphons, as structural objects of a Petri net, are closely related to its liveness. Many deadlock control policies for flexible manufacturing systems (FMS) modelled by Petri nets are implemented via siphon control. Most of the existing methods design liveness-enforcing supervisors by adding control places for siphons based on their controllability conditions. To compute a liveness-enforcing supervisor with as much as permissive behaviour, it is both theoretically and practically significant to find an exact controllability condition for siphons. However, the existing conditions, max, max‧, and max″-controllability of siphons are all overly restrictive and generally sufficient only. This paper develops a new condition called max*-controllability of the siphons in generalised systems of simple sequential processes with resources (GS3PR), which are a net subclass that can model many real-world automated manufacturing systems. We show that a GS3PR is live if all its strict minimal siphons (SMS) are max*-controlled. Compared with the existing conditions, i.e., max-, max‧-, and max″-controllability of siphons, max*-controllability of the SMS is not only sufficient but also necessary. An example is used to illustrate the proposed method.

Secondhand Smoke-Prevalent Polycyclic Aromatic Hydrocarbon Binary Mixture-Induced Specific Mitogenic and Pro-inflammatory Cell Signaling Events in Lung Epithelial Cells

PubMed Central

Osgood, Ross S.; Upham, Brad L.; Bushel, Pierre R.; Velmurugan, Kalpana; Xiong, Ka-Na

2017-01-01

Abstract Low molecular weight polycyclic aromatic hydrocarbons (LMW PAHs; < 206.3 g/mol) are prevalent and ubiquitous environmental contaminants, presenting a human health concern, and have not been as thoroughly studied as the high MW PAHs. LMW PAHs exert their pulmonary effects, in part, through P38-dependent and -independent mechanisms involving cell-cell communication and the production of pro-inflammatory mediators known to contribute to lung disease. Specifically, we determined the effects of two representative LMW PAHs, 1-methylanthracene (1-MeA) and fluoranthene (Flthn), individually and as a binary PAH mixture on the dysregulation of gap junctional intercellular communication (GJIC) and connexin 43 (Cx43), activation of mitogen activated protein kinases (MAPK), and induction of inflammatory mediators in a mouse non-tumorigenic alveolar type II cell line (C10). Both 1-MeA, Flthn, and the binary PAH mixture of 1-MeA and Flthn dysregulated GJIC in a dose and time-dependent manner, reduced Cx43 protein, and activated the following MAPKs: P38, ERK1/2, and JNK. Inhibition of P38 MAPK prevented PAH-induced dysregulation of GJIC, whereas inhibiting ERK and JNK did not prevent these PAHs from dysregulating GJIC indicating a P38-dependent mechanism. A toxicogenomic approach revealed significant P38-dependent and -independent pathways involved in inflammation, steroid synthesis, metabolism, and oxidative responses. Genes in these pathways were significantly altered by the binary PAH mixture when compared with 1-MeA and Flthn alone suggesting interactive effects. Exposure to the binary PAH mixture induced the production and release of cytokines and metalloproteinases from the C10 cells. Our findings with a binary mixture of PAHs suggest that combinations of LMW PAHs may elicit synergistic or additive inflammatory responses which warrant further investigation and confirmation. PMID:28329830

Complete genome sequence of Streptococcus mutans GS-5, a serotype c strain.

PubMed

Biswas, Saswati; Biswas, Indranil

2012-09-01

Streptococcus mutans, a principal causative agent of dental caries, is considered to be the most cariogenic among all oral streptococci. Of the four S. mutans serotypes (c, e, f, and k), serotype c strains predominate in the oral cavity. Here, we present the complete genome sequence of S. mutans GS-5, a serotype c strain originally isolated from human carious lesions, which is extensively used as a laboratory strain worldwide.

Natural variation in maternal care and cross-tissue patterns of oxytocin receptor gene methylation in rats

PubMed Central

Beery, Annaliese K.; McEwen, Lisa M.; MacIsaac, Julia L; Francis, Darlene D.; Kobor, Michael S.

2015-01-01

Since the first report of maternal care effects on DNA methylation in rats, epigenetic modifications of the genome in response to life experience have become the subject of intense focus across many disciplines. Oxytocin receptor expression varies in response to early experience, and both oxytocin signaling and methylation status of the oxytocin receptor gene (Oxtr) in blood have been related to disordered social behavior. It is unknown whether Oxtr methylation varies in response to early life experience, and whether currently employed peripheral measures of Oxtr methylation reflect variation in the brain. We examined the effects of early life rearing experience via natural variation in maternal licking and grooming during the first week of life on behavior, physiology, gene expression, and epigenetic regulation of Oxtr across blood and brain tissues (mononucleocytes, hippocampus, striatum, and hypothalamus). Rats reared by “high” licking-grooming (HL) and “low” licking-grooming (LL) rat dams exhibited differences across study outcomes: LL offspring were more active in behavioral arenas, exhibited lower body mass in adulthood, and showed reduced corticosterone responsivity to a stressor. Oxtr methylation was significantly lower at multiple CpGs in the blood of LL versus HL rats, but no differences were found in the brain. Across groups, Oxtr transcript levels in the hypothalamus were associated with reduced corticosterone secretion in response to stress, congruent with the role of oxytocin signaling in this region. Methylation of specific CpGs at a high or low level was consistent across tissues, especially within the brain. However, individual variation in methylation relative to these global patterns was not consistent across tissues. These results suggest that blood Oxtr methylation may reflect early experience of maternal care, and that Oxtr methylation across tissues is highly concordant for specific CpGs, but that inferences across tissues are not

Our trial to develop a risk assessment tool for CO2 geological storage (GERAS-CO2GS)

NASA Astrophysics Data System (ADS)

Tanaka, A.; Sakamoto, Y.; Komai, T.

2012-12-01

We will introduce our researches about to develop a risk assessment tool named 'GERAS-CO2GS' (Geo-environmental Risk Assessment System, CO2 Geological Storage Risk Assessment System) for 'Carbon Dioxide Geological Storage (Geological CCS)'. It aims to facilitate understanding of size of impact of risks related with upper migration of injected CO2. For gaining public recognition about feasibility of Geological CCS, quantitative estimation of risks is essential, to let public knows the level of the risk: whether it is negligible or not. Generally, in preliminary hazard analysis procedure, potential hazards could be identified within Geological CCS's various facilities such as: reservoir, cap rock, upper layers, CO2 injection well, CO2 injection plant and CO2 transport facilities. Among them, hazard of leakage of injected C02 is crucial, because it is the clue to estimate risks around a specific injection plan in terms of safety, environmental protection effect and economy. Our risk assessment tool named GERAS-CO2GS evaluates volume and rate of retention and leakage of injected CO2 in relation with fractures and/or faults, and then it estimates impact of seepages on the surface of the earth. GERAS-CO2GS has four major processing segments: (a) calculation of CO2 retention and leakage volume and rate, (b) data processing of CO2 dispersion on the surface and ambient air, (c) risk data definition and (d) evaluation of risk. Concerning to the injection site, we defined a model, which is consisted from an injection well and a geological strata model: which involves a reservoir, a cap rock, an upper layer, faults, seabed, sea, the surface of the earth and the surface of the sea. For retention rate of each element of CO2 injection site model, we use results of our experimental and numerical studies on CO2 migration within reservoirs and faults with specific lithological conditions. For given CO2 injection rate, GERAS-CO2GS calculates CO2 retention and leakage of each segment

Changes in Air CO₂ Concentration Differentially Alter Transcript Levels of NtAQP1 and NtPIP2;1 Aquaporin Genes in Tobacco Leaves.

PubMed

Secchi, Francesca; Schubert, Andrea; Lovisolo, Claudio

2016-04-14

The aquaporin specific control on water versus carbon pathways in leaves is pivotal in controlling gas exchange and leaf hydraulics. We investigated whether Nicotiana tabacum aquaporin 1 (NtAQP1) and Nicotiana tabacum plasma membrane intrinsic protein 2;1 (NtPIP2;1) gene expression varies in tobacco leaves subjected to treatments with different CO₂ concentrations (ranging from 0 to 800 ppm), inducing changes in photosynthesis, stomatal regulation and water evaporation from the leaf. Changes in air CO₂ concentration ([CO₂]) affected net photosynthesis (Pn) and leaf substomatal [CO₂] (Ci). Pn was slightly negative at 0 ppm air CO₂; it was one-third that of ambient controls at 200 ppm, and not different from controls at 800 ppm. Leaves fed with 800 ppm [CO₂] showed one-third reduced stomatal conductance (gs) and transpiration (E), and their gs was in turn slightly lower than in 200 ppm- and in 0 ppm-treated leaves. The 800 ppm air [CO₂] strongly impaired both NtAQP1 and NtPIP2;1 gene expression, whereas 0 ppm air [CO₂], a concentration below any in vivo possible conditions and specifically chosen to maximize the gene expression alteration, increased only the NtAQP1 transcript level. We propose that NtAQP1 expression, an aquaporin devoted to CO₂ transport, positively responds to CO₂ scarcity in the air in the whole range 0-800 ppm. On the contrary, expression of NtPIP2;1, an aquaporin not devoted to CO₂ transport, is related to water balance in the leaf, and changes in parallel with gs. These observations fit in a model where upregulation of leaf aquaporins is activated at low Ci, while downregulation occurs when high Ci saturates photosynthesis and causes stomatal closure.

Chitosan-thioglycolic acid as a versatile antimicrobial agent.

PubMed

Geisberger, Georg; Gyenge, Emina Besic; Hinger, Doris; Käch, Andres; Maake, Caroline; Patzke, Greta R

2013-04-08

As functionalized chitosans hold great potential for the development of effective and broad-spectrum antibiotics, representative chitosan derivatives were tested for antimicrobial activity in neutral media: trimethyl chitosan (TMC), carboxy-methyl chitosan (CMC), and chitosan-thioglycolic acid (TGA; medium molecular weight: MMW-TGA; low molecular weight: LMW-TGA). Colony forming assays indicated that LMW-TGA displayed superior antimicrobial activity over the other derivatives tested: a 30 min incubation killed 100% Streptococcus sobrinus (Gram-positive bacteria) and reduced colony counts by 99.99% in Neisseria subflava (Gram-negative bacteria) and 99.97% in Candida albicans (fungi). To elucidate LMW-TGA effects at the cellular level, microscopic studies were performed. Use of fluorescein isothiocyanate (FITC)-labeled chitosan derivates in confocal microscopy showed that LMW-TGA attaches to microbial cell walls, while transmission electron microscopy indicated that this derivative severely affects cell wall integrity and intracellular ultrastructure in all species tested. We therefore propose LMW-TGA as a promising and effective broad-band antimicrobial compound.

Gas stunning with CO2 affected meat color, lipid peroxidation, oxidative stress, and gene expression of mitogen-activated protein kinases, glutathione S-transferases, and Cu/Zn-superoxide dismutase in the skeletal muscles of broilers.

PubMed

Xu, Lei; Zhang, Haijun; Yue, Hongyuan; Wu, Shugeng; Yang, Haiming; Wang, Zhiyue; Qi, Guanghai

2018-01-01

Meat color and lipid peroxidation are important traits related to meat quality. CO 2 concentration is a critical factor that can affect meat quality in the commercial use of gas stunning (GS). However, the effect and mechanism of CO 2 stunning on meat color and lipid peroxidation during long-term storage remain poorly studied. We aimed to study the effects of GS methods, especially CO 2 concentration, on meat color and meat lipid peroxidation in broilers during long-term storage at 4 °C and to explore the potential mechanism of meat color change via lipid peroxidation and the inner lipid peroxide scavenging system. Eighteen broilers were sacrificed after exposure to one of the following gas mixtures for 90 s: 40% CO 2  + 21% O 2  + 39% N 2 (G40%), 79% CO 2  + 21% O 2 (G79%), or no stunning (0% CO 2 , control). Meat color, serum variables, enzyme activities, and the gene expression of mitogen-activated protein kinase ( MAPK ), nuclear factor-erythroid 2-related factor 2 ( Nrf2 ), glutathione S-transferase ( GST ) and superoxide dismutase ( SOD ) were determined. The concentrations of serum triiodothyronine (T3, P  = 0.03) and the ratio of serum free triiodothyronine/free thyroxine (FT3/FT4, P  <  0.01) were decreased, whereas levels of serum cortisol ( P  <  0.01) were increased in the 40% CO 2 group compared with the control group. Additionally, the thiobarbituric acid-reactive substances (TBARS) 3 d ( P  <  0.01) and TBARS 6 d ( P  = 0.01) in breast meat and the TBARS 3 d in thigh meat ( P  <  0.01) were increased in the 40% CO 2 group compared with the control group. Serum T3 was negatively correlated with TBARS 6 d both in the breast and thigh meat ( r  = - 0.63, P  <  0.01 and r  = - 0.47, P  = 0.05 respectively). T3/T4 was negatively correlated with TBARS 6 d in the breast meat and in the thigh meat ( r  = - 0.57, P  = 0.01; and r  = - 0.53, P  = 0.03 respectively). Compared

Oxycodone plus ultra-low-dose naltrexone attenuates neuropathic pain and associated mu-opioid receptor-Gs coupling.

PubMed

Largent-Milnes, Tally M; Guo, Wenhong; Wang, Hoau-Yan; Burns, Lindsay H; Vanderah, Todd W

2008-08-01

Both peripheral nerve injury and chronic opioid treatment can result in hyperalgesia associated with enhanced excitatory neurotransmission at the level of the spinal cord. Chronic opioid administration leads to a shift in mu-opioid receptor (MOR)-G protein coupling from G(i/o) to G(s) that can be prevented by cotreatment with an ultra-low-dose opioid antagonist. In this study, using lumbar spinal cord tissue from rats with L(5)/L(6) spinal nerve ligation (SNL), we demonstrated that SNL injury induces MOR linkage to G(s) in the damaged (ipsilateral) spinal dorsal horn. This MOR-G(s) coupling occurred without changing G(i/o) coupling levels and without changing the expression of MOR or Galpha proteins. Repeated administration of oxycodone alone or in combination with ultra-low-dose naltrexone (NTX) was assessed on the SNL-induced MOR-G(s) coupling as well as on neuropathic pain behavior. Repeated spinal oxycodone exacerbated the SNL-induced MOR-G(s) coupling, whereas ultra-low-dose NTX cotreatment slightly but significantly attenuated this G(s) coupling. Either spinal or oral administration of oxycodone plus ultra-low-dose NTX markedly enhanced the reductions in allodynia and thermal hyperalgesia produced by oxycodone alone and minimized tolerance to these effects. The MOR-G(s) coupling observed in response to SNL may in part contribute to the excitatory neurotransmission in spinal dorsal horn in neuropathic pain states. The antihyperalgesic and antiallodynic effects of oxycodone plus ultra-low-dose NTX (Oxytrex, Pain Therapeutics, Inc., San Mateo, CA) suggest a promising new treatment for neuropathic pain. The current study investigates whether Oxytrex (oxycodone with an ultra-low dose of naltrexone) alleviates mechanical and thermal hypersensitivities in an animal model of neuropathic pain over a period of 7 days, given locally or systemically. In this report, we first describe an injury-induced shift in mu-opioid receptor coupling from G(i/o) to G(s), suggesting

A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators.

PubMed

Xie, Wensheng; Silvers, Robert; Ouellette, Michael; Wu, Zining; Lu, Quinn; Li, Hu; Gallagher, Kathleen; Johnson, Kathy; Montoute, Monica

2016-01-01

Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic β-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase.

Glycation of polyclonal IgGs: Effect of sugar excipients during stability studies.

PubMed

Leblanc, Y; Bihoreau, N; Jube, M; Andre, M-H; Tellier, Z; Chevreux, G

2016-05-01

A number of intravenous immunoglobulin preparations are stabilized with sugar additives that may lead over time to undesirable glycation reactions especially in liquid formulation. This study aimed to evaluate the reactivity of sugar excipients on such preparations in condition of temperature, formulation and concentration commonly used for pharmaceutical products. Through an innovative LC-MS method reported to characterize post-translational modifications of IgGs Fc/2 fragments, a stability study of IVIg formulated with reducing and non-reducing sugars has been undertaken. The rate of polyclonal IgGs glycation was investigated during 6months at 5, 25, 30 and 40°C. High levels of glycation were observed with reducing sugars such as glucose and maltose in the first months of the stability study from 25°C. Non-reducing sugars presented a low reactivity even at the highest tested temperature (40°C). Furthermore, a site by site analysis was performed by MS/MS to determine the glycation sites which were mainly identified at Lys246, Lys248 and Lys324. This work points out the high probability of glycation reactions in some commercialized products and describes a useful method to characterize IVIg glycated products issued from reducing sugar excipients. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Genomics Study of the Exposure Effect of Gymnodinium catenatum, a Paralyzing Toxin Producer, on Crassostrea gigas' Defense System and Detoxification Genes

PubMed Central

García-Lagunas, Norma; Romero-Geraldo, Reyna; Hernández-Saavedra, Norma Y.

2013-01-01

Background Crassostrea gigas accumulates paralytic shellfish toxins (PST) associated with red tide species as Gymnodinium catenatum. Previous studies demonstrated bivalves show variable feeding responses to toxic algae at physiological level; recently, only one study has reported biochemical changes in the transcript level of the genes involved in C. gigas stress response. Principal Findings We found that 24 h feeding on toxic dinoflagellate cells (acute exposure) induced a significant decrease in clearance rate and expression level changes of the genes involved in antioxidant defense (copper/zinc superoxide dismutase, Cu/Zn-SOD), cell detoxification (glutathione S-transferase, GST and cytochrome P450, CPY450), intermediate immune response activation (lipopolysaccharide and beta glucan binding protein, LGBP), and stress responses (glutamine synthetase, GS) in Pacific oysters compared to the effects with the non-toxic microalga Isochrysis galbana. A sub-chronic exposure feeding on toxic dinoflagellate cells for seven and fourteen days (30×103 cells mL−1) showed higher gene expression levels. A significant increase was observed in Cu/Zn-SOD, GST, and LGBP at day 7 and a major increase in GS and CPY450 at day 14. We also observed that oysters fed only with G. catenatum (3×103 cells mL−1) produced a significant increase on the transcription level than in a mixed diet (3×103 cells mL−1 of G. catenatum+0.75×106 cells mL−1 I. galbana) in all the analyzed genes. Conclusions Our results provide gene expression data of PST producer dinoflagellate G. catenatum toxic effects on C. gigas, a commercially important bivalve. Over expressed genes indicate the activation of a potent protective mechanism, whose response depends on both cell concentration and exposure time against these toxic microalgae. Given the importance of dinoflagellate blooms in coastal environments, these results provide a more comprehensive overview of how oysters respond to stress generated by

Changes in Air CO2 Concentration Differentially Alter Transcript Levels of NtAQP1 and NtPIP2;1 Aquaporin Genes in Tobacco Leaves

PubMed Central

Secchi, Francesca; Schubert, Andrea; Lovisolo, Claudio

2016-01-01

The aquaporin specific control on water versus carbon pathways in leaves is pivotal in controlling gas exchange and leaf hydraulics. We investigated whether Nicotiana tabacum aquaporin 1 (NtAQP1) and Nicotiana tabacum plasma membrane intrinsic protein 2;1 (NtPIP2;1) gene expression varies in tobacco leaves subjected to treatments with different CO2 concentrations (ranging from 0 to 800 ppm), inducing changes in photosynthesis, stomatal regulation and water evaporation from the leaf. Changes in air CO2 concentration ([CO2]) affected net photosynthesis (Pn) and leaf substomatal [CO2] (Ci). Pn was slightly negative at 0 ppm air CO2; it was one-third that of ambient controls at 200 ppm, and not different from controls at 800 ppm. Leaves fed with 800 ppm [CO2] showed one-third reduced stomatal conductance (gs) and transpiration (E), and their gs was in turn slightly lower than in 200 ppm– and in 0 ppm–treated leaves. The 800 ppm air [CO2] strongly impaired both NtAQP1 and NtPIP2;1 gene expression, whereas 0 ppm air [CO2], a concentration below any in vivo possible conditions and specifically chosen to maximize the gene expression alteration, increased only the NtAQP1 transcript level. We propose that NtAQP1 expression, an aquaporin devoted to CO2 transport, positively responds to CO2 scarcity in the air in the whole range 0–800 ppm. On the contrary, expression of NtPIP2;1, an aquaporin not devoted to CO2 transport, is related to water balance in the leaf, and changes in parallel with gs. These observations fit in a model where upregulation of leaf aquaporins is activated at low Ci, while downregulation occurs when high Ci saturates photosynthesis and causes stomatal closure. PMID:27089333

Immunocytochemical localization of glutamic acid decarboxylase (GAD) and glutamine synthetase (GS) in the area postrema of the cat. Light and electron microscopy

NASA Technical Reports Server (NTRS)

D'Amelio, Fernando E.; Mehler, William R.; Gibbs, Michael A.; Eng, Lawrence F.; Wu, Jang-Yen

1987-01-01

Morphological evidence is presented of the existence of the putative neurotransmitter gamma-aminobutyric acid (GABA) in axon terminals and of glutamine synthetase (GS) in ependymoglial cells and astroglial components of the area postrema (AP) of the cat. Purified antiserum directed against the GABA biosynthetic enzyme glutamic acid decarboxylase (GAD) and GS antiserum were used. The results showed that punctate structures of variable size corresponding to axon terminals exhibited GAD-immunoreactivity and were distributed in varying densities. The greatest accumulation occurred in the caudal and middle segment of the AP and particularly in the area subpostrema, where the aggregation of terminals was extremely dense. The presence of both GAD-immunoreactive profiles and GS-immunostained ependymoglial cells and astrocytes in the AP provide further evidence of the functional correlation between the two enzymes.

Polycomb repressive complex 1 modifies transcription of active genes

PubMed Central

Pherson, Michelle; Misulovin, Ziva; Gause, Maria; Mihindukulasuriya, Kathie; Swain, Amanda; Dorsett, Dale

2017-01-01

This study examines the role of Polycomb repressive complex 1 (PRC1) at active genes. The PRC1 and PRC2 complexes are crucial for epigenetic silencing during development of an organism. They are recruited to Polycomb response elements (PREs) and establish silenced domains over several kilobases. Recent studies show that PRC1 is also directly recruited to active genes by the cohesin complex. Cohesin participates broadly in control of gene transcription, but it is unknown whether cohesin-recruited PRC1 also plays a role in transcriptional control of active genes. We address this question using genome-wide RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq). The results show that PRC1 influences transcription of active genes, and a significant fraction of its effects are likely direct. The roles of different PRC1 subunits can also vary depending on the gene. Depletion of PRC1 subunits by RNA interference alters phosphorylation of RNA polymerase II (Pol II) and occupancy by the Spt5 pausing-elongation factor at most active genes. These effects on Pol II phosphorylation and Spt5 are likely linked to changes in elongation and RNA processing detected by nascent RNA-seq, although the mechanisms remain unresolved. The experiments also reveal that PRC1 facilitates association of Spt5 with enhancers and PREs. Reduced Spt5 levels at these regulatory sequences upon PRC1 depletion coincide with changes in Pol II occupancy and phosphorylation. Our findings indicate that, in addition to its repressive roles in epigenetic gene silencing, PRC1 broadly influences transcription of active genes and may suppress transcription of nonpromoter regulatory sequences. PMID:28782042

Expression of glnB and a glnB-Like Gene (glnK) in a Ribulose Bisphosphate Carboxylase/Oxygenase-Deficient Mutant of Rhodobacter sphaeroides

PubMed Central

Qian, Yilei; Tabita, F. Robert

1998-01-01

In a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient mutant of Rhodobacter sphaeroides, strain 16PHC, nitrogenase activity was derepressed in the presence of ammonia under photoheterotrophic growth conditions. Previous studies also showed that reintroduction of a functional RubisCO and Calvin-Benson-Bassham (CBB) pathway suppressed the deregulation of nitrogenase synthesis in this strain. In this study, the derepression of nitrogenase synthesis in the presence of ammonia in strain 16PHC was further explored by using a glnB::lacZ fusion, since the product of the glnB gene is known to have a negative effect on ammonia-regulated nif control. It was found that glnB expression was repressed in strain 16PHC under photoheterotrophic growth conditions with either ammonia or glutamate as the nitrogen source; glutamine synthetase (GS) levels were also affected in this strain. However, when cells regained a functional CBB pathway by trans complementation of the deleted genes, wild-type levels of GS and glnB expression were restored. Furthermore, a glnB-like gene, glnK, was isolated from this organism, and its expression was found to be under tight nitrogen control in the wild type. Surprisingly, glnK expression was found to be derepressed in strain 16PHC under photoheterotrophic conditions in the presence of ammonia. PMID:9721307

A deep intronic mutation in the SLC12A3 gene leads to Gitelman syndrome.

