Glycogenin protein and mRNA expression in response to changing glycogen concentration in exercise and recovery

Glycogenin (GN-1) is essential for the formation of a glycogen granule; however, rarely has it been studied when glycogen concentration changes in exercise and recovery. It is unclear whether GN-1 is degraded or is liberated and exists as apoprotein (apo)-GN-1 (unglycosylated). To examine this, we measured GN-1 protein and mRNA level at rest, at exhaustion (EXH), and during 5 h of recovery in which the rate of glycogen restoration was influenced by carbohydrate (CHO) provision. Ten males cycled (65% V̇o2 max) to volitional EXH (117.8 ± 4.2 min) on two separate occasions. Subjects were administered carbohydrate (CHO; 1 g·kg−1·h−1 Gatorlode) or water [placebo (PL)] during 5 h of recovery. Muscle biopsies were taken at rest, at EXH, and following 30, 60, 120, and 300 min of recovery. At EXH, total glycogen concentration was reduced ( P < 0.05). However, GN-1 protein and mRNA content did not change. By 5 h of recovery, glycogen was resynthesized to ∼60% of rest in the CHO trial and remained unchanged in the PL trial. GN-1 protein and mRNA level did not increase during recovery in either trial. We observed modest amounts of apo-GN-1 at EXH, suggesting complete degradation of some granules. These data suggest that GN-1 is conserved, possibly as very small, or nascent, granules when glycogen concentration is low. This would provide the ability to rapidly restore glycogen during early recovery.

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Regulation of mdr2 P-glycoprotein expression by bile salts

Biochemical Journal ◽

10.1042/bj3210389 ◽

1997 ◽

Vol 321 (2) ◽

pp. 389-395 ◽

Cited By ~ 39

Author(s):

Charles M. G. FRIJTERS ◽

Roelof OTTENHOFF ◽

Michel J. A. van WIJLAND ◽

Carin M. J. van NIEUWKERK ◽

Albert K. GROEN ◽

...

Keyword(s):

Bile Salt ◽

Bile Salts ◽

Control Diet ◽

Mrna Level ◽

Northern Blotting ◽

Mrna Levels ◽

Male Mice ◽

Complete Replacement ◽

P Glycoprotein ◽

Mrna Content

The phosphatidyl translocating activity of the mdr2 P-glycoprotein (Pgp) in the canalicular membrane of the mouse hepatocyte is a rate-controlling step in the biliary secretion of phospholipid. Since bile salts also regulate the secretion of biliary lipids, we investigated the influence of the type of bile salt in the circulation on mdr2 Pgp expression and activity. Male mice were fed a purified diet to which either 0.1% (w/w) cholate or 0.5% (w/w) ursodeoxycholate was added. This led to a near-complete replacement of the endogenous bile salt pool (mainly tauromuricholate) by taurocholate or tauroursodeoxycholate respectively. The phospholipid secretion capacity was then determined by infusion of increasing amounts of tauroursodeoxycholate. Cholate feeding resulted in a 55% increase in maximal phospholipid secretion compared with that in mice on the control diet. Northern blotting revealed that cholate feeding increased mdr2 Pgp mRNA levels by 42%. Feeding with ursodeoxycholate did not influence the maximum rate of phospholipid output or the mdr2 mRNA content. Female mice had a higher basal mdr2 Pgp mRNA level than male mice, and this was also correlated with a higher phospholipid secretion capacity. This could be explained by the 4-fold higher basal cholate content in the bile of female compared with male mice. Our results suggest that the type of bile salts in the circulation influences the expression of the mdr2 gene.

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Relevance of IL7R genotype and mRNA expression in Dutch patients with multiple sclerosis

Multiple Sclerosis Journal ◽

10.1177/1352458511402411 ◽

2011 ◽

Vol 17 (8) ◽

pp. 922-930 ◽

Cited By ~ 9

Author(s):

MH Sombekke ◽

LF van der Voort ◽

JJ Kragt ◽

JM Nielsen ◽

H Guzel ◽

...

