Comparative Analysis of CREB3 and CREB3L2 Protein Expression in HEK293 Cells

We performed a comparative analysis of two ER-resident CREB3 family proteins, CREB3 and CREB3L2, in HEK293 cells using pharmacological and genome editing approaches and identified several differences between the two. Treatment with brefeldin A (BFA) and monensin induced the cleavage of full-length CREB3 and CREB3L2; however, the level of the full-length CREB3 protein, but not CREB3L2 protein, was not noticeably reduced by the monensin treatment. On the other hand, treatment with tunicamycin (Tm) shifted the molecular weight of the full-length CREB3L2 protein downward but abolished CREB3 protein expression. Thapsigargin (Tg) significantly increased the expression of only full-length CREB3L2 protein concomitant with a slight increase in the level of its cleaved form. Treatment with cycloheximide and MG132 revealed that both endogenous CREB3 and CREB3L2 are proteasome substrates. In addition, kifunensine, an α-mannosidase inhibitor, significantly increased the levels of both full-length forms. Consistent with these findings, cells lacking SEL1L, a crucial ER-associated protein degradation (ERAD) component, showed increased expression of both full-length CREB3 and CREB3L2; however, cycloheximide treatment downregulated full-length CREB3L2 protein expression more rapidly in SEL1L-deficient cells than the full-length CREB3 protein. Finally, we investigated the induction of the expression of several CREB3 and CREB3L2 target genes by Tg and BFA treatments and SEL1L deficiency. In conclusion, this study suggests that both endogenous full-length CREB3 and CREB3L2 are substrates for ER-associated protein degradation but are partially regulated by distinct mechanisms, each of which contributes to unique cellular responses that are distinct from canonical ER signals.

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Quantifying the Protein Levels of All Nuclear Hormone Receptors by Mass Spectrometry

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.1660 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A815-A815

Author(s):

Michael Fadi Saikali ◽

Carolyn L Cummins

Keyword(s):

Mass Spectrometry ◽

Protein Expression ◽

Hormone Receptors ◽

Target Genes ◽

Mrna Level ◽

Fold Increase ◽

Hek293 Cells ◽

Endogenous Expression ◽

Mouse Tissues ◽

Wide Range

Abstract Nuclear Receptors (NRs) are a family of ligand-activated transcription factors that control the expression of genes involved in a wide range of physiological processes. An atlas detailing the expression of all NRs at the mRNA level was completed in 2006 using quantitative PCR [Bookout et al. Cell 2006]. The comparative measurement of NRs at the protein level, however, has been hindered by the poor quality of commercially available antibodies, as well as the absence of a high throughput method for quantitation. To address this need, we are developing a mass spectrometry-based targeted proteomic assay to quantify the absolute amounts of NR protein in a panel of mouse tissues. NRs were overexpressed in HEK293 cells by transient transfection and protein was isolated. The cell lysates were digested with a combination of trypsin and Lys-C following the Multi-Enzyme Digestion Filter Aided Sample Preparation protocol. The peptides were desalted using an in-house made C18 tip, separated on an EASY-Spray C18 column (75 um x 50 cm, 3Å), and analyzed on a Thermo QExactive HF in Top20 data-dependent acquisition mode. Protein identifications were made using MaxQuant software, and the identifications were mined for members of the NR family. The NR peptides detected were searched against an in silico generated list of optimal NR peptides (filtered for uniqueness, length, absence of post translational modifications, and conservation between human and mouse). The matching peptides were validated by parallel reaction monitoring (PRM) and purchased as synthetic isotopes with a heavy terminal arginine or lysine. Peptide linearity, and lower limits of detection (LLOD) were estimated by spiking digests from a C57Bl/6 mouse liver lysate with increasing amounts of the labeled peptides and analyzing by PRM. Peptides that displayed non-linear behavior were excluded for quantitation. The LLOD were between 100 amol and 1.5 fmol on column. A test panel of tissues (cerebrum, hippocampus, cerebellum, liver, spleen, brown/white adipose, and kidney) showed that we could detect endogenous expression of NRs. To date, we have purchased and validated peptides for 44 of the 49 receptors. We used this assay to quantify the changes in NR protein expression in mouse livers in response to 16 hours of fasting. We found significant changes in the nuclear expression of CAR (3.1-fold increase), RXRβ (1.8-fold increase), SHP (3.9-fold decrease) and RARβ (2.0-fold decrease) in the fasted vs. fed state. Increased CAR activity with fasting was further supported by label-free quantitative proteomics on the same lysates which revealed 210 differentially expressed proteins (2-fold change, p<0.05), with 61 (29%) identified as known CAR target genes. Once complete, this assay will provide researchers with a robust quantitative tool to investigate changes in NR protein expression that will be widely applicable to endocrine research.