PubMed

Nozu, Kandai; Iijima, Kazumoto; Nozu, Yoshimi; Ikegami, Ei; Imai, Takehide; Fu, Xue Jun; Kaito, Hiroshi; Nakanishi, Koichi; Yoshikawa, Norishige; Matsuo, Masafumi

2009-11-01

Many mutations have been detected in the SLC12A3 gene of Gitelman syndrome (GS, OMIM 263800) patients. In previous studies, only one mutant allele was detected in approximately 20 to 41% of patients with GS; however, the exact reason for the nonidentification has not been established. In this study, we used RT-PCR using mRNA to investigate for the first time transcript abnormalities caused by deep intronic mutation. Direct sequencing analysis of leukocyte DNA identified one base insertion in exon 6 (c.818_819insG), but no mutation was detected in another allele. We analyzed RNA extracted from leukocytes and urine sediments and detected unknown sequence containing 238bp between exons 13 and 14. The genomic DNA analysis of intron 13 revealed a single-base substitution (c.1670-191C>T) that creates a new donor splice site within the intron resulting in the inclusion of a novel cryptic exon in mRNA. This is the first report of creation of a splice site by a deep intronic single-nucleotide change in GS and the first report to detect the onset mechanism in a patient with GS and missing mutation in one allele. This molecular onset mechanism may partly explain the poor success rate of mutation detection in both alleles of patients with GS.

Lipase Activity in Insect Oral Secretions Mediates Defense Responses in Arabidopsis1[C][W][OA

PubMed Central

Schäfer, Martin; Fischer, Christine; Meldau, Stefan; Seebald, Eileen; Oelmüller, Ralf; Baldwin, Ian T.

2011-01-01

How plants perceive herbivory is not yet well understood. We investigated early responses of the model plant Arabidopsis (Arabidopsis thaliana) to attack from the generalist grasshopper herbivore, Schistocerca gregaria (Caelifera). When compared with wounding alone, S. gregaria attack and the application of grasshopper oral secretions (GS) to puncture wounds elicited a rapid accumulation of various oxylipins, including 13-hydroperoxy octadecatrienoic acid, 12-oxo-phytodienoic acid (OPDA), jasmonic acid, and jasmonic acid-isoleucine. Additionally, GS increased cytosolic calcium levels, mitogen-activated protein kinase (MPK3 and MPK6) activity, and ethylene emission but not the accumulation of hydrogen peroxide. Although GS contain caeliferin A16:0, a putative elicitor of caeliferan herbivores, treatment with pure, synthetic caeliferin A16:0 did not induce any of the observed responses. With mutant plants, we demonstrate that the observed changes in oxylipin levels are independent of MPK3 and MPK6 activity but that MPK6 is important for the GS-induced ethylene release. Biochemical and pharmacological analyses revealed that the lipase activity of GS plays a central role in the GS-induced accumulation of oxylipins, especially OPDA, which could be fully mimicked by treating puncture wounds only with a lipase from Rhizopus arrhizus. GS elicitation increased the levels of OPDA-responsive transcripts. Because the oral secretions of most insects used to study herbivory-induced responses in Arabidopsis rapidly elicit similar accumulations of OPDA, we suggest that lipids containing OPDA (arabidopsides) play an important role in the activation of herbivory-induced responses. PMID:21546453

Transgenerational effects of the endocrine disruptor vinclozolin on the methylation pattern of imprinted genes in the mouse sperm.

PubMed

Stouder, Christelle; Paoloni-Giacobino, Ariane

2010-02-01

Endocrine-disrupting chemicals (EDCs), among which is the antiandrogen vinclozolin (VCZ), have been reported to affect the male reproductive system. In this study, VCZ was administered to pregnant mice at the time of embryo sex determination, and its possible effects on the differentially methylated domains (DMDs) of two paternally (H19 and Gtl2) and three maternally (Peg1, Snrpn, and Peg3) imprinted genes were tested in the male offspring. The CpGs methylation status within the five gene DMDs was analyzed in the sperm, tail, liver, and skeletal muscle DNAs by pyrosequencing. In the sperm of controls, the percentages of methylated CpGs were close to the theoretical values of 100 and 0% in paternally or maternally imprinted genes respectively. VCZ decreased the percentages of methylated CpGs of H19 and Gtl2 (respective values 83.1 and 91.5%) and increased those of Peg1, Snrpn, and Peg3 (respective values 11.3, 18.3, and 11.2%). The effects of VCZ were transgenerational, but they disappeared gradually from F1 to F3. The mean sperm concentration of the VCZ-administered female offspring was only 56% of that of the controls in the F1 offspring, and it was back to normal values in the F2 and F3 offspring. In the somatic cells of controls, the percentages of methylated CpGs were close to the theoretical value of 50% and, surprisingly, VCZ altered the methylation of Peg3. We propose that the deleterious effects of VCZ on the male reproductive system are mediated by imprinting defects in the sperm. The reported effects of EDCs on human male spermatogenesis might be mediated by analogous imprinting alterations.

MAXI/GSC detection of a hard-to-soft transition of NS-LMXB GS 1826-238

NASA Astrophysics Data System (ADS)

Nakahira, S.; Mihara, T.; Sugizaki, M.; Serino, M.; Morii, M.; Sugimoto, J.; Takagi, T.; Yoshikawa, A.; Matsuoka, M.; Kawai, N.; Yoshii, T.; Tachibana, Y.; Ueno, S.; Tomida, H.; Kimura, M.; Ishikawa, M.; Nakagawa, Y. E.; Negoro, H.; Nakajima, M.; Fukushima, K.; Onodera, T.; Suzuki, K.; Fujita, M.; Namba, T.; Honda, F.; Yoshida, A.; Sakamoto, T.; Kawakubo, Y.; Ohtsuki, H.; Tsunemi, H.; Uchida, D.; Ueda, Y.; Shidatsu, M.; Kawamuro, T.; Hori, T.; Kawagoe, A.; Tsuboi, Y.; Yamauchi, M.; Morooka, Y.; Yamaoka, K.

2014-06-01

We report on a hard-to-soft spectral transition of an X-ray burster GS 1826-238 detected with MAXI/GSC. This source had been consistently observed in the hard state for more than 25 years since the discovery in 1988 (Tanaka 1989, Barret et al. ...

Identification and analysis of low molecular weight dissolved organic carbon in subglacial basal ice ecosystems by ion chromatography

NASA Astrophysics Data System (ADS)

Lawson, E. C.; Wadham, J. L.; Lis, G. P.; Tranter, M.; Pickard, A. E.; Stibal, M.; Dewsbury, P.; Fitzsimons, S.

2015-08-01

Glacial runoff is an important source of dissolved organic carbon (DOC) for downstream heterotrophic activity, despite the low overall DOC concentrations. This is because of the abundance of bioavailable, low molecular weight (LMW) DOC species. However, the provenance and character of LMW-DOC is not fully understood. We investigated the abundance and composition of DOC in subglacial environments via a molecular level DOC analysis of basal ice, which forms by water/sediment freeze-on to the glacier sole. Spectrofluorometry and a novel ion chromatographic method, which has been little utilised in glacial science for LMW-DOC determinations, were employed to identify and quantify the major LMW fractions (free amino acids, carbohydrates and carboxylic acids) in basal ice from four glaciers, each with a different basal debris type. Basal ice from Joyce Glacier (Antarctica) was unique in that 98 % of the LMW-DOC was derived from the extremely diverse FAA pool, comprising 14 FAAs. LMW-DOC concentrations in basal ice were dependent on the bioavailability of the overridden organic carbon (OC), which in turn, was influenced by the type of overridden material. Mean LMW-DOC concentrations in basal ice from Russell Glacier (Greenland), Finsterwalderbreen (Svalbard) and Engabreen (Norway) were low (0-417 nM C), attributed to the relatively refractory nature of the OC in the overridden paleosols and bedrock. In contrast, mean LMW-DOC concentrations were an order of magnitude higher (4430 nM C) in basal ice from Joyce Glacier, a reflection of the high bioavailability of the overridden lacustrine material (>17 % of the sediment OC comprised extractable carbohydrates, a proxy for bioavailable OC). We find that the overridden material may act as a direct (via abiotic leaching) and indirect (via microbial cycling) source of DOC to the subglacial environment and provides a range of LMW-DOC compounds that may stimulate microbial activity in wet sediments in current subglacial

Low molecular weight hyaluronic acid increases the self-defense of skin epithelium by induction of beta-defensin 2 via TLR2 and TLR4.

PubMed

Gariboldi, Silvia; Palazzo, Marco; Zanobbio, Laura; Selleri, Silvia; Sommariva, Michele; Sfondrini, Lucia; Cavicchini, Stefano; Balsari, Andrea; Rumio, Cristiano

2008-08-01

In sites of inflammation or tissue injury, hyaluronic acid (HA), ubiquitous in the extracellular matrix, is broken down into low m.w. HA (LMW-HA) fragments that have been reported to activate immunocompetent cells. We found that LMW-HA induces activation of keratinocytes, which respond by producing beta-defensin 2. This production is mediated by TLR2 and TLR4 activation and involves a c-Fos-mediated, protein kinase C-dependent signaling pathway. LMW-HA-induced activation of keratinocytes seems not to be accompanied by an inflammatory response, because no production of IL-8, TNF-alpha, IL-1beta, or IL-6 was observed. Ex vivo and in vivo treatments of murine skin with LMW-HA showed a release of mouse beta-defensin 2 in all layers of the epidermal compartment. Therefore, the breakdown of extracellular matrix components, for example after injury, stimulates keratinocytes to release beta-defensin 2, which protects cutaneous tissue at a time when it is particularly vulnerable to infection. In addition, our observation might be important to open new perspectives in the development of possible topical products containing LMW-HA to improve the release of beta-defensins by keratinocytes, thus ameliorating the self-defense of the skin for the protection of cutaneous tissue from infection by microorganisms.

Identification of Three Alcohol Dehydrogenase Genes Involved in the Stereospecific Catabolism of Arylglycerol-β-Aryl Ether by Sphingobium sp. Strain SYK-6▿ †

PubMed Central

Sato, Yusuke; Moriuchi, Hideki; Hishiyama, Shojiro; Otsuka, Yuichiro; Oshima, Kenji; Kasai, Daisuke; Nakamura, Masaya; Ohara, Seiji; Katayama, Yoshihiro; Fukuda, Masao; Masai, Eiji

2009-01-01

Degradation of arylglycerol-β-aryl ether is the most important process in bacterial lignin catabolism. Sphingobium sp. strain SYK-6 degrades guaiacylglycerol-β-guaiacyl ether (GGE) to α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV), and then the ether linkage of MPHPV is cleaved to generate α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol. We have characterized three enantioselective glutathione S-transferase genes, including two genes that are involved in the ether cleavage of two enantiomers of MPHPV and one gene that is involved in the elimination of glutathione from a GS-HPV enantiomer. However, the first step in the degradation of four different GGE stereoisomers has not been characterized. In this study, three alcohol dehydrogenase genes, ligL, ligN, and ligO, which conferred GGE transformation activity in Escherichia coli, were isolated from SYK-6 and characterized, in addition to the previously cloned ligD gene. The levels of amino acid sequence identity of the four GGE dehydrogenases, which belong to the short-chain dehydrogenase/reductase family, ranged from 32% to 39%. Each gene was expressed in E. coli, and the stereospecificities of the gene products with the four GGE stereoisomers were determined by using chiral high-performance liquid chromatography with recently synthesized authentic enantiopure GGE stereoisomers. LigD and LigO converted (αR,βS)-GGE and (αR,βR)-GGE into (βS)-MPHPV and (βR)-MPHPV, respectively, while LigL and LigN transformed (αS,βR)-GGE and (αS,βS)-GGE to (βR)-MPHPV and (βS)-MPHPV, respectively. Disruption of the genes indicated that ligD is essential for the degradation of (αR,βS)-GGE and (αR,βR)-GGE and that both ligL and ligN contribute to the degradation of the two other GGE stereoisomers. PMID:19542348

Genetic divergence for high-molecular weight glutenin subunits (HMW-GS) in indigenous landraces and commercial cultivars of bread wheat of Pakistan.

PubMed

Yasmeen, F; Khurshid, H; Ghafoor, A

2015-05-11

Wheat flour quality is an important consideration in the breeding and development of new cultivars. A strong association between high-molecular weight glutenin subunits (HMW-GS) and bread making quality has resulted in the widespread utilization of HMW-GS in wheat breeding. In this study, we analyzed 242 lines of wheat, including landraces from the provinces of Punjab and Baluchistan, as well as the commercial varieties of Pakistan, to determine allelic variation in the Glu-A1, Glu-B1, and Glu-D1 loci encoding HMW-GS. Higher genetic diversity was observed for HMW-GS in landraces from Baluchistan, followed by landraces collected from Punjab and then commercial varieties. Rare and uncommon subunits were observed in Glu-B1, whereas Glu-A1 was less polymorphic. However, Glu-B1 was the highest contributor to overall diversity (78%), with a total of 31 rare alleles, followed by Glu-D1 (20%) with the high quality 5+10 allele and other variants. Commercial cultivars possessed favorable alleles, potentially from indirect selection for wheat flour quality by the breeders; however, this indirect selection has decreased the pedigree base of commercial cultivars. The allelic combinations, including 2*, 5+10, and 17+18, showing high quality scores were frequent among landraces, indicating their usefulness in future crop improvement and breeding programs.

Investigation of the multifunctional gene AOP3 expands the regulatory network fine-tuning glucosinolate production in Arabidopsis

PubMed Central

Jensen, Lea M.; Kliebenstein, Daniel J.; Burow, Meike

2015-01-01

Quantitative trait loci (QTL) mapping studies enable identification of loci that are part of regulatory networks controlling various phenotypes. Detailed investigations of genes within these loci are required to ultimately understand the function of individual genes and how they interact with other players in the network. In this study, we use transgenic plants in combination with natural variation to investigate the regulatory role of the AOP3 gene found in GS-AOP locus previously suggested to contribute to the regulation of glucosinolate defense compounds. Phenotypic analysis and QTL mapping in F2 populations with different AOP3 transgenes support that the enzymatic function and the AOP3 RNA both play a significant role in controlling glucosinolate accumulation. Furthermore, we find different loci interacting with either the enzymatic activity or the RNA of AOP3 and thereby extend the regulatory network controlling glucosinolate accumulation. PMID:26442075

Lengsin is a survivor of an ancient family of class I glutamine synthetases in eukaryotes that has undergone evolutionary re-engineering for a role in the vertebrate eye lens.

PubMed Central

Wyatt, Keith; White, Helen E.; Wang, Luchun; Bateman, Orval A.; Slingsby, Christine; Orlova, Elena V.; Wistow, Graeme

2006-01-01

Summary Lengsin is a member of the glutamine synthetase (GS) superfamily expressed in the vertebrate eye lens. While the GS homology domains of lengsin are well conserved in all species examined, the N-terminal domain shows evidence of dynamic evolutionary changes. Compared with zebrafish and chicken, most mammals have an additional exon in the lengsin gene corresponding to part of the N-terminal domain, however in human this is a non-functional pseudoexon. Lengsin belongs to the hitherto purely prokaryotic GS I branch of the GS superfamily. Cryo-EM and modeling studies of lengsin show a dodecamer structure with important similarities and differences with GS I structures. There are sequence changes in GS catalytic motifs of lengsin and in multiple enzyme assays recombinant mouse lengsin showed no activity. Like the taxon-specific crystallins, lengsin may be the result of the recruitment of an ancient enzyme to a different, non-catalytic role in the vertebrate lens. Genomics data show that protein-coding genes related to lengsin are present in the Sea urchin, suggesting that this branch of the GS I family has an ancient role in metazoans. PMID:17161372

Environmental Conditions Influence Induction of Key ABC-Transporter Genes Affecting Glyphosate Resistance Mechanism in Conyza canadensis.

PubMed

Tani, Eleni; Chachalis, Demosthenis; Travlos, Ilias S; Bilalis, Dimitrios

2016-04-20

Conyza canadensis has been reported to be the most frequent weed species that evolved resistance to glyphosate in various parts of the world. The objective of the present study was to investigate the effect of environmental conditions (temperature and light) on the expression levels of the EPSPS gene and two major ABC-transporter genes (M10 and M11) on glyphosate susceptible (GS) and glyphosate resistant (GR) horseweed populations, collected from several regions across Greece. Real-time PCR was conducted to determine the expression level of the aforementioned genes when glyphosate was applied at normal (1×; 533 g·a.e.·ha(-1)) and high rates (4×, 8×), measured at an early one day after treatment (DAT) and a later stage (four DAT) of expression. Plants were exposed to light or dark conditions, at three temperature regimes (8, 25, 35 °C). GR plants were made sensitive when exposed to 8 °C with light; those sensitized plants behaved biochemically (shikimate accumulation) and molecularly (expression of EPSPS and ABC-genes) like the GS plants. Results from the current study show the direct link between the environmental conditions and the induction level of the above key genes that likely affect the efficiency of the proposed mechanism of glyphosate resistance.

Environmental Conditions Influence Induction of Key ABC-Transporter Genes Affecting Glyphosate Resistance Mechanism in Conyza canadensis

PubMed Central

Tani, Eleni; Chachalis, Demosthenis; Travlos, Ilias S.; Bilalis, Dimitrios

2016-01-01

Conyza canadensis has been reported to be the most frequent weed species that evolved resistance to glyphosate in various parts of the world. The objective of the present study was to investigate the effect of environmental conditions (temperature and light) on the expression levels of the EPSPS gene and two major ABC-transporter genes (M10 and M11) on glyphosate susceptible (GS) and glyphosate resistant (GR) horseweed populations, collected from several regions across Greece. Real-time PCR was conducted to determine the expression level of the aforementioned genes when glyphosate was applied at normal (1×; 533 g·a.e.·ha−1) and high rates (4×, 8×), measured at an early one day after treatment (DAT) and a later stage (four DAT) of expression. Plants were exposed to light or dark conditions, at three temperature regimes (8, 25, 35 °C). GR plants were made sensitive when exposed to 8 °C with light; those sensitized plants behaved biochemically (shikimate accumulation) and molecularly (expression of EPSPS and ABC-genes) like the GS plants. Results from the current study show the direct link between the environmental conditions and the induction level of the above key genes that likely affect the efficiency of the proposed mechanism of glyphosate resistance. PMID:27104532

Rapid actions of calcitriol and its side chain analogues CB1093 and GS1500 on intracellular calcium levels in skeletal muscle cells: a comparative study

PubMed Central

Vazquez, Guillermo; Sellés, Juana; de Boland, Ana Russo; Boland, Ricardo

1999-01-01

The ability of synthetic analogues of the secosteroid hormone 1α,25-dihydroxy-vitamin-D3 [calcitriol, CT; 1,25(OH)2D3] to exert non-genomic (rapid) effects on target cells has been scarcely studied. To evaluate the pharmacological potential of the CT side-chain analogues CB1093 and GS1500, we compared their fast effects on intracellular calcium concentration ([Ca2+]i) in chick skeletal muscle cells with those elicited by the natural hormone.Both analogues, similarly to CT, specifically induced rapid (30–60 s) and sustained rises in [Ca2+]i levels. CB1093 and GS1500 were more potent than the natural hormone at concentrations as low as 10−13 M (4.5 fold stimulation) and 10−12 M (2.5 fold), respectively, whereas higher concentrations (10−9–10−8 M) of CT were more effective than the analogues in elevating [Ca2+]i. Cyclic AMP was markedly increased by both analogues pointing for a role of this messenger in the fast actions of the synthetic compounds.In Ca2+ free medium CT and analogues elicited a transient elevation in [Ca2+]i. The PLC inhibitors U73122 (2 μM) and neomycin (0.5 mM), as well as depletion of intracellular stores with thapsigargin (1 μM), completely prevented CB1093/GS1500-dependent changes in [Ca2+]i suggesting that, similarly to CT, these analogues mobilized Ca2+ from an IP3/thapsigargin-sensitive store.The voltage-dependent calcium channel (VDCC) blocker nifedipine (2 μM) reduced by 50–60% the influx phase of the [Ca2+]i response to CB1093 and GS1500, indicating that VDCC contributed partially to Ca2+ entry. The Ca2+ readdition protocol suggested that analogue-dependent activation of a SOC entry pathway accounted, to the same extent as for CT, for the remaining non-VDCC mediated Ca2+ influx. PMID:10372825

The toxic effects of diethyl phthalate on the activity of glutamine synthetase in greater duckweed (Spirodela polyrhiza L.).

PubMed

Cheng, Tai-Sheng

2012-11-15

The toxic effects of diethyl phthalate (DEP), a potent allelochemical, on the enzyme activity and polypeptide accumulation of glutamine synthetase (GS) in greater duckweed were investigated. In our previous studies, DEP induced oxidative responses at concentrations from 0.5 to 2 mM in greater duckweed and the antioxidant enzymes played important roles in the defense strategy against DEP stress. In this study, DAB-H(2)O(2) and NBT stain for superoxide radicals (O(2)(·-)), lipid peroxidation, HSP70, and ammonia accumulation in DEP-treated duckweed tissues revealed adverse effect of DEP in plant growth. Biochemical analysis and physiological methods were combined to investigate GS activity and polypeptide accumulation under DEP-induced stress. The results showed that GS activity was reduced with the increasing concentration of DEP, indicative of enhanced toxic effect. Immunoblot analysis with chloroplast soluble fractions indicated that the chloroplastic GS (GS2) polypeptide from greater duckweed was degraded under DEP stress conditions. The response of GS2 to the DEP stress may be modulated by means of redox change in plant tissues, chloroplasts, and chloroplast lysates. The results suggest that DEP is toxic to the greater duckweed by inhibition of the GS isoenzymes in nitrogen assimilation and the GS2 plays important roles in the adaptation strategy against DEP toxicity. Copyright © 2012 Elsevier B.V. All rights reserved.

THE FAST DECLINING TYPE Ia SUPERNOVA 2003gs, AND EVIDENCE FOR A SIGNIFICANT DISPERSION IN NEAR-INFRARED ABSOLUTE MAGNITUDES OF FAST DECLINERS AT MAXIMUM LIGHT

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

Krisciunas, Kevin; Marion, G. H.; Suntzeff, Nicholas B.

