A preliminary characterization of the cytosolic glutathione transferase proteome from Drosophila melanogaster

2012 ◽  
Vol 442 (1) ◽  
pp. 181-190 ◽  
Author(s):  
Chonticha Saisawang ◽  
Jantana Wongsantichon ◽  
Albert J. Ketterman
Keyword(s):  
Drosophila Melanogaster ◽  
Cell Line ◽  
Glutathione Transferase ◽  
Embryonic Cell ◽  
Kinetic Properties ◽  
Genome Database ◽  
Reverse Transcription Pcr ◽  
Unique Character ◽  
Cytosolic Glutathione ◽  
Processed Pseudogene

The cytosolic GST (glutathione transferase) superfamily has been annotated in the Drosophila melanogaster genome database. Of 36 genes, four undergo alternative splicing to yield a total of 41 GST proteins. In the present study, we have obtained the 41 transcripts encoding proteins by RT (reverse transcription)–PCR using RNA template from Drosophila S2 cells, an embryonic cell line. This observation suggests that all of the annotated DmGSTs (D. melanogaster GSTs) in the proteome are expressed in the late embryonic stages of D. melanogaster. To avoid confusion in naming these numerous DmGSTs, we have designated them following the universal GST nomenclature as well as previous designations that fit within this classification. Furthermore, in the cell line, we identified an apparent processed pseudogene, gste8, in addition to two isoforms from the Delta class that have been published previously. Only approximately one-third of the expressed DmGSTs could be purified by conventional GSH affinity chromatography. The diverse kinetic properties as well as physiological substrate specificity of the DmGSTs are such that each individual enzyme displayed a unique character even compared with members from the same class.

scholarly journals Kinetic independence of the subunits of cytosolic glutathione transferase from the rat

1985 ◽  
Vol 231 (2) ◽  
pp. 263-267 ◽  
Author(s):  
U H Danielson ◽  
B Mannervik
Keyword(s):  
Glutathione Transferase ◽  
Ethacrynic Acid ◽  
Glutathione Transferases ◽  
Kinetic Properties ◽  
Subunit Interactions ◽  
Steady State Kinetics ◽  
Binding Isotherms ◽  
Cytosolic Glutathione ◽  
Kinetics Of ◽  
Rate Behaviour

The steady-state kinetics of the dimeric glutathione transferases deviate from Michaelis-Menten kinetics, but have hyperbolic binding isotherms for substrates and products of the enzymic reaction. The possibility of subunit interactions during catalysis as an explanation for the rate behaviour was investigated by use of rat isoenzymes composed of subunits 1, 2, 3 and 4, which have distinct substrate specificities. The kinetic parameter kcat./Km was determined with 1-chloro-2,4-dinitrobenzene, 4-hydroxyalk-2-enals, ethacrynic acid and trans-4-phenylbut-3-en-2-one as electrophilic substrates for six isoenzymes: rat glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4. It was found that the kcat./Km values for the heterodimeric transferases 1-2 and 3-4 could be predicted from the kcat./Km values of the corresponding homodimers. Likewise, the initial velocities determined with transferases 3-3, 3-4 and 4-4 at different degrees of saturation with glutathione and 1-chloro-2,4-dinitrobenzene demonstrated that the kinetic properties of the subunits are additive. These results show that the subunits of glutathione transferase are kinetically independent.

scholarly journals Enlisting the Ixodes scapularis Embryonic ISE6 Cell Line to Investigate the Neuronal Basis of Tick—Pathogen Interactions

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 70
Author(s):  
Lourdes Mateos-Hernández ◽  
Natália Pipová ◽  
Eléonore Allain ◽  
Céline Henry ◽  
Clotilde Rouxel ◽  
...  
Keyword(s):  
Cell Line ◽  
Peripheral Nerves ◽  
Ixodes Scapularis ◽  
Embryonic Cell ◽  
Cell Subpopulation ◽  
In Vivo Experiments

Neuropeptides are small signaling molecules expressed in the tick central nervous system, i.e., the synganglion. The neuronal-like Ixodes scapularis embryonic cell line, ISE6, is an effective tool frequently used for examining tick–pathogen interactions. We detected 37 neuropeptide transcripts in the I. scapularis ISE6 cell line using in silico methods, and six of these neuropeptide genes were used for experimental validation. Among these six neuropeptide genes, the tachykinin-related peptide (TRP) of ISE6 cells varied in transcript expression depending on the infection strain of the tick-borne pathogen, Anaplasma phagocytophilum. The immunocytochemistry of TRP revealed cytoplasmic expression in a prominent ISE6 cell subpopulation. The presence of TRP was also confirmed in A. phagocytophilum-infected ISE6 cells. The in situ hybridization and immunohistochemistry of TRP of I. scapularis synganglion revealed expression in distinct neuronal cells. In addition, TRP immunoreaction was detected in axons exiting the synganglion via peripheral nerves as well as in hemal nerve-associated lateral segmental organs. The characterization of a complete Ixodes neuropeptidome in ISE6 cells may serve as an effective in vitro tool to study how tick-borne pathogens interact with synganglion components that are vital to tick physiology. Therefore, our current study is a potential stepping stone for in vivo experiments to further examine the neuronal basis of tick–pathogen interactions.

scholarly journals Transactivation of Latent Marek's Disease Herpesvirus Genes in QT35, a Quail Fibroblast Cell Line, by Herpesvirus of Turkeys

