ABSTRACT
Endurance training is accompanied by important adaptations in both cardiovascular and autonomic nervous systems. Previous works have shown that the main component of gap junctions in the ventricular myocardium (connexin 43 (Cx43) can be regulated by adrenergic stimulus. On the other hand, training raises vagal and decreases sympathetic tone, while augmenting myocardial sensitivity to sympathetic stimulation during exercise. We therefore evaluated the regulation of Cx43 expression by sympathetic tone during exercise in trained and sedentary mice. Training induced an increase in the protein level of Cx43 by 45-70% under resting conditions. The expression of Cx43 was inhibited in trained but not in untrained mice in response to a 60 min exercise bout. Normal basal expression was restored after 60 min of resting. Cx43 reached a minimum that was not different from basal expression in untrained mice. In accordance, electrocardiography and action potential analysis did not reveal major electrophysiological implications for the drop in Cx43 abundance in trained-exercise mice. We prevented Cx43 inhibition using propranolol, and observed increased basal mRNA levels of ß-adrenergic receptors without significant changes in the ratio ß1 to ß2. In conclusion, we showed that Cx43 expression is transiently inhibited by ß-adrenergic stimulus in trained mice during acute exercise.
Subject(s)
Adaptation, Physiological , Connexin 43/metabolism , Myocardium/metabolism , Physical Conditioning, Animal/physiology , Physical Exertion/physiology , Sympathetic Nervous System/physiology , Action Potentials , Adrenergic beta-Antagonists/pharmacology , Animals , Connexin 43/genetics , Electrocardiography , Exercise Test , Male , Mice, Inbred BALB C , Physical Endurance/physiology , Propranolol/pharmacology , Protein Biosynthesis/drug effects , RNA, Messenger/metabolism , Receptors, Adrenergic, beta-1/genetics , Receptors, Adrenergic, beta-2/genetics , Sympathetic Nervous System/drug effectsABSTRACT
The cell--cell adhesion molecule 1 (C-CAM1) plays an important role as a tumor suppressor for prostate cancer. Decreased expression of C-CAM1 was detected in prostate, breast, and colon carcinoma. Reexpression of C-CAM1 in prostate and breast cancer cell lines was able to suppress tumorigenicity in vivo. These observations suggest that C-CAM1 may be used as a marker for cancer detection or diagnosis. To generate monoclonal antibodies specific to C-CAM1, we have overexpressed full-length human C-CAM1 in Sf9 cells using a baculovirus expression system. The protein was purified 104-fold using nickel affinity chromatography. About 0.4 mg purified C-CAM1 was obtained from 200 mg of infected cells. When the purified protein was digested with peptidyl-N-glycosidase, the apparent mobility of the protein on SDS--PAGE changed from 90 to 58 kDa, which is close to the molecular weight predicted from the cloned cDNA sequence. This observation suggests that C-CAM1 was glycosylated on asparagine residues when expressed in Sf9 cells. Western blotting and internal protein sequencing analysis confirmed that the purified protein is human C-CAM1. Biochemical and functional assays indicate that this protein expressed in Sf9 cells displays characteristics similar to those of native protein, including adhesion function and glycosylation modification. Using this protocol, sufficient quantity of this protein can be produced with purity suitable for monoclonal antibody generation and biochemical study.
Subject(s)
Adenosine Triphosphatases/isolation & purification , Adenosine Triphosphatases/metabolism , Cell Adhesion Molecules/isolation & purification , Cell Adhesion Molecules/metabolism , Spodoptera , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Amidohydrolases/metabolism , Amino Acid Sequence , Animals , Antigens, CD , Baculoviridae/genetics , Blotting, Western , Cell Adhesion , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/genetics , Cell Line , Cell Size , Chromatography, Affinity , Glycosylation , Humans , Nickel/metabolism , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Analysis, Protein , Spodoptera/cytology , Spodoptera/metabolism , Spodoptera/virologyABSTRACT
The mechanism by which (CTG)n expansion in the 3' UTR of the DMPK gene causes myotonic dystrophy (DM) is unknown. We identified four RNA splicing factors--hnRNP C, U2AF (U2 auxiliary factor), PTB (polypyrimidine tract binding protein), and PSF (PTB associated splicing factor)--that bind to two short regions 3' of the (CUG)n, and found a novel 3' DMPK exon resulting in an mRNA lacking the repeats. We propose that the (CUG)n is an essential cis acting element for this splicing event. In contrast to (CUG)n containing mRNAs, the novel isoform is not retained in the nucleus in DM cells, resulting in imbalances in relative levels of cytoplasmic DMPK mRNA isoforms and a new dominant effect of the mutation on DMPK.
Subject(s)
Exons/genetics , Myotonic Dystrophy/genetics , Protein Serine-Threonine Kinases/genetics , RNA Splicing/genetics , RNA, Messenger/genetics , Trinucleotide Repeats/genetics , 3' Untranslated Regions/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Nucleus/genetics , Cells, Cultured , Cytoplasm/genetics , Humans , Introns/genetics , Mice , Molecular Sequence Data , Mutation , Myotonic Dystrophy/enzymology , Myotonin-Protein Kinase , Protein Binding , RNA-Binding Proteins/metabolism , Regulatory Sequences, Nucleic Acid/genetics , Ultraviolet RaysABSTRACT
Centrifugation through sucrose gradients was adapted to separate spore-forming cells of B. megaterium deficient in poly-beta-hydroxybutyrate synthesis from wild type cells. The conditions described allowed the detection of mutant clones screening a low percentage of the mutagenized population.
Subject(s)
Bacillus megaterium/genetics , Hydroxybutyrates/metabolism , Polyesters/metabolism , Bacillus megaterium/isolation & purification , Bacillus megaterium/metabolism , Centrifugation, Density Gradient , Spores, BacterialABSTRACT
Centrifugation through sucrose gradients was adapted to separate spore-forming cells of B. megaterium deficient in poly-beta-hydroxybutyrate synthesis from wild type cells. The conditions described allowed the detection of mutant clones screening a low percentage of the mutagenized population.
