Regulation of HSP25 expression and phosphorylation in functionally overloaded rat plantaris and soleus muscles

Functional overload (FO) is a powerful inducer of muscle hypertrophy and both oxidative and mechanical stress in muscle fibers. Heat shock protein 25 (HSP25) may protect against both of these stressors, and its expression can be regulated by changes in muscle loading and activation. The primary purpose of the present study was to test the hypothesis that chronic FO increases HSP25 expression and phosphorylation (pHSP25) in hypertrophying rat hindlimb muscle. HSP25 and pHSP25 levels were quantified in soluble and insoluble fractions of the soleus and plantaris to determine whether 3 or 7 days of FO increase translocation of HSP25 and/or pHSP25 to the insoluble fraction. p38 protein and phosphorylation (p-p38) was measured to determine its association with changes in pHSP25. HSP25 mRNA showed time-dependent increases in both the soleus and plantaris with FO. Three or seven days of FO increased HSP25 and pHSP25 in the soluble fraction in both muscles, with a greater response in the plantaris. In the insoluble fraction, HSP25 was increased after 3 or 7 days in both muscles, whereas pHSP25 was only increased in the 7-day plantaris. p38 and p-p38 increased in the plantaris at both time points. In the soleus, p-p38 only increased after 7 days. These results show that FO is associated with changes in HSP25 expression and phosphorylation and suggest its role in the remodeling that occurs during muscle hypertrophy. Increases in HSP25 in the insoluble fraction suggest that it may help to stabilize actin and/or other cytoskeletal proteins during the stress of muscle remodeling.

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Modulation of HSP25 and TNF-α during the early stages of functional overload of a rat slow and fast muscle

Journal of Applied Physiology ◽

10.1152/japplphysiol.00021.2007 ◽

2007 ◽

Vol 102 (6) ◽

pp. 2307-2314 ◽

Cited By ~ 24

Author(s):

Kimberly A. Huey ◽

Gary E. McCall ◽

Hui Zhong ◽

Roland R. Roy

Keyword(s):

Heat Shock ◽

Tumor Necrosis ◽

Tumor Necrosis Factor Α ◽

Soluble Fraction ◽

Insoluble Fraction ◽

Mrna Levels ◽

Tnf Α ◽

Muscle Loading ◽

Factor Α ◽

Necrosis Factor

Early events in response to abrupt increases in activation and loading with muscle functional overload (FO) are associated with increased damage and inflammation. Heat shock protein 25 (HSP25) may protect against these stressors, and its expression can be regulated by muscle loading and activation. The purpose of this study was to investigate the responses of HSP25, phosphorylated HSP25 (pHSP25), and tumor necrosis factor-α (TNF-α) during FO of the slow soleus and fast plantaris. We compared the HSP25 mRNA, HSP25 protein, pHSP25, and TNF-α responses in the soleus and plantaris after 0.5, 1, 2, 3, and 7 days of FO. HSP25 and pHSP25 were quantified in soluble and insoluble fractions. HSP25 mRNA increased immediately in both muscles and decreased with continued FO. However, HSP25 mRNA levels were consistently higher in the muscles of FO than control rats. In the soluble fraction, HSP25 increased in the plantaris after 2–7 days of FO with the greatest response at 3 and 7 days. The pHSP25 response to FO was greater in the plantaris than soleus at all points in the soluble fraction and at 0.5 days in the insoluble fraction. TNF-α levels in the plantaris, but not soleus, were higher than control at 0.5–2 days of FO. This may have contributed to the greater FO response in pHSP25 in the plantaris than soleus as TNF-α increased pHSP25 in C2C12 myotubes. These results suggest that the initial responses of pHSP25 and TNF-α to mechanical stress and inflammation associated with FO are greater in a fast than slow extensor muscle.

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INTERACTION OF MEMBRANE GLYCOPROTEIN GPIIb AND Ilia WITH CYTOSKELETAL PROTEINS DURING PLATELET ACTIVATION

10.1055/s-0038-1643515 ◽

1987 ◽

Author(s):

K Fujimura ◽

T Fujimoto ◽

M Takemoto ◽

K Oda ◽

S Maehama ◽

...

