scholarly journals Cloning, expression and characterization of three types of 17β-hydroxysteroid dehydrogenases from the Nile tilapia, Oreochromis niloticus

2005 ◽  
Vol 35 (1) ◽  
pp. 103-116 ◽  
Author(s):  
L Y Zhou ◽  
D S Wang ◽  
B Senthilkumaran ◽  
M Yoshikuni ◽  
Y Shibata ◽  
...  
Keyword(s):  
Tissue Distribution ◽  
Oreochromis Niloticus ◽  
Nile Tilapia ◽  
Enzyme Characterization ◽  
Open Reading Frames ◽  
Rt Pcr ◽  
Hek 293 ◽  
E Coli ◽  
Hek 293 Cells ◽  
293 Cells

In order to elucidate the roles of 17β-HSDs in fish gonadal steroidogenesis, three types of 17β-HSDs (17β-HSD1, 17β-HSD8 and putative 17β-HSD12) were cloned and characterized from the Nile tilapia, Oreochromis niloticus. The cloned cDNAs of 17β-HSD type 1, 8 and 12 were 1504, 1006 and 1930 bp long, with open reading frames encoding proteins of 289, 256 and 314 aminoacids, respectively. Tissue distribution pattern analyzed by RT-PCR and Northern blot showed that 17β-HSD1 was dominantly expressed in the ovary, while the putative 17β-HSD12, one of the two duplicates found in fish, is a male specific enzyme and expressed exclusively in testis (detected by RT-PCR only). On the other hand, 17β-HSD8 was expressed in the brain, gill, heart, liver, intestine, gonad, kidney and muscle of both male and female. Enzymatic assays of the three types of 17β-HSDs were performed using recombinant proteins expressed in E. coli or HEK 293 cells. Tilapia 17β-HSD1 expressed in E. coli had the preference for NADP(H) as cofactor and could catalyze the inter-conversion between estrone and estradiol efficiently as well as the inter-conversion between androstenedione and testosterone, but less efficiently. Tilapia 17β-HSD8 recombinant protein expressed in HEK 293 cells could catalyze the conversion of testosterone to androstenedione, as well as the inter-conversion between estrone and estradiol. However, the putative 17β-HSD12 expressed in E. coli or in HEK 293 cells showed no conversion to any of the four substrates tested in this study. Based on enzyme characterization and tissue distribution, it is plausible to attribute crucial roles to 17β-HSDs in the gonadal steroidogenesis of teleosts.

Molecular cloning and expression of pulmonary lipid phosphate phosphohydrolases

2001 ◽  
Vol 281 (6) ◽  
pp. L1484-L1493 ◽  
Author(s):  
Meera Nanjundan ◽  
Fred Possmayer
Keyword(s):  
Plasma Membranes ◽  
Cloning And Expression ◽  
Rat Lung ◽  
Rt Pcr ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Mammalian Expression ◽  
293 Cells ◽  
Lipid Phosphate ◽  
Novel Isoforms

Pulmonary lipid phosphate phosphohydrolase (LPP) was shown previously to hydrolyze phosphatidic acid and lysophosphatidic acid in purified rat lung plasma membranes. To better investigate the nature of pulmonary LPP isoforms and their role in the lung, LPPs were cloned by RT-PCR from both adult rat lung and type II cell RNA. The RT-PCR generated LPP1 (849 bp), up to three LPP1 variants, and LPP3 (936 bp) cDNAs. The three LPP1 variants include LPP1a (852 bp) and two novel isoforms, LPP1b (697 bp) and LPP1c (1004 bp). The pulmonary LPP1 and LPP3 isoforms are essentially identical to the previously cloned rat liver and intestinal LPPs, respectively, and the LPP1a isoform has 80% sequence identity to the human homolog. The LPP2 isoform was not detected in lung by RT-PCR. Northern analyses revealed that the mRNAs for LPP1 and LPP3 increase in fetal rat lung in late gestation to day 1 after birth. These mRNAs decrease somewhat during the neonatal period but increase slightly during postnatal development. Expression of LPP1, LPP1a, and LPP3 cDNAs in HEK 293 cells established that they encode functional LPP. In contrast, the novel isoforms LPP1b and LPP1c contain frameshifts that would result in premature termination, producing putative catalytically inactive polypeptides of 30 and 76 amino acids, respectively. Further investigation of the LPP1b isoform revealed that it was present across a variety of tissues, although at lower levels than LPP1/1a. Transient mammalian expression of LPP1b failed to increase phosphatidate phosphohydrolase activity in HEK 293 cells.

scholarly journals Transient Expression in HEK 293 Cells: An Alternative to E. coli for the Production of Secreted and Intracellular Mammalian Proteins

2014 ◽  
pp. 209-222 ◽  
Author(s):  
Joanne E. Nettleship ◽  
Peter J. Watson ◽  
Nahid Rahman-Huq ◽  
Louise Fairall ◽  
Mareike G. Posner ◽  
...  
Keyword(s):  
Transient Expression ◽  
Hek 293 ◽  
E Coli ◽  
Hek 293 Cells ◽  
Mammalian Proteins ◽  
293 Cells

scholarly journals Recombinant swine beta interferon protects swine alveolar macrophages and MARC-145 cells from infection with Porcine reproductive and respiratory syndrome virus

2007 ◽  
Vol 88 (3) ◽  
pp. 925-931 ◽  
Author(s):  
C. Overend ◽  
R. Mitchell ◽  
D. He ◽  
G. Rompato ◽  
M. J. Grubman ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Real Time ◽  
Alveolar Macrophages ◽  
Viral Rna ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Beta Interferon ◽  
293 Cells