Keyword(s):

Multiple Sclerosis ◽

Mrna Expression ◽

Mrna Level ◽

Susceptibility Gene ◽

Disease Phenotype ◽

Single Nucleotide ◽

Relative Expression ◽

Interleukin 7 ◽

Membrane Bound ◽

Magnetic Resonance Imaging Mri

Background: The interleukin 7 receptor (IL7R) has been recognized as a susceptibility gene for Multiple Sclerosis (MS). Analysis of rs6897932 (the most strongly MS-associated single nucleotide polymorphism (SNP)), showed effects of genotype on the relative expression of membrane-bound to total amount of IL7R mRNA. Objective: We assessed the relevance of IL7R on MS phenotype (including clinical and magnetic resonance imaging (MRI) parameters) at DNA and mRNA level in Dutch patients with MS. Methods: The genotype of rs6897932 was analyzed in 697 patients with MS and 174 healthy controls. The relevance of genotype and carriership of the C allele on MS phenotype (disease activity and severity, using clinical and MRI parameters) was assessed. In addition, relative gene expression of membrane-bound to total IL7R mRNA was analyzed with respect to disease phenotype in a subgroup of 95 patients with early relapsing MS. Results: In particular, homozygosity for the risk allele is a risk factor for MS in our population (ORCC vs CT and TT = 1.65 (95% CI: 1.18–2.30), two-sided p = 0.004). However, no effect of genotype or the relative expression of membrane-bound IL7R (presence of exon 6–7) to total amount of IL7R mRNA (presence of exon 4–5) was found on MS phenotype. Discussion: Homozygosity for the IL7R exon 6 rs6897932 C allele is associated with a higher risk for MS in our Dutch population. No effect was found of genotype or mRNA expression on disease phenotype.

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Lipopolysaccharide regulates proinflammatory cytokine expression in mouse myoblasts and skeletal muscle

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00039.2002 ◽

2002 ◽

Vol 283 (3) ◽

pp. R698-R709 ◽

Cited By ~ 145

Author(s):

Robert A. Frost ◽

Gerald J. Nystrom ◽

Charles H. Lang

Keyword(s):

Skeletal Muscle ◽

Cell Wall ◽

Mrna Expression ◽

Nuclear Factor Κb ◽

Cytokine Expression ◽

Mrna Accumulation ◽

C2c12 Myoblasts ◽

Tnf Α ◽

Mrna Content

The purpose of the present study was to examine the regulation of tumor necrosis factor (TNF)-α and interleukin (IL)-6 by lipopolysaccharide (LPS) in C2C12 myoblasts and mouse skeletal muscle. LPS produced dose- and time-dependent increases in TNF-α and IL-6 mRNA content in C2C12 myoblasts. The LPS-induced cytokine response could be mimicked by peptidoglycan from the cell wall of Staphylococcus aureus but not by zymosan A, a cell wall component from Saccharomyces cerevisiae. Ongoing protein synthesis was not necessary for the increase in the two cytokine mRNAs. The transcriptional inhibitor 5,6-dichloro-β-d-ribofuranosyl-benzimidazole blocked LPS-stimulated IL-6 mRNA expression without changing its mRNA half-life. The anti-inflammatory glucocorticoid dexamethasone selectively blocked LPS-stimulated IL-6 mRNA accumulation but not TNF-α. In contrast, the proteasomal inhibitor MG-132 blocked TNF-α mRNA expression but not IL-6. Exposure of myoblasts to LPS was associated with a rapid decrease in the inhibitor of nuclear factor-κB (I κB, α, and ε), and this response was also blocked by MG-132. Treatment of myocytes with IL-1 or TNF-α also increased IL-6 mRNA content, but the increase in IL-6 mRNA due to LPS could not be prevented by pretreatment with antagonists to either IL-1 or TNF. Under in vivo conditions, LPS increased the plasma concentration of TNF-α and IL-6 and stimulated the accumulation of their mRNAs in multiple tissues including skeletal muscle from wild-type mice. In contrast, the ability of LPS to stimulate the same cytokines was markedly decreased in mice that harbor a mutation in the Toll-like receptor 4. Our data suggest that LPS stimulates cytokine expression not only in classical immune tissues but also in skeletal muscle.