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Soluble Fraction ◽

Insoluble Fraction ◽

Cytoskeletal Proteins ◽

Molecular Weight Protein ◽

High Molecular Weight Protein ◽

Weight Protein ◽

Triton X ◽

Molecular Weight Protein Fraction

Experiments were designed and performed to analyse the cytoskeleton assembly and the interaction of glycoprotein (GP)IIb, IIIa and cytoskeletal proteins during platelet activation. A23187 stimulated 125I labeled platelets were solubilised with Triton X-100 solution and centrifuged. The insoluble fraction were analysed by two dimensional electrophoresis and the soluble fraction were fractionated with 5-25% sucrose gradient centrifugation and analysed by SDS PAGE. In Triton X-100 insoluble fraction, high molecular weight protein fraction(MW > 106) was present after stimulation which were consisted of actin binding protein(ABP), myosin heavy chain(MHC), actin and GPIIb and IIIa. And some of the ABP and MHC formed dimer. ABP and actin in this fraction were decreased with 1 mM CaCl2 treatment but the reduction of ABP was inhibited by leupeptm. In Triton X-100 soluble fraction after stimulation, some of the ABP, MHC, P235 protein, actin and small amount of GPIIb, IIIa were sedimented in the same high density fraction but most proteins were sedimented as a monomer form or GPIIb-IIIa complex form. The GPIIb, IIIa incorporation in high molecular weight protein fraction or high density fraction was absent in Ca++ chelating condition or the presence of competitive fibrinogen binding inhibitor which blocked the platelet aggregation. It is concluded that cytoskeletal proteins and GPIIb, IIIa are assembled each other and formed high molecular weight protein fraction or dimer formation during activation. In stimulated platelets these assembled cytoskeletal proteins containing GPIIb, IIIa were also found in Triton X-100 soluble fraction as a precursor of high molecular weight fraction in Triton X-100 insoluble fraction. The binding of fibrinogen to GPIIb-IIIa complex induce the linkage of GPIIb, IIIa to assembled cytoskeletal proteins.

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Heat shock increases cytosolic free Ca2+ concentration via Na(+)-Ca2+ exchange in human epidermoid A 431 cells

AJP Cell Physiology ◽

10.1152/ajpcell.1992.263.1.c30 ◽

1992 ◽

Vol 263 (1) ◽

pp. C30-C38 ◽

Cited By ~ 37

Author(s):

J. G. Kiang ◽

M. L. Koenig ◽

R. C. Smallridge

Keyword(s):

Heat Treatment ◽

Heat Shock ◽

Time Dependent ◽

Maximal Velocity ◽

Dependent Manner ◽

Michaelis Constant ◽

Exchange System ◽

Maximal Increase ◽

Normal Cells ◽

Dimethyl Amiloride

This study characterized cytosolic free Ca2+ concentration ([Ca2+]i) in normal and thermally injured human epidermoid A 431 cells. The resting [Ca2+]i in normal cells at 37 degrees C was 87 +/- 5 nM (n = 105). When cells were subjected to hyperthermia (40-50 degrees C), [Ca2+]i increased in a temperature- and time-dependent manner. The maximal increase in cells exposed to 45 degrees C was observed at 20 min; [Ca2+]i returned to normal within 1 h. The heat-induced [Ca2+]i increase depended on the presence of external Ca2+. La3+ and Cd2+ but not Co2+, verapamil, or nifedipine attenuated the heat-induced [Ca2+]i increase. TMB-8 partially blocked the increase in [Ca2+]i but pertussis toxin and cholera toxin pretreatment did not. The magnitude of the heat-induced [Ca2+]i increase or 45Ca2+ uptake depended on the presence of extracellular Na+. Heat treatment reduced the apparent Michaelis constant for external Ca2+ from 490 +/- 91 to 210 +/- 60 microM, whereas the maximal velocity remained the same. The intracellular Na+ concentration decreased 62.5% after heating. The heat-induced [Ca2+]i increase was completely blocked by amiloride (5 microM) and 5'-(N,N-dimethyl)-amiloride (1 microM). These results suggest heat activates the Na(+)-Ca2+ exchange system so as to increase [Ca2+]i and reduce [Na+]i.

