The inhibition of bovine and rat parotid deoxyribonuclease I by skeletal muscle actin. A biochemical and immunocytochemical study.
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ABSTRACT:
SUBMITTER: Mannherz HG
PROVIDER: S-EPMC1153861 | biostudies-other | 1982 Nov
REPOSITORIES: biostudies-other
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Project description:Using biochemical and immunocytochemical techniques, we have assessed both the protein expression and the cellular localization of the GLUT5 transporter in human skeletal muscle. Human muscle membranes, prepared by subcellular fractionation, were subjected to SDS/PAGE and Western-blot analyses using antiserum raised against a specific C-terminal amino acid sequence of the human GLUT5 transporter. GLUT5 was detected as a discrete 49 kDa protein band in a plasma-membrane-enriched fraction prepared from either soleus or gracilis muscle. In contrast, GLUT5 protein was not detectable to any significant extent in fractions which were devoid of muscle plasma membranes (mean GLUT5 abundance in intracellular fractions from three muscle preparations amounted to approximately 10% of that in the plasma-membrane-enriched fraction). Immunofluorescence studies using cryostat sections of human triceps muscle supported the biochemical observations and revealed that GLUT5 antibody selectivity labelled the plasma membrane of muscle cells. This immuno-labelling was significantly suppressed after tissue incubation with antiserum in the presence of a 14-amino-acid synthetic peptide corresponding to a specific C-terminus sequence of human GLUT5. These results indicate that human skeletal muscle expresses the GLUT5 transporter and that it is specifically localized to the plasma membrane, where it may participate in regulating hexose transfer across the sarcolemma.
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Project description:Skeletal muscle actin mice (Crawford et al., (2002) Mol Cell Biol 22, 5587) were crossed with cardiac actin transgenic mice (termed "ACTC^Coco" or "Coco" for short), to produce mice that had cardiac actin instead of skeletal muscle actin in their skeletal muscles (termed "ACTC^Co/KO" or for short "Coco/KO"). Microarray analysis using the Illumina mouse-6 v1.1 expression beadchip was performed on RNA extraced from the soleus muscle of Coco/KO mice and wildtype mice, to confirm the swith in actin isoform expression, and to determine what other differences might exist between wildtype mice and the Coco/KO mice. Keywords: genetic modification 3 RNA samples (each being the pool of two individual samples extracted from different soleus muscles from different individual mice) per genotype (either wildtype or Coco/KO) were used. The total 6 RNA samples were processed using an Illumina mouse-6 v1.1expression beadchip and then the differentially expressed genes determined.
2009-05-24 | E-GEOD-12882 | biostudies-arrayexpress
Project description:Skeletal muscle actin mice (Crawford et al., (2002) Mol Cell Biol 22, 5587) were crossed with cardiac actin transgenic mice (termed "ACTC^Coco" or "Coco" for short), to produce mice that had cardiac actin instead of skeletal muscle actin in their skeletal muscles (termed "ACTC^Co/KO" or for short "Coco/KO"). Microarray analysis using the Illumina mouse-6 v1.1 expression beadchip was performed on RNA extraced from the soleus muscle of Coco/KO mice and wildtype mice, to confirm the swith in actin isoform expression, and to determine what other differences might exist between wildtype mice and the Coco/KO mice. Keywords: genetic modification
2009-05-25 | GSE12882 | GEO
Project description:The molecular components largely responsible for muscle attributes such as passive tension development (titin and collagen), active tension development (myosin heavy chain, MHC) and mechanosensitive signaling (titin) have been well studied in animals but less is known about their roles in humans. The purpose of this study was to perform a comprehensive analysis of titin, collagen and MHC isoform distributions in a large number of human muscles, to search for common themes and trends in the muscular organization of the human body. In this study, 599 biopsies were obtained from six human cadaveric donors (mean age 83 years). Three assays were performed on each biopsy - titin molecular mass determination, hydroxyproline content (a surrogate for collagen content) and MHC isoform distribution. Titin molecular mass was increased in more distal muscles of the upper and lower limbs. This trend was also observed for collagen. Percentage MHC-1 data followed a pattern similar to collagen in muscles of the upper extremity but this trend was reversed in the lower extremity. Titin molecular mass was the best predictor of anatomical region and muscle functional group. On average, human muscles had more slow myosin than other mammals. Also, larger titins were generally associated with faster muscles. These trends suggest that distal muscles should have higher passive tension than proximal ones, and that titin size variability may potentially act to 'tune' the protein's mechanotransduction capability.
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Project description:Deoxyribonuclease 1 (DNASE1, DNase I) and deoxyribonuclease 1-like 3 (DNASE1L3, DNase gamma, DNase Y, LS-DNase) are members of a DNASE1 protein family that is defined by similar biochemical properties such as Ca2+/Mg2+-dependency and an optimal pH of about 7.0 as well as by a high similarity in their nucleic acid and amino acid sequences. In the present study we describe the recombinant expression of rat Dnase1 and murine Dnase1l3 as fusion proteins tagged by their C-terminus to green fluorescent protein in NIH-3T3 fibroblasts and bovine lens epithelial cells. Both enzymes were translocated into the rough endoplasmic reticulum, transported along the entire secretory pathway and finally secreted into the cell culture medium. No nuclear occurrence of the nucleases was detectable. However, deletion of the N-terminal signal peptide of both nucleases resulted in a cytoplasmic and nuclear distribution of both fusion proteins. Dnase1 preferentially hydrolysed 'naked' plasmid DNA, whereas Dnase1l3 cleaved nuclear DNA with high activity. Dnase1l3 was able to cleave chromatin in an internucleosomal manner without proteolytic help. By contrast, Dnase1 was only able to achieve this cleavage pattern in the presence of proteases that hydrolysed chromatin-bound proteins. Detailed analysis of murine sera derived from Dnase1 knockout mice revealed that serum contains, besides the major serum nuclease Dnase1, an additional Dnase1l3-like nucleolytic activity, which, in co-operation with Dnase1, might help to suppress anti-DNA autoimmunity by degrading nuclear chromatin released from dying cells.