Swine beta interferon (swIFN-β) produced in HEK 293 cells infected with a recombinant, replication-defective human adenovirus 5 (Ad5) encoding the swIFN-β gene was tested for antiviral activity against Porcine reproductive and respiratory syndrome virus (PRRSV). MARC-145 cells were incubated overnight with dilutions of supernatant fluids from HEK 293 cells infected with Ad5-swIFN-β or with an Ad5 control virus (Ad5-Blue). Treated cells were infected with PRRSV; MARC-145 cells incubated with Ad5-Blue supernatants developed cytopathic effects (CPE), whereas those incubated with swIFN-β showed no CPE. To confirm the antiviral activity of swIFN-β, culture fluids from Ad5-swIFN-β-infected cells were affinity-purified on a Sepharose–anti-swIFN-β matrix, and the resulting fractions exhibited antiviral activity upon infection with PRRSV. The antiviral effects were specific, as they were blocked by mAbs against swIFN-β. Additional cultures of MARC-145 cells treated with swIFN-β-containing supernatants or affinity-purified swIFN-β were infected with PRRSV and tested by real-time RT-PCR for viral RNA in culture supernatants at various times post-inoculation. These experiments confirmed the protective effects of swIFN-β. swIFN-β was also tested for antiviral activity on porcine alveolar macrophages (PAMs) obtained by bronchoalveolar lavage from PRRSV-negative swine. PAMs were treated with dilutions of swIFN-β or Ad5-Blue culture fluids, infected with PRRSV and tested for viral RNA by real-time RT-PCR. The viral load data showed a dose-dependent protection in swIFN-β-treated PAMs, whereas no protection was evident from Ad5-Blue culture fluids. The data demonstrate that swIFN-β protects both MARC-145 cells and PAMs from PRRSV infection.

scholarly journals Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽  
2013 ◽  
Vol 9 (9) ◽  
pp. 1407-1417 ◽  
Author(s):  
Patience Musiwaro ◽  
Matthew Smith ◽  
Maria Manifava ◽  
Simon A. Walker ◽  
Nicholas T. Ktistakis
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

2005 ◽  
Vol 103 (6) ◽  
pp. 1156-1166 ◽  
Author(s):  
Kevin J. Gingrich ◽  
Son Tran ◽  
Igor M. Nikonorov ◽  
Thomas J. Blanck
Keyword(s):  
Charge Transfer ◽  
Calcium Channels ◽  
Dependent Manner ◽  
Current Time ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Dependent Inactivation ◽  
Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

Micropatterned surfaces of PDMS as growth templates for HEK 293 cells

2007 ◽  
Vol 9 (4) ◽  
pp. 475-485 ◽  
Author(s):  
R. M. Johann ◽  
Ch. Baiotto ◽  
Ph. Renaud
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Calcium Induces Expression of Cytoplasmic Gelsolin in SH-SY5Y and HEK-293 Cells

2010 ◽  
Vol 35 (7) ◽  
pp. 1075-1082 ◽  
Author(s):  
Lina Ji ◽  
Abha Chauhan ◽  
Ved Chauhan
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Cytoplasmic Gelsolin

Characteristics of rat downregulated in adenoma (rDRA) expressed in HEK 293 cells

2007 ◽  
Vol 454 (3) ◽  
pp. 441-450 ◽  
Author(s):  
Christian Barmeyer ◽  
Jeff Huaqing Ye ◽  
Shafik Sidani ◽  
John Geibel ◽  
Henry J. Binder ◽  
...  
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Abstract 5316: hERG 1b as a Potential Target for Inherited and Acquired Long QT Syndrome

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Gail A Robertson ◽  
Harinath Sale ◽  
David Tester ◽  
Thomas J O’Hara ◽  
Pallavi Phartiyal ◽  
...  
Keyword(s):  
Action Potential ◽  
Long Qt Syndrome ◽  
Time Course ◽  
Drug Sensitivity ◽  
Long Qt ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Acquired Long Qt Syndrome ◽  
293 Cells ◽  
Qt Syndrome

Cardiac I Kr is a critical repolarizing current in the heart and a target for inherited and acquired long QT syndrome. Biochemical studies show that native I Kr channels are heteromers composed of both hERG 1a and 1b subunits, yet our current understanding of I Kr functional properties derives primarily from studies of homo-oligomers of the original hERG 1a isolate. The hERG 1a and 1b subunits are identical except at the amino (NH2) terminus, which in hERG 1b is much shorter and has a unique primary sequence. We compared the biophysical properties of currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells at near-physiological temperatures. We found that heteromeric hERG 1a/1b currents are much larger than hERG 1a currents and conduct 80% more charge during an action potential. This surprising difference corresponds to a two-fold increase in the apparent rates of activation and recovery from inactivation, which reduces rectification and facilitates current rebound during repolarization. Kinetic modeling shows these gating differences account quantitatively for the differences in current amplitude between the two channel types. Depending on the action potential model used, loss of 1b predicts an increase in action potential duration of 27 ms (7%) or 41 ms (17%), respectively. Drug sensitivity was also different. Compared to homomeric 1a channels, heteromeric 1a/1b channels were inhibited by E-4031 with a slower time course and a corresponding four-fold positive shift in the IC 50 . Differences in current kinetics and drug sensitivity were modeled by “NH2 mode” gating with conformational states bound by the amino terminus in hERG 1a homomers but not 1a/1b heteromers. The importance of hERG 1b in vivo is supported by the identification of a 1b-specific A8V missense mutation in 1/269 unrelated genotype-negative LQTS patients and absent in 400 control alleles. Mutant 1bA8V expressed alone or with hERG 1a in HEK-293 cells nearly eliminated 1b protein. Thus, mutations specifically disrupting hERG 1b function are expected to reduce cardiac I Kr , prolong QT interval and enhance drug sensitivity, thus representing a potential mechanism underlying inherited or acquired LQTS.

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