Diaphragm assessment in mice overexpressing phospholamban in slow-twitch type I muscle fibers.

AIMS: Phospholamban (PLN) and sarcolipin (SLN) are small inhibitory proteins that regulate the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump. Previous work from our laboratory revealed that in the soleus and gluteus minimus muscles of mice overexpressing PLN (Pln (OE)), SERCA function was impaired, dynamin 2 (3-5 fold) and SLN (7-9 fold) were upregulated, and features of human centronuclear myopathy (CNM) were observed. Here, we performed structural and functional experiments to evaluate whether the diaphragm muscles of the Pln (OE) mouse would exhibit CNM pathology and muscle weakness. METHODS: Diaphragm muscles from Pln (OE) and WT mice were subjected to histological/histochemical/immunofluorescent staining, Ca(2+)-ATPase and Ca(2+) uptake assays, Western blotting, and in vitro electrical stimulation. RESULTS: Our results demonstrate that PLN overexpression reduced SERCA's apparent affinity for Ca(2+) but did not reduce maximal SERCA activity or rates of Ca(2+) uptake. SLN was upregulated 2.5-fold, whereas no changes in dynamin 2 expression were found. With respect to CNM, we did not observe type I fiber predominance, central nuclei, or central aggregation of oxidative activity in diaphragm, although type I fiber hypotrophy was present. Furthermore, in vitro contractility assessment of Pln (OE) diaphragm strips revealed no reductions in force-generating capacity, maximal rates of relaxation or force development, but did indicate that ½ relaxation time was prolonged. CONCLUSIONS: Therefore, the effects of PLN overexpression on skeletal muscle phenotype differ between diaphragm and the postural soleus and gluteus minimus muscles. Our findings here point to differences in SLN expression and type I fiber distribution as potential contributing factors.

Sarcolipin Is a Key Determinant of the Basal Metabolic Rate, and Its Overexpression Enhances Energy Expenditure and Resistance against Diet-induced Obesity.

Sarcolipin (SLN) is a novel regulator of sarcoplasmic reticulum Ca(2+) ATPase (SERCA) in muscle. SLN binding to SERCA uncouples Ca(2+) transport from ATP hydrolysis. By this mechanism, SLN promotes the futile cycling of SERCA, contributing to muscle heat production. We recently showed that SLN plays an important role in cold- and diet-induced thermogenesis. However, the detailed mechanism of how SLN regulates muscle metabolism remains unclear. In this study, we used both SLN knockout (Sln(-/-)) and skeletal muscle-specific SLN overexpression (Sln(OE)) mice to explore energy metabolism by pair feeding (fixed calories) and high-fat diet feeding (ad libitum). Our results show that, upon pair feeding, Sln(OE) mice lost weight compared with the WT, but Sln(-/-) mice gained weight. Interestingly, when fed with a high-fat diet, Sln(OE) mice consumed more calories but gained less weight and maintained a normal metabolic profile in comparison with WT and Sln(-/-) mice. We found that oxygen consumption and fatty acid oxidation were increased markedly in Sln(OE) mice. There was also an increase in both mitochondrial number and size in Sln(OE) muscle, together with increased expression of peroxisome proliferator-activated receptor δ (PPARδ) and PPAR γ coactivator 1 α (PGC1α), key transcriptional activators of mitochondrial biogenesis and enzymes involved in oxidative metabolism. These results, taken together, establish an important role for SLN in muscle metabolism and energy expenditure. On the basis of these data we propose that SLN is a novel target for enhancing whole-body energy expenditure.

Reconstitution of functional mycobacterial arabinosyltransferase AftC proteoliposome and assessment of decaprenylphosphorylarabinose analogues as arabinofuranosyl donors.