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Project description:Skeletal muscle alpha-actin (ACTA1) is the major actin in postnatal skeletal muscle. Mutations of ACTA1 cause mostly fatal congenital myopathies. Cardiac alpha-actin (ACTC) is the major striated actin in adult heart and fetal skeletal muscle. It is unknown why ACTC and ACTA1 expression switch during development. We investigated whether ACTC can replace ACTA1 in postnatal skeletal muscle. Two ACTC transgenic mouse lines were crossed with Acta1 knockout mice (which all die by 9 d after birth). Offspring resulting from the cross with the high expressing line survive to old age, and their skeletal muscles show no gross pathological features. The mice are not impaired on grip strength, rotarod, or locomotor activity. These findings indicate that ACTC is sufficiently similar to ACTA1 to produce adequate function in postnatal skeletal muscle. This raises the prospect that ACTC reactivation might provide a therapy for ACTA1 diseases. In addition, the mouse model will allow analysis of the precise functional differences between ACTA1 and ACTC.
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Project description:Skeletal muscle provides the contractile force necessary for movement, swallowing, and breathing and, consequently, is necessary for survival. Skeletal muscle cells are unique in that they are extremely large cells containing thousands of nuclei. These nuclei must all work in concert to maintain skeletal muscle function and thereby maintain life. The nucleus is a major site of signaling integration and gene expression regulation. However, examining nuclear processes in skeletal muscle can be difficult because myonuclei are challenging to isolate. We optimized a protocol to purify myonuclei from whole muscle tissue using ultracentrifugation over a discontinuous sucrose gradient to separate the nuclear fraction. We used these purified nuclei for downstream applications including flow cytometry and mass spectrometry. We used this method to compare the myonuclear proteome of young and old mouse hindlimb muscles (Cutler et al., 2017). This protocol may be applied to isolating myonuclei for a variety of downstream analyses such as flow cytometry, microscopy, Western blot, and proteomics.
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Project description:We present the application of large-scale multivariate mixed-model equations to the joint analysis of nine gene expression experiments in beef cattle muscle and fat tissues with a total of 147 hybridizations, and exploring 47 experimental conditions or treatments. Using a correlation-based method, we constructed a gene network for 822 genes. Modules of muscle structural proteins and enzymes, extracellular matrix, fat metabolism and protein synthesis were clearly evident. Detailed analysis of the network identified groupings of proteins on the basis of physical association. For example, expression of three components of the z-disk, MYOZ1, TCAP and PDLIM3, was significantly correlated. In contrast, expression of these z-disk proteins was not highly correlated with the expression of a cluster of thick (myosins) and thin (actin and tropomyosins) filament proteins, or titin, the third major filament system. However, expression of titin was itself not significantly correlated with the cluster of thick and thin filament proteins and enzymes. Correlation in expression of many fast twitch muscle structural proteins and enzymes was observed, but slow twitch specific proteins were not correlated with the fast twitch proteins or with each other. In addition, a number of significant associations between genes and transcription factors were also identified. Our results not only recapitulate the known biology of muscle, but have also started to reveal some of the underlying associations between and within the structural components of skeletal muscle. Keywords: gene network
2006-08-30 | GSE5659 | GEO
Project description:Previous work has demonstrated that human skeletal muscle and adipose tissue both express the GLUT5 fructose transporter, but to date the issue of whether this protein is also expressed in skeletal muscle and adipose tissue of rodents has remained unresolved. In the present study we have used a combination of biochemical and molecular approaches to ascertain whether rat skeletal muscle expresses GLUT5 protein and, if so, whether it possesses the capacity to transport fructose. An isoform-specific antibody against rat GLUT5 reacted positively with crude membranes prepared from rat skeletal muscle. A single immunoreactive band of approx. 50 kDa was visualized on immunoblots which was lost when using anti-(rat GLUT5) serum that had been pre-adsorbed with the antigenic peptide. Subcellular fractionation of skeletal muscle localized this immunoreactivity to a single membrane fraction that was enriched with sarcolemma. Plasma membranes, but not low-density microsomes, from rat adipose tissue also displayed a single protein band of equivalent molecular mass to that seen in muscle. Reverse transcription-PCR analyses, using rat-specific GLUT5 primers, of muscle and jejunal RNA revealed a single PCR fragment of the expected size in jejunum and in four different skeletal muscle types. Sarcolemmal vesicles from rat muscle displayed fructose and glucose uptake. Vesicular uptake of glucose was inhibited by nearly 90% in the presence of cytochalasin B, whereas that of fructose was unaffected. To determine whether fructose could regulate GLUT5 expression in skeletal muscle, rats were maintained on a fructose-enriched diet for 4 days. This procedure increased jejunal and renal GLUT5 protein expression by approx. 4- and 2-fold respectively, but had no detectable effects on the abundance of GLUT5 protein in skeletal muscle or on fructose uptake in rat adipocytes. The present results show that GLUT5 is expressed in the sarcolemma of rat skeletal muscle and that it is likely to mediate fructose uptake in this tissue. Furthermore, unlike the situation in absorptive and re-absorptive epithelia, GLUT5 expression in insulin-sensitive tissues is not regulated by increased substrate supply.
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