Determination of Relative Protein Abundance by Internally Normalized Ratio Algorithm with Antibody Arrays

Abstract. The objectives of this study were to investigate the expression and localization of myostatin (MSTN) and decorin (DCN) in bovine skeletal muscle and to find associations with muscle fibre and adipocyte development. Samples of two muscles, known for differences in meat quality and fibre composition, namely longissimus muscle (LD) and semitendinosus muscle (ST), were obtained from 18 months old bulls of the F2 generation of a Charolais×Holstein cross. Individual muscle sections were stained for determination of size and type of muscle fibres and immunohistochemical detection of the proteins. The mRNA abundance and protein expression of MSTN and DCN were quantified by real-time PCR and Western blot, respectively. As expected, the ST had more fast fibres, less fibres of the intermediate and the slow type, and less intramuscular fat than the LD. Despite these differences, the mRNA and protein abundance of MSTN was comparable in both muscles. The protein abundance of MSTN inhibitors, namely MSTN propeptide and DCN, was greater in LD, which may have affected the biological activity of mature MSTN. Myostatin propeptide was detected in all muscle fibres; however the mature MSTN was detected to a much lower extent and mainly in slow fibres. Furthermore, MSTN was localized in close proximity to DCN in intermyocellular space, suggesting possible interactions between both proteins and effects on muscle structure and meat quality. The role of MSTN and DCN as well as their interactions in the determination of muscle composition needs to be further elucidated.

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Intestinal function of gene-targeted mice lacking serum- and glucocorticoid-inducible kinase 1

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00231.2005 ◽

2006 ◽

Vol 290 (6) ◽

pp. G1114-G1123 ◽

Cited By ~ 39

Author(s):

Florian Grahammer ◽

Guido Henke ◽

Ciprian Sandu ◽

Rexhep Rexhepaj ◽

Azeemudeen Hussain ◽

...

Keyword(s):

Day Treatment ◽

Ussing Chamber ◽

Intestinal Transport ◽

Protein Abundance ◽

Transcript Levels ◽

Basic Functions ◽

Induced Currents

In vitro experiments have revealed the ability of serum- and glucocorticoid-inducible kinase 1 (SGK1) to stimulate intestinal Na+-coupled glucose cotransporter 1 (SGLT1) and intestinal Na+/H+ exchanger 3 (NHE3). The present study explored the contribution of SGK1 to the regulation of intestinal transport in vivo. SGK1 transcript levels were determined by real-time PCR and glucose-induced currents ( Ig) reflecting SGLT1 activity by Ussing chamber experiments. BCECF fluorescence was utilized for the determination of Na+-dependent pH recovery from an ammonium pulse (ΔpHNHE) reflecting NHE activity. As a result, intestinal SGK1 transcript levels were significantly enhanced by a 4-day treatment with 10 μg·mg body wt−1·day−1 dexamethasone (Dex). Ig was, under control conditions, virtually identical in sgk1 knockout mice ( sgk1−/−) and their wild type littermates ( sgk1+/+). A 4-day treatment with Dex, however, increased Ig approximately threefold in sgk1+/+ mice but not in sgk1−/− mice. ΔpHNHE was similar in sgk1−/− and sgk1+/+ mice before treatment. Dex increased ΔpHNHE approximately threefold in sgk1+/+ mice and approximately twofold in sgk1−/−mice, an effect significantly blunted in the presence of the specific NHE3 blocker S-3226 (10 μM). According to Western blot analysis, Dex significantly enhanced SGLT1 and NHE3 protein abundance in brush-border membranes of sgk1+/+ mice but not of sgk1−/−mice. In conclusion, basic functions of SGLT1 and NHE3 in the intestine do not require stimulation by SGK1. However, the effects of glucocorticoids on SGLT1 are fully, and on NHE3 partially, dependent on SGK1.