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Tissue Factor Expression in Vital Organs during Murine Traumatic Shock

Anesthesiology ◽

10.1097/00000542-199912000-00039 ◽

1999 ◽

Vol 91 (6) ◽

pp. 1844-1844 ◽

Cited By ~ 24

Author(s):

Valerie E. Armstead ◽

Irina L. Opentanova ◽

Alexander G. Minchenko ◽

Allan M. Lefer

Keyword(s):

Small Intestine ◽

Mrna Expression ◽

Tissue Factor ◽

Messenger Rna ◽

Mrna Level ◽

Regulatory Elements ◽

Traumatic Shock ◽

Surface Glycoprotein ◽

Cell Surface Glycoprotein ◽

Nf Kappab

Background Tissue factor (TF) is a cell-surface glycoprotein responsible for initiating the extrinsic pathway of coagulation that has been shown to have a role in the pathophysiology of sepsis and reperfusion injury. The purpose of this study was to investigate TF expression in vital organs and to determine possible regulatory mechanisms of TF expression in the lung during traumatic shock in rats. Methods Noble-Collip drum trauma was induced in anesthetized Sprague-Dawley rats. Anesthetized rats without trauma served as controls. TF activity was measured in plasma and lung tissue. TF messenger RNA (mRNA) was measured in the lung, liver, and small intestine using ribonuclease protection assays. Electromobility shift assays were used to quantify binding of nuclear extracts from lung to TF-specific consensus domains for transcription factors NF-kappaB and AP-1. Results TF activity in plasma increased up to 14-fold and +232% in the lung (P < 0.001 for plasma and lung) 2 h after trauma. TF mRNA level was significantly increased in the lungs (P < 0.01), small intestine (P < 0.01), and liver (P < 0.05) 1 h after trauma compared to sham-operated control rats. TF mRNA expression continued to increase in the lungs and the liver (both, P < 0.001) 2 h after trauma TF sequence-specific complex binding to AP-1 and NF-kappaB domains was enhanced in the lungs of trauma rats (+395%, P < 0.001 and +168%, P < 0.001, respectively). Conclusions These results suggest that TF may play an important role in the pathophysiology of severe trauma and that regulatory elements AP-1 and NF-kappaB may be involved in the regulation of TF mRNA expression in traumatic shock.

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Children with oligoarticular Juvenile Idiopathic Arthritis have skewed synovial monocyte polarization pattern with functional impairment – a distinct inflammatory pattern for oligoarticular juvenile arthritis

10.21203/rs.3.rs-29879/v2 ◽

2020 ◽

Author(s):

Tobias Schmidt ◽

Elisabet Berthold ◽

Sabine Arve-Butler ◽

Birgitta Gullstrand ◽

Anki Mossberg ◽

...

Keyword(s):