2009-12-15

We obtained optical photometry of SN 2003gs on 49 nights, from 2 to 494 days after T(B {sub max}). We also obtained near-IR photometry on 21 nights. SN 2003gs was the first fast declining Type Ia SN that has been well observed since SN 1999by. While it was subluminous in optical bands compared to more slowly declining Type Ia SNe, it was not subluminous at maximum light in the near-IR bands. There appears to be a bimodal distribution in the near-IR absolute magnitudes of Type Ia SNe at maximum light. Those that peak in the near-IR after T(B {sub max})more » are subluminous in the all bands. Those that peak in the near-IR prior to T(B {sub max}), such as SN 2003gs, have effectively the same near-IR absolute magnitudes at maximum light regardless of the decline rate {delta}m {sub 15}(B). Near-IR spectral evidence suggests that opacities in the outer layers of SN 2003gs are reduced much earlier than for normal Type Ia SNe. That may allow {gamma} rays that power the luminosity to escape more rapidly and accelerate the decline rate. This conclusion is consistent with the photometric behavior of SN 2003gs in the IR, which indicates a faster than normal decline from approximately normal peak brightness.« less

Determination of the Michaelis-Menten kinetics and the genes expression involved in phyto-degradation of cyanide and ferri-cyanide.

PubMed

Yu, Xiao-Zhang; Zhang, Xue-Hong

2016-07-01

Hydroponic experiments were conducted with different species of plants (rice, maize, soybean and willow) exposed to ferri-cyanide to investigate the half-saturation constant (K M ) and the maximal metabolic capacity (v max ) involved in phyto-assimilation. Three varieties for each testing species were collected from different origins. Measured concentrations show that the uptake rates responded biphasically to ferri-cyanide treatments by showing increases linearly at low and almost constant at high concentrations from all treatments, indicating that phyto-assimilation of ferri-cyanide followed the Michaelis-Menten kinetics. Using non-linear regression, the highest v max was by rice, followed by willows. The lowest v max was found for soybean. All plants, except maize (DY26) and rice (XJ12), had a similar K M value, suggesting the same enzyme was active in phyto-assimilation of ferri-cyanide. Transcript level, by real-time quantitative PCR, of enzymes involved in degradation of cyanides showed that the analyzed genes were differently expressed during different cyanides exposure. The expression of CAS and ST genes responded positively to KCN exposure, suggesting that β-CAS and ST pathways were two possible pathways for cyanide detoxification in rice. The transcript level of NIT and ASPNASE genes also showed a remarkable up-regulation to KCN, implying the contribution to the pool of amino acid aspartate, which is an end product of CN metabolism. Up-regulation of GS genes suggests that acquisition of ammonium released from cyanide degradation may be an additional nitrogen source for plant nutrition. Results also revealed that the expressions of these genes, except for GS, were relatively constant during iron cyanide exposure, suggesting that they are likely metabolized by plants through a non-defined pathway rather than the β-CAS pathway.

Beta-catenin interacts with low-molecular-weight protein tyrosine phosphatase leading to cadherin-mediated cell-cell adhesion increase.

PubMed

Taddei, Maria Letizia; Chiarugi, Paola; Cirri, Paolo; Buricchi, Francesca; Fiaschi, Tania; Giannoni, Elisa; Talini, Doriana; Cozzi, Giacomo; Formigli, Lucia; Raugei, Giovanni; Ramponi, Giampietro

2002-11-15

Beta-catenin plays a dual role as a major constituent of cadherin-based adherens junctions and also as a transcriptional coactivator. In normal ephitelial cells, at adherens junction level, beta-catenin links cadherins to the actin cytoskeleton. The structure of adherens junctions is dynamically regulated by tyrosine phosphorylation. In particular, cell-cell adhesion can be negatively regulated through the tyrosine phosphorylation of beta-catenin. Furthermore, the loss of beta-catenin-cadherin association has been correlated with the transition from a benign tumor to an invasive, metastatic cancer. Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is a ubiquitous PTP implicated in the regulation of mitosis and cytoskeleton rearrangement. Here we demonstrate that the amount of free cytoplasmic beta-catenin is decreased in NIH3T3, which overexpresses active LMW-PTP, and this results in a stronger association between cadherin complexes and the actin-based cytoskeleton with respect to control cells. Confocal microscopy analysis shows that beta-catenin colocalizes with LMW-PTP at the plasma membrane. Furthermore, we provide evidence that beta-catenin is able to associate with LMW-PTP both in vitro and in vivo. Moreover, overexpression of active LMW-PTP strongly potentiates cadherin-mediated cell-cell adhesion, whereas a dominant-negative form of LMW-PTP induces the opposite phenotype, both in NIH3T3 and in MCF-7 carcinoma cells. On the basis of these results, we propose that the stability of cell-cell contacts at the adherens junction level is positively influenced by LMW-PTP expression, mainly because of the beta-catenin and LMW-PTP interaction at the plasma membrane level with consequent dephosphorylation.

Assessment of leg muscle activity using toe tapping in patients with Parkinson's disease: comparison of two types of toe tapping.

PubMed

Taniguchi, Seira; Peper, Ferdinand; Shimokawa, Tetsuya

2018-05-01

[Purpose] This study investigates two types of toe tapping, i.e., "closed," with both feet on the floor, and "open," in which the foot does not touch the ground, and evaluates their usefulness in combination with monitoring of muscle activity during toe tapping. [Subjects and Methods] The study enrolled 11 patients with Parkinson's disease (PD) and 9 controls (Controls). The tibialis anterior (TA) and gastrocnemius (GS) muscle activity during toe tapping was measured using surface electromyography. [Results] In closed tapping, the minima in GS activation with the first tap was significantly higher in patients with PD than in Controls. In open tapping, the coefficient of variation (CV) of local maxima in TA activation was significantly higher in patients with PD than in Controls. In both types of tapping, the CV of extrema in GS activities increased with disease duration, but this may be due to the long-term administration of Levodopa, which itself tends to cause excessive GS activities. [Conclusion] Closed tapping is suitable for the assessment of GS activity and can detect excessive activities, which is observed as visible movement. Open tapping, on the other hand, is suitable for assessment of TA activity.

A Sequence of Outbursts from the Transient X-Ray Pulsar GS 0834-430

NASA Technical Reports Server (NTRS)

Wilson, Colleen A.; Finger, Mark H.; Harmon, B.Alan; Scott, D. Matthew; Wilson, Robert B.; Bildsten, Lars; Chakrabarty, Deepto; Prince, Thomas A.

1997-01-01

GS 0834-430, a 12.3 s accretion-powered pulsar, has been observed in seven outbursts with the BATSE large-area detectors on the Compton Gamma Ray Observatory. The first five outbursts observed by BATSE occurred at intervals of about 107 days, while the final two outbursts were separated by about 140 days. The photon energy spectrum, measured by Earth occultation in the 20 100 keV band, can be fitted by a power law with photon index alpha approximately equals -3.7 or by an exponential spectrum with temperature kT approximately equals 15 keV, with some variations within outbursts. The source has a low pulse fraction, less than or equal to 0.15 in the 20-50 keV band. We have observed significant temporal and energy-dependent variations in epoch folded pulse profiles. Because the intrinsic torque effects for this system are at least comparable to orbital effects, pulse timing analysis did not produce a unique orbital solution. However, confidence regions for the orbital elements yielded the following 1 sigma limits: orbital period P(sub orb) = 105.8 +/- 0.4 days and eccentricity 0.10 less than or approximately equals epsilon less than or approximately equals 0.17. GS 0834-430 is most likely a Be/X-ray binary.

A 10 GS/s time-interleaved ADC in 0.25 micrometer CMOS technology

NASA Astrophysics Data System (ADS)

Aytar, Oktay; Tangel, Ali; Afacan, Engin

2017-11-01

This paper presents design and simulation of a 4-bit 10 GS/s time interleaved ADC in 0.25 micrometer CMOS technology. The designed TI-ADC has 4 channels including 4-bit flash ADC in each channel, in which area and power efficiency are targeted. Therefore, basic standard cell logic gates are preferred. Meanwhile, the aspect ratios in the gate designs are kept as small as possible considering the speed performance. In the literature, design details of the timing control circuits have not been provided, whereas the proposed timing control process is comprehensively explained and design details of the proposed timing control process are clearly presented in this study. The proposed circuits producing consecutive pulses for timing control of the input S/H switches (ie the analog demultiplexer front-end circuitry) and the very fast digital multiplexer unit at the output are the main contributions of this study. The simulation results include +0.26/-0.22 LSB of DNL and +0.01/-0.44 LSB of INL, layout area of 0.27 mm2, and power consumption of 270 mW. The provided power consumption, DNL and INL measures are observed at 100 MHz input with 10 GS/s sampling rate.

Constitutive androstane receptor activation evokes the expression of glycolytic genes

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

Yarushkin, Andrei A.; Kazantseva, Yuliya A.; Prokopyeva, Elena A.

It is well-known that constitutive androstane receptor (CAR) activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) increases the liver-to-body weight ratio. CAR-mediated liver growth is correlated with increased expression of the pleiotropic transcription factor cMyc, which stimulates cell cycle regulatory genes and drives proliferating cells into S phase. Because glycolysis supports cell proliferation and cMyc is essential for the activation of glycolytic genes, we hypothesized that CAR-mediated up-regulation of cMyc in mouse livers might play a role in inducing the expression of glycolytic genes. The aim of the present study was to examine the effect of long-term CAR activation on glycolytic genes in amore » mouse model not subjected to metabolic stress. We demonstrated that long-term CAR activation by TCPOBOP increases expression of cMyc, which was correlated with reduced expression of gluconeogenic genes and up-regulation of glucose transporter, glycolytic and mitochondrial pyruvate metabolising genes. These changes in gene expression after TCPOBOP treatment were strongly correlated with changes in levels of glycolytic intermediates in mouse livers. Moreover, we demonstrated a significant positive regulatory effect of TCPOBOP-activated CAR on both mRNA and protein levels of Pkm2, a master regulator of glucose metabolism and cell proliferation. Thus, our findings provide evidence to support the conclusion that CAR activation initiates a transcriptional program that facilitates the coordinated metabolic activities required for cell proliferation. - Highlights: • CAR-mediated liver growth is correlated with increased expression of cMyc. • CAR activation increased the expression of glycolytic genes in mouse livers. • CAR activation increased the level of Pkm2 in mouse livers.« less

Mechanisms of specificity in neuronal activity-regulated gene transcription

PubMed Central

Lyons, Michelle R.; West, Anne E.

2011-01-01

The brain is a highly adaptable organ that is capable of converting sensory information into changes in neuronal function. This plasticity allows behavior to be accommodated to the environment, providing an important evolutionary advantage. Neurons convert environmental stimuli into long-lasting changes in their physiology in part through the synaptic activity-regulated transcription of new gene products. Since the neurotransmitter-dependent regulation of Fos transcription was first discovered nearly 25 years ago, a wealth of studies have enriched our understanding of the molecular pathways that mediate activity-regulated changes in gene transcription. These findings show that a broad range of signaling pathways and transcriptional regulators can be engaged by neuronal activity to sculpt complex programs of stimulus-regulated gene transcription. However, the shear scope of the transcriptional pathways engaged by neuronal activity raises the question of how specificity in the nature of the transcriptional response is achieved in order to encode physiologically relevant responses to divergent stimuli. Here we summarize the general paradigms by which neuronal activity regulates transcription while focusing on the molecular mechanisms that confer differential stimulus-, cell-type-, and developmental-specificity upon activity-regulated programs of neuronal gene transcription. In addition, we preview some of the new technologies that will advance our future understanding of the mechanisms and consequences of activity-regulated gene transcription in the brain. PMID:21620929

20. FOUR 4B17Gs BEING CONVERTED TO F9Cs. Photographic copy of ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

20. FOUR 4B-17Gs BEING CONVERTED TO F-9Cs. Photographic copy of historic photograph. Jan.-June 1947 OAMA (original print located at Ogden Air Logistics Center, Hill Air Force Base, Utah). Photographer unknown. - Hill Field, Airplane Repair Hangars No. 1-No. 4, 5875 Southgate Avenue, Layton, Davis County, UT

Expression profiles of sugarcane under drought conditions: Variation in gene regulation.

PubMed

Andrade, Júlio César Farias de; Terto, Jackeline; Silva, José Vieira; Almeida, Cícero

2015-12-01

Drought is a major factor in decreased sugarcane productivity because of the resulting morphophysiological effects that it causes. Gene expression studies that have examined the influence of water stress in sugarcane have yielded divergent results, indicating the absence of a fixed pattern of changes in gene expression. In this work, we investigated the expression profiles of 12 genes in the leaves of a drought-tolerant genotype (RB72910) of sugarcane and compared the results with those of other studies. The genotype was subjected to 80-100% water availability (control condition) and 0-20% water availability (simulated drought). To analyze the physiological status, the SPAD index, Fv/Fm ratio, net photosynthesis (A), stomatal conductance (gs) and stomatal transpiration (E) were measured. Total RNA was extracted from leaves and the expression of SAMDC, ZmPIP2-1 protein, ZmTIP4-2 protein, WIP protein, LTP protein, histone H3, DNAj, ferredoxin I, β-tubulin, photosystem I, gene 1 and gene 2 was analyzed by quantitative real-time PCR (RT-PCR). Important differences in the expression profiles of these genes were observed when compared with other genotypes, suggesting that complex defense mechanisms are activated in response to water stress. However, there was no recognizable pattern for the changes in expression of the different proteins associated with tolerance to drought stress.

Gene Transfer in Eukaryotic Cells Using Activated Dendrimers

NASA Astrophysics Data System (ADS)

Dennig, Jörg

Gene transfer into eukaryotic cells plays an important role in cell biology. Over the last 30 years a number of transfection methods have been developed to mediate gene transfer into eukaryotic cells. Classical methods include co-precipitation of DNA with calcium phosphate, charge-dependent precipitation of DNA with DEAE-dextran, electroporation of nucleic acids, and formation of transfection complexes between DNA and cationic liposomes. Gene transfer technologies based on activated PAMAM-dendrimers provide another class of transfection reagents. PAMAM-dendrimers are highly branched, spherical molecules. Activation of newly synthesized dendrimers involves hydrolytic removal of some of the branches, and results in a molecule with a higher degree of flexibility. Activated dendrimers assemble DNA into compact structures via charge interactions. Activated dendrimer - DNA complexes bind to the cell membrane of eukaryotic cells, and are transported into the cell by non-specific endocytosis. A structural model of the activated dendrimer - DNA complex and a potential mechanism for its uptake into cells will be discussed.

Glutamine synthetase and alanine transaminase expression are decreased in livers of aged vs. young beef cows and GS can be upregulated by 17β-estradiol implants.

PubMed

Miles, E D; McBride, B W; Jia, Y; Liao, S F; Boling, J A; Bridges, P J; Matthews, J C

2015-09-01

Aged beef cows (≥ 8 yr of age) produce calves with lower birth and weaning weights. In mammals, aging is associated with reduced hepatic expression of glutamine synthetase (GS) and alanine transaminase (ALT), thus impaired hepatic Gln-Glu cycle function. To determine if the relative protein content of GS, ALT, aspartate transaminase (AST), glutamate transporters (EAAC1, GLT-1), and their regulating protein (GTRAP3-18) differed in biopsied liver tissue of (a) aged vs. young (3 to 4 yr old) nonlactating, nongestating Angus cows (Exp. 1 and 2) and (b) aged mixed-breed cows with and without COMPUDOSE (17β-estradiol) ear implants (Exp. 3), Western blot analyses were performed. In Exp. 1, 12 young (3.62 ± 0.01 yr) and 13 aged (10.08 ± 0.42 yr) cows grazed the same mixed forage for 42 d (August-October). In Exp. 2, 12 young (3.36 ± 0.01 yr) and 12 aged (10.38 ± 0.47 yr) cows were individually fed (1.03% of BW) a corn-silage-based diet to maintain BW for 20 d. For both Exp. 1 and 2, the effect of cow age was assessed by ANOVA using the MIXED procedure of SAS. Cow BW did not change ( ≥ 0.17). Hepatic ALT (78% and 61%) and GS (52% and 71%) protein content (Exp. 1 and 2, respectively) was decreased ( ≤ 0.01), whereas GTRAP3-18 (an inhibitor of EAAC1 activity) increased ( ≤ 0.01; 170% and 136%) and AST, GLT-1, and EAAC1 contents did not differ ( ≥ 0.17) in aged vs. young cows. In Exp. 2, free concentrations (nmol/g) of Glu, Ala, Gln, Arg, and Orn in liver homogenates were determined. Aged cows tended to have less ( = 0.10) free Gln (15.0%) than young cows, whereas other AA concentrations did not differ ( 0.26). In Exp. 3, 14 aged (> 10 yr) cows were randomly allotted ( = 7) to sham or COMPUDOSE (25.7 mg of 17β-estradiol) implant treatment (TRT), and had ad libitum access to alfalfa hay for 28 d. Blood and liver biopsies were collected 14 and 28 d after implant treatment. Treatment, time after implant (DAY), and TRT × DAY effects were assessed by ANOVA using

Transcriptional regulation of host NH₄⁺ transporters and GS/GOGAT pathway in arbuscular mycorrhizal rice roots.

PubMed

Pérez-Tienda, Jacob; Corrêa, Ana; Azcón-Aguilar, Concepción; Ferrol, Nuria

2014-02-01

Arbuscular mycorrhizal (AM) fungi play a key role in the nutrition of many land plants. AM roots have two pathways for nutrient uptake, directly through the root epidermis and root hairs and via AM fungal hyphae into root cortical cells, where arbuscules or hyphal coils provide symbiotic interfaces. Recent studies demonstrated that the AM symbiosis modifies the expression of plant transporter genes and that NH₄⁺ is the main form of N transported in the symbiosis. The aim of the present work was to get insights into the mycorrhizal N uptake pathway in Oryza sativa by analysing the expression of genes encoding ammonium transporters (AMTs), glutamine synthase (GS) and glutamate synthase (GOGAT) in roots colonized by the AM fungus Rhizophagus irregularis and grown under two N regimes. We found that the AM symbiosis down-regulated OsAMT1;1 and OsAMT1;3 expression at low-N, but not at high-N conditions, and induced, independently of the N status of the plant, a strong up-regulation of OsAMT3;1 expression. The AM-inducible NH₄⁺ transporter OsAMT3;1 belongs to the family 2 of plant AMTs and is phylogenetically related to the AM-inducible AMTs of other plant species. Moreover, for the first time we provide evidence of the specific induction of a GOGAT gene upon colonization with an AM fungus. These data suggest that OsAMT3;1 is likely involved in the mycorrhizal N uptake pathway in rice roots and that OsGOGAT2 plays a role in the assimilation of the NH₄⁺ supplied via the OsAMT3;1 AM-inducible transporter. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Field gamma-ray spectrometer GS256: measurements stability

NASA Astrophysics Data System (ADS)

Mojzeš, Andrej

2009-01-01

The stability of in situ readings of the portable gamma-ray spectrometer GS256 during the field season of 2006 was studied. The instrument is an impulse detector of gamma rays based on NaI(Tl) 3" × 3" scintillation unit and 256-channel spectral analyzer which allows simultaneous assessment of up to 8 radioisotopes in rocks. It is commonly used in surface geophysical survey for the measurement of natural 40K, 238U and 232Th but also artificial 137Cs quantities. The statistical evaluation is given of both repeated measurements - in the laboratory and at several field control points in different survey areas. The variability of values shows both the instrument stability and also the relative influence of some meteorological factors, mainly rainfalls. The analysis shows an acceptable level of instrument measurements stability, the necessity to avoid measurement under unfavourable meteorological conditions and to keep detailed field book information about time, position and work conditions.

Horizontal gene transfer confers adaptive advantages to phytopathogenic fungi: a case study of catalase-peroxidase in Fusarium verticillioides

USDA-ARS?s Scientific Manuscript database

Horizontal gene transfer (HGT), the exchange and stable integration of genetic material between different evolutionary lineages, is widely observed in fungi. We hypothesize that successful stabilization of HGT elements provides adaptive advantages (e.g., virulence). Catalase/peroxidases (KatGs) are ...

Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine.

PubMed

Gersbach, Charles A; Perez-Pinera, Pablo

2014-08-01

New technologies have recently been developed to control the expression of human genes in their native genomic context by engineering synthetic transcription factors that can be targeted to any DNA sequence. The ability to precisely regulate any gene as it occurs naturally in the genome provides a means to address a variety of diseases and disorders. This approach also circumvents some of the traditional challenges of gene therapy. In this editorial, we review the technologies that have enabled targeted human gene activation, including the engineering of transcription factors based on zinc finger proteins, transcription activator-like effectors and the CRISPR/Cas9 system. Additionally, we highlight examples in which these methods have been developed for therapeutic applications and discuss challenges and opportunities.

Comparison of Antibodies with Amylase Activity from Cerebrospinal Fluid and Serum of Patients with Multiple Sclerosis.

PubMed

Doronin, Vasilii B; Parkhomenko, Taisiya A; Castellazzi, Massimiliano; Cesnik, Edward; Buneva, Valentina N; Granieri, Enrico; Nevinsky, Georgy A

2016-01-01

We have recently shown that IgGs from serum and cerebrospinal fluid (CSF) of MS patients are active in hydrolysis of DNA and myelin basic protein. According to literature data, anti-DNA and anti-MBP abzymes may promote important neuropathologic mechanisms in this chronic inflammatory disorder and in MS pathogenesis development. At the same time, the involvement of antibodies with amylase activity in the pathogenesis of any autoimmune disease has not yet been identified. Electrophoretically and immunologically homogeneous IgGs were obtained by a sequential affinity chromatography of the CSF proteins on protein G-Sepharose and FPLC gel filtration. We are able to present the first unpredictable evidence showing that IgGs from CSF possess amylase activity and efficiently hydrolyze maltoheptaose; their average specific Ab activity is ~30-fold higher than that of antibodies from sera of the same MS patients. Specific average RA (SAA) for IgGs from healthy volunteers was approximately ~1000 lower than that for MS patients. In addition, it was shown that a relative SAA of total proteins of CSF (including Abs) ~15-fold lower than that for purified IgGs, while the relative SAA of the total sera protein is higher than that of sera IgGs by a factor of 1033. This result speaks in favor of the fact that amylolytic activity of CSF proteins is mainly caused by the activity of amylase abzymes. One cannot exclude, that amylase abzymes of CSF can play a, as yet unknown, role in the pathogenesis of MS. Some possible reasons of these findings are discussed.

L-Theanine Content and Related Gene Expression: Novel Insights into Theanine Biosynthesis and Hydrolysis among Different Tea Plant (Camellia sinensis L.) Tissues and Cultivars

PubMed Central

Liu, Zhi-Wei; Wu, Zhi-Jun; Li, Hui; Wang, Yong-Xin; Zhuang, Jing

2017-01-01

L-Theanine content has tissues and cultivars specificity in tea plant (Camellia sinensis L.), the correlations of theanine metabolic related genes expression profiles with theanine contents were explored in this study. L-theanine contents in the bud and 1st leaf, 2nd leaf, 3rd leaf, old leaf, stem, and lateral root were determined by HPLC from three C. sinensis cultivars, namely ‘Huangjinya’, ‘Anjibaicha’, and ‘Yingshuang’, respectively. The theanine contents in leaves and root of ‘Huangjinya’ were the highest, followed by ‘Anjibaicha’, and ‘Yingshuang’. The theanine contents in the leaves reduced as the leaf mature gradually, and in stem were the least. Seventeen genes encoding enzymes involved in theanine metabolism were identified from GenBank and our tea transcriptome database, including CsTS1, CsTS2, CsGS1, CsGS2, CsGOGAT-Fe, CsGOGAT-NAD(P)H, CsGDH1, CsGDH2, CsALT, CsSAMDC, CsADC, CsCuAO, CsPAO, CsNiR, CsNR, CsGGT1, and CsGGT3. The transcript profiles of those seventeen genes in the different tissues of three tea plant cultivars were analyzed comparatively. Among the different cultivars, the transcript levels of most selected genes in ‘Huangjinya’ were significantly higher than that in the ‘Anjibaicha’ and ‘Yingshuang’. Among the different tissues, the transcript levels of CsTS2, CsGS1, and CsGDH2 almost showed positive correlation with the theanine contents, while the other genes showed negative correlation with the theanine contents in most cases. The theanine contents showed correlations with related genes expression levels among cultivars and tissues of tea plant, and were determined by the integrated effect of the metabolic related genes. PMID:28439281

Methylation of an alpha-foetoprotein gene intragenic site modulates gene activity.

