scholarly journals Characterization of γ-Glutamyl Peptidases and γ-Glutamyl Cyclotransferases for Glutathione Degradation in Arabidopsis

2021 ◽  
Author(s):  
Takehiro Ito ◽  
Taisuke Kitaiwa ◽  
Kosuke Nishizono ◽  
Minori Umahashi ◽  
Shunsuke Miyaji ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Energy Cost ◽  
Expression Patterns ◽  
The Novel ◽  
Activity Assay ◽  
Sulfur Deficiency ◽  
Nutritional Conditions ◽  
Knockout Mutants ◽  
Efficient Route

Organic sulfur is stored as glutathione (GSH) in plants. In Arabidopsis, γ-glutamyl cyclotransferases (GGCT2;1, GGCT2;2, and GGCT2;3) degrade cytosolic GSH, but they do not fully explain the rapid GSH turnover. Here, we demonstrate that γ-glutamyl peptidases, GGP1 and GGP3, play a substantial role in degrading GSH in the cytosol. We conducted yeast complementation assay and activity assay of recombinant proteins to identify the novel GSH degradation enzymes. The expression patterns were investigated by RT-qPCR. GSH concentrations in the mutants were also analyzed. GGP1 complemented the yeast phenotype. Recombinant GGP1 and GGP3 showed reasonable Km values considering cytosolic GSH concentration, and their activity was comparable to that of GGCTs. The GGP1 transcript was highly abundant in mature organs such as rosette leaves. The expression of GGCT2;1 was conspicuously enhanced under sulfur deficiency. GSH concentration was higher in ggp1 knockout mutants regardless of nutritional conditions; the concentration was higher in ggct2;1 knockout mutants under sulfur-deficient conditions. We propose a model wherein cytosolic GSH is degraded fundamentally by GGP1. The degradation is accelerated by GGCT2;1 under sulfur deficiency. Given the energy cost throughout the reactions, GGPs could render a more efficient route for GSH degradation than GGCTs.

Characterization of the Novel Human AGE1hn Cell Line for Production of Recombinant Proteins

2010 ◽  
pp. 693-697 ◽  
Author(s):  
S. Northoff ◽  
H. Büntemeyer ◽  
V. Sandig ◽  
S. Zietze ◽  
T. Noll
Keyword(s):  
Cell Line ◽  
Recombinant Proteins ◽  
The Novel

scholarly journals The Novel Leptospiral Surface Adhesin Lsa20 Binds Laminin and Human Plasminogen and Is Probably Expressed during Infection

2011 ◽  
Vol 79 (11) ◽  
pp. 4657-4667 ◽  
Author(s):  
Renata Siqueira Mendes ◽  
Marina Von Atzingen ◽  
Zenaide Maria de Morais ◽  
Amane Paldes Gonçales ◽  
Solange M. T. Serrano ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Outer Membrane Proteins ◽  
Proteinase K ◽  
The Novel ◽  
Pathogenic Species ◽  
Purification And Characterization ◽  
Serum Samples ◽  
Content Type ◽  
Human Plasminogen

ABSTRACTLeptospirosis is an emerging infectious disease caused by pathogenic species ofLeptospira. In this work, we report the cloning, expression, purification, and characterization of two predicted leptospiral outer membrane proteins, LIC11469 and LIC11030. The LIC11469 protein is well conserved among leptospiral strains, while LIC11030 was identified only inLeptospirainterrogans. We confirmed by surface proteolysis of intact leptospires with proteinase K that these proteins are most likely new surface leptospiral proteins. The recombinant proteins were evaluated for their capacity to attach to extracellular matrix (ECM) components and to plasminogen. The leptospiral protein encoded by LIC11469, named Lsa20 (leptospiral surface adhesin of 20 kDa), binds to laminin and to plasminogen. The binding with both components was not detected when Lsa20 was previously denatured or blocked with anti-Lsa20 antibodies. Moreover, Lsa20 binding to laminin was also confirmed by surface plasmon resonance (SPR). Laminin competes with plasminogen for binding to Lsa20, suggesting the same ligand-binding site. Lsa20-bound plasminogen could be converted to enzymatically active plasmin, capable of cleaving plasmin substrated-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Lsa20 was recognized by antibodies in confirmed-leptospirosis serum samples, suggesting that this protein is expressed during infection. Taken together, our results indicate that Lsa20 is a novel leptospiral adhesin that in concert with the host-derived plasmin may help the bacteria to adhere and to spread through the hosts.