Juvenile Idiopathic Arthritis ◽

Synovial Fluid ◽

Mrna Expression ◽

Mrna Level ◽

Western World ◽

Polarization Pattern ◽

Oligoarticular Juvenile Idiopathic Arthritis ◽

Monocytes And Macrophages ◽

Anti Inflammatory

Abstract Background Juvenile idiopathic arthritis (JIA) is an umbrella term of inflammatory joint diseases in children. Oligoarthritis is the most common form in the Western world, representing roughly 60% of all patients. Monocytes and macrophages play an important role in adult arthritides, but their role in oligoarticular JIA is less studied. Polarization highly influences monocytes’ and macrophages’ effector functions, broadly separated into pro-inflammatory M1 or anti-inflammatory M2 phenotypes. Here, we set out to investigate the polarization pattern and functional aspects of synovial monocytes in oligoarticular juvenile idiopathic arthritis (JIA). Methods Paired synovial fluid, blood samples (n=13) and synovial biopsies (n=3) were collected from patients with untreated oligoarticular JIA. Monocytes were analyzed for polarization markers by flow cytometry and qPCR. Effector function was analyzed by a phagocytosis assay. Polarization of healthy monocytes was investigated by stimulation with synovial fluid in vitro . Monocyte/macrophage distribution, polarization and mRNA expression were investigated in biopsies by immunohistochemistry, immunofluorescence and in situ hybridization. Results Children with oligoarticular JIA have polarized synovial fluid monocytes of a specific M1(IFNγ)/M2(IL-4)-like pattern. This was evidenced by increased surface expression of CD40 (p<0.001), CD86 (p<0.001) and CD206 (p<0.001), but not CD163, as compared to paired circulating monocytes. Additionally, polarization was extensively explored at the mRNA level and synovial fluid monocytes differentially expressed classical markers of M1(IFNγ)/M2(IL-4) polarization compared to circulating monocytes. Synovial fluid monocytes were functionally affected, as assessed by reduced capacity to phagocytose (p<0.01). Synovial fluid induced M2 markers (CD16 and CD206), but not M1 (CD40) or CD86 in healthy monocytes and did not induce cytokine production. Single and co-expression of surface CD40 and CD206, as well as mRNA expression of IL-10 and TNF, was observed in monocytes/macrophages in synovial biopsies. Conclusion Children with untreated oligoarticular JIA have similar and distinct synovial fluid monocyte polarization pattern of mixed pro- and anti-inflammatory features. This pattern was not exclusively a result of the synovial fluid milieu as monocytes/macrophages in the synovial membrane show similar patterns. Our study highlights a distinct polarization pattern in oligoarticular JIA, which could be utilized for future treatment strategies.

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Tumor cell total mRNA expression shapes the molecular and clinical phenotype of cancer

10.21203/rs.3.rs-600171/v1 ◽

2021 ◽

Author(s):

Shaolong Cao ◽

Jennifer Wang ◽

Shuangxi Ji ◽

Peng Yang ◽

Matthew Montierth ◽

...

Keyword(s):

Tumor Cell ◽

Mrna Expression ◽

Genetic Alterations ◽

Individual Variability ◽

Cancer Type ◽

Sequencing Data ◽

Increased Risk ◽

Mrna Content ◽

Risk Of Disease ◽

Pan Cancer

Abstract Cancers can vary greatly in their transcriptomes. In contrast to alterations in specific genes or pathways, differences in tumor cell total mRNA content have not been comprehensively assessed. Technical and analytical challenges have impeded examination of total mRNA expression at scale across cancers. To address this, we developed a model for quantifying tumor-specific total mRNA expression (TmS) from bulk sequencing data, which performs transcriptomic deconvolution while adjusting for mixed genomes. We used single-cell RNA sequencing data to demonstrate total mRNA expression as a feature of tumor phenotype. We estimated and validated TmS in 5,015 patients across 15 cancer types identifying significant inter-individual variability. At a pan-cancer level, high TmS is associated with increased risk of disease progression and death. Cancer type-specific patterns of genetic alterations, intra-tumor genetic heterogeneity, as well as pan-cancer trends in metabolic dysregulation and hypoxia contribute to TmS. Taken together, our results suggest that measuring cell-type specific total mRNA expression offers a broader perspective of tracking cancer transcriptomes, which has important biological and clinical implications.

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Osteogenic protein-1 mRNA expression is selectively modulated after acute ischemic renal injury.

Journal of the American Society of Nephrology ◽

10.1681/asn.v981456 ◽

1998 ◽

Vol 9 (8) ◽

pp. 1456-1463

Author(s):

M M Almanzar ◽

K S Frazier ◽

P H Dube ◽

A I Piqueras ◽

W K Jones ◽

...

Keyword(s):