PubMed Central

Opdecamp, K; Rivière, M; Molné, M; Szpirer, J; Szpirer, C

1992-01-01

By comparing the methylation pattern of Mspl/Hpall sites in the 5' region of the mouse alpha-foetoprotein (AFP) gene of different cells (hepatoma cells, foetal and adult liver, fibroblasts), we found a correlation between gene expression and unmethylation of a site located in the first intron of the gene. Other sites did not show this correlation. In transfection experiments of unmethylated and methylated AFP-CAT chimeric constructions, we then showed that methylation of the intronic site negatively modulates expression of CAT activity. We also found that a DNA segment centered on this site binds nuclear proteins; however methylation did not affect protein binding. Images PMID:1371343

Natural variation in maternal care and cross-tissue patterns of oxytocin receptor gene methylation in rats.

PubMed

Beery, Annaliese K; McEwen, Lisa M; MacIsaac, Julia L; Francis, Darlene D; Kobor, Michael S

2016-01-01

This article is part of a Special Issue "Parental Care". Since the first report of maternal care effects on DNA methylation in rats, epigenetic modifications of the genome in response to life experience have become the subject of intense focus across many disciplines. Oxytocin receptor expression varies in response to early experience, and both oxytocin signaling and methylation status of the oxytocin receptor gene (Oxtr) in blood have been related to disordered social behavior. It is unknown whether Oxtr DNA methylation varies in response to early life experience, and whether currently employed peripheral measures of Oxtr methylation reflect variation in the brain. We examined the effects of early life rearing experience via natural variation in maternal licking and grooming during the first week of life on behavior, physiology, gene expression, and epigenetic regulation of Oxtr across blood and brain tissues (mononucleocytes, hippocampus, striatum, and hypothalamus). Rats reared by "high" licking-grooming (HL) and "low" licking-grooming (LL) rat dams exhibited differences across study outcomes: LL offspring were more active in behavioral arenas, exhibited lower body mass in adulthood, and showed reduced corticosterone responsivity to a stressor. Oxtr DNA methylation was significantly lower at multiple CpGs in the blood of LL versus HL males, but no differences were found in the brain. Across groups, Oxtr transcript levels in the hypothalamus were associated with reduced corticosterone secretion in response to stress, congruent with the role of oxytocin signaling in this region. Methylation of specific CpGs at a high or low level was consistent across tissues, especially within the brain. However, individual variation in DNA methylation relative to these global patterns was not consistent across tissues. These results suggest that blood Oxtr DNA methylation may reflect early experience of maternal care, and that Oxtr methylation across tissues is highly concordant

Carcinogen-induced trans activation of gene expression.

PubMed Central

Kleinberger, T; Flint, Y B; Blank, M; Etkin, S; Lavi, S

1988-01-01

We report a new mechanism of carcinogen action by which the expression of several genes was concomitantly enhanced. This mechanism involved the altered activity of cellular factors which modulate the expression of genes under their control. The increased expression was regulated at least in part on the transcriptional level and did not require amplification of the overexpressed genes. This phenomenon was transient; it was apparent as early as 24 h after carcinogen treatment and declined a few days later. Images PMID:2835673

Tumor suppressor genes are larger than apoptosis-effector genes and have more regions of active chromatin: Connection to a stochastic paradigm for sequential gene expression programs.

PubMed

Garcia, Marlene; Mauro, James A; Ramsamooj, Michael; Blanck, George

2015-08-03

Apoptosis- and proliferation-effector genes are substantially regulated by the same transactivators, with E2F-1 and Oct-1 being notable examples. The larger proliferation-effector genes have more binding sites for the transactivators that regulate both sets of genes, and proliferation-effector genes have more regions of active chromatin, i.e, DNase I hypersensitive and histone 3, lysine-4 trimethylation sites. Thus, the size differences between the 2 classes of genes suggest a transcriptional regulation paradigm whereby the accumulation of transcription factors that regulate both sets of genes, merely as an aspect of stochastic behavior, accumulate first on the larger proliferation-effector gene "traps," and then accumulate on the apoptosis effector genes, thereby effecting sequential activation of the 2 different gene sets. As IRF-1 and p53 levels increase, tumor suppressor proteins are first activated, followed by the activation of apoptosis-effector genes, for example during S-phase pausing for DNA repair. Tumor suppressor genes are larger than apoptosis-effector genes and have more IRF-1 and p53 binding sites, thereby likewise suggesting a paradigm for transcription sequencing based on stochastic interactions of transcription factors with different gene classes. In this report, using the ENCODE database, we determined that tumor suppressor genes have a greater number of open chromatin regions and histone 3 lysine-4 trimethylation sites, consistent with the idea that a larger gene size can facilitate earlier transcriptional activation via the inclusion of more transactivator binding sites.

A massive parallel sequencing workflow for diagnostic genetic testing of mismatch repair genes

PubMed Central

Hansen, Maren F; Neckmann, Ulrike; Lavik, Liss A S; Vold, Trine; Gilde, Bodil; Toft, Ragnhild K; Sjursen, Wenche

2014-01-01

The purpose of this study was to develop a massive parallel sequencing (MPS) workflow for diagnostic analysis of mismatch repair (MMR) genes using the GS Junior system (Roche). A pathogenic variant in one of four MMR genes, (MLH1, PMS2, MSH6, and MSH2), is the cause of Lynch Syndrome (LS), which mainly predispose to colorectal cancer. We used an amplicon-based sequencing method allowing specific and preferential amplification of the MMR genes including PMS2, of which several pseudogenes exist. The amplicons were pooled at different ratios to obtain coverage uniformity and maximize the throughput of a single-GS Junior run. In total, 60 previously identified and distinct variants (substitutions and indels), were sequenced by MPS and successfully detected. The heterozygote detection range was from 19% to 63% and dependent on sequence context and coverage. We were able to distinguish between false-positive and true-positive calls in homopolymeric regions by cross-sample comparison and evaluation of flow signal distributions. In addition, we filtered variants according to a predefined status, which facilitated variant annotation. Our study shows that implementation of MPS in routine diagnostics of LS can accelerate sample throughput and reduce costs without compromising sensitivity, compared to Sanger sequencing. PMID:24689082

Secondhand Smoke-Prevalent Polycyclic Aromatic Hydrocarbon Binary Mixture-Induced Specific Mitogenic and Pro-inflammatory Cell Signaling Events in Lung Epithelial Cells.

PubMed

Osgood, Ross S; Upham, Brad L; Bushel, Pierre R; Velmurugan, Kalpana; Xiong, Ka-Na; Bauer, Alison K

2017-05-01

Low molecular weight polycyclic aromatic hydrocarbons (LMW PAHs; < 206.3 g/mol) are prevalent and ubiquitous environmental contaminants, presenting a human health concern, and have not been as thoroughly studied as the high MW PAHs. LMW PAHs exert their pulmonary effects, in part, through P38-dependent and -independent mechanisms involving cell-cell communication and the production of pro-inflammatory mediators known to contribute to lung disease. Specifically, we determined the effects of two representative LMW PAHs, 1-methylanthracene (1-MeA) and fluoranthene (Flthn), individually and as a binary PAH mixture on the dysregulation of gap junctional intercellular communication (GJIC) and connexin 43 (Cx43), activation of mitogen activated protein kinases (MAPK), and induction of inflammatory mediators in a mouse non-tumorigenic alveolar type II cell line (C10). Both 1-MeA, Flthn, and the binary PAH mixture of 1-MeA and Flthn dysregulated GJIC in a dose and time-dependent manner, reduced Cx43 protein, and activated the following MAPKs: P38, ERK1/2, and JNK. Inhibition of P38 MAPK prevented PAH-induced dysregulation of GJIC, whereas inhibiting ERK and JNK did not prevent these PAHs from dysregulating GJIC indicating a P38-dependent mechanism. A toxicogenomic approach revealed significant P38-dependent and -independent pathways involved in inflammation, steroid synthesis, metabolism, and oxidative responses. Genes in these pathways were significantly altered by the binary PAH mixture when compared with 1-MeA and Flthn alone suggesting interactive effects. Exposure to the binary PAH mixture induced the production and release of cytokines and metalloproteinases from the C10 cells. Our findings with a binary mixture of PAHs suggest that combinations of LMW PAHs may elicit synergistic or additive inflammatory responses which warrant further investigation and confirmation. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology

NICER Discovers mHz Oscillations and Marginally Stable Burning in GS 1826-24

NASA Astrophysics Data System (ADS)

Strohmayer, Tod E.; Gendreau, Keith C.; Keek, Laurens; Bult, Peter; Mahmoodifar, Simin; Chakrabarty, Deepto; Arzoumanian, Zaven; NICER Science Team

2018-01-01

To date, marginally stable thermonuclear burning, evidenced as mHz X-ray flux oscillations, has been observed in only five accreting neutron star binaries, 4U 1636-536, 4U 1608-52, Aql X-1, 4U 1323-619 and Terzan 5 X-2. Here we report the discovery with NASA's Neutron Star Interior Composition Explorer (NICER) of such oscillations from the well-known X-ray burster GS 1826-24. NICER observed GS 1826-24 on 9 September, 2017 for a total exposure of about 4 ksec. Timing analysis revealed highly significant oscillations at a frequency of 8.2 mHz in two successive pointings. The oscillations have a fractional modulation amplitude of approximately 3% for photon energies less than 6 keV. The observed frequency is consistent with the range observed in the other mHz QPO systems, and indeed is slightly higher than the frequency measured in 4U 1636-536 below which mHz oscillations ceased and unstable burning (X-ray bursts) resumed. We discuss the mass accretion rate dependence of the oscillations as well as the X-ray spectrum as a function of pulsation phase. We place the observations in the context of the current theory of marginally stable burning and briefly discuss the potential for constraining neutron star properties using mHz oscillations.

Congenital chloride diarrhea needs to be distinguished from Bartter and Gitelman syndrome.

PubMed

Matsunoshita, Natsuki; Nozu, Kandai; Yoshikane, Masahide; Kawaguchi, Azusa; Fujita, Naoya; Morisada, Naoya; Ishimori, Shingo; Yamamura, Tomohiko; Minamikawa, Shogo; Horinouchi, Tomoko; Nakanishi, Keita; Fujimura, Junya; Ninchoji, Takeshi; Morioka, Ichiro; Nagase, Hiroaki; Taniguchi-Ikeda, Mariko; Kaito, Hiroshi; Iijima, Kazumoto

2018-05-30

Pseudo-Bartter/Gitelman syndrome (p-BS/GS) encompasses a clinically heterogeneous group of inherited or acquired disorders similar to Bartter syndrome (BS) or Gitelman syndrome (GS), both renal salt-losing tubulopathies. Phenotypic overlap frequently occurs between p-BS/GS and BS/GS, which are difficult to diagnose based on their clinical presentation and require genetic tests for accurate diagnosis. In addition, p-BS/GS can occur as a result of other inherited diseases such as cystic fibrosis, autosomal dominant hypocalcemia, Dent disease, or congenital chloride diarrhea (CCD). However, the detection of the variants in genes other than known BS/GS-causing genes by conventional Sanger sequencing requires substantial time and resources. We studied 27 cases clinically diagnosed with BS/GS, but with negative genetic tests for known BS/GS genes. We conducted targeted sequencing for 22 genes including genes responsible for tubulopathies and other inherited diseases manifesting with p-BS/GS symptoms. We detected the SLC26A3 gene variants responsible for CCD in two patients. In Patient 1, we found the SLC26A3 compound heterozygous variants: c.354delC and c.1008insT. In Patient 2, we identified the compound heterozygous variants: c.877G > A, p.(Glu293Lys), and c.1008insT. Our results suggest that a comprehensive genetic screening system using targeted sequencing is useful for the diagnosis of patients with p-BS/GS with alternative genetic origins.

A truncated form of CD9-partner 1 (CD9P-1), GS-168AT2, potently inhibits in vivo tumour-induced angiogenesis and tumour growth

PubMed Central

Colin, S; Guilmain, W; Creoff, E; Schneider, C; Steverlynck, C; Bongaerts, M; Legrand, E; Vannier, J P; Muraine, M; Vasse, M; Al-Mahmood, S

2011-01-01

Background: Tetraspanins are transmembrane proteins known to contribute to angiogenesis. CD9 partner-1 (CD9P-1/EWI-F), a glycosylated type 1 transmembrane immunoglobulin, is a member of the tetraspanin web, but its role in angiogenesis remains to be elucidated. Methods: We measured the expression of CD9P-1 under angiogenic and angiostatic conditions, and the influence of its knockdown onto capillary structures formation by human endothelial cells (hECs). A truncated form of CDP-1, GS-168AT2, was produced and challenged vs hEC proliferation, migration and capillaries' formation. Its association with CD9P-1, CD9, CD81 and CD151 and the expressions of these later at hEC surface were analysed. Finally, its effects onto in vivo tumour-induced angiogenesis and tumour growth were investigated. Results: Vascular endothelial growth factor (VEGF)-induced capillary tube-like formation was inhibited by tumour necrosis factor α and was associated with a rise in CD9P-1 mRNA expression (P<0.05); accordingly, knockdown of CD9P-1 inhibited VEGF-dependent in vitro angiogenesis. GS-168AT2 dose-dependently inhibited in vitro angiogenesis, hEC migration and proliferation (P<0.05). Co-precipitation experiments suggest that GS-168AT2 corresponds to the sequence by which CD9P-1 physiologically associates with CD81. GS-168AT2 induced the depletion of CD151, CD9 and CD9P-1 from hEC surface, correlating with GS-168AT2 degradation. Finally, in vivo injections of GS-168AT2 inhibited tumour-associated angiogenesis by 53.4±9.5% (P=0.03), and reduced tumour growth of Calu 6 tumour xenografts by 73.9±16.4% (P=0.007) without bodyweight loss. Conclusion: The truncated form of CD9P-1, GS-168AT2, potently inhibits angiogenesis and cell migration by at least the downregulation of CD151 and CD9, which provides the first evidences for the central role of CD9P-1 in tumour-associated angiogenesis and tumour growth. PMID:21863033

Behavioral science and the study of gene-nutrition and gene-physical activity interactions in obesity research.

PubMed

Faith, Myles S

2008-12-01

This report summarizes emerging opportunities for behavioral science to help advance the field of gene-environment and gene-behavior interactions, based on presentations at The National Cancer Institute (NCI) Workshop, "Gene-Nutrition and Gene-Physical Activity Interactions in the Etiology of Obesity." Three opportunities are highlighted: (i) designing potent behavioral "challenges" in experiments, (ii) determining viable behavioral phenotypes for genetics studies, and (iii) identifying specific measures of the environment or environmental exposures. Additional points are underscored, including the need to incorporate novel findings from neuroimaging studies regarding motivation and drive for eating and physical activity. Advances in behavioral science theory and methods can play an important role in advancing understanding of gene-brain-behavior relationships in obesity onset.

Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression.

EPA Science Inventory

Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression Exposure to many drugs and environmentally-relevant chemicals can cause adverse outcomes. These adverse outcomes, such as cancer, have been linked to mol...

Activity-Dependent Human Brain Coding/Noncoding Gene Regulatory Networks

PubMed Central

Lipovich, Leonard; Dachet, Fabien; Cai, Juan; Bagla, Shruti; Balan, Karina; Jia, Hui; Loeb, Jeffrey A.

2012-01-01

While most gene transcription yields RNA transcripts that code for proteins, a sizable proportion of the genome generates RNA transcripts that do not code for proteins, but may have important regulatory functions. The brain-derived neurotrophic factor (BDNF) gene, a key regulator of neuronal activity, is overlapped by a primate-specific, antisense long noncoding RNA (lncRNA) called BDNFOS. We demonstrate reciprocal patterns of BDNF and BDNFOS transcription in highly active regions of human neocortex removed as a treatment for intractable seizures. A genome-wide analysis of activity-dependent coding and noncoding human transcription using a custom lncRNA microarray identified 1288 differentially expressed lncRNAs, of which 26 had expression profiles that matched activity-dependent coding genes and an additional 8 were adjacent to or overlapping with differentially expressed protein-coding genes. The functions of most of these protein-coding partner genes, such as ARC, include long-term potentiation, synaptic activity, and memory. The nuclear lncRNAs NEAT1, MALAT1, and RPPH1, composing an RNAse P-dependent lncRNA-maturation pathway, were also upregulated. As a means to replicate human neuronal activity, repeated depolarization of SY5Y cells resulted in sustained CREB activation and produced an inverse pattern of BDNF-BDNFOS co-expression that was not achieved with a single depolarization. RNAi-mediated knockdown of BDNFOS in human SY5Y cells increased BDNF expression, suggesting that BDNFOS directly downregulates BDNF. Temporal expression patterns of other lncRNA-messenger RNA pairs validated the effect of chronic neuronal activity on the transcriptome and implied various lncRNA regulatory mechanisms. lncRNAs, some of which are unique to primates, thus appear to have potentially important regulatory roles in activity-dependent human brain plasticity. PMID:22960213

Exploring bacterial diversity in hospital environments by GS-FLX Titanium pyrosequencing.

PubMed

Poza, Margarita; Gayoso, Carmen; Gómez, Manuel J; Rumbo-Feal, Soraya; Tomás, María; Aranda, Jesús; Fernández, Ana; Bou, Germán

2012-01-01

Understanding microbial populations in hospital environments is crucial for improving human health. Hospital-acquired infections are an increasing problem in intensive care units (ICU). In this work we present an exploration of bacterial diversity at inanimate surfaces of the ICU wards of the University Hospital A Coruña (Spain), as an example of confined hospital environment subjected to selective pressure, taking the entrance hall of the hospital, an open and crowded environment, as reference. Surface swab samples were collected from both locations and recovered DNA used as template to amplify a hypervariable region of the bacterial 16S rRNA gene. Sequencing of the amplicons was performed at the Roche 454 Sequencing Center using GS-FLX Titanium procedures. Reads were pre-processed and clustered into OTUs (operational taxonomic units), which were further classified. A total of 16 canonical bacterial phyla were detected in both locations. Members of the phyla Firmicutes (mainly Staphylococcus and Streptococcus) and Actinobacteria (mainly Micrococcaceae, Corynebacteriaceae and Brevibacteriaceae) were over-represented in the ICU with respect to the Hall. The phyllum Proteobacteria was also well represented in the ICU, mainly by members of the families Enterobacteriaceae, Methylobacteriaceae and Sphingomonadaceae. In the Hall sample, the phyla Proteobacteria, Bacteroidetes, Deinococcus-Thermus and Cyanobacteria were over-represented with respect to the ICU. Over-representation of Proteobacteria was mainly due to the high abundance of Enterobacteriaceae members. The presented results demonstrate that bacterial diversity differs at the ICU and entrance hall locations. Reduced diversity detected at ICU, relative to the entrance hall, can be explained by its confined character and by the existence of antimicrobial selective pressure. This is the first study using deep sequencing techniques made in hospital wards showing substantial hospital microbial diversity.

The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops.

PubMed

Miflin, Ben J; Habash, Dimah Z

2002-04-01

This short review outlines the central role of glutamine synthetase (GS) in plant nitrogen metabolism and discusses some possibilities for crop improvement. GS functions as the major assimilatory enzyme for ammonia produced from N fixation, and nitrate or ammonia nutrition. It also reassimilates ammonia released as a result of photorespiration and the breakdown of proteins and nitrogen transport compounds. GS is distributed in different subcellular locations (chloroplast and cytoplasm) and in different tissues and organs. This distribution probably changes as a function of the development of the tissue, for example, GS1 appears to play a key role in leaf senescence. The enzyme is the product of multiple genes with complex promoters that ensure the expression of the genes in an organ- and tissue-specific manner and in response to a number of environmental variables affecting the nutritional status of the cell. GS activity is also regulated post-translationally in a manner that involves 14-3-3 proteins and phosphorylation. GS and plant nitrogen metabolism is best viewed as a complex matrix continually changing during the development cycle of plants. Along with GS, a number of other enzymes play key roles in maintaining the balance of carbon and nitrogen. It is proposed that one of these is glutamate dehydrogenase (GDH). There is considerable evidence for a GDH shunt to return the carbon in amino acids back into reactions of carbon metabolism and the tri-carboxylic acid cycle. Results with transgenic plants containing transferred GS genes suggest that there may be ways in which it is possible to improve the efficiency with which crop plants use nitrogen. Marker-assisted breeding may also bring about such improvements.

A canopy radiative transfer scheme with explicit FAPAR for the ISBA-A-gs land surface model: impact on carbon fluxes

NASA Astrophysics Data System (ADS)

Calvet, Jean-Christophe; Carrer, Dominique; Roujean, Jean-Louis; Lafont, Sébastien

2013-04-01

The ISBA-A-gs land surface model is a component of the SURFEX modeling platform developed by Meteo-France, used for research and operational applications in meteorology, hydrology, and climate modeling. ISBA-A-gs simulates hourly water and CO2 fluxes together with soil moisture. An option of the model permits the simulation of the vegetation biomass and of the leaf area index (LAI). The simulated photosynthesis depends on atmospheric CO2 concentration, air temperature and humidity, soil moisture, radiant solar energy, the photosynthetic capacity of the leaves and on factors that condition the distribution of solar radiation over the leaves. In the original version of the model (Jacobs et al. (Agr. Forest Meteorol., 1996), Calvet et al. (Agr. Forest Meteorol., 1998)), the radiative transfer scheme within the canopy was implemented according to a self shading approach. The incident fluxes at the top of the canopy go through a multi-layer vegetation cover. Then, the attenuated flux in the PAR wavelength domain of each layer is used by the photosynthesis model to calculate the leaf net assimilation of CO2 (An). The leaf-level An values are then integrated at the canopy level. In this study, an upgraded version of the radiative transfer model is implemented. An assessment of the vegetation transmittance functions and of various canopy light-response curves is made. The fluxes produced by the new version of ISBA-A-gs are evaluated using data from a number of FLUXNET forest sites. The new model presents systematically better scores than the previous version. Moreover, ISBA-A-gs is now able to simulate prognostic values of the fraction of absorbed PAR (FAPAR). As FAPAR can be observed from space, this new capability permits the validation of the model simulations at a global scale, and the integration of measured FAPAR values in the model through data assimilation techniques.

Selection of full-length IgGs by tandem display on filamentous phage particles and Escherichia coli fluorescence-activated cell sorting screening.