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The radical scavenging activity of 2-2’ diphenyl -1- picrylhydrazil (DPPH) on the methanol extracts and Ethyl acetate fractions of red dragon fruit peel (Hylocereus polyrhizus (F.A.C.Weber) Britton dan Rose)

International Journal of Phytomedicine ◽

10.5138/09750185.1945 ◽

2017 ◽

Vol 9 (1) ◽

pp. 79

Author(s):

Sri Wahdaningsih ◽

Subagus Wahyuono ◽

Sugeng Riyanto ◽

Retno Murwanti

Keyword(s):

Antioxidant Activity ◽

Ethyl Acetate ◽

Methanol Extract ◽

Soluble Fraction ◽

Radical Scavenging Activity ◽

Radical Scavenging ◽

Insoluble Fraction ◽

Fruit Peel ◽

Activity Test ◽

Dragon Fruit

Red dragon fruit (H. Polyrhizus) is one of the the plants that has a great potential as natural antioxidant. This study tested the activity of radical scavenging of 2-2' diphenyl -1- pikril hidrazil (DPPH) in the methanol extract, as well as in the soluble and insoluble fractions of ethyl acetate of red dragon fruit peel. This research is carried out through various stages, such as: extraction and fractionation to obtain both insoluble fraction and soluble fractions of ethyl acetate. Antioxidant activity test is conducted by the method of thin layer chromatography and spectrophotometry. Antioxidant activity test, IC50 values of methanol extract, ethyl acetate soluble fraction, and insoluble fraction of ethyl acetate had been obtained consecutively as much as 241.19 µg /mL, 8.34 µg/mL, 46.84 µg/mL. The soluble fraction of ethyl acetate had greater antioxidant activity compared to the methanol extract and the insoluble fractions of ethyl acetate.

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A novel murine muscle loading model to investigate Achilles musculotendinous adaptation

Journal of Applied Physiology ◽

10.1152/japplphysiol.00638.2020 ◽

2021 ◽

Vol 130 (4) ◽

pp. 1043-1051

Author(s):

Sabah N. Rezvani ◽

Anne E. C. Nichols ◽

Robert W. Grange ◽

Linda A. Dahlgren ◽

P. Gunnar Brolinson ◽

...

Keyword(s):

Mouse Model ◽

Achilles Tendon ◽

Tendon Injuries ◽

Clinical Treatment ◽

Ankle Dorsiflexion ◽

Therapeutic Exercise ◽

Hindlimb Muscle ◽

Innovative Model ◽

Muscle Loading ◽

Targeted Therapeutic

We introduce a novel mouse model of hindlimb muscle loading designed to achieve a tissue-targeted therapeutic exercise. This innovative model allows for application of muscle loading “doses,” coupled with ankle dorsiflexion and plantarflexion, inspired by human loading clinical treatment. Our model facilitates future investigation of mechanisms whereby rehabilitative muscle loading promotes healing of Achilles tendon injuries.

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Invariant Leu Preceding Turn Motif Phosphorylation Site Controls the Interaction of Protein Kinase C with Hsp70

Journal of Biological Chemistry ◽

10.1074/jbc.m604076200 ◽

2006 ◽

Vol 281 (43) ◽

pp. 32461-32468 ◽

Cited By ~ 26

Author(s):

Tianyan Gao ◽

Alexandra C. Newton

Keyword(s):