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Comparison of blood serum protein analysis by MALDI-MS from either conventional frozen samples or storage disc-deposited samples: A study with human serum from pregnant donors and from patients with intrauterine growth restriction

European Journal of Mass Spectrometry ◽

10.1177/1469066718820991 ◽

2018 ◽

Vol 25 (4) ◽

pp. 381-390

Author(s):

Manja Wölter ◽

Manuela Russ ◽

Charles A Okai ◽

Werner Rath ◽

Ulrich Pecks ◽

...

Keyword(s):

Intrauterine Growth Restriction ◽

Room Temperature ◽

Serum Proteins ◽

Protein Analysis ◽

Mass Spectrometric ◽

Growth Restriction ◽

Intact Protein ◽

Protein Abundance ◽

Intrauterine Growth

Mass spectrometric profiling of intact serum proteins, i.e. determination of relative protein abundance differences, was performed using two different serum sample preparation methods: one with frozen and thawed serum, the other with at room temperature deposited and dried serum. Since in a typical clinical setting freezing of serum is difficult to achieve, sampling at room temperature is preferred and can be met when using the Noviplex™ card system. Once deposited and dried, serum proteins can be stored and shipped at room temperature. After resolubilization of serum proteins from “dried serum spots”, mass spectra of high quality have been recorded comparable to those that were obtained using fresh-frozen and subsequently thawed serum samples. Differentiation between patients with intrauterine growth restriction and control individuals was achievable, independent from the sample work-up procedure. Having at hand a reliable and robust method for serum storage and shipment which works at room temperature bridges the gap between the clinics and the protein analysis laboratory. Our novel serum handling protocol reduces costs for both, storage and shipping, and ultimately enables clinical risk assessment based on mass spectrometric determination of intact protein abundance profiles.

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ELOVL5 Participates in Embryonic Lipid Determination of Cellular Membranes and Cytoplasmic Droplets

International Journal of Molecular Sciences ◽

10.3390/ijms22031311 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1311

Author(s):

Franciele Lanzarini ◽

Fernanda Alves Pereira ◽

Janine de Camargo ◽

Andressa Minozzo Oliveira ◽

Katia Roberta Anacleto Belaz ◽

...

Keyword(s):

Biological Control ◽

Embryonic Development ◽

Lipid Droplet ◽

Cell Number ◽

Protein Abundance ◽

Droplet Deposition ◽

Cytoplasmic Lipid Droplet ◽

Technical Control ◽

Lipid Species

Embryonic lipids are crucial for the formation of cellular membranes and dynamically participate in metabolic pathways. Cells can synthesize simple fatty acids, and the elongation of fatty acids facilitates the formation of complex lipids. The aim of this work was to investigate the involvement of the elongation of very long chain fatty acid enzyme 5 (ELOVL5) in embryonic development and lipid determination. Bovine embryos were produced in vitro using a standard protocol and randomly divided to receive one of three treatments at Day 4: morpholino (Mo) gene expression knockdown assay for ELOVL5 (ELOVL5-Mo), Mo antisense oligonucleotides for the thalassemic β-globulin human mRNA (technical control Mo), and placebo (biological control). The phenotypes of embryonic development, cell number, ELOVL5 protein abundance, lipid droplet deposits, and lipid fingerprint were investigated. No detrimental effects (p > 0.05) were observed on embryo development in terms of cleavage (59.4 ± 3.5%, 63.6 ± 4.1%, and 65.4 ± 2.2%), blastocyst production (31.3 ± 4.2%, 28.1 ± 4.9%, and 36.1 ± 2.1%), and blastocyst cell number (99.6 ± 7.7, 100.2 ± 6.2, 86.8 ± 5.6), respectively, for biological control, technical control Mo, and ELOVL5-Mo. ELOVL5 protein abundance and cytoplasmic lipid droplet deposition were increased (p < 0.05) in ELOVL5-Mo–derived blastocysts compared with the controls. However, seven lipid species, including phosphatidylcholines, phosphatidylethanolamines, and triacylglycerol, were downregulated in the ELOVL5-Mo–derived blastocysts compared with the biological control. Therefore, ELOVL5 is involved in the determination of embryonic lipid content and composition. Transient translational blockage of ELOVL5 reduced the expression of specific lipid species and promoted increased cytoplasmic lipid droplet deposition, but with no apparent deleterious effect on embryonic development and blastocyst cell number.