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The Kidney and Fibrinolysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654207 ◽

1970 ◽

Vol 24 (01/02) ◽

pp. 026-032 ◽

Cited By ~ 2

Author(s):

N. A Marsh

Keyword(s):

Molecular Weight ◽

Plasminogen Activator ◽

Enzyme System ◽

Normal Animal ◽

Fibrinolytic Enzyme ◽

The Other ◽

Exclusion Chromatography ◽

Normal Urine ◽

Renal Origin ◽

Molecular Exclusion Chromatography

SummaryMolecular exclusion chromatography was performed on samples of urine from normal and aminonucleoside nephrotic rats. Normal urine contained 2 peaks of urokinase activity, one having a molecular weight of 22,000 and the other around 200,000. Nephrotic urine contained three peaks of activity with MW’s 126,000, 60,000 and 30,000. Plasma activator determined from euglobulin precipitate had a MW. in excess of 200,000. The results indicate that in the normal animal, plasma plasminogen activator does not escape into the urine in substantial quantities but under the conditions of extreme proteinuria there may be some loss through the kidney. The alteration in urokinase output in nephrotic animals indicates a greatly disordered renal fibrinolytic enzyme system.The findings of this study largely support the hypothesis that plasma plasminogen activator of renal origin and urinary plasminogen activator (urokinase) are different molecular species.

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Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657167 ◽

1982 ◽

Vol 47 (03) ◽

pp. 197-202 ◽

Cited By ~ 23

Author(s):

Kurt Huber ◽

Johannes Kirchheimer ◽

Bernd R Binder

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Human Urine ◽

Gel Filtration ◽

Chain Structure ◽

The Other ◽

Single Chain ◽

Apparent Homogeneity ◽

Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

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Barricyclin D1—a dimeric ellagitannin with a macrocyclic structure—and accompanying tannins from Barringtonia racemosa

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab073 ◽

2021 ◽

Author(s):

Shinji Yoshikawa ◽

Lih-Geeng Chen ◽

Morio Yoshimura ◽

Yoshiaki Amakura ◽

Tsutomu Hatano ◽

...

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Natural Resource ◽

The Other ◽

Hydrolysable Tannin ◽

The Family ◽

Macrocyclic Structure

Abstract Our examination of high molecular weight polyphenolic constituents in the leaves of Barringtonia racemosa of the family Lecythidaceae uncovered five previously undescribed ellagitannins. One, barringtin M1 (1), among them was a hydrolysable tannin monomer, while remaining four, barringtins D1 (2), D2 (3), D3 (4) and barricyclin D1 (5), were all dimers. Barricyclin D1 had a first macrocyclic structure formed from casuarictin (6) and tellimagrandin I (7), and the other ellagitannins had structures related to 5. Two additional known phenolics, valoneic acid dilactone (8) and schimawalin A (9), were also isolated from the leaves. These results suggested that the leaves of B. racemosa is a natural resource rich in hydrolysable tannin oligomers.

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Representing the victorious past: Chinese revolutionary TV drama between propaganda and marketization

Media Culture & Society ◽

10.1177/01634437211022721 ◽

2021 ◽

pp. 016344372110227

Author(s):

Yingzi Wang ◽

Thoralf Klein

Keyword(s):

Comparative Analysis ◽

The Other ◽

Market Reforms ◽

Fictional Characters ◽

Production Environment ◽

Cultural Transformations ◽

The One ◽

Political Economic ◽

Revolutionary History ◽

Over Time

This paper examines the changes and continuities in TV representations of Chinese Communist Party’s revolutionary history and interprets them within the broader context of China’s political, economic and cultural transformations since the 1990s. Drawing on a comparative analysis of three state-sponsored TV dramas produced between the late 1990s and mid-2010s, it traces how the state-sanctioned revolutionary narratives have changed over time in response to the Party’s propaganda imperatives on the one hand, and to the market-oriented production environment on the other. The paper argues that while recent TV productions in the new century have made increasing concessions to audience taste by adopting visually stimulating depictions and introducing fictional characters as points of identification for the audience, the revolutionary narratives were still aligned with the Party’s propaganda agenda at different times. This shows the ongoing competition between ideological and commercial interests in Chinese TV production during the era of market reforms.

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Comparative analysis and phylogenetic investigation of Hong Kong Ilex chloroplast genomes

Scientific Reports ◽

10.1038/s41598-021-84705-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Bobby Lim-Ho Kong ◽

Hyun-Seung Park ◽

Tai-Wai David Lau ◽

Zhixiu Lin ◽

Tae-Jin Yang ◽

...

