ABSTRACT
A benign, clonable tag for the localization of proteins by electron microscopy of cells would be valuable, especially if it provided labelling with high signal-to-noise ratio and good spatial resolution. Here we explore the use of metallothionein as such a localization marker. We have achieved good success with desmin labelled in vitro and with a component of the yeast spindle pole body labelled in cells. Heavy metals added after fixation and embedding or during the process of freeze-substitution fixation provide readily visible signals with no concern that the heavy atoms are affecting the behaviour of the protein in its physiological environment. However, our methods did not work with protein components of the nuclear pore complex, suggesting that this approach is not yet universally applicable. We provide a full description of our optimal labelling conditions and other conditions tried, hoping that our work will allow others to label their own proteins of interest and/or improve on the methods we have defined.
Subject(s)
Cytoskeletal Proteins/analysis , Desmin/analysis , Metallothionein , Microscopy, Electron, Transmission/methods , Phosphoproteins/analysis , Saccharomyces cerevisiae Proteins/analysis , Cytoskeletal Proteins/genetics , Metallothionein/chemistry , Metallothionein/metabolism , Microscopy, Electron/methods , Nanoparticles , Phosphoproteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Signal-To-Noise Ratio , Tissue Embedding , Tissue FixationABSTRACT
We have examined the effect of male sexual hormones on the regeneration of skeletal muscles. Degeneration/regeneration of the left soleus and extensor digitorum longus muscles (EDL) of Wistar male rats was induced by an injection of snake venom (2 microg, Notechis scutatus scutatus). During the muscle regeneration (25 days), rats were treated with either oil (CON), nandrolone (NAN), NAN combined with exercise (NAN + EXE) or were castrated (CAS). Muscle growth and myosin heavy chain (MyHC) isoform content of regenerating muscles were studied. Castration altered the concentrations of MyHC in venom-treated EDL (P < 0.01) and soleus (P < 0.05). NAN increased the mass (P < 0.01) of regenerating soleus and decreased the relative amount of fast MyHC protein (% of total, P < 0.05). The effect of NAN + EXE on the fast MyHC proteins of venom-treated soleus was opposite (P < 0.05). NAN and NAN + EXE were without effect on the regenerating EDL (P > 0.05). In conclusion, it is possible that male sexual hormones play a role in the growth (synthesis of contractile proteins) of regenerating muscles in rat. In addition, contrary to NAN + EXE, NAN could be beneficial to soleus regeneration.
Subject(s)
Anabolic Agents/pharmacology , Muscle, Skeletal/physiology , Nandrolone/pharmacology , Regeneration/drug effects , Animals , Elapid Venoms/toxicity , Male , Muscle, Skeletal/drug effects , Muscle, Skeletal/pathology , Myosin Heavy Chains/genetics , Myosin Heavy Chains/metabolism , Orchiectomy , Physical Conditioning, Animal , RNA/biosynthesis , Rats , Rats, WistarABSTRACT
Methyl fluoride and dimethyl ether were previously identified as inhibitors of ammonia oxidation and N2O production in autotrophic nitrifying bacteria. We demonstrate that methyl fluoride and dimethyl ether are substrates for ammonia monooxygenase and are converted to formaldehyde and a mixture of methanol and formaldehyde, respectively.