2000 ◽  
Vol 74 (21) ◽  
pp. 10176-10186 ◽  
Author(s):  
T. Yamaguchi ◽  
S. L. Kaplan ◽  
P. Wakenell ◽  
K. A. Schat
Keyword(s):  
Cell Line ◽  
Fibroblast Cell ◽  
Northern Blotting ◽  
Marek's Disease ◽  
Pcr Analysis ◽  
Marek’S Disease ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Herpesvirus Of Turkeys ◽  
Serotype 1

ABSTRACT The QT35 cell line was established from a methylcholanthrene-induced tumor in Japanese quail (Coturnix coturnix japonica) (C. Moscovici, M. G. Moscovici, H. Jimenez, M. M. Lai, M. J. Hayman, and P. K. Vogt, Cell 11:95–103, 1977). Two independently maintained sublines of QT35 were found to be positive for Marek's disease virus (MDV)-like genes by Southern blotting and PCR assays. Sequence analysis of fragments of the ICP4, ICP22, ICP27, VP16, meq, pp14, pp38, open reading frame (ORF) L1, and glycoprotein B (gB) genes showed a strong homology with the corresponding fragments of MDV genes. Subsequently, a serotype 1 MDV-like herpesvirus, tentatively name QMDV, was rescued from QT35 cells in chicken kidney cell (CKC) cultures established from 6- to 9-day-old chicks inoculated at 8 days of embryonation with QT35 cells. Transmission electron microscopy failed to show herpesvirus particles in QT35 cells, but typical intranuclear herpesvirus particles were detected in CKCs. Reverse transcription-PCR analysis showed that the following QMDV transcripts were present in QT35 cells: sense and antisense meq, ORF L1, ICP4, and latency-associated transcripts, which are antisense to ICP4. A transcript of approximately 4.5 kb was detected by Northern blotting using total RNA from QT35 cells. Inoculation of QT35 cells with herpesvirus of turkeys (HVT)-infected chicken embryo fibroblasts (CEF) but not with uninfected CEF resulted in the activation of ICP22, ICP27, VP16, pp38, and gB. In addition, the level of ICP4 mRNA was increased compared to that in QT35 cells. The activation by HVT resulted in the production of pp38 protein. It was not possible to detect if the other activated genes were translated due to the lack of serotype 1-specific monoclonal antibodies.

Characterization of mutant spectra generated by a forward mutational assay for geneA of ?X174 from ENU-treated transgenic mouse embryonic cell line PX-2

2002 ◽  
Vol 39 (1) ◽  
pp. 55-68 ◽  
Author(s):  
Carrie R. Valentine ◽  
Beverly A. Montgomery ◽  
Scott G. Miller ◽  
Robert R. Delongchamp ◽  
Bentley A. Fane ◽  
...  
Keyword(s):  
Cell Line ◽  
Transgenic Mouse ◽  
Embryonic Cell ◽  
Embryonic Cell Line

scholarly journals Genome-wide identification of the 14-3-3 gene family and its participation in floral transition by interacting with TFL1/FT in Apple

2020 ◽  
Author(s):  
Xiya Zuo ◽  
Shixiang Wang ◽  
Wen Xiang ◽  
Huiru Yang ◽  
Muhammad Mobeen Tahir ◽  
...  
Keyword(s):  
Gene Family ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Floral Transition ◽  
Bimolecular Fluorescence Complementation ◽  
Pcr Analysis ◽  
Transcriptional Responses ◽  
Genome Database ◽  
Reverse Transcription Pcr ◽  
Conserved Core

Abstract Background: Apple (Malus domestica Borkh.) is one of the most popular cultivated fruit crops in China. Apple floral transition is an important process but liable to be affected by various environmental factors. The 14-3-3 proteins are involved in regulating diverse biological processes in plants, and some 14-3-3 members play vital roles in flowering. However, little information was available about the 14-3-3 members in apple.Results: In the current study, we identified eighteen 14-3-3 gene family members from the apple genome database, designated MdGF14a to MdGF14r. The isoforms possess a conserved core region comprising nine antiparallel α-helices and divergent N and C termini. According to their structural and phylogenetic features, Md14-3-3 proteins could be classified into two major evolutionary branches, the epsilon (ɛ) group and the non-epsilon (non-ɛ) group. Moreover, expression profiles derived from transcriptome data and quantitative real-time reverse transcription PCR analysis showed diverse expression patterns of Md14-3-3 genes in various tissues and in response to different sugars and hormone treatments during the floral transition phase. Four Md14‑3-3 isoforms (MdGF14a, MdGF14d, MdGF14i, and MdGF14j) exhibiting prominent transcriptional responses to sugars and hormones were selected for further investigation. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments showed that the four Md14-3-3 proteins interact with key floral integrators, MdTFL1 (TERMINAL FLOWER1) and MdFT (FLOWERING LOCUS T). Subcellular localization of four selected Md14-3-3 proteins demonstrated their localization in both the cytoplasm and nucleus.Conclusion: We identified the Md14-3-3s family in apple comprehensively. Certain Md14-3-3 genes are expressed predominantly during the apple floral transition stage, and may participate in the regulation of flowering through association with flower control genes. Our results provide a preliminary framework for further investigation into the roles of Md14-3-3s in floral transition.

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