PubMed

Mazor, Yariv; Van Blarcom, Thomas; Carroll, Sean; Georgiou, George

2010-05-01

Phage display of antibody libraries is a powerful tool for antibody discovery and evolution. Recombinant antibodies have been displayed on phage particles as scFvs or Fabs, and more recently as bivalent F(ab')(2). We recently developed a technology (E-clonal) for screening of combinatorial IgG libraries using bacterial periplasmic display and selection by fluorescence-activated cell sorting (FACS) [Mazor Y et al. (2007) Nat Biotechnol 25, 563-565]. Although, as a single-cell analysis technique, FACS is very powerful, especially for the isolation of high-affinity binders, even with state of the art instrumentation the screening of libraries with diversity > 10(8) is technically challenging. We report here a system that takes advantage of display of full-length IgGs on filamentous phage particles as a prescreening step to reduce library size and enable subsequent rounds of FACS screening in Escherichia coli. For the establishment of an IgG phage display system, we utilized phagemid-encoded IgG with the fUSE5-ZZ phage as a helper phage. These phage particles display the Fc-binding ZZ protein on all copies of the phage p3 coat protein, and are exploited as both helper phages and anchoring surfaces for the soluble IgG. We demonstrate that tandem phage selection followed by FACS allows the selection of a highly diversified profile of binders from antibody libraries without undersampling, and at the same time capitalizes on the advantages of FACS for real-time monitoring and optimization of the screening process.

Determination of Flux rope axis for GS reconstruction

NASA Astrophysics Data System (ADS)

Tian, A.; Shi, Q.; Bai, S.; Zhang, S.

2016-12-01

It is important to give the axis direction and velocity of a magnetic flux ropes before employing Grad-Shafranov reconstruction. The ability of single-satellite based MVA (MVAB and CMVA) and multi-satellite based MDD methods in finding the invariant axis are tested by a model. The choice of principal axis given by MVA along the aimed direction is dependent on the distance of the path from the flux-rope axis. The MDD results are influenced by the ratio of Noise level/separation to the gradient of the structure. An accurate axial direction will be obtained when the ratio is less than 1. By a model, an example with failed HT method is displayed indicating the importance of the STD method in obtaining the velocity of such a structure. The applicability of trial and error method by Hu and Sonnerup(2012) was also used and discussed. Finally, all above methods were applied to a flux-rope observed by Cluster. It shows that the GS method can be easily carried out in the case of clearly known dimensionality and velocity.

Spearfishing regulation benefits artisanal fisheries: the ReGS indicator and its application to a multiple-use Mediterranean marine protected area.

PubMed

Rocklin, Delphine; Tomasini, Jean-Antoine; Culioli, Jean-Michel; Pelletier, Dominique; Mouillot, David

2011-01-01

The development of fishing efficiency coupled with an increase of fishing effort led to the overexploitation of numerous natural marine resources. In addition to this commercial pressure, the impact of recreational activities on fish assemblages remains barely known. Here we examined the impact of spearfishing limitation on resources in a marine protected area (MPA) and the benefit it provides for the local artisanal fishery through the use of a novel indicator. We analysed trends in the fish assemblage composition using artisanal fisheries data collected in the Bonifacio Strait Natural Reserve (BSNR), a Mediterranean MPA where the spearfishing activity has been forbidden over 15% of its area. Fish species were pooled into three response groups according to their target level by spearfishing. We developed the new flexible ReGS indicator reflecting shifts in species assemblages according to the relative abundance of each response group facing external pressure. The catch per unit effort (CPUE) increased by ca. 60% in the BSNR between 2000 and 2007, while the MPA was established in 1999. The gain of CPUE strongly depended on the considered response group: for the highly targeted group, the CPUE doubled while the CPUE of the untargeted group increased by only 15.5%. The ReGS value significantly increased from 0.31 to 0.45 (on a scale between 0 and 1) in the general perimeter of this MPA while it has reached a threshold of 0.43, considered as a reference point, in the area protected from spearfishing since 1982. Our results demonstrated that limiting recreational fishing by appropriate zoning in multiple-use MPAs represents a real benefit for artisanal fisheries. More generally we showed how our new indicator may reveal a wide range of impacts on coastal ecosystems such as global change or habitat degradation.

Dynamic release of gentamicin sulfate (GS) from alginate dialdehyde (AD)-crosslinked casein (CAS) films for antimicrobial applications

PubMed Central

Bajpai, S. K.; Shah, Farhan Ferooz; Bajpai, M.

2017-01-01

Abstract In the present work, antibiotic drug gentamicin sulfate (GS) has been loaded into alginate dialdehyde-crosslinked casein (CAS) films for wound dressing applications. The films have been characterized by Fourier transform infrared spectroscopy, X-ray diffraction analysis and scanning electron microscopy. The dynamic release of model drug GS has been investigated in the physiological fluid at 37 °C. The drug release data has been interpreted in the terms of various kinetic models such as Power function model, first order model and Schott model. The release data was found to be well fitted by Schott model. The various diffusion coefficients are also evaluated. The adsorption of model therapeutic protein BSA on the film has been investigated. The maximum adsorption is found to be 5.7 mg/cm2.The films were tested for their antibacterial and anti-fungal action. Finally, the in vivo wound healing study was carried out on Albino wistar rats. PMID:29491776

A novel glycogen synthase kinase-3 inhibitor optimized for acute myeloid leukemia differentiation activity

PubMed Central

Stetson, Lindsay; Ignatz-Hoover, James; Moreton, Stephen; Chakrabarti, Amit; Xia, Zhiqiang; Karan, Goutam; de Lima, Marcos; Agrawal, Mukesh K; Wald, David N

2016-01-01

Standard therapies used for the treatment of Acute Myeloid Leukemia (AML) are cytotoxic agents that target rapidly proliferating cells. Unfortunately, this therapeutic approach has limited efficacy and significant toxicity and the majority of AML patients still die of their disease. In contrast to the poor prognosis of most AML patients, most individuals with a rare subtype of AML, Acute Promyelocytic Leukemia (APL), can be cured by differentiation therapy using regimens containing all-trans retinoic acid. GSK3 has previously been identified as a therapeutic target in AML where its inhibition can lead to the differentiation and growth arrest of leukemic cells. Unfortunately, existing GSK3 inhibitors lead to suboptimal differentiation activity making them less useful as clinical AML differentiation agents. Here we describe the discovery of a novel GSK3 inhibitor, GS87. GS87 was discovered in efforts to optimize GSK3 inhibition for AML differentiation activity. Despite GS87's dramatic ability to induce AML differentiation, kinase profiling reveals its high specificity in targeting GSK3 as compared to other kinases. GS87 demonstrates high efficacy in a mouse AML model system and unlike current AML therapeutics, exhibits little effect on normal bone marrow cells. GS87 induces potent differentiation by more effectively activating GSK3-dependent signaling components including MAPK signaling as compared to other GSK3 inhibitors. GS87 is a novel GSK3 inhibitor with therapeutic potential as a differentiation agent for non-promyelocytic AML. PMID:27196775

The positive impact of simultaneous implementation of the BD FocalPoint GS Imaging System and lean principles on the operation of gynecologic cytology.

PubMed

Wong, Rebecca; Levi, Angelique W; Harigopal, Malini; Schofield, Kevin; Chhieng, David C

2012-02-01

Our cytology laboratory, like many others, is under pressure to improve quality and provide test results faster while decreasing costs. We sought to address these issues by introducing new technology and lean principles. To determine the combined impact of the FocalPoint Guided Screener (GS) Imaging System (BD Diagnostics-TriPath, Burlington, North Carolina) and lean manufacturing principles on the turnaround time (TAT) and productivity of the gynecologic cytology operation. We established a baseline measure of the TAT for Papanicolaou tests. We then compared that to the performance after implementing the FocalPoint GS Imaging System and lean principles. The latter included value-stream mapping, workflow modification, and a first in-first out policy. The mean (SD) TAT for Papanicolaou tests before and after the implementation of FocalPoint GS Imaging System and lean principles was 4.38 (1.28) days and 3.20 (1.32) days, respectively. This represented a 27% improvement in the average TAT, which was statistically significant (P < .001). In addition, the productivity of staff improved 17%, as evidenced by the increase in slides screened from 8.85/h to 10.38/h. The false-negative fraction decreased from 1.4% to 0.9%, representing a 36% improvement. In our laboratory, the implementation of FocalPoint GS Imaging System in conjunction with lean principles resulted in a significant decrease in the average TAT for Papanicolaou tests and a substantial increase in the productivity of cytotechnologists while maintaining the diagnostic quality of gynecologic cytology.

Activation of silenced cytokine gene promoters by the synergistic effect of TBP-TALE and VP64-TALE activators.

PubMed

Anthony, Kim; More, Abhijit; Zhang, Xiaoliu

2014-01-01

Recent work has shown that the combinatorial use of multiple TALE activators can selectively activate certain cellular genes in inaccessible chromatin regions. In this study, we aimed to interrogate the activation potential of TALEs upon transcriptionally silenced immune genes in the context of non-immune cells. We designed a unique strategy, in which a single TALE fused to the TATA-box binding protein (TBP-TALE) is coupled with multiple VP64-TALE activators. We found that our strategy is significantly more potent than multiple TALE activators alone in activating expression of IL-2 and GM-CSF in diverse cell origins in which both genes are otherwise completely silenced. Chromatin analysis revealed that the gene activation was due in part to displacement of a distinctly positioned nucleosome. These studies provide a novel epigenetic mechanism for artificial gene induction and have important implications for targeted cancer immunotherapy, DNA vaccine development, as well as rational design of TALE activators.

Activation of Silenced Cytokine Gene Promoters by the Synergistic Effect of TBP-TALE and VP64-TALE Activators

PubMed Central

Anthony, Kim; More, Abhijit; Zhang, Xiaoliu

2014-01-01

Recent work has shown that the combinatorial use of multiple TALE activators can selectively activate certain cellular genes in inaccessible chromatin regions. In this study, we aimed to interrogate the activation potential of TALEs upon transcriptionally silenced immune genes in the context of non-immune cells. We designed a unique strategy, in which a single TALE fused to the TATA-box binding protein (TBP-TALE) is coupled with multiple VP64-TALE activators. We found that our strategy is significantly more potent than multiple TALE activators alone in activating expression of IL-2 and GM-CSF in diverse cell origins in which both genes are otherwise completely silenced. Chromatin analysis revealed that the gene activation was due in part to displacement of a distinctly positioned nucleosome. These studies provide a novel epigenetic mechanism for artificial gene induction and have important implications for targeted cancer immunotherapy, DNA vaccine development, as well as rational design of TALE activators. PMID:24755922

Reconstructing Dynamic Promoter Activity Profiles from Reporter Gene Data.

PubMed

Kannan, Soumya; Sams, Thomas; Maury, Jérôme; Workman, Christopher T

2018-03-16

Accurate characterization of promoter activity is important when designing expression systems for systems biology and metabolic engineering applications. Promoters that respond to changes in the environment enable the dynamic control of gene expression without the necessity of inducer compounds, for example. However, the dynamic nature of these processes poses challenges for estimating promoter activity. Most experimental approaches utilize reporter gene expression to estimate promoter activity. Typically the reporter gene encodes a fluorescent protein that is used to infer a constant promoter activity despite the fact that the observed output may be dynamic and is a number of steps away from the transcription process. In fact, some promoters that are often thought of as constitutive can show changes in activity when growth conditions change. For these reasons, we have developed a system of ordinary differential equations for estimating dynamic promoter activity for promoters that change their activity in response to the environment that is robust to noise and changes in growth rate. Our approach, inference of dynamic promoter activity (PromAct), improves on existing methods by more accurately inferring known promoter activity profiles. This method is also capable of estimating the correct scale of promoter activity and can be applied to quantitative data sets to estimate quantitative rates.

A statistical method for measuring activation of gene regulatory networks.

PubMed

Esteves, Gustavo H; Reis, Luiz F L

2018-06-13

Gene expression data analysis is of great importance for modern molecular biology, given our ability to measure the expression profiles of thousands of genes and enabling studies rooted in systems biology. In this work, we propose a simple statistical model for the activation measuring of gene regulatory networks, instead of the traditional gene co-expression networks. We present the mathematical construction of a statistical procedure for testing hypothesis regarding gene regulatory network activation. The real probability distribution for the test statistic is evaluated by a permutation based study. To illustrate the functionality of the proposed methodology, we also present a simple example based on a small hypothetical network and the activation measuring of two KEGG networks, both based on gene expression data collected from gastric and esophageal samples. The two KEGG networks were also analyzed for a public database, available through NCBI-GEO, presented as Supplementary Material. This method was implemented in an R package that is available at the BioConductor project website under the name maigesPack.

Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells.

PubMed

Mariscal, Ana M; Kakizawa, Shigeyuki; Hsu, Jonathan Y; Tanaka, Kazuki; González-González, Luis; Broto, Alicia; Querol, Enrique; Lluch-Senar, Maria; Piñero-Lambea, Carlos; Sun, Lijie; Weyman, Philip D; Wise, Kim S; Merryman, Chuck; Tse, Gavin; Moore, Adam J; Hutchison, Clyde A; Smith, Hamilton O; Tomita, Masaru; Venter, J Craig; Glass, John I; Piñol, Jaume; Suzuki, Yo

2018-05-22

Functional genomics studies in minimal mycoplasma cells enable unobstructed access to some of the most fundamental processes in biology. Conventional transposon bombardment and gene knockout approaches often fail to reveal functions of genes that are essential for viability, where lethality precludes phenotypic characterization. Conditional inactivation of genes is effective for characterizing functions central to cell growth and division, but tools are limited for this purpose in mycoplasmas. Here we demonstrate systems for inducible repression of gene expression based on clustered regularly interspaced short palindromic repeats-mediated interference (CRISPRi) in Mycoplasma pneumoniae and synthetic Mycoplasma mycoides, two organisms with reduced genomes actively used in systems biology studies. In the synthetic cell, we also demonstrate inducible gene expression for the first time. Time-course data suggest rapid kinetics and reversible engagement of CRISPRi. Targeting of six selected endogenous genes with this system results in lowered transcript levels or reduced growth rates that agree with lack or shortage of data in previous transposon bombardment studies, and now produces actual cells to analyze. The ksgA gene encodes a methylase that modifies 16S rRNA, rendering it vulnerable to inhibition by the antibiotic kasugamycin. Targeting the ksgA gene with CRISPRi removes the lethal effect of kasugamycin and enables cell growth, thereby establishing specific and effective gene modulation with our system. The facile methods for conditional gene activation and inactivation in mycoplasmas open the door to systematic dissection of genetic programs at the core of cellular life.

Massive activation of archaeal defense genes during viral infection.

PubMed

Quax, Tessa E F; Voet, Marleen; Sismeiro, Odile; Dillies, Marie-Agnes; Jagla, Bernd; Coppée, Jean-Yves; Sezonov, Guennadi; Forterre, Patrick; van der Oost, John; Lavigne, Rob; Prangishvili, David

2013-08-01

Archaeal viruses display unusually high genetic and morphological diversity. Studies of these viruses proved to be instrumental for the expansion of knowledge on viral diversity and evolution. The Sulfolobus islandicus rod-shaped virus 2 (SIRV2) is a model to study virus-host interactions in Archaea. It is a lytic virus that exploits a unique egress mechanism based on the formation of remarkable pyramidal structures on the host cell envelope. Using whole-transcriptome sequencing, we present here a global map defining host and viral gene expression during the infection cycle of SIRV2 in its hyperthermophilic host S. islandicus LAL14/1. This information was used, in combination with a yeast two-hybrid analysis of SIRV2 protein interactions, to advance current understanding of viral gene functions. As a consequence of SIRV2 infection, transcription of more than one-third of S. islandicus genes was differentially regulated. While expression of genes involved in cell division decreased, those genes playing a role in antiviral defense were activated on a large scale. Expression of genes belonging to toxin-antitoxin and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems was specifically pronounced. The observed different degree of activation of various CRISPR-Cas systems highlights the specialized functions they perform. The information on individual gene expression and activation of antiviral defense systems is expected to aid future studies aimed at detailed understanding of the functions and interplay of these systems in vivo.

Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat

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

Kenney, S.; Kamine, J.; Markovitz, D.

Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, the authors demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBVmore » gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.« less

Oral nano-delivery of anticancer ginsenoside 25-OCH3-PPD, a natural inhibitor of the MDM2 oncogene: Nanoparticle preparation, characterization, in vitro and in vivo anti-prostate cancer activity, and mechanisms of action.

PubMed

Voruganti, Sukesh; Qin, Jiang-Jiang; Sarkar, Sushanta; Nag, Subhasree; Walbi, Ismail A; Wang, Shu; Zhao, Yuqing; Wang, Wei; Zhang, Ruiwen

2015-08-28

The Mouse Double Minute 2 (MDM2) oncogene plays a critical role in cancer development and progression through p53-dependent and p53-independent mechanisms. Both natural and synthetic MDM2 inhibitors have been shown anticancer activity against several human cancers. We have recently identified a novel ginsenoside, 25-OCH3-PPD (GS25), one of the most active anticancer ginsenosides discovered thus far, and have demonstrated its MDM2 inhibition and anticancer activity in various human cancer models, including prostate cancer. However, the oral bioavailability of GS25 is limited, which hampers its further development as an oral anticancer agent. The present study was designed to develop a novel nanoparticle formulation for oral delivery of GS25. After GS25 was successfully encapsulated into PEG-PLGA nanoparticles (GS25NP) and its physicochemical properties were characterized, the efficiency of MDM2 targeting, anticancer efficacy, pharmacokinetics, and safety were evaluated in in vitro and in vivo models of human prostate cancer. Our results indicated that, compared with the unencapsulated GS25, GS25NP demonstrated better MDM2 inhibition, improved oral bioavailability and enhanced in vitro and in vivo activities. In conclusion, the validated nano-formulation for GS25 oral delivery improves its molecular targeting, oral bioavailability and anticancer efficacy, providing a basis for further development of GS25 as a novel agent for cancer therapy and prevention.

Phenylethanoid Glycosides: Research Advances in Their Phytochemistry, Pharmacological Activity and Pharmacokinetics.

PubMed

Xue, Zhenzhen; Yang, Bin

2016-07-29

Phenylethanoid glycosides (PhGs) are widely distributed in traditional Chinese medicines as well as in other medicinal plants, and they were characterized by a phenethyl alcohol (C₆-C₂) moiety attached to a β-glucopyranose/β-allopyranose via a glycosidic bond. The outstanding activity of PhGs in diverse diseases proves their importance in medicinal chemistry research. This review summarizes new findings on PhGs over the past 10 years, concerning the new structures, their bioactivities, including neuroprotective, anti-inflammatory, antioxidant, antibacterial and antivirus, cytotoxic, immunomodulatory, and enzyme inhibitory effects, and pharmacokinetic properties.

Virus-induced gene silencing suggests (1,3;1,4)-β-glucanase is a susceptibility factor in the compatible russian wheat aphid-wheat interaction.

PubMed

Anderson, Victoria A; Haley, Scott D; Peairs, Frank B; van Eck, Leon; Leach, Jan E; Lapitan, Nora L V

2014-09-01

The Russian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), is a significant insect pest of wheat (Triticum aestivum L.) and has a major economic impact worldwide, especially on winter wheat in the western United States. The continuing emergence of new RWA biotypes virulent to existing resistance genes reinforces the need for more durable resistance. Studies have indicated that resistance in previously susceptible plants can be produced by knock-down of susceptibility genes or other genes involved in host plant susceptibility. Therefore, investigation into genes involved in compatible RWA-wheat interactions could be a feasible approach to achieving durable RWA resistance. The objective of this study was to test whether silencing (1,3;1,4)-β-glucanase, previously observed to be highly induced in susceptible compared with resistant wheat during aphid infestation, would confer resistance to a susceptible wheat genotype. Barley stripe mosaic virus-mediated virus-induced gene silencing was employed to test whether (1,3;1,4)-β-glucanase is involved in the susceptible reaction of 'Gamtoos-S' (GS). Controlled infestation with U.S. biotype RWA2 was done to assess aphid reproduction and host symptom development. Aphids on (1,3;1,4)-β-glucanase-silenced plants reproduced less per day and had longer prenymphipositional periods than those on control GS plants. Furthermore, the (1,3;1,4)-β-glucanase-silenced plants exhibited less chlorosis and greater dry weight compared with GS. Aphid reproduction and host plant symptom development showed linear relationships with (1,3;1,4)-β-glucanase transcript levels. Our results suggest that (1,3;1,4)-β-glucanase is required for successful infestation by the RWA and may be a susceptibility factor that could be exploited as a potential target for RWA resistance breeding.

Detoxification of Atrazine by Low Molecular Weight Thiols in Alfalfa (Medicago sativa).

PubMed

Zhang, Jing Jing; Xu, Jiang Yan; Lu, Feng Fan; Jin, She Feng; Yang, Hong

2017-10-16

Low molecular weight (LMW) thiols in higher plants are a group of sulfur-rich nonprotein compounds and play primary and multiple roles in cellular redox homeostasis, enzyme activities, and xenobiotics detoxification. This study focused on identifying thiols-related protein genes from the legume alfalfa exposed to the herbicide atrazine (ATZ) residues in environment. Using high-throughput RNA-sequencing, a set of ATZ-responsive thiols-related protein genes highly up-regulated and differentially expressed in alfalfa was identified. Most of the differentially expressed genes (DEGs) were involved in regulation of biotic and abiotic stress responses. By analyzing the genes involved in thiols-mediated redox homeostasis, we found that many of them were thiols-synthetic enzymes such as γ-glutamylcysteine synthase (γECS), homoglutathione synthetase (hGSHS), and glutathione synthetase (GSHS). Using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), we further characterized a group of ATZ-thiols conjugates, which are the detoxified forms of ATZ in plants. Cysteine S-conjugate ATZ-HCl+Cys was the most important metabolite detected by MS. Several other ATZ-conjugates were also examined as ATZ-detoxified metabolites. Such results were validated by characterizing their analogs in rice. Our data showed that some conjugates under ATZ stress were detected in both plants, indicating that some detoxified mechanisms and pathways can be shared by the two plant species. Overall, these results indicate that LMW thiols play critical roles in detoxification of ATZ in the plants.

The kinetics of oocyst shedding and sporulation in two immunologically distinct strains of Eimeria maxima, GS and M6.

PubMed

Al-Badri, Riadh; Barta, John Robert

2012-11-01

The kinetics of oocyst shedding and sporulation of two immunologically distinct strains of Eimeria maxima (GS and M6) were compared. Both strains had a prepatent period of approximately 120 h followed by peak oocyst shedding at 144-150 h post inoculation. Mean total oocyst output determined for each strain demonstrated that the fecundity of the M6 strain (12.8 × 10(3) ± 1.95) of E. maxima was roughly twice that of the GS strain (6.9 × 10(3) ± 3.33) when inoculated at the rate of 1,000 infective oocysts per bird. The process of oocyst sporulation was followed by repetitive sampling of sporulating oocysts at 26 °C with aeration over a 138 hour period. Sporulation was divided into five morphologically distinguishable stages whose abundance peaked at the following times during sporulation: unsporulated oocysts at 0 h; sporoblast anlagen at 18 h; sporoblasts without sporocyst walls at 22 h; and sporocysts without mature sporozoites at 38 h. The time to 50 % sporulation of E. maxima oocysts observed in the present study was approximately 53 h for both strains and all viable oocysts had completed sporulation by 60 h. In the present study, the prepatent periods, duration of oocyst shedding, and the relative kinetics of sporulation of the GS and M6 strains of E. maxima were found to be virtually identical despite the immunological distinctiveness of these two parasite strains.

The influence of aging on the methylation status of brain-derived neurotrophic factor gene in blood.