scholarly journals Heterogeneity Among Ly-49C Natural Killer (NK) Cells: Characterization of Highly Related Receptors with Differing Functions and Expression Patterns

1996 ◽  
Vol 184 (6) ◽  
pp. 2085-2090 ◽  
Author(s):  
Jack Brennan ◽  
Suzanne Lemieux ◽  
J. Douglas Freeman ◽  
Dixie L. Mager ◽  
Fumio Takei
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Expression Patterns ◽  
Flow Cytometric Analysis ◽  
Cdna Clones ◽  
The Novel ◽  
Color Flow ◽  
Flow Cytometric

Ly-49C is a member of the polymorphic family of murine NK cell inhibitory receptors. The 5E6 antibody that defines a subset of NK cells responsible for the rejection of parental H-2d bone marrow by F1 mice has been shown previously to react with Ly-49C. Here, the 5E6 antibody was found to detect two Ly-49C-related molecules in B6 mice. Two cDNA clones were isolated from B6 NK cells, one identical to previously reported Ly-49CB6 and the other a novel cDNA. The deduced amino acid sequence of the latter differs from that of Ly-49CBALB at only 4 residues, whereas the previously reported Ly-49CB6 differs at 22 residues. Flow cytometric analyses of COS cells transfected with the two cDNAs showed that the 5E6 antibody binds to both Ly-49 molecules, while another anti-Ly-49C antibody, 4LO3311, binds to the newly described Ly-49C but not the previously reported Ly-49CB6. Two-color flow cytometric analysis detected 5E6+4LO3311− as well as 5E6+4LO3311+ subsets of NK cells from B6, but not BALB/c, mice. The level of Ly-49C expression on B6 NK cells detected by the 4LO3311 antibody was substantially lower than that on BALB/c NK cells. Binding specificity of the novel Ly-49CB6 was indistinguishable from that of Ly-49CBALB, whereas no binding was detectable with previously reported Ly-49CB6. These results demonstrate that the newly described Ly-49CB6, not the previously reported Ly-49CB6, is the probable B6 allelic form of Ly49C. The previously reported Ly-49CB6 must be encoded by a separate gene and should be renamed Ly-49I. The implication of these results with respect to the role of Ly-49C in hybrid resistance is discussed.

scholarly journals Development of an ELISA-Based HDAC Activity Assay for Characterization of Isoform-Selective Inhibitors

2015 ◽  
Vol 20 (10) ◽  
pp. 1277-1285 ◽  
Author(s):  
Geetha Padige ◽  
Ahmed T. Negmeldin ◽  
Mary Kay H. Pflum
Keyword(s):  
Mammalian Cell ◽  
Recombinant Proteins ◽  
Hdac Inhibitors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Activity Assay ◽  
Hdac Activity ◽  
Anticancer Properties ◽  
The Individual ◽  
Hdac Proteins

Histone deacetylase (HDAC) proteins are promising targets for cancer treatment, with several HDAC inhibitors used clinically as anticancer drugs. Most HDAC inhibitors nonspecifically interact with all or many of the 11 HDAC isoforms. Isoform-selective HDAC inhibitors would be useful tools to dissect the individual functions of HDAC proteins in cancer formation, in addition to potentially displaying effective anticancer properties. We report here a robust HDAC activity assay for screening selective HDAC inhibitors, which is inspired by the traditional enzyme-linked immunosorbent assay (ELISA). The key feature of the ELISA-based HDAC activity assay is use of mammalian cell–derived HDAC isoforms instead of recombinant proteins. Importantly, the assay was validated with several known HDAC inhibitors. The ELISA-based HDAC activity assay will facilitate the characterization of isoform-selective HDAC inhibitors against mammalian cell–derived HDAC proteins, which will enhance HDAC-centered cancer research and provide a foundation for anticancer drug development.