Keyword(s):

Hong Kong ◽

Comparative Analysis ◽

Phylogenetic Relationships ◽

Species Level ◽

The Other ◽

Trna Genes ◽

Protein Coding ◽

Chloroplast Genomes ◽

Plant Group ◽

Ilex Rotunda

AbstractIlex is a monogeneric plant group (containing approximately 600 species) in the Aquifoliaceae family and one of the most commonly used medicinal herbs. However, its taxonomy and phylogenetic relationships at the species level are debatable. Herein, we obtained the complete chloroplast genomes of all 19 Ilex types that are native to Hong Kong. The genomes are conserved in structure, gene content and arrangement. The chloroplast genomes range in size from 157,119 bp in Ilex graciliflora to 158,020 bp in Ilex kwangtungensis. All these genomes contain 125 genes, of which 88 are protein-coding and 37 are tRNA genes. Four highly varied sequences (rps16-trnQ, rpl32-trnL, ndhD-psaC and ycf1) were found. The number of repeats in the Ilex genomes is mostly conserved, but the number of repeating motifs varies. The phylogenetic relationship among the 19 Ilex genomes, together with eight other available genomes in other studies, was investigated. Most of the species could be correctly assigned to the section or even series level, consistent with previous taxonomy, except Ilex rotunda var. microcarpa, Ilex asprella var. tapuensis and Ilex chapaensis. These species were reclassified; I. rotunda was placed in the section Micrococca, while the other two were grouped with the section Pseudoaquifolium. These studies provide a better understanding of Ilex phylogeny and refine its classification.

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PCV81 THE COMPARATIVE ANALYSIS OF THE EFFECTIVENESS AND THE COSTS OF USING THE LOW-MOLECULAR-WEIGHT HEPARINS AND THE ORAL ANTICOAGULANTS FOR THE TREATMENT OF THE VENOUS THROMBOEMBOLISM IN POLAND

Value in Health ◽

10.1016/s1098-3015(11)72424-x ◽

2010 ◽

Vol 13 (7) ◽

pp. A356

Author(s):

T Bochenek ◽

R Nizankowski

Keyword(s):

Molecular Weight ◽

Comparative Analysis ◽

Venous Thromboembolism ◽

Oral Anticoagulants ◽

Low Molecular Weight ◽

Low Molecular Weight Heparins

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Multiple Domains Within the Cauliflower mosaic virus Gene VI Product Interact with the Full-Length Protein

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.10.1050 ◽

2002 ◽

Vol 15 (10) ◽

pp. 1050-1057 ◽

Cited By ~ 26

Author(s):

Yongzhong Li ◽

Scott M. Leisner

Keyword(s):

Mosaic Virus ◽

Systemic Infection ◽

Full Length ◽

Cauliflower Mosaic Virus ◽

The Other ◽

Multifunctional Protein ◽

Viral Propagation ◽

Self Association ◽

Multiple Domains ◽

Two Hybrid

The Cauliflower mosaic virus (CaMV) gene VI product (P6) is a multifunctional protein essential for viral propagation. It is likely that at least some of these functions require P6 self-association. The work described here was performed to confirm that P6 self-associates and to identify domains involved in this interaction. Yeast two-hybrid analyses indicated that full-length P6 self-associates and that this interaction is specific. Additional analyses indicated that at least four independent domains bind to full-length P6. When a central domain (termed domain D3) was removed, these interactions were abolished. However, this deleted P6 was able to bind to the full-length wild-type protein and to isolated domain D3. Viruses lacking domain D3 were incapable of producing a systemic infection. Isolated domain D3 was capable of binding to at least two of the other domains but was unable to self-associate. This suggests that domain D3 facilitates P6 self-association by binding to the other domains but not itself. The presence of multiple domains involved in P6 self-association may help explain the ability of this protein to form the intracellular inclusions characteristic of caulimoviruses.

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CO-ORDINATION COMPLEXES OF TITANIUM (IV) HALIDES: II. PREPARATION AND INFRARED SPECTRA OF THE COMPLEXES OF TITANIUM TETRACHLORIDE WITH DIESTERS OF α,ω-DICARBOXYLIC ACIDS AND COMPARISON WITH ANALOGOUS COMPLEXES OF ZIRCONIUM TETRACHLORIDE AND TIN TETRACHLORIDE

Canadian Journal of Chemistry ◽

10.1139/v61-312 ◽

1961 ◽

Vol 39 (11) ◽

pp. 2343-2352 ◽

Cited By ~ 12

Author(s):

Ernest Rivet ◽

Real Aubin ◽

Roland Rivest

Keyword(s):

Molecular Weight ◽

Infrared Spectra ◽

Titanium Tetrachloride ◽

Complex Molecule ◽

Dicarboxylic Acids ◽

The Other ◽

Zirconium Tetrachloride ◽

Melting Points ◽

Zirconium Complexes ◽

Tin Tetrachloride

Co-ordination complexes between diesters of α,ω-dicarboxylic acids and titanium tetrachloride, tin tetrachloride, and zirconium tetrachloride have been prepared. The analytical results, the infrared spectra, the melting points, and the molecular-weight determinations indicate that for the titanium and zirconium complexes, two types of complexes are obtained, one having a general formula MX4•1 diester in which chelate rings from five to nine atoms are formed and the other one, 2MX4•1 diester in which there are two 4-membered rings per complex molecule. With tin tetrachloride only one type of complex is formed, which has two tin tetrachlorides and two diesters per complex molecule.

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