PubMed

Ihara, Kazushige; Fuchikami, Manabu; Hashizume, Masahiro; Okada, Satoshi; Kawai, Hisashi; Obuchi, Shuichi; Hirano, Hirohiko; Fujiwara, Yoshinori; Hachisu, Mitsugu; Hongyong, Kim; Morinobu, Shigeru

2018-06-28

Brain-derived neurotrophic factor (BDNF) is involved in the pathophysiology of psychiatric disorders in adults and elderly individuals, and as a result, the DNA methylation (DNAm) of the BDNF gene in peripheral tissues including blood has been extensively examined to develop a useful biomarker for psychiatric disorders. However, studies to date have not previously investigated the effect of age on DNAm of the BDNF gene in blood. In this context, we measured DNAm of 39 CpG units in the CpG island at the promoter of exon I of the BDNF gene. We analyzed genomic DNA from peripheral blood of 105 health Japanese women 20 to 80 years of age to identify aging-associated change in DNAm of the BDNF gene. In addition, we examined the relationship between total MMSE scores, numbers of stressful life events, and serum BDNF levels on DNAm of the BDNF gene. The DNAm rate at each CpG unit was measured using a MassArray ® system (Agena Bioscience), and serum BDNF levels were measured by ELISA. There was a significant correlation between DNAm and age in 13 CpGs. However, there was no significant correlation between DNAm and total MMSE scores, numbers of life events, or serum BDNF levels. Despite the small number of subjects and the inclusion of only female subjects, our results suggest that DNAm of 13 CpGs of the BDNF gene may be an appropriate biomarker for aging and useful for predicting increased susceptibility to age-related psychiatric disorders. © 2018 John Wiley & Sons, Ltd.

Controlling nuclear JAKs and STATs for specific gene activation by IFNγ.

PubMed

Noon-Song, Ezra N; Ahmed, Chulbul M; Dabelic, Rea; Canton, Johnathan; Johnson, Howard M

2011-07-08

We previously showed that gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interacted with the promoter region of IFNγ-activated genes along with transcription factor STAT1α. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFNγ. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFNγ treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The β-actin gene, which is not activated by IFNγ, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFNγ treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFNγ treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFNγ treatment resulted in its disassociation and then re-association as pSTAT1. The results suggest a novel role for activated JAKs in epigenetic events for specific gene activation. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification and analysis of low-molecular-weight dissolved organic carbon in subglacial basal ice ecosystems by ion chromatography

NASA Astrophysics Data System (ADS)

O'Donnell, Emily C.; Wadham, Jemma L.; Lis, Grzegorz P.; Tranter, Martyn; Pickard, Amy E.; Stibal, Marek; Dewsbury, Paul; Fitzsimons, Sean

2016-07-01

Determining the concentration and composition of dissolved organic carbon (DOC) in glacial ecosystems is important for assessments of in situ microbial activity and contributions to wider biogeochemical cycles. Nonetheless, there is limited knowledge of the abundance and character of DOC in basal ice and the subglacial environment and a lack of quantitative data on low-molecular-weight (LMW) DOC components, which are believed to be highly bioavailable to microorganisms. We investigated the abundance and composition of DOC in basal ice via a molecular-level DOC analysis. Spectrofluorometry and a novel ion chromatographic method, which has been little utilized in glacial science for LMW-DOC determinations, were employed to identify and quantify the major LMW fractions (free amino acids, carbohydrates, and carboxylic acids) in basal ice from four glaciers, each with a different type of overridden material (i.e. the pre-entrainment sedimentary type such as lacustrine material or palaeosols). Basal ice from Joyce Glacier (Antarctica) was unique in that 98 % of the LMW-DOC was derived from the extremely diverse free amino acid (FAA) pool, comprising 14 FAAs. LMW-DOC concentrations in basal ice were dependent on the bioavailability of the overridden organic carbon (OC), which in turn was influenced by the type of overridden material. Mean LMW-DOC concentrations in basal ice from Russell Glacier (Greenland), Finsterwalderbreen (Svalbard), and Engabreen (Norway) were low (0-417 nM C), attributed to the relatively refractory nature of the OC in the overridden palaeosols and bedrock. In contrast, mean LMW-DOC concentrations were an order of magnitude higher (4430 nM C) in basal ice from Joyce Glacier, a reflection of the high bioavailability of the overridden lacustrine material (> 17 % of the sediment OC comprised extractable carbohydrates, a proxy for bioavailable OC). We find that the overridden material may act as a direct (via abiotic leaching) and indirect (via

Promoter polymorphisms in genes involved in porcine myogenesis influence their transcriptional activity.

PubMed

Bongiorni, Silvia; Tilesi, Francesca; Bicorgna, Silvia; Iacoponi, Francesca; Willems, Daniela; Gargani, Maria; D'Andrea, MariaSilvia; Pilla, Fabio; Valentini, Alessio

2014-11-07

Success of meat production and selection for improvement of meat quality is among the primary aims in animal production. Meat quality traits are economically important in swine; however, the underlying genetic nature is very complex. Therefore, an improved pork production strongly depends on identifying and studying how genetic variations contribute to modulate gene expression. Promoters are key regions in gene modulation as they harbour several binding motifs to transcription regulatory factors. Therefore, polymorphisms in these regions are likely to deeply affect RNA levels and consequently protein synthesis. In this study, we report the identification of single nucleotide polymorphisms (SNPs) in promoter regions of candidate genes involved in development, cellular differentiation and muscle growth in Sus scrofa. We identified SNPs in the promoter regions of genes belonging to the Myogenic Regulatory Factors (MRF) gene family (the Myogenic Differentiation gene, MYOD1) and to Growth and Differentiation Factors (GDF) gene family (Myostatin gene, MSTN, GDF8), in Casertana and Large White breeds. The purpose of this study was to investigate if polymorphisms in the promoters could affect the transcriptional activity of these genes. With this aim, we evaluated in vitro the functional activity of the luciferase reporter gene luc2 activity, driven by two constructs carrying different promoter haplotypes. We tested the effects of the G302A (U12574) transition on the promoter efficiency in MYOD1 gene. We ascertained a difference in transcription efficiency for the two variants. A stronger activity of the A-carrying construct is more evident in C2C12. The luciferase expression driven by the MYOD1-A allelic variant displayed a 3.8-fold increased transcriptional activity. We investigated the activity of two haplotype variants (AY527152) in the promoter of GDF8 gene. The haploptype-1 (A435-A447-A879) up-regulated the expression of the reporter gene by a two-fold increase, and

Critical roles of DNA demethylation in the activation of ripening-induced genes and inhibition of ripening-repressed genes in tomato fruit

PubMed Central

Lang, Zhaobo; Wang, Yihai; Tang, Kai; Tang, Dengguo; Datsenka, Tatsiana; Cheng, Jingfei; Zhang, Yijing; Handa, Avtar K.

2017-01-01

DNA methylation is a conserved epigenetic mark important for genome integrity, development, and environmental responses in plants and mammals. Active DNA demethylation in plants is initiated by a family of 5-mC DNA glycosylases/lyases (i.e., DNA demethylases). Recent reports suggested a role of active DNA demethylation in fruit ripening in tomato. In this study, we generated loss-of-function mutant alleles of a tomato gene, SlDML2, which is a close homolog of the Arabidopsis DNA demethylase gene ROS1. In the fruits of the tomato mutants, increased DNA methylation was found in thousands of genes. These genes included not only hundreds of ripening-induced genes but also many ripening-repressed genes. Our results show that SlDML2 is critical for tomato fruit ripening and suggest that active DNA demethylation is required for both the activation of ripening-induced genes and the inhibition of ripening-repressed genes. PMID:28507144

Removal of organic compounds from shale gas flowback water.

PubMed

Butkovskyi, Andrii; Faber, Ann-Hélène; Wang, Yue; Grolle, Katja; Hofman-Caris, Roberta; Bruning, Harry; Van Wezel, Annemarie P; Rijnaarts, Huub H M

2018-07-01

Ozonation, sorption to granular activated carbon and aerobic degradation were compared as potential treatment methods for removal of dissolved organic carbon (DOC) fractions and selected organic compounds from shale gas flowback water after pre-treatment in dissolved air flotation unit. Flowback water was characterised by high chemical oxygen demand and DOC. Low molecular weight (LMW) acids and neutral compounds were the most abundant organic fractions, corresponding to 47% and 35% of DOC respectively. Ozonation did not change distribution of organic carbon fractions and concentrations of detected individual organic compounds significantly. Sorption to activated carbon targeted removal of individual organic compounds with molecular weight >115 Da, whereas LMW compounds remained largely unaffected. Aerobic degradation was responsible for removal of LMW compounds and partial ammonium removal, whereas formation of intermediates with molecular weight of 200-350 Da was observed. Combination of aerobic degradation for LMW organics removal with adsorption to activated carbon for removal of non-biodegradable organics is proposed to be implemented between pre-treatment (dissolved air floatation) and desalination (thermal or membrane desalination) steps. Copyright © 2018 Elsevier Ltd. All rights reserved.

The ginseng's fireness is associated with the lowering activity of liver Na(+)-K(+)-ATPase.

PubMed

Xu, Xu; Dou, Deqiang

2016-08-22

Ginseng is an herbal medicine used worldwide that possesses a wide range of pharmacological activities. However, its side effects are rarely discussed. The experience of Chinese medicine has revealed that taking ginseng at a high dose chronically can cause fireness, i.e., the ginseng-abuse syndrome. Here, we explored the mechanism of ginseng's fireness by comparing the energy metabolism of mice affected by red ginseng (RG), ginseng (GS), ginseng leaves (GL) and American ginseng (AG), which exhibit different drug properties according to the theory of TCM. KM mice were randomly divided into five groups (n≥30 per group) and administered distilled water or drugs, respectively. Mice receiving RG, GS, or GL received 4.5g/(kgday), while the mice receiving AG received 3g/(kgday). Control mice received distilled water. The duration of exposure for all groups was 31 days. The mice's physical characteristics, such as eye condition, rectal temperature, saliva secretion, urine, stool weight, blood coagulation time and swimming time, were measured at different times after administration. Energy metabolism indexes were measured via TSE phenoMaster/LabMaster animal monitoring system, including the mice' 24h oxygen consumption (VO2), carbon dioxide production (VCO2), heat production (H) and energy expenditure (EE). Biochemical indices were measured by ultraviolet spectrophotometer and microplate reader, including pyruvic acid content in serum and succinate dehydrogenase (SDH) activity, lactate dehydrogenase (LDH) activity, the Na(+)-K(+)-ATPase activity and the content of glycogen in the liver tissue. After 31 days of drug administration, mice in the RG and GS groups exhibited obviously more eye secretions, less saliva secretion and less urine. Compared with the control group, the swimming times of mice in the GS, AG and GL groups were significantly prolonged; the clotting time of mice in the GL was extended significantly; VCO2, H and EE of mice in the GS group were obviously

An Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat.

PubMed Central

Kenney, S; Kamine, J; Markovitz, D; Fenrick, R; Pagano, J

1988-01-01

Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, we demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses. Images PMID:2830625

An Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat.

PubMed

Kenney, S; Kamine, J; Markovitz, D; Fenrick, R; Pagano, J

1988-03-01

Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, we demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.

BRAIN NETWORKS. Correlated gene expression supports synchronous activity in brain networks.

PubMed

Richiardi, Jonas; Altmann, Andre; Milazzo, Anna-Clare; Chang, Catie; Chakravarty, M Mallar; Banaschewski, Tobias; Barker, Gareth J; Bokde, Arun L W; Bromberg, Uli; Büchel, Christian; Conrod, Patricia; Fauth-Bühler, Mira; Flor, Herta; Frouin, Vincent; Gallinat, Jürgen; Garavan, Hugh; Gowland, Penny; Heinz, Andreas; Lemaître, Hervé; Mann, Karl F; Martinot, Jean-Luc; Nees, Frauke; Paus, Tomáš; Pausova, Zdenka; Rietschel, Marcella; Robbins, Trevor W; Smolka, Michael N; Spanagel, Rainer; Ströhle, Andreas; Schumann, Gunter; Hawrylycz, Mike; Poline, Jean-Baptiste; Greicius, Michael D

2015-06-12

During rest, brain activity is synchronized between different regions widely distributed throughout the brain, forming functional networks. However, the molecular mechanisms supporting functional connectivity remain undefined. We show that functional brain networks defined with resting-state functional magnetic resonance imaging can be recapitulated by using measures of correlated gene expression in a post mortem brain tissue data set. The set of 136 genes we identify is significantly enriched for ion channels. Polymorphisms in this set of genes significantly affect resting-state functional connectivity in a large sample of healthy adolescents. Expression levels of these genes are also significantly associated with axonal connectivity in the mouse. The results provide convergent, multimodal evidence that resting-state functional networks correlate with the orchestrated activity of dozens of genes linked to ion channel activity and synaptic function. Copyright © 2015, American Association for the Advancement of Science.

Characterization of two catalase-peroxidase-encoding genes in Fusarium verticillioides reveals differential responses to in vitro versus in planta oxidative challenges

USDA-ARS?s Scientific Manuscript database

Catalase/peroxidases (KatGs) are a superfamily of reactive oxygen species (ROS)-degrading enzymes believed to be horizontally acquired by ancient Ascomycota from bacteria. Subsequent gene duplication resulted in two KatG paralogs in ascomycetes: the widely distributed intracellular KatG1 group, and ...

Activation of endothelial-leukocyte adhesion molecule 1 (ELAM-1) gene transcription

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

Montgomery, K.F.; Tarr, P.I.; Bomsztyk, K.

1991-08-01

Leukocyte adherence to endothelium is in part mediated by the transient expression of endothelial-leukocyte adhesion molecule 1 (ELAM-1) on endothelial surfaces stimulated by tumor necrosis factor {alpha} (TNF), interleukin (IL) 1, or bacterial lipopolysaccharide (LPS). The intracellular factors controlling induction of ELAM-1 mRNA and protein are unknown. In nuclear runoff experiments with cultured human umbilical vein endothelial cells (HUVEC), the authors demonstrate that transcriptional activation of the ELAM-1 gene occurs following stimulation with TNF. Sequence analysis of the 5{prime} flanking region of the ELAM-1 gene reveals consensus DNA-binding sequences for two known transcription factors, NF-{kappa}B and AP-1. Gel mobility shiftmore » assays demonstrate that TNF, IL-1, or LPS induces activation of NF-{kappa}B-like DNA binding activity in HUVEC. Phorbol 12-myristate 13-acetate, a known activator of protein kinase C (PKC), weakly induces NF-{kappa}B-like activity, ELAM-1 mRNA, and ELAM-1 surface expression in HUVEC. However, TNF, IL-1, and LPS do not activate PKC in HUVEC at doses that strongly induce NF-{kappa}B-like protein activation and ELAM-1 gene expression. PKC blockade with H7 does not inhibit activation of these NF-kB-like proteins but does inhibit ELAM-1 gene transcription. They conclude that PKC-independent activation of NF-{kappa}B in HUVEC with TNF, IL-1, or LPS is associated with, but not sufficient for, activation of ELAM-1 gene transcription.« less

The impact of generic substitution on the activities of pharmaceutical companies - a survey from the companies' perspective one year and five years after the introduction of generic substitution in finland

PubMed Central

2010-01-01

Background Mandatory generic substitution (GS) was introduced in Finland on 1 April 2003. The aim of this study was to explore and compare the impacts of GS on the activities of pharmaceutical companies representing mainly original or generic pharmaceutical products in Finland. The self-reported impact of GS from pharmaceutical companies' perspective was explored with a focus on the number of employees, the range of sales packages on the market, the marketing activities, the research and development of new pharmaceutical products and storage of pharmaceuticals. Methods A cross-sectional postal survey was conducted among pharmaceutical companies with an office in Finland and substitutable medicines in the Finnish pharmaceutical market one year (2004) and five years (2008) after the introduction of GS. Completed questionnaires were returned by 16 original and 7 generic product companies in 2004 (response rate 56%, n = 41) and by 16 original and 6 generic product companies in 2008 (response rate 56%, n = 39). Descriptive statistical analyses were performed. Results The number of employees (2004: n = 6/16, 2008: n = 7/16) and the amount of prescription medicine marketing (2004: n = 7/16, 2008: n = 8/16) decreased in many of the original product companies after the introduction of GS. GS resulted in problems related to the storage of pharmaceuticals in the original product companies shortly after GS was introduced (p = 0.032 between 2004 and 2008). In the generic product companies, the prescription medicine representatives' visits to pharmacies increased at the beginning of GS (p = 0.021 between 2004 and 2008). In addition, GS caused problems with the storage of pharmaceuticals one year and five years after the reform (2004: n = 4/7, 2008: n = 3/6). The differences between original and generic product companies regarding the impacts of GS were not, however, statistically significant. GS did not affect on the range of sales packages on the market or the research

The impact of generic substitution on the activities of pharmaceutical companies - a survey from the companies' perspective one year and five years after the introduction of generic substitution in finland.

PubMed

Timonen, Johanna; Bengtström, Marina; Karttunen, Pekka; Ahonen, Riitta

2010-10-22

Mandatory generic substitution (GS) was introduced in Finland on 1 April 2003. The aim of this study was to explore and compare the impacts of GS on the activities of pharmaceutical companies representing mainly original or generic pharmaceutical products in Finland. The self-reported impact of GS from pharmaceutical companies' perspective was explored with a focus on the number of employees, the range of sales packages on the market, the marketing activities, the research and development of new pharmaceutical products and storage of pharmaceuticals. A cross-sectional postal survey was conducted among pharmaceutical companies with an office in Finland and substitutable medicines in the Finnish pharmaceutical market one year (2004) and five years (2008) after the introduction of GS. Completed questionnaires were returned by 16 original and 7 generic product companies in 2004 (response rate 56%, n = 41) and by 16 original and 6 generic product companies in 2008 (response rate 56%, n = 39). Descriptive statistical analyses were performed. The number of employees (2004: n = 6/16, 2008: n = 7/16) and the amount of prescription medicine marketing (2004: n = 7/16, 2008: n = 8/16) decreased in many of the original product companies after the introduction of GS. GS resulted in problems related to the storage of pharmaceuticals in the original product companies shortly after GS was introduced (p = 0.032 between 2004 and 2008). In the generic product companies, the prescription medicine representatives' visits to pharmacies increased at the beginning of GS (p = 0.021 between 2004 and 2008). In addition, GS caused problems with the storage of pharmaceuticals one year and five years after the reform (2004: n = 4/7, 2008: n = 3/6). The differences between original and generic product companies regarding the impacts of GS were not, however, statistically significant. GS did not affect on the range of sales packages on the market or the research activities of the majority of

Controlling nuclear JAKs and STATs for specific gene activation by IFN{gamma}

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

Noon-Song, Ezra N.; Ahmed, Chulbul M.; Dabelic, Rea

2011-07-08

Highlights: {yields} Gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interact with the promoter region of IFN{gamma}-associated genes along with transcription factor STAT1{alpha}. {yields} We show that activated Janus kinases pJAK2 and pJAK1 also associate with IFNGR1 in the nucleus. {yields} The activated Janus kinases are responsible for phosphorylation of tyrosine 41 on histone H3, an important epigenetic event for specific gene activation. -- Abstract: We previously showed that gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interacted with the promoter region of IFN{gamma}-activated genes along with transcription factor STAT1{alpha}. Recent studies have suggested that activated Janus kinases pJAK2 andmore » pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFN{gamma}. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFN{gamma} treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The {beta}-actin gene, which is not activated by IFN{gamma}, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFN{gamma} treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFN{gamma} treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFN

Reassortant H5N1 avian influenza viruses containing PA or NP gene from an H9N2 virus significantly increase the pathogenicity in mice.

PubMed

Hao, Xiaoli; Hu, Jiao; Wang, Jiongjiong; Xu, Jing; Cheng, Hao; Xu, Yunpeng; Li, Qunhui; He, Dongchang; Liu, Xiaowen; Wang, Xiaoquan; Gu, Min; Hu, Shunlin; Xu, Xiulong; Liu, Huimou; Chen, Sujuan; Peng, Daxin; Liu, Xiufan

2016-08-30

Reassortment between different influenza viruses is a crucial way to generate novel influenza viruses with unpredictable virulence and transmissibility, which may threaten the public health. As currently in China, avian influenza viruses (AIVs) of H9N2 and H5N1 subtypes are endemic in poultry in many areas, while they are prone to reassort with each other naturally. In order to evaluate the risk of the reassortment to public health, A/Goose/Jiangsu/k0403/2010 [GS/10(H5N1)] virus was used as a backbone to generate a series of reassortants, each contained a single internal gene derived from the predominant S genotype of the A/Chicken/Jiangsu/WJ57/2012 [WJ/57(H9N2)]. We next assessed the biological characteristics of these assortments, including pathogenicity, replication efficiency and polymerase activity. We found that the parental WJ/57(H9N2) and GS/10(H5N1) viruses displayed high genetic compatibility. Notably, the H5N1 reassortants containing the PA or NP gene from WJ/57(H9N2) virus significantly increased virulence and replication ability in mice, as well as markedly enhanced polymerase activity. Our results indicate that the endemicity of H9N2 and H5N1 in domestic poultry greatly increases the possibility of generating new viruses by reassortment that may pose a great threat to poultry industry and public health. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of HMW-GS Ax1 or Dx2 absence on the glutenin polymerization and gluten micro structure of wheat (Triticum aestivum L.).

PubMed

Gao, Xin; Liu, Tianhong; Ding, Mengyun; Wang, Jun; Li, Chunlian; Wang, Zhonghua; Li, Xuejun

2018-02-01

Wheat (Triticum aestivum L.) dough strength and extensibility are mainly determined by the polymerization of glutenin. The number of high-molecular-weight glutenin subunits (HMW-GS) differs in various wheat varieties due to the silencing of some genes. The effects of Ax1 or Dx2 subunit absence on glutenin polymerization, dough mixing properties and gluten micro structure were investigated with two groups of near-isogenic lines. The results showed that Ax1 or Dx2 absence decreased the accumulation rate of glutenin polymers and thus delayed the rapid increase period for glutenin polymerization by at least ten days, which led to lower percentage of polymeric protein in mature grain. Ax1 or Dx2 absence significantly decreased the dough development time and dough stability, but increased the uniformity of micro structure. Lacunarity, derived from quantitative analysis of gluten network, is suggested as a new indicator for wheat quality. Copyright © 2017 Elsevier Ltd. All rights reserved.

Heterogeneous activation of the TGFβ pathway in glioblastomas identified by gene expression-based classification using TGFβ-responsive genes

PubMed Central

Xu, Xie L; Kapoun, Ann M

2009-01-01

Background TGFβ has emerged as an attractive target for the therapeutic intervention of glioblastomas. Aberrant TGFβ overproduction in glioblastoma and other high-grade gliomas has been reported, however, to date, none of these reports has systematically examined the components of TGFβ signaling to gain a comprehensive view of TGFβ activation in large cohorts of human glioma patients. Methods TGFβ activation in mammalian cells leads to a transcriptional program that typically affects 5–10% of the genes in the genome. To systematically examine the status of TGFβ activation in high-grade glial tumors, we compiled a gene set of transcriptional response to TGFβ stimulation from tissue culture and in vivo animal studies. These genes were used to examine the status of TGFβ activation in high-grade gliomas including a large cohort of glioblastomas. Unsupervised and supervised classification analysis was performed in two independent, publicly available glioma microarray datasets. Results Unsupervised and supervised classification using the TGFβ-responsive gene list in two independent glial tumor gene expression data sets revealed various levels of TGFβ activation in these tumors. Among glioblastomas, one of the most devastating human cancers, two subgroups were identified that showed distinct TGFβ activation patterns as measured from transcriptional responses. Approximately 62% of glioblastoma samples analyzed showed strong TGFβ activation, while the rest showed a weak TGFβ transcriptional response. Conclusion Our findings suggest heterogeneous TGFβ activation in glioblastomas, which may cause potential differences in responses to anti-TGFβ therapies in these two distinct subgroups of glioblastomas patients. PMID:19192267

Identification of fifteen novel mutations in the SLC12A3 gene encoding the Na-Cl Co-transporter in Italian patients with Gitelman syndrome.