Protein Kinase C ◽

Heat Shock ◽

Protein Kinase ◽

Phosphorylation Site ◽

Insoluble Fraction ◽

Down Regulation ◽

Kinase C ◽

Shock Proteins ◽

Invariant Residue ◽

Autophosphorylation Site

Heat shock proteins play important roles in regulating signal transduction in cells by associating with, and stabilizing, diverse signaling molecules, including protein kinases. Previously, we have shown that heat shock protein Hsp70 associates with protein kinase C (PKC) via an interaction that is triggered by dephosphorylation at the turn phosphorylation motif. Here we have identified an invariant residue in the carboxyl terminus of PKC that mediates the binding to Hsp70. Specifically, we show that Hsp70 binds to Leu (Leu-640) immediately preceding the conserved turn motif autophosphorylation site (Thr-641) in PKC βII. Co-immunoprecipitation experiments reveal that mutation of Leu-640 to Gly decreases the interaction of Hsp70 with PKC βII. This weakened interaction between Hsp70 and the mutant PKCs results in accumulation of dephosphorylated PKC in the detergent-insoluble fraction of cells. In addition, the Hsp70-binding mutant is considerably more sensitive to down-regulation compared with WT PKC: disruption of Hsp70 binding leads to accelerated dephosphorylation and enhanced ubiquitination of mutant PKC upon phorbol ester treatment. Last, pulse-chase experiments demonstrate that Hsp70 preferentially binds the species of mature PKC that has become dephosphorylated compared with the newly synthesized protein that has yet to be phosphorylated. Thus, Hsp70 binds a hydrophobic residue preceding the turn motif, protecting PKC from down-regulation and sustaining the signaling lifetime of the kinase.

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Fractionation of Rubber

Rubber Chemistry and Technology ◽

10.5254/1.3540010 ◽

1941 ◽

Vol 14 (1) ◽

pp. 1-14 ◽

Cited By ~ 1

Author(s):

George F. Bloomfield ◽

Ernest Harold Farmer

Keyword(s):

Molecular Weight ◽

Soluble Fraction ◽

Insoluble Fraction ◽

The Other ◽

Major Fraction ◽

Low Concentrations ◽

Fractional Dissolution ◽

Judicious Choice ◽

Hydrocarbon Molecules ◽

Soluble Fractions

Abstract Latex rubber which has been purified to the point at which it contains an insignificant amount of nitrogen can be separated by fractional dissolution in a mixture of petroleum and acetone into a series of hydrocarbon fractions of decreasing solubility and increasing molecular magnitude. All these fractions except the highest are soluble in petroleum and in benzene. Crepe rubber, on the other hand, appears invariably to contain a small, most-soluble fraction of oxygenated rubber, and a small similar quite insoluble fraction of material of high molecular weight. Between these extremes the rubber can be divided into fractions of increasing molecular weight, although, up to the present, about 70 per cent of the total rubber has appeared in a single fraction. It may be possible later, by judicious choice of another pair of solvents, to resolve this major fraction into a series of subfractions. Kemp and Peters refer to the effect of polar nonsolvents in reducing the viscosity of rubber solutions and also in assisting to bring gel rubber into solution, phenomena to which the polar molecules conceivably contribute by countering the forces of association between the rubber molecules. The present series of fractionations was conducted throughout in the presence of a polar nonsolvent (acetone), and hence may be considered to approach towards a separation of true rubber molecules as distinct from molecular aggregates. It is found, however, that, whereas the more soluble fractions of acetone-extracted crepe rubber contain small proportions of nitrogen, the least soluble fractions contain substantial proportions. Any effect which the nitrogenous material may have in assisting to link together hydrocarbon molecules to which it is attached, i. e., in contributing to the high-molecular condition of a portion of natural rubber, remains at present uncertain in character. The fractions of rubber, and especially the higher ones, show a strong tendency to become insoluble when they have once been freed from the last traces of solvent. It seems doubtful whether the decreased solubility is due to oxygen as it would require to be effective at exceedingly low concentrations.

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2 GLOBAL PEPTIDE SEQUENCING AND QUANTIFICATION OF PROTEINS IN PORCINE PARTHENOTES BY PROTEOMICS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab2 ◽

2007 ◽

Vol 19 (1) ◽

pp. 119

Author(s):

J. M. Jang ◽

M. K. Gupta ◽

J. W. Jung ◽

S. J. Uhm ◽

K. P. Kim ◽

...