Mrna Expression ◽

Renal Injury ◽

Tissue Repair ◽

Western Blots ◽

Time Points ◽

Mrna Content ◽

Repair Mechanisms ◽

Osteogenic Protein ◽

Ischemic Renal Injury ◽

Deficient Mice

Osteogenic protein-1 (OP-1) is a morphogenetic factor highly expressed in the kidney and involved in tissue repair and development. Homozygous OP-1-deficient mice die shortly after birth due mainly to arrest of renal growth and differentiation. Because postischemic injury involves several repair mechanisms, this study examined whether kidney OP-1 mRNA expression is modulated after ischemia. Acute ischemic renal injury was achieved in rats by unilateral clamping of the renal pedicle followed by reperfusion. Rats were killed at 3, 6, 12, 24, and 48 h and 7 d after reperfusion, and kidneys were microdissected and analyzed by histology and Northern and Western blots. Changes in OP-1 mRNA were determined by measuring the ratio of OP-1/glyceraldehyde 3-phosphate dehydrogenase signals for each OP-1 transcript (4.0 and 2.4 kb) from ischemic, opposite, and sham-operated rats. The OP-1 mRNA content for transcript 4.0 kb was fivefold lower in the whole ischemic kidney compared with that in sham animals 24 h after reperfusion. In the ischemic medulla, OP-1 mRNA was strikingly downregulated 20-fold when compared with the ischemic cortex. Results for transcript 2.4 kb and for the other time points were comparable. OP-1 mRNA expression was also affected in the opposite medulla compared with the sham medulla. However, only in the ischemic medulla was the relative OP-1 content significantly lower at all time points. Similar results were obtained when analyzing OP-1 protein by Western blot at 24 h after reperfusion. Seven days after reperfusion, the levels of OP-1 mRNA returned to baseline. In conclusion, kidney OP-1 mRNA and protein are selectively downregulated in the medulla after acute ischemic renal injury. OP-1 modulation may be a key element for kidney repair.

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AMP-activated protein kinase agonists increase mRNA content of the muscle-specific ubiquitin ligases MAFbx and MuRF1 in C2C12 cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00622.2006 ◽

2007 ◽

Vol 292 (6) ◽

pp. E1555-E1567 ◽

Cited By ~ 93

Author(s):

Brian J. Krawiec ◽

Gerald J. Nystrom ◽

Robert A. Frost ◽

Leonard S. Jefferson ◽

Charles H. Lang

Keyword(s):

Skeletal Muscle ◽

Protein Kinase ◽

Mrna Expression ◽

Muscle Atrophy ◽

Ubiquitin Ligases ◽

C2c12 Cells ◽

Skeletal Muscle Atrophy ◽

Compound C ◽

Mrna Content ◽

Amp Activated Protein Kinase

The hypothesis of the present study was that exposure of differentiated muscle cells to agonists of the AMP-activated protein kinase (AMPK) would increase the mRNA content of the muscle-specific ubiquitin ligases muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). C2C12 cells were incubated with incremental doses of 5-aminoimidazol-4-carboximide ribonucleoside (AICAR) or metformin for 24 h. Both MAFbx and MuRF1 mRNA increased dose dependently in response to these AMPK activators. AICAR, metformin, and 2-deoxy-d-glucose produced time-dependent alterations in ubiquitin ligase expression, typified by a biphasic pattern of expression marked by an acute repression followed by a sustained induction. AMPK-activating treatments in conjunction with dexamethasone produced a pronounced synergistic effect on ligase mRNA expression at later time points. This cooperative response occurred in the absence of a dexamethasone-dependent increase in AMPK expression or activity, as determined by immunoblotting for phosphorylation and expression of AMPKα and its downstream target acetyl-CoA carboxylase (ACC). These responses elicited by AMPK activation singly or in combination with dexamethasone did not extend to the mRNA expression of the UBR box family E3s UBR1/E3αI and UBR2/E3αII. Treatment with the AMPK inhibitor compound C prevented increases in MAFbx and MuRF1 mRNA in response to serum deprivation, as well as AICAR and dexamethasone treatment individually or jointly. Stimulation of AMPK activity in vivo via AICAR injection increased both MAFbx and MuRF1 mRNA in murine skeletal muscle. These data suggest that activation of AMPK in skeletal muscle results in a specific upregulation of MAFbx and MuRF1, responses that are reminiscent of the proposed atrophic transcriptional program executed under various conditions of skeletal muscle wasting. Therefore, AMPK may be a critical component of the intercalated network of signaling pathways governing skeletal muscle atrophy, where its input acts to modify anti- and proatrophic signals to influence gene expression in reaction to catabolic perturbations.