PubMed

Syrén, Marie-Louise; Tedeschi, Silvana; Cesareo, Laila; Bellantuono, Rosa; Colussi, Giacomo; Procaccio, Mirella; Alì, Anna; Domenici, Raffaele; Malberti, Fabio; Sprocati, Monica; Sacco, Michele; Miglietti, Nunzia; Edefonti, Alberto; Sereni, Fabio; Casari, Giorgio; Coviello, Domenico A; Bettinelli, Alberto

2002-07-01

The SLC12A3 gene encodes the thiazide-sensitive Na-Cl co-transporter (NCCT) expressed in the apical membrane of the distal convoluted tubule of the kidney. Inactivating mutations of this gene are responsible for Gitelman syndrome (GS), a disorder inherited as an autosomal recessive trait. We searched for SLC12A3 gene mutations in 21 Italian patients with the clinical and biochemical features of GS (hypokalemia, hypomagnesemia, metabolic alkalosis, hypocalciuria, and the absence of nephrocalcinosis). All coding regions with their intron-exon boundaries were analyzed using PCR and SSCP techniques followed by sequencing analysis. We identified 21 different mutations evenly distributed throughout the gene without any mutation hot-spot. Fifteen are novel variants, including 12 missense mutations, one deletion, one deletion-insertion and one splice site mutation: R158Q, T163M, W172R, G316V, G374V, G463E, A464T, S615W, V677M, R852S, R958G, C985Y, 2114-2120delACCAAGT, 2144-2158delGCCTTCTACTCGGATinsTG, and 531-2A>G. Copyright 2002 Wiley-Liss, Inc.

Tissue plasminogen activator (tPA) as a reporter gene in transient gene expression.

PubMed

Cheng, S M; Lee, S G; Kalyan, N K; McCloud, S; Levner, M; Hung, P P

1987-01-01

Using the gene coding for tissue plasminogen activator (tPA) as a reporter gene, a transient gene expression system has been established. Vectors containing the full-length cDNA of tPA with its signal sequences were introduced into mammalian recipient cells by a modified gene transfer procedure. Thirty hours after transfection, the secreted tPA was found in serum-free medium and measured by a fibrin-agarose plate assay (FAPA). In this assay, tPA converts plasminogen into plasmin which then degrades high-Mr fibrin to produce cleared zones. The sizes of these zones correspond to quantities of tPA. The combination of transient tPA expression system and the FAPA provides a quick, sensitive, quantitative and non-destructive method to examine the strength of eukaryotic regulatory elements in tissue-culture cells.

MafK/NF-E2 p18 is required for beta-globin genes activation by mediating the proximity of LCR and active beta-globin genes in MEL cell line.

PubMed

Du, Mei-Jun; Lv, Xiang; Hao, De-Long; Zhao, Guo-Wei; Wu, Xue-Song; Wu, Feng; Liu, De-Pei; Liang, Chih-Chuan

2008-01-01

Evidences indicate that locus control region (LCR) of beta-globin spatially closes to the downstream active gene promoter to mediate the transcriptional activation by looping. DNA binding proteins may play an important role in the looping formation. NF-E2 is one of the key transcription factors in beta-globin gene transcriptional activation. To shed light on whether NF-E2 is involved in this process, DS19MafKsiRNA cell pools were established by specifically knocked down the expression of MafK/NF-E2 p18, one subunit of NF-E2 heterodimer. In the above cell pools, it was observed that the occupancy efficiency of NF-E2 on beta-globin gene locus and the expression level of beta-globin genes were decreased. H3 acetylation, H3-K4 methylation and the deposition of RNA polymerase II, but not the recruitment of GATA-1, were also found reduced at the beta-globin gene cluster. Chromosome Conformation Capture (3C) assay showed that the cross-linking frequency between the main NF-E2 binding site HS2 and downstream structural genes was reduced compared to the normal cell. This result demonstrated that MafK/NF-E2 p18 recruitment was involved in the physical proximity of LCR and active beta-globin genes upon beta-globin gene transcriptional activation.

Website for popularization of meteorology

NASA Astrophysics Data System (ADS)

Špoler Čanić, K.; Rasol, D.

2012-04-01

Little meteorological workshop (LMW) is an educational project that has started in 2007 at the Science Festival in Zagreb, Croatia. In 2009 began a new phase of the project which was introduction of the LMW as an extracurricular school activity for pupils. To reach more users the authors of the LMW published a booklet of experiments which were conducted at the workshops in schools. Furthermore, a website (www.malameteo.com) that shows how to make those experiments was developed. The website has some more educational information as well. Here, the content of the website will be presented.

Genome Sequence of Aeribacillus pallidus Strain GS3372, an Endospore-Forming Bacterium Isolated in a Deep Geothermal Reservoir

PubMed Central

Filippidou, Sevasti; Jaussi, Marion; Junier, Thomas; Wunderlin, Tina; Jeanneret, Nicole; Regenspurg, Simona; Li, Po-E; Lo, Chien-Chi; McMurry, Kim; Gleasner, Cheryl D.; Vuyisich, Momchilo; Chain, Patrick S.

2015-01-01

The genome of strain GS3372 is the first publicly available strain of Aeribacillus pallidus. This endospore-forming thermophilic strain was isolated from a deep geothermal reservoir. The availability of this genome can contribute to the clarification of the taxonomy of the closely related Anoxybacillus, Geobacillus, and Aeribacillus genera. PMID:26316637

Evolutionary relationships between miRNA genes and their activity.

PubMed

Zhu, Yan; Skogerbø, Geir; Ning, Qianqian; Wang, Zhen; Li, Biqing; Yang, Shuang; Sun, Hong; Li, Yixue

2012-12-22

The emergence of vertebrates is characterized by a strong increase in miRNA families. MicroRNAs interact broadly with many transcripts, and the evolution of such a system is intriguing. However, evolutionary questions concerning the origin of miRNA genes and their subsequent evolution remain unexplained. In order to systematically understand the evolutionary relationship between miRNAs gene and their function, we classified human known miRNAs into eight groups based on their evolutionary ages estimated by maximum parsimony method. New miRNA genes with new functional sequences accumulated more dynamically in vertebrates than that observed in Drosophila. Different levels of evolutionary selection were observed over miRNA gene sequences with different time of origin. Most genic miRNAs differ from their host genes in time of origin, there is no particular relationship between the age of a miRNA and the age of its host genes, genic miRNAs are mostly younger than the corresponding host genes. MicroRNAs originated over different time-scales are often predicted/verified to target the same or overlapping sets of genes, opening the possibility of substantial functional redundancy among miRNAs of different ages. Higher degree of tissue specificity and lower expression level was found in young miRNAs. Our data showed that compared with protein coding genes, miRNA genes are more dynamic in terms of emergence and decay. Evolution patterns are quite different between miRNAs of different ages. MicroRNAs activity is under tight control with well-regulated expression increased and targeting decreased over time. Our work calls attention to the study of miRNA activity with a consideration of their origin time.

Cdk5 Regulates Activity-Dependent Gene Expression and Dendrite Development.

PubMed

Liang, Zhuoyi; Ye, Tao; Zhou, Xiaopu; Lai, Kwok-On; Fu, Amy K Y; Ip, Nancy Y

2015-11-11

The proper growth and arborization of dendrites in response to sensory experience are essential for neural connectivity and information processing in the brain. Although neuronal activity is important for sculpting dendrite morphology, the underlying molecular mechanisms are not well understood. Here, we report that cyclin-dependent kinase 5 (Cdk5)-mediated transcriptional regulation is a key mechanism that controls activity-dependent dendrite development in cultured rat neurons. During membrane depolarization, Cdk5 accumulates in the nucleus to regulate the expression of a subset of genes, including that of the neurotrophin brain-derived neurotrophic factor, for subsequent dendritic growth. Furthermore, Cdk5 function is mediated through the phosphorylation of methyl-CpG-binding protein 2, a key transcriptional repressor that is mutated in the mental disorder Rett syndrome. These findings collectively suggest that the nuclear import of Cdk5 is crucial for activity-dependent dendrite development by regulating neuronal gene transcription during neural development. Neural activity directs dendrite development through the regulation of gene transcription. However, how molecular signals link extracellular stimuli to the transcriptional program in the nucleus remains unclear. Here, we demonstrate that neuronal activity stimulates the translocation of the kinase Cdk5 from the cytoplasmic compartment into the nucleus; furthermore, the nuclear localization of Cdk5 is required for dendrite development in cultured neurons. Genome-wide transcriptome analysis shows that Cdk5 deficiency specifically disrupts activity-dependent gene transcription of bdnf. The action of Cdk5 is mediated through the modulation of the transcriptional repressor methyl-CpG-binding protein 2. Therefore, this study elucidates the role of nuclear Cdk5 in the regulation of activity-dependent gene transcription and dendritic growth. Copyright © 2015 the authors 0270-6474/15/3515127-08$15.00/0.

Kinetic commitment in the catalysis of glutamine synthesis by GS1 from Arabidopsis using 14N/15N and solvent isotope effects.

PubMed

Mauve, Caroline; Giraud, Nicolas; Boex-Fontvieille, Edouard R A; Antheaume, Ingrid; Tea, Illa; Tcherkez, Guillaume

2016-11-01

Glutamine synthetase (GS, EC 6.3.1.2) catalyzes the production of glutamine from glutamate, ammonium and ATP. Although being essential in plants for N assimilation and recycling, kinetic commitments and transition states of the reaction have not been clearly established yet. Here, we examined 12 C/ 13 C, 14 N/ 15 N and H 2 O/D 2 O isotope effects in Arabidopsis GS1 catalysis and compared to the prokaryotic (Escherichia coli) enzyme. A 14 N/ 15 N isotope effect ( 15 V/K ≈ 1.015, with respect to substrate NH 4 + ) was observed in the prokaryotic enzyme, indicating that ammonium utilization (deprotonation and/or amidation) was partially rate-limiting. In the plant enzyme, the isotope effect was inverse ( 15 V/K = 0.965), suggesting that the reaction intermediate is involved in an amidation-deamidation equilibrium favoring 15 N. There was no 12 C/ 13 C kinetic isotope effect ( 13 V/K = 1.000), suggesting that the amidation step of the catalytic cycle involves a transition state with minimal alteration of overall force constants at the C-5 carbon. Surprisingly, the solvent isotope effect was found to be inverse, that is, with a higher turn-over rate in heavy water ( D V ≈ 0.5), showing that restructuration of the active site due to displacement of H 2 O by D 2 O facilitates the processing of intermediates. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Diagnostic value of immunoglobulin κ light chain gene rearrangement analysis in B-cell lymphomas.

PubMed

Kokovic, Ira; Jezersek Novakovic, Barbara; Novakovic, Srdjan

2015-03-01

Analysis of the immunoglobulin κ light chain (IGK) gene is an alternative method for B-cell clonality assessment in the diagnosis of mature B-cell proliferations in which the detection of clonal immunoglobulin heavy chain (IGH) gene rearrangements fails. The aim of the present study was to evaluate the added value of standardized BIOMED-2 assay for the detection of clonal IGK gene rearrangements in the diagnostic setting of suspected B-cell lymphomas. With this purpose, 92 specimens from 80 patients with the final diagnosis of mature B-cell lymphoma (37 specimens), mature T-cell lymphoma (26 specimens) and reactive lymphoid proliferation (29 specimens) were analyzed for B-cell clonality. B-cell clonality analysis was performed using the BIOMED-2 IGH and IGK gene clonality assays. The determined sensitivity of the IGK assay was 67.6%, while the determined sensitivity of the IGH assay was 75.7%. The sensitivity of combined IGH+IGK assay was 81.1%. The determined specificity of the IGK assay was 96.2% in the group of T-cell lymphomas and 96.6% in the group of reactive lesions. The determined specificity of the IGH assay was 84.6% in the group of lymphomas and 86.2% in the group of reactive lesions. The comparison of GeneScan (GS) and heteroduplex pretreatment-polyacrylamide gel electrophoresis (HD-PAGE) methods for the analysis of IGK gene rearrangements showed a higher efficacy of GS analysis in a series of 27 B-cell lymphomas analyzed by both methods. In the present study, we demonstrated that by applying the combined IGH+IGK clonality assay the overall detection rate of B-cell clonality was increased by 5.4%. Thus, we confirmed the added value of the standardized BIOMED-2 IGK assay for assessment of B-cell clonality in suspected B-cell lymphomas with inconclusive clinical and cyto/histological diagnosis.

Association of Body Mass Index with DNA Methylation and Gene Expression in Blood Cells and Relations to Cardiometabolic Disease: A Mendelian Randomization Approach

PubMed Central

Joehanes, Roby; Liu, Chunyu; Aslibekyan, Stella; Demerath, Ellen W.; Guan, Weihua; Zhi, Degui; Willinger, Christine; Courchesne, Paul; Multhaup, Michael; Irvin, Marguerite R.; Schadt, Eric E.; Bressler, Jan; North, Kari; Sundström, Johan; Gustafsson, Stefan; Shah, Sonia; McRae, Allan F.; Harris, Sarah E.; Gibson, Jude; Redmond, Paul; Corley, Janie; Starr, John M.; Visscher, Peter M.; Wray, Naomi R.; Krauss, Ronald M.; Feinberg, Andrew; Fornage, Myriam; Pankow, James S.; Lind, Lars; Fox, Caroline; Ingelsson, Erik; Arnett, Donna K.; Boerwinkle, Eric; Liang, Liming; Levy, Daniel; Deary, Ian J.

2017-01-01

Background The link between DNA methylation, obesity, and adiposity-related diseases in the general population remains uncertain. Methods and Findings We conducted an association study of body mass index (BMI) and differential methylation for over 400,000 CpGs assayed by microarray in whole-blood-derived DNA from 3,743 participants in the Framingham Heart Study and the Lothian Birth Cohorts, with independent replication in three external cohorts of 4,055 participants. We examined variations in whole blood gene expression and conducted Mendelian randomization analyses to investigate the functional and clinical relevance of the findings. We identified novel and previously reported BMI-related differential methylation at 83 CpGs that replicated across cohorts; BMI-related differential methylation was associated with concurrent changes in the expression of genes in lipid metabolism pathways. Genetic instrumental variable analysis of alterations in methylation at one of the 83 replicated CpGs, cg11024682 (intronic to sterol regulatory element binding transcription factor 1 [SREBF1]), demonstrated links to BMI, adiposity-related traits, and coronary artery disease. Independent genetic instruments for expression of SREBF1 supported the findings linking methylation to adiposity and cardiometabolic disease. Methylation at a substantial proportion (16 of 83) of the identified loci was found to be secondary to differences in BMI. However, the cross-sectional nature of the data limits definitive causal determination. Conclusions We present robust associations of BMI with differential DNA methylation at numerous loci in blood cells. BMI-related DNA methylation and gene expression provide mechanistic insights into the relationship between DNA methylation, obesity, and adiposity-related diseases. PMID:28095459

Increased urinary prostaglandin E2 metabolite: A potential therapeutic target of Gitelman syndrome.

PubMed

Peng, Xiaoyan; Jiang, Lanping; Chen, Chen; Qin, Yan; Yuan, Tao; Wang, Ou; Xing, Xiaoping; Li, Xuemei; Nie, Min; Chen, Limeng

2017-01-01

Gitelman syndrome (GS), an inherited autosomal recessive salt-losing renal tubulopathy caused by mutations in SLC12A3 gene, has been associated with normal prostaglandin E2 (PGE2) levels since 1995 by a study involving 11 clinically diagnosed patients. However, it is difficult to explain why cyclooxygenase-2 (COX2) inhibitors, which pharmacologically reduce PGE2 synthesis, are helpful to patients with GS, and few studies performed in the last 20 years have measured PGE2 levels. The relationships between the clinical manifestations and PGE2 levels were never thoroughly analyzed. This study involved 39 GS patients diagnosed by SLC12A3 gene sequencing. Plasma and 24-h urine samples as well as the clinical data were collected at admission. PGE2 and PGEM levels were detected in plasma and urine samples by enzyme immunoassays. The in vivo function of the sodium-chloride co-transporter (NCC) in GS patients was evaluated using a modified thiazide test. The association among PGE2 levels, clinical manifestations and the function of NCC in GS patients were analyzed. Significantly higher levels of urinary and plasma PGEM were observed in GS patients than in the healthy volunteers. Higher urinary PGEM levels indicated more severe clinical manifestations and NCC dysfunction estimated by the increase of Cl- clearance. A higher PGEM level was found in male GS patients, who showed earlier onset age and more severe hypokalemia, hypochloremia and metabolic alkalosis than female GS patients. No relationship between renin angiotensin aldosterone system activation and PGEM level was observed. Higher urinary PGEM levels indicated more severe clinical manifestations and NCC dysfunction in GS patients. COX2 inhibition might be a potential therapeutic target in GS patients with elevated PGEM levels.

Profilin is associated with transcriptionally active genes

PubMed Central

Söderberg, Emilia; Hessle, Viktoria; von Euler, Anne; Visa, Neus

2012-01-01

We have raised antibodies against the profilin of Chironomus tentans to study the location of profilin relative to chromatin and to active genes in salivary gland polytene chromosomes. We show that a fraction of profilin is located in the nucleus, where profilin is highly concentrated in the nucleoplasm and at the nuclear periphery. Moreover, profilin is associated with multiple bands in the polytene chromosomes. By staining salivary glands with propidium iodide, we show that profilin does not co-localize with dense chromatin. Profilin associates instead with protein-coding genes that are transcriptionally active, as revealed by co-localization with hnRNP and snRNP proteins. We have performed experiments of transcription inhibition with actinomycin D and we show that the association of profilin with the chromosomes requires ongoing transcription. However, the interaction of profilin with the gene loci does not depend on RNA. Our results are compatible with profilin regulating actin polymerization in the cell nucleus. However, the association of actin with the polytene chromosomes of C. tentans is sensitive to RNase, whereas the association of profilin is not, and we propose therefore that the chromosomal location of profilin is independent of actin. PMID:22572953

Inhibition of nitrogen-fixing activity of the cyanobiont affects the localization of glutamine synthetase in hair cells of Azolla.

PubMed

Uheda, Eiji; Maejima, Kazuhiro

2009-10-15

In the Azolla-Anabaena association, the host plant Azolla efficiently incorporates and assimilates ammonium ions that are released from the nitrogen-fixing cyanobiont, probably via glutamine synthetase (GS; EC 6.3.1.2) in hair cells, which are specialized cells protruding into the leaf cavity. In order to clarify the regulatory mechanism underlying ammonium assimilation in the Azolla-Anabaena association, Azolla plants were grown under an argon environment (Ar), in which the nitrogen-fixing activity of the cyanobiont was inhibited specifically and completely. The localization of GS in hair cells was determined by immunoelectron microscopy and quantitative analysis of immunogold labeling. Azolla plants grew healthily under Ar when nitrogen sources, such as NO(3)(-) and NH(4)(+), were provided in the growth medium. Both the number of cyanobacterial cells per leaf and the heterocyst frequency of the plants under Ar were similar to those of plants in a nitrogen environment (N(2)). In hair cells of plants grown under Ar, regardless of the type of nitrogen source provided, only weak labeling of GS was observed in the cytoplasm and in chloroplasts. In contrast, in hair cells of plants grown under N(2), abundant labeling of GS was observed in both sites. These findings indicate that specific inhibition of the nitrogen-fixing activity of the cyanobiont affects the localization of GS isoenzymes. Ammonium fixed and released by the cyanobiont could stimulate GS synthesis in hair cells. Simultaneously, the abundant GS, probably GS1, in these cells, could assimilate ammonium rapidly.

A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: process development and product quality assessment.

PubMed

Rajendra, Yashas; Hougland, Maria D; Alam, Riazul; Morehead, Teresa A; Barnard, Gavin C

2015-05-01

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS-KO host cell line. By creating a bi-allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS-KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)-mediated transfections were performed at high cell density (4 × 10(6) cells/mL) followed by immediate growth arrest at 32 °C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre-complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7-day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc-fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7-day process. Titers

Effects of Lys to Glu mutations in GsMTx4 on membrane binding, peptide orientation, and self-association propensity, as analyzed by molecular dynamics simulations.

PubMed

Nishizawa, Kazuhisa; Nishizawa, Manami; Gnanasambandam, Radhakrishnan; Sachs, Frederick; Sukharev, Sergei I; Suchyna, Thomas M

2015-11-01

GsMTx4, a gating modifier peptide acting on cationic mechanosensitive channels, has a positive charge (+5e) due to six Lys residues. The peptide does not have a stereospecific binding site on the channel but acts from the boundary lipids within a Debye length of the pore probably by changing local stress. To gain insight into how these Lys residues interact with membranes, we performed molecular dynamics simulations of Lys to Glu mutants in parallel with our experimental work. In silico, K15E had higher affinity for 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine bilayers than wild-type (WT) peptide or any other mutant tested, and showed deeper penetration than WT, a finding consistent with the experimental data. Experimentally, the inhibitory activities of K15E and K25E were most compromised, whereas K8E and K28E inhibitory activities remained similar to WT peptide. Binding of WT in an interfacial mode did not influence membrane thickness. With interfacial binding, the direction of the dipole moments of K15E and K25E was predicted to differ from WT, whereas those of K8E and K28E oriented similarly to that of WT. These results support a model in which binding of GsMTx4 to the membrane acts like an immersible wedge that serves as a membrane expansion buffer reducing local stress and thus inhibiting channel activity. In simulations, membrane-bound WT attracted other WT peptides to form aggregates. This may account for the positive cooperativity observed in the ion channel experiments. The Lys residues seem to fine-tune the depth of membrane binding, the tilt angle, and the dipole moments. Copyright © 2015 Elsevier B.V. All rights reserved.

Genome Sequence of Aeribacillus pallidus Strain GS3372, an Endospore-Forming Bacterium Isolated in a Deep Geothermal Reservoir.

PubMed

Filippidou, Sevasti; Jaussi, Marion; Junier, Thomas; Wunderlin, Tina; Jeanneret, Nicole; Regenspurg, Simona; Li, Po-E; Lo, Chien-Chi; Johnson, Shannon; McMurry, Kim; Gleasner, Cheryl D; Vuyisich, Momchilo; Chain, Patrick S; Junier, Pilar

2015-08-27

The genome of strain GS3372 is the first publicly available strain of Aeribacillus pallidus. This endospore-forming thermophilic strain was isolated from a deep geothermal reservoir. The availability of this genome can contribute to the clarification of the taxonomy of the closely related Anoxybacillus, Geobacillus, and Aeribacillus genera. Copyright © 2015 Filippidou et al.