Keyword(s):

Heat Shock ◽

Ribosomal Proteins ◽

Dna Methyltransferase ◽

Electric Pulse ◽

Peptide Sequencing ◽

Cytoskeletal Proteins ◽

Mitochondrial Enzymes ◽

Relative Quantification ◽

Protein Database

Mass spectrometry (MS) is a powerful emerging tool in proteomics for global identification and quantification of proteins and their differential analysis. The aim of this study was to utilize MS for global peptide sequencing and the relative quantification of peptide sequences in porcine embryos produced by parthenogenesis. Oocytes (n = 6000) recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro for 42–44 h and parthenogenetically activated by a single electric pulse of 1.36 kV cm-1. After 24 h of in vitro culture in NCSU23 medium supplemented with 0.4% BSA, presumptive zygotes were sampled in Laemmli buffer and stored in liquid nitrogen until analysis. SDS-PAGE was used as the first step to separate intact proteins into 16 fractions. Each fraction was then in-gel tryptic-digested and analyzed by nanoLC-nano-ESI-MS/MS to sequence the peptide components in each fraction by LTQ ion-trap mass spectrometer (Finnigan, Fresno, CA, USA) after peptide purification by C18 ZipTips (Millipore, Billerica, MA, USA). Synthetic peptides from Myoglobin (LFTGHPETL*EK) and MAP3 Kinase (IPTGTV*HNQAK) protein having an isotope-labeled carbon (*) were used as internal standards for relative quantification. In total, 54 060 peptides were sequenced. Of these, 259 proteins were identified in the NCBI protein database for Sus scrofa using Spectrum Mill software (Agilent Technologies, Palo Alto, CA, USA). Of these, 199 proteins were identified with high confidence having at least 2 peptide sequences matching each protein. These included heat shock protein families, ribosomal proteins, cytoskeletal proteins, mitochondrial enzymes, disulfide isomerase proteins, glutathione S-transferase, DNA methyltransferase, STAT1, Ras-related protein Rab 1A, voltage-dependent ion channel proteins, zinc finger proteins and elongation factors, etc., which are involved in several important metabolic and cell signaling pathways. Several proteins including DNA methyltransferase 1, peroxidoxin 2, ubiquitin carboxyl-terminal hydrolase L1, serpin, heat shock proteins, cytoskeletal proteins, mitochondrial enzymes, elongation factor 1, bone morphogenetic protein, glucose-related proteins, etc., were among the highly abundant proteins. Our results thus showed that proteomic strategy can be successfully applied to analyze porcine embryo proteome. To our knowledge, this is the first time that several proteins have been sequenced and quantified in porcine parthenotes by proteomic means. In the long run, this study may help the understanding of mechanisms underlying embryonic development and identification of biomarkers of embryo quality. This work was supported by grants from the Research Project on the Production of Bio-organs (No. 200503030202), Ministry of Agriculture and Forestry, and Korea Health 21 R&D Project (No. A060769), Ministry of Health and Welfare, Republic of Korea.

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Making Mice Mighty: recent advances in translational models of load-induced muscle hypertrophy

Journal of Applied Physiology ◽

10.1152/japplphysiol.00319.2020 ◽

2020 ◽

Vol 129 (3) ◽

pp. 516-521 ◽

Cited By ~ 1

Author(s):

Kevin A. Murach ◽

John J. McCarthy ◽

Charlotte A. Peterson ◽

Cory M. Dungan

Keyword(s):

Skeletal Muscle ◽

Muscle Hypertrophy ◽

Muscle Growth ◽

Therapeutic Interventions ◽

Loss Of Function ◽

Hypertrophic Response ◽

Advantages And Disadvantages ◽

Muscle Loading ◽

Specific Factors

The ability to genetically manipulate mice allows for gain- and loss-of-function in vivo, making them an ideal model for elucidating mechanisms of skeletal muscle mass regulation. Combining genetic models with mechanical muscle loading enables identification of specific factors involved in the hypertrophic response as well as the ability to test the requirement of those factors for adaptation, thereby informing performance and therapeutic interventions. Until recently, approaches for inducing mechanically mediated muscle hypertrophy (i.e., resistance-training analogs) have been limited and considered “nontranslatable” to humans. This mini-review outlines recent translational advances in loading-mediated strategies for inducing muscle hypertrophy in mice, and highlights the advantages and disadvantages of each method. The skeletal muscle field is poised for new breakthroughs in understanding mechanisms regulating load-induced muscle growth given the numerous murine tools that have very recently been described.

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