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The level of cytoskeleton remodeling proteins in the blood serum and the expression of their mRNA in the tumor tissue in metastasis of the larynx and hypopharynx cancer

10.21203/rs.3.rs-315722/v1 ◽

2021 ◽

Author(s):

Gelena Kakurina ◽

Olga V Cheremisina ◽

Elena E Sereda ◽

Elena S Kolegova ◽

Irina V Kondakova ◽

...

Keyword(s):

Serum Level ◽

Blood Serum ◽

Mrna Expression ◽

Tumor Tissue ◽

Mrna Level ◽

Actin Binding ◽

Tissue Samples ◽

Signaling Systems ◽

Cytoskeleton Remodeling ◽

The Relationship

Abstract Purpose: Actin-binding proteins (ABPs) and various signaling systems are involved in the metastasis of squamous cell carcinoma of the larynx and hypopharynx (SCCLH). The clinical significance of these proteins has not yet been determined. We analyzed the relationship between the mRNA level of cofilin 1 (CFL1), profilin 1 (PFN1), adenylyl cyclase-associated protein 1 (CAP1), SNAIL and RND3 with metastasis in the SCCLH tissue. The serum level of the listed ABPs was estimated and the relationship of them with the expression of the corresponding mRNA was carried out. Materials and methods: The expression level of ABPs mRNA was measured by real-time RT-PCR in paired tissue samples taken from 54 patients with SCCLH (T 1-4 N 0-1 M 0 ). Expression analysis was performed using the 2 - ΔΔ CT method. The level of ABPs in the blood serum was measured by ELISA. Statistical analysis was carried out using the SPSS Statistica 20.0 software package. Results: The mRNA expression of the studied genes in tumor tissue of patients with SCCLH T 1-3 N 0 M 0 and T 2-4 N 1-2 M 0 did not differ significantly. High expression of RND3 mRNA was accompanied by an increase in mRNA expression of all studied ABPs. In the blood serum of T 2-4 N 1-2 M 0 patients the level of PFN1 was significantly lower by 21% and the level of CAP1 was higher by 75% compared with the group of patients with T 1-4 N 0 M 0 stage. Conclusion: According to our data RND3 is involved in the regulation of molecular cascades SCCLH metastasis. PFN1 and CAP1 serum level can be a good classifier of metastases in patients with SCCLH.

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Chronic intrauterine pulmonary hypertension decreases calcium-sensitive potassium channel mRNA expression

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2000.279.5.l857 ◽

2000 ◽

Vol 279 (5) ◽

pp. L857-L862 ◽

Cited By ~ 12

Author(s):

David N. Cornfield ◽

Ernesto R. Resnik ◽

Jean M. Herron ◽

Steven H. Abman

Keyword(s):

Gene Expression ◽

Pulmonary Hypertension ◽

Mrna Expression ◽

Internal Control ◽

Vascular Reactivity ◽

Ductus Arteriosus ◽

Critical Role ◽

Mrna Levels ◽

Channel Gene ◽

Mrna Content

Calcium-sensitive potassium (KCa) channels play a critical role in mediating perinatal pulmonary vasodilation. Because infants with persistent pulmonary hypertension of the newborn (PPHN) have blunted vasodilator responses to birth-related stimuli, we hypothesized that lung KCachannel gene expression is decreased in PPHN. To test this hypothesis, we measured KCa channel gene expression in distal lung homogenates from both fetal lambs with severe pulmonary hypertension caused by prolonged compression of the ductus arteriosus and age-matched, sham-operated animals (controls). After at least 9 days of compression of the ductus arteriosus, fetal lambs were killed. To determine lung KCa channel mRNA levels, primers were designed against the known sequence of the KCa channel and used in semiquantitative RT-PCR, with lung 18S rRNA content as an internal control. Compared to that in control lambs, lung KCa channel mRNA content in the PPHN group was reduced by 26 ± 6% ( P < 0.02), whereas lung voltage-gated K+ 2.1 mRNA content was unchanged. We conclude that lung KCa channel mRNA expression is decreased in an ovine model of PPHN. Decreased KCa channel gene expression may contribute to the abnormal pulmonary vascular reactivity associated with PPHN.

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