Characterization of the SIRT genes and their distinct expression patterns in Manila clams (Ruditapes philippinarum) exposed to air

Meta Gene ◽  
2021 ◽  
Vol 28 ◽  
pp. 100892
Author(s):  
Jingtian Wang ◽  
He Zhang ◽  
Shuang Xu ◽  
Ze Liu ◽  
Lu Yang ◽  
...  
Keyword(s):  
Expression Patterns ◽  
Ruditapes Philippinarum

Characterization of the novel HLA‐C *07:944 allele by next‐generation sequencing

HLA ◽  
2021 ◽  
Author(s):  
Maria Loginova ◽  
Olga Makhova ◽  
Daria Smirnova ◽  
Igor Paramonov ◽  
Maksim Zarubin
Keyword(s):  
Next Generation Sequencing ◽  
The Novel ◽  
Next Generation ◽  
Generation Sequencing

Characterization of the novel HLA‐B *51:296 allele by next‐generation sequencing

HLA ◽  
2020 ◽  
Author(s):  
Steve Genebrier ◽  
Vincent Elsermans ◽  
Emeric Texeraud ◽  
Gerald Bertrand ◽  
Virginie Renac
Keyword(s):  
Next Generation Sequencing ◽  
The Novel ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals Identification and Characterization of Abiotic Stress Responsive CBL-CIPK Family Genes in Medicago

2021 ◽  
Vol 22 (9) ◽  
pp. 4634
Author(s):  
Wenxuan Du ◽  
Junfeng Yang ◽  
Lin Ma ◽  
Qian Su ◽  
Yongzhen Pang
Keyword(s):  
Abiotic Stress ◽  
Abiotic Stresses ◽  
Expression Patterns ◽  
Interacting Protein ◽  
Forage Crop ◽  
Cis Acting ◽  
Protein Motifs ◽  
Plant Signal Transduction ◽  
Identification And Characterization

The calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK) play important roles in plant signal transduction and response to abiotic stress. Plants of Medicago genus contain many important forages, and their growth is often affected by a variety of abiotic stresses. However, studies on the CBL and CIPK family member and their function are rare in Medicago. In this study, a total of 23 CBL and 58 CIPK genes were identified from the genome of Medicago sativa as an important forage crop, and Medicaog truncatula as the model plant. Phylogenetic analysis suggested that these CBL and CIPK genes could be classified into five and seven groups, respectively. Moreover, these genes/proteins showed diverse exon-intron organizations, architectures of conserved protein motifs. Many stress-related cis-acting elements were found in their promoter region. In addition, transcriptional analyses showed that these CBL and CIPK genes exhibited distinct expression patterns in various tissues, and in response to drought, salt, and abscisic acid treatments. In particular, the expression levels of MtCIPK2 (MsCIPK3), MtCIPK17 (MsCIPK11), and MtCIPK18 (MsCIPK12) were significantly increased under PEG, NaCl, and ABA treatments. Collectively, our study suggested that CBL and CIPK genes play crucial roles in response to various abiotic stresses in Medicago.

Characterization of the novel HLA‐DRB1*11:282 allele by sequencing‐based typing

HLA ◽  
2021 ◽  
Author(s):  
Marine Cargou ◽  
Vincent Elsermans ◽  
Isabelle Top ◽  
Laura Blouin ◽  
Jonathan Visentin
Keyword(s):  
The Novel
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