Comparison of the effect of the GABAB receptor agonist, baclofen, and the positive allosteric modulator of the GABAB receptor, GS39783, on alcohol self-administration in three different lines of alcohol-preferring rats

PubMed Central

Maccioni, Paola; Zaru, Alessandro; Loi, Barbara; Lobina, Carla; Carai, Mauro A.M.; Gessa, Gian Luigi; Capra, Alessandro; Mugnaini, Claudia; Pasquini, Serena; Corelli, Federico; Hyytiä, Petri; Lumeng, Lawrence; Colombo, Giancarlo

2012-01-01

Background Administration of the GABAB receptor agonist, baclofen, and positive allosteric modulator (PAM), GS39783, has been repeatedly reported to suppress multiple alcohol-related behaviors, including operant oral alcohol self-administration, in rats. The present study was designed to compare the effect of baclofen and GS39783 on alcohol self-administration in three lines of selectively bred, alcohol-preferring rats: Indiana alcohol-preferring (P), Sardinian alcohol-preferring (sP), and Alko Alcohol (AA). Methods Rats of each line were initially trained to respond on a lever, on a fixed ratio (FR) 4 (FR4) schedule of reinforcement, to orally self-administer alcohol (15%, v/v) in daily 30-min sessions. Once responding reached stable levels, rats were exposed to a sequence of experiments testing baclofen (0, 1, 1.7, and 3 mg/kg; i.p.) and GS39783 (0, 25, 50, and 100 mg/kg; i.g.) on FR4 and progressive ratio (PR) schedules of reinforcement. Finally, to assess the specificity of baclofen and GS39783 action, rats were slightly food-deprived and trained to lever-respond for food pellets. Results The rank of order of the reinforcing and motivational properties of alcohol was: P>sP>AA rats. Under both FR and PR schedules of reinforcement, the rank of order of potency and efficacy of baclofen and GS39783 in suppressing alcohol self-administration was: P>sP>AA rats. Only the highest dose of baclofen reduced lever-responding for food pellets; this effect was common to all three rat lines. Conversely, no dose of GS39783 altered lever-responding for food in any rat line. Conclusions These results suggest that: (a) the strength of the reinforcing and motivational properties of alcohol differ among P, sP, and AA rats; (b) the reinforcing and motivational properties of alcohol in P, sP, and AA rats are differentially sensitive to treatment with baclofen and GS39783; (c) the heterogeneity in sensitivity to baclofen and GS39783 of alcohol self-administration in P, sP, and AA rats

Predicting home and community walking activity in people with stroke.

PubMed

Fulk, George D; Reynolds, Chelsea; Mondal, Sumona; Deutsch, Judith E

2010-10-01

To determine the ability of the 6-minute walk test (6MWT) and other commonly used clinical outcome measures to predict home and community walking activity in high-functioning people with stroke. Cross-sectional. Outpatient physical therapy clinic. Participants (N=32) with chronic stroke (n=19; >6mo poststroke) with self-selected gait speed (GS) faster than .40m/s and age-matched healthy participants (n=13). Not applicable. 6MWT, self-selected GS, Berg Balance Scale (BBS), lower extremity motor section of the Fugl-Meyer Assessment, and Stroke Impact Scale. Dependent variable: average steps taken per day during a 7-day period, measured using an accelerometer. 6MWT, self-selected GS, and BBS were moderately related to home and community walking activity. The 6MWT was the only predictor of average steps taken per day; it explained 46% of the variance in steps per day. The 6MWT is a useful outcome measure in higher functioning people with stroke to guide intervention and assess community walking activity. Copyright © 2010 American Congress of Rehabilitation Medicine. Published by Elsevier Inc. All rights reserved.

Glutamine synthetase in Medicago truncatula, unveiling new secrets of a very old enzyme

PubMed Central

Seabra, Ana R.; Carvalho, Helena G.

2015-01-01

Glutamine synthetase (GS) catalyzes the first step at which nitrogen is brought into cellular metabolism and is also involved in the reassimilation of ammonium released by a number of metabolic pathways. Due to its unique position in plant nitrogen metabolism, GS plays essential roles in all aspects of plant development, from germination to senescence, and is a key component of nitrogen use efficiency (NUE) and plant yield. Understanding the mechanisms regulating GS activity is therefore of utmost importance and a great effort has been dedicated to understand how GS is regulated in different plant species. The present review summarizes exciting recent developments concerning the structure and regulation of GS isoenzymes, using the model legume Medicago truncatula. These include the understanding of the structural determinants of both the cytosolic and plastid located isoenzymes, the existence of a seed-specific GS gene unique to M. truncatula and closely related species and the discovery that GS isoenzymes are regulated by nitric oxide at the post-translational level. The data is discussed and integrated with the potential roles of the distinct GS isoenzymes within the whole plant context. PMID:26284094

Chronic exposure to glufosinate-ammonium induces spatial memory impairments, hippocampal MRI modifications and glutamine synthetase activation in mice.

PubMed

Calas, André-Guilhem; Richard, Olivier; Même, Sandra; Beloeil, Jean-Claude; Doan, Bich-Thuy; Gefflaut, Thierry; Même, William; Crusio, Wim E; Pichon, Jacques; Montécot, Céline

2008-07-01

Glufosinate-ammonium (GLA), the active compound of a worldwide-used herbicide, acts by inhibiting the plant glutamine synthetase (GS) leading to a lethal accumulation of ammonia. GS plays a pivotal role in the mammalian brain where it allows neurotransmitter glutamate recycling within astroglia. Clinical studies report that an acute GLA ingestion induces convulsions and memory impairment in humans. Toxicological studies performed at doses used for herbicidal activity showed that GLA is probably harmless at short or medium range periods. However, effects of low doses of GLA on chronically exposed subjects are not known. In our study, C57BL/6J mice were treated during 10 weeks three times a week with 2.5, 5 and 10mg/kg of GLA. Effects of this chronic treatment were assessed at behavioral, structural and metabolic levels by using tests of spatial memory, locomotor activity and anxiety, hippocampal magnetic resonance imaging (MRI) texture analysis, and hippocampal GS activity assay, respectively. Chronic GLA treatments have effects neither on anxiety nor on locomotor activity of mice but at 5 and 10mg/kg induce (1) mild memory impairments, (2) a modification of hippocampal texture and (3) a significant increase in hippocampal GS activity. It is suggested that these modifications may be causally linked one to another. Since glutamate is the main neurotransmitter in hippocampus where it plays a crucial role in spatial memory, hippocampal MRI texture and spatial memory alterations might be the consequences of hippocampal glutamate homeostasis modification revealed by increased GS activity in hippocampus. The present study provides the first data that show cerebral alterations after chronic exposure to GLA.

B(IS4; 0{GS/+}→4{/γ +}) systematics in rare-earth nuclei: SUsdg (3) description

NASA Astrophysics Data System (ADS)

Devi, Y. D.; Kota, V. K. B.

1993-12-01

The observed variation of B(IS4; 0{GS/+}→{/γ +}) with mass number A, that gives information about hexadecupole component in γ-vibration, in rare-earth nuclei is studied in the SUsdg (3) limit of sdg interacting boson model employing IBM-2 to IBM-1 projected hexadecupole transition operator with effective charges determined using a multi- j shell mapping procedure. The SUsdg (3) limit provides a reasonably good description of the data.

Replication-Competent Controlled Herpes Simplex Virus

PubMed Central

Bloom, David C.; Feller, Joyce; McAnany, Peterjon; Vilaboa, Nuria

2015-01-01

ABSTRACT We present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn+ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model. IMPORTANCE The alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate

Release of skeletal muscle peptide fragments identifies individual proteins degraded during insulin deprivation in type 1 diabetic humans and mice.

PubMed

Robinson, Matthew M; Dasari, Surendra; Karakelides, Helen; Bergen, H Robert; Nair, K Sreekumaran

2016-09-01

Insulin regulates skeletal muscle protein degradation, but the types of proteins being degraded in vivo remain to be determined due to methodological limitations. We present a method to assess the types of skeletal muscle proteins that are degraded by extracting their degradation products as low-molecular weight (LMW) peptides from muscle samples. High-resolution mass spectrometry was used to identify the original intact proteins that generated the LMW peptides, which we validated in rodents and then applied to humans. We deprived insulin from insulin-treated streptozotocin (STZ) diabetic mice for 6 and 96 h and for 8 h in type 1 diabetic humans (T1D) for comparison with insulin-treated conditions. Protein degradation was measured using activation of autophagy and proteasome pathways, stable isotope tracers, and LMW approaches. In mice, insulin deprivation activated proteasome pathways and autophagy in muscle homogenates and isolated mitochondria. Reproducibility analysis of LMW extracts revealed that ∼80% of proteins were detected consistently. As expected, insulin deprivation increased whole body protein turnover in T1D. Individual protein degradation increased with insulin deprivation, including those involved in mitochondrial function, proteome homeostasis, nDNA support, and contractile/cytoskeleton. Individual mitochondrial proteins that generated more LMW fragment with insulin deprivation included ATP synthase subunit-γ (+0.5-fold, P = 0.007) and cytochrome c oxidase subunit 6 (+0.305-fold, P = 0.03). In conclusion, identifying LMW peptide fragments offers an approach to determine the degradation of individual proteins. Insulin deprivation increases degradation of select proteins and provides insight into the regulatory role of insulin in maintaining proteome homeostasis, especially of mitochondria. Copyright © 2016 the American Physiological Society.

Release of skeletal muscle peptide fragments identifies individual proteins degraded during insulin deprivation in type 1 diabetic humans and mice

PubMed Central

Robinson, Matthew M.; Dasari, Surendra; Karakelides, Helen; Bergen, H. Robert

2016-01-01

Insulin regulates skeletal muscle protein degradation, but the types of proteins being degraded in vivo remain to be determined due to methodological limitations. We present a method to assess the types of skeletal muscle proteins that are degraded by extracting their degradation products as low-molecular weight (LMW) peptides from muscle samples. High-resolution mass spectrometry was used to identify the original intact proteins that generated the LMW peptides, which we validated in rodents and then applied to humans. We deprived insulin from insulin-treated streptozotocin (STZ) diabetic mice for 6 and 96 h and for 8 h in type 1 diabetic humans (T1D) for comparison with insulin-treated conditions. Protein degradation was measured using activation of autophagy and proteasome pathways, stable isotope tracers, and LMW approaches. In mice, insulin deprivation activated proteasome pathways and autophagy in muscle homogenates and isolated mitochondria. Reproducibility analysis of LMW extracts revealed that ∼80% of proteins were detected consistently. As expected, insulin deprivation increased whole body protein turnover in T1D. Individual protein degradation increased with insulin deprivation, including those involved in mitochondrial function, proteome homeostasis, nDNA support, and contractile/cytoskeleton. Individual mitochondrial proteins that generated more LMW fragment with insulin deprivation included ATP synthase subunit-γ (+0.5-fold, P = 0.007) and cytochrome c oxidase subunit 6 (+0.305-fold, P = 0.03). In conclusion, identifying LMW peptide fragments offers an approach to determine the degradation of individual proteins. Insulin deprivation increases degradation of select proteins and provides insight into the regulatory role of insulin in maintaining proteome homeostasis, especially of mitochondria. PMID:27436610

Pollen-mediated gene flow from glyphosate-resistant common waterhemp (Amaranthus rudis Sauer): consequences for the dispersal of resistance genes

PubMed Central

Sarangi, Debalin; Tyre, Andrew J.; Patterson, Eric L.; Gaines, Todd A.; Irmak, Suat; Knezevic, Stevan Z.; Lindquist, John L.; Jhala, Amit J.

2017-01-01

Gene flow is an important component in evolutionary biology; however, the role of gene flow in dispersal of herbicide-resistant alleles among weed populations is poorly understood. Field experiments were conducted at the University of Nebraska-Lincoln to quantify pollen-mediated gene flow (PMGF) from glyphosate-resistant (GR) to -susceptible (GS) common waterhemp using a concentric donor-receptor design. More than 130,000 common waterhemp plants were screened and 26,199 plants were confirmed resistant to glyphosate. Frequency of gene flow from all distances, directions, and years was estimated with a double exponential decay model using Generalized Nonlinear Model (package gnm) in R. PMGF declined by 50% at <3 m distance from the pollen source, whereas 90% reduction was found at 88 m (maximum) depending on the direction of the pollen-receptor blocks. Amplification of the target site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), was identified as the mechanism of glyphosate resistance in parent biotype. The EPSPS gene amplification was heritable in common waterhemp and can be transferred via PMGF, and also correlated with glyphosate resistance in pseudo-F2 progeny. This is the first report of PMGF in GR common waterhemp and the results are critical in explaining the rapid dispersal of GR common waterhemp in Midwestern United States. PMID:28327669

System-Wide Associations between DNA-Methylation, Gene Expression, and Humoral Immune Response to Influenza Vaccination.

PubMed

Zimmermann, Michael T; Oberg, Ann L; Grill, Diane E; Ovsyannikova, Inna G; Haralambieva, Iana H; Kennedy, Richard B; Poland, Gregory A

2016-01-01

Failure to achieve a protected state after influenza vaccination is poorly understood but occurs commonly among aged populations experiencing greater immunosenescence. In order to better understand immune response in the elderly, we studied epigenetic and transcriptomic profiles and humoral immune response outcomes in 50-74 year old healthy participants. Associations between DNA methylation and gene expression reveal a system-wide regulation of immune-relevant functions, likely playing a role in regulating a participant's propensity to respond to vaccination. Our findings show that sites of methylation regulation associated with humoral response to vaccination impact known cellular differentiation signaling and antigen presentation pathways. We performed our analysis using per-site and regionally average methylation levels, in addition to continuous or dichotomized outcome measures. The genes and molecular functions implicated by each analysis were compared, highlighting different aspects of the biologic mechanisms of immune response affected by differential methylation. Both cis-acting (within the gene or promoter) and trans-acting (enhancers and transcription factor binding sites) sites show significant associations with measures of humoral immunity. Specifically, we identified a group of CpGs that, when coordinately hypo-methylated, are associated with lower humoral immune response, and methylated with higher response. Additionally, CpGs that individually predict humoral immune responses are enriched for polycomb-group and FOXP2 transcription factor binding sites. The most robust associations implicate differential methylation affecting gene expression levels of genes with known roles in immunity (e.g. HLA-B and HLA-DQB2) and immunosenescence. We believe our data and analysis strategy highlight new and interesting epigenetic trends affecting humoral response to vaccination against influenza; one of the most common and impactful viral pathogens.

Nuclear Calcium Signaling Controls Expression of a Large Gene Pool: Identification of a Gene Program for Acquired Neuroprotection Induced by Synaptic Activity

PubMed Central

Zhang, Sheng-Jia; Zou, Ming; Lu, Li; Lau, David; Ditzel, Désirée A. W.; Delucinge-Vivier, Celine; Aso, Yoshinori; Descombes, Patrick; Bading, Hilmar

2009-01-01

Synaptic activity can boost neuroprotection through a mechanism that requires synapse-to-nucleus communication and calcium signals in the cell nucleus. Here we show that in hippocampal neurons nuclear calcium is one of the most potent signals in neuronal gene expression. The induction or repression of 185 neuronal activity-regulated genes is dependent upon nuclear calcium signaling. The nuclear calcium-regulated gene pool contains a genomic program that mediates synaptic activity-induced, acquired neuroprotection. The core set of neuroprotective genes consists of 9 principal components, termed Activity-regulated Inhibitor of Death (AID) genes, and includes Atf3, Btg2, GADD45β, GADD45γ, Inhibin β-A, Interferon activated gene 202B, Npas4, Nr4a1, and Serpinb2, which strongly promote survival of cultured hippocampal neurons. Several AID genes provide neuroprotection through a common process that renders mitochondria more resistant to cellular stress and toxic insults. Stereotaxic delivery of AID gene-expressing recombinant adeno-associated viruses to the hippocampus confers protection in vivo against seizure-induced brain damage. Thus, treatments that enhance nuclear calcium signaling or supplement AID genes represent novel therapies to combat neurodegenerative conditions and neuronal cell loss caused by synaptic dysfunction, which may be accompanied by a deregulation of calcium signal initiation and/or propagation to the cell nucleus. PMID:19680447

Age-dependent DNA methylation of genes that are suppressed in stem cells is a hallmark of cancer.

PubMed

Teschendorff, Andrew E; Menon, Usha; Gentry-Maharaj, Aleksandra; Ramus, Susan J; Weisenberger, Daniel J; Shen, Hui; Campan, Mihaela; Noushmehr, Houtan; Bell, Christopher G; Maxwell, A Peter; Savage, David A; Mueller-Holzner, Elisabeth; Marth, Christian; Kocjan, Gabrijela; Gayther, Simon A; Jones, Allison; Beck, Stephan; Wagner, Wolfgang; Laird, Peter W; Jacobs, Ian J; Widschwendter, Martin

2010-04-01

Polycomb group proteins (PCGs) are involved in repression of genes that are required for stem cell differentiation. Recently, it was shown that promoters of PCG target genes (PCGTs) are 12-fold more likely to be methylated in cancer than non-PCGTs. Age is the most important demographic risk factor for cancer, and we hypothesized that its carcinogenic potential may be referred by irreversibly stabilizing stem cell features. To test this, we analyzed the methylation status of over 27,000 CpGs mapping to promoters of approximately 14,000 genes in whole blood samples from 261 postmenopausal women. We demonstrate that stem cell PCGTs are far more likely to become methylated with age than non-targets (odds ratio = 5.3 [3.8-7.4], P < 10(-10)), independently of sex, tissue type, disease state, and methylation platform. We identified a specific subset of 69 PCGT CpGs that undergo hypermethylation with age and validated this methylation signature in seven independent data sets encompassing over 900 samples, including normal and cancer solid tissues and a population of bone marrow mesenchymal stem/stromal cells (P < 10(-5)). We find that the age-PCGT methylation signature is present in preneoplastic conditions and may drive gene expression changes associated with carcinogenesis. These findings shed substantial novel insights into the epigenetic effects of aging and support the view that age may predispose to malignant transformation by irreversibly stabilizing stem cell features.

Whole Genome and Tandem Duplicate Retention Facilitated Glucosinolate Pathway Diversification in the Mustard Family

PubMed Central

Hofberger, Johannes A.; Lyons, Eric; Edger, Patrick P.; Chris Pires, J.; Eric Schranz, M.

2013-01-01

Plants share a common history of successive whole-genome duplication (WGD) events retaining genomic patterns of duplicate gene copies (ohnologs) organized in conserved syntenic blocks. Duplication was often proposed to affect the origin of novel traits during evolution. However, genetic evidence linking WGD to pathway diversification is scarce. We show that WGD and tandem duplication (TD) accelerated genetic versatility of plant secondary metabolism, exemplified with the glucosinolate (GS) pathway in the mustard family. GS biosynthesis is a well-studied trait, employing at least 52 biosynthetic and regulatory genes in the model plant Arabidopsis. In a phylogenomics approach, we identified 67 GS loci in Aethionema arabicum of the tribe Aethionemae, sister group to all mustard family members. All but one of the Arabidopsis GS gene families evolved orthologs in Aethionema and all but one of the orthologous sequence pairs exhibit synteny. The 45% fraction of duplicates among all protein-coding genes in Arabidopsis was increased to 95% and 97% for Arabidopsis and Aethionema GS pathway inventory, respectively. Compared with the 22% average for all protein-coding genes in Arabidopsis, 52% and 56% of Aethionema and Arabidopsis GS loci align to ohnolog copies dating back to the last common WGD event. Although 15% of all Arabidopsis genes are organized in tandem arrays, 45% and 48% of GS loci in Arabidopsis and Aethionema descend from TD, respectively. We describe a sequential combination of TD and WGD events driving gene family extension, thereby expanding the evolutionary playground for functional diversification and thus potential novelty and success. PMID:24171911

TALE activators regulate gene expression in a position- and strand-dependent manner in mammalian cells.

PubMed

Uhde-Stone, Claudia; Cheung, Edna; Lu, Biao

2014-01-24

Transcription activator-like effectors (TALEs) are a class of transcription factors that are readily programmable to regulate gene expression. Despite their growing popularity, little is known about binding site parameters that influence TALE-mediated gene activation in mammalian cells. We demonstrate that TALE activators modulate gene expression in mammalian cells in a position- and strand-dependent manner. To study the effects of binding site location, we engineered TALEs customized to recognize specific DNA sequences located in either the promoter or the transcribed region of reporter genes. We found that TALE activators robustly activated reporter genes when their binding sites were located within the promoter region. In contrast, TALE activators inhibited the expression of reporter genes when their binding sites were located on the sense strand of the transcribed region. Notably, this repression was independent of the effector domain utilized, suggesting a simple blockage mechanism. We conclude that TALE activators in mammalian cells regulate genes in a position- and strand-dependent manner that is substantially different from gene activation by native TALEs in plants. These findings have implications for optimizing the design of custom TALEs for genetic manipulation in mammalian cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Analysis of Ly49 gene transcripts in mature NK cells supports a role for the Pro1 element in gene activation, not gene expression.

PubMed

McCullen, M V; Li, H; Cam, M; Sen, S K; McVicar, D W; Anderson, S K

2016-09-01

The variegated expression of murine Ly49 loci has been associated with the probabilistic behavior of an upstream promoter active in immature cells, the Pro1 element. However, recent data suggest that Pro1 may be active in mature natural killer (NK) cells and function as an enhancer element. To assess directly if Pro1 transcripts are present in mature Ly49-expressing NK cells, RNA-sequencing of the total transcript pool was performed on freshly isolated splenic NK cells sorted for expression of either Ly49G or Ly49I. No Pro1 transcripts were detected from the Ly49a, Ly49c or Ly49i genes in mature Ly49(+) NK cells that contained high levels of Pro2 transcripts. Low levels of Ly49g Pro1 transcripts were found in both Ly49G(+) and Ly49G(-) populations, consistent with the presence of a small population of mature NK cells undergoing Ly49g gene activation, as previously demonstrated by culture of splenic NK cells in interleukin-2. Ly49 gene reporter constructs containing Pro1 failed to show any enhancer activity of Pro1 on Pro2 in a mature Ly49-expressing cell line. Taken together, the results are consistent with Pro1 transcription having a role in gene activation in developing NK, and argue against a role for Pro1 in Ly49 gene transcription by mature NK cells.

Validation study of genes with hypermethylated promoter regions associated with prostate cancer recurrence

PubMed Central

Stott-Miller, Marni; Zhao, Shanshan; Wright, Jonathan L.; Kolb, Suzanne; Bibikova, Marina; Klotzle, Brandy; Ostrander, Elaine A.; Fan, Jian-Bing; Feng, Ziding; Stanford, Janet L.

2014-01-01

Background One challenge in prostate cancer (PCa) is distinguishing indolent from aggressive disease at diagnosis. DNA promoter hypermethylation is a frequent epigenetic event in PCa, but few studies of DNA methylation in relation to features of more aggressive tumors or PCa recurrence have been completed. Methods We used the Infinium® HumanMethylation450 BeadChip to assess DNA methylation in tumor tissue from 407 patients with clinically localized PCa who underwent radical prostatectomy. Recurrence status was determined by follow-up patient surveys, medical record review, and linkage with the SEER registry. The methylation status of 14 genes for which promoter hypermethylation was previously correlated with advanced disease or biochemical recurrence was evaluated. Average methylation level for promoter region CpGs in patients who recurred compared to those with no evidence of recurrence was analyzed. For two genes with differential methylation, time to recurrence was examined. Results During an average follow-up of 11.7 years, 104 (26%) patients recurred. Significant promoter hypermethylation in at least 50% of CpG sites in two genes, ABHD9 and HOXD3, was found in tumors from patients who recurred compared to those without recurrence. Evidence was strongest for HOXD3 (lowest P = 9.46x10−6), with higher average methylation across promoter region CpGs associated with reduced recurrence-free survival (P = 2×10−4). DNA methylation profiles did not differ by recurrence status for the other genes. Conclusions These results validate the association between promoter hypermethylation of ADHB9 and HOXD3 and PCa recurrence. Impact Tumor DNA methylation profiling may help distinguish PCa patients at higher risk for disease recurrence. PMID:24718283