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Antibody Arrays for Determination of Relative Protein Abundances

Methods in Molecular Biology - Tissue Proteomics ◽

10.1007/978-1-60327-047-2_9 ◽

2008 ◽

pp. 129-151 ◽

Cited By ~ 8

Author(s):

Grigoriy S. Chaga

Keyword(s):

Antibody Arrays

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Effects of live high, train low hypoxic exposure on lactate metabolism in trained humans

Journal of Applied Physiology ◽

10.1152/japplphysiol.00799.2003 ◽

2004 ◽

Vol 96 (2) ◽

pp. 517-525 ◽

Cited By ~ 42

Author(s):

Sally A. Clark ◽

Robert J. Aughey ◽

Christopher J. Gore ◽

Allan G. Hahn ◽

Nathan E. Townsend ◽

...

Keyword(s):

Ph Regulation ◽

Whole Body ◽

Hypoxic Exposure ◽

Protein Abundance ◽

Lactate Metabolism ◽

Hypoxia Exposure ◽

Before And After ◽

Resting Muscle

We determined the effect of 20 nights of live high, train low (LHTL) hypoxic exposure on lactate kinetics, monocarboxylate lactate transporter proteins (MCT1 and MCT4), and muscle in vitro buffering capacity (βm) in 29 well-trained cyclists and triathletes. Subjects were divided into one of three groups: 20 consecutive nights of hypoxic exposure (LHTLc), 20 nights of intermittent hypoxic exposure [four 5-night blocks of hypoxia, each interspersed with 2 nights of normoxia (LHTLi)], or control (Con). Rates of lactate appearance (Ra), disappearance (Rd), and oxidation (Rox) were determined from a primed, continuous infusion of l-[U-14C]lactic acid tracer during 90 min of steady-state exercise [60 min at 65% peak O2 uptake (V̇o2 peak) followed by 30 min at 85% V̇o2 peak]. A resting muscle biopsy was taken before and after 20 nights of LHTL for the determination of βm and MCT1 and MCT4 protein abundance. Ra during the first 60 min of exercise was not different between groups. During the last 25 min of exercise at 85% V̇o2 peak, Ra was higher compared with exercise at 65% of V̇o2 peak and was decreased in LHTLc ( P < 0.05) compared with the other groups. Rd followed a similar pattern to Ra. Although Rox was significantly increased during exercise at 85% compared with 65% of V̇o2 peak, there were no differences between the three groups or across trials. There was no effect of hypoxic exposure on βm or MCT1 and MCT4 protein abundance. We conclude that 20 consecutive nights of hypoxia exposure decreased whole body Ra during intense exercise in well-trained athletes. However, muscle markers of lactate metabolism and pH regulation were unchanged by the LHTL intervention.

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Galactic orbits of stars

Symposium - International Astronomical Union ◽

10.1017/s0074180900105340 ◽

1966 ◽

Vol 25 ◽

pp. 93-97

Author(s):

Richard Woolley

Keyword(s):

Globular Cluster ◽

Potential Field ◽

High Velocity ◽

Velocity Vector ◽

Variable Stars ◽

The Sun ◽

Proper Motions ◽

Rr Lyrae ◽

The Galaxy

It is now possible to determine proper motions of high-velocity objects in such a way as to obtain with some accuracy the velocity vector relevant to the Sun. If a potential field of the Galaxy is assumed, one can compute an actual orbit. A determination of the velocity of the globular clusterωCentauri has recently been completed at Greenwich, and it is found that the orbit is strongly retrograde in the Galaxy. Similar calculations may be made, though with less certainty, in the case of RR Lyrae variable stars.

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