Arabinosyltransferases are a family of membrane-bound glycosyltransferases involved in the biosynthesis of the arabinan segment of two key glycoconjugates, arabinogalactan and lipoarabinomannan, in the mycobacterial cell wall. All arabinosyltransferases identified have been found to be essential for the growth of Mycobcterium tuberculosis and are potential targets for developing new antituberculosis drugs. Technical bottlenecks in designing enzyme assays for screening for inhibitors of these enzymes are (1) the enzymes are membrane proteins and refractory to isolation; and (2) the sole arabinose donor, decaprenylphosphoryl-d-arabinofuranose is sparingly produced and difficult to isolate, and commercial substrates are not available. In this study, we have synthesized several analogues of decaprenylphosphoryl-d-arabinofuranose by varying the chain length and investigated their arabinofuranose (Araf) donating capacity. In parallel, an essential arabinosyltransferase (AftC), an enzyme that introduces α-(1→3) branch points in the internal arabinan domain in both arabinogalactan and lipoarabinomannan synthesis, has been expressed, solubilized, and purified for the first time. More importantly, it has been shown that the AftC is active only when reconstituted in a proteoliposome using mycobacterial phospholipids and has a preference for diacylated phosphatidylinositoldimannoside (Ac(2)PIM(2)), a major cell wall associated glycolipid. α-(1→3) branched arabinans were generated when AftC-liposome complex was used in assays with the (Z,Z)-farnesylphosphoryl d-arabinose and linear α-d-Araf-(1→5)(3-5) oligosaccharide acceptors and not with the acceptor that had a α-(1→3) branch point preintroduced.

Monte Carlo folding of trans-membrane helical peptides in an implicit generalized Born membrane.

An efficient Monte Carlo (MC) algorithm using concerted backbone rotations is combined with a recently developed implicit membrane model to simulate the folding of the hydrophobic transmembrane domain M2TM of the M2 protein from influenza A virus and Sarcolipin at atomic resolution. The implicit membrane environment is based on generalized Born theory and has been calibrated against experimental data. The MC sampling has previously been used to fold several small polypeptides and been shown to be equivalent to molecular dynamics (MD). In combination with a replica exchange algorithm, M2TM is found to form continuous membrane spanning helical conformations for low temperature replicas. Sarcolipin is only partially helical, in agreement with the experimental NMR structures in lipid bilayers and detergent micelles. Higher temperature replicas exhibit a rapidly decreasing helicity, in agreement with expected thermodynamic behavior. To exclude the possibility of an erroneous helical bias in the simulations, the model is tested by sampling a synthetic Alanine-rich polypeptide of known helicity. The results demonstrate there is no overstabilization of helical conformations, indicating that the implicit model captures the essential components of the native membrane environment for M2TM and Sarcolipin.

Assessment of transcriptional gene activity in situ by application of HOPE-fixed, paraffin-embedded tissues.

We report the use of HOPE-fixation (HOPE = Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) for specimens utilized for in situ hybridization targeting mRNA. For this purpose, an optimized protocol was developed and repeatedly tested on HOPE-fixed lung specimens. We observed that neither pretreatment, permeabilizing the cells, nor prehybridization is necessary to generate signals. After deparaffinizing, the random primed digoxigenin-labeled probes are directly hybridized together with yeast tRNA for blocking unspecific signals. Detection was performed using anti digoxigenin antibodies conjugated with alkaline phosphatase and new-fuchsine or NBT/BCIP as substrates. The results were verified by RT-PCR and adequate negative controls. Signals for human surfactant protein-A and interferon-gamma-inducible protein-10 developed rapidly within 10 min, accompanied by high signal intensities comparable to those observed in immunohistochemistry. Signal enhancement by biotinyl-tyramide, although giving suitable results as well, did not lead to higher signal intensities, and thus was not necessary in conjunction with the probes tested so far. These experiments were performed with material stored under appropriate conditions (at +4 degrees C) up to five years. To sum up, these initial results, obtained with the novel HOPE-fixative, are promising as regards the enhancement of the capabilities of in situ hybridization in the future.

Interaction of 7-n-alkoxycoumarins with cytochrome P-450(2) and their partitioning into liposomal membranes. Assessment of methods for determination of membrane partition coefficients.

A study was made of the binding of 7-ethoxy-, 7-n-propoxy- and 7-n-pentoxy-coumarin to cytochrome P-450(2) reconstituted into large unilamellar liposomes composed of a mixture of egg L-alpha-phosphatidylcholine, egg phosphatidylethanolamine and dipalmitoyl phosphatidic acid (2:1:0.06, by weight). The apparent spectral dissociation constants Ksapp. increased linearly with increasing proteoliposomal concentration. When both cytochrome P-450(2) and NADPH:cytochrome P-450 reductase were reconstituted into liposomes, the apparent Michaelis constants Kmapp. for O-dealkylation of 7-methoxy-, 7-ethoxy- and 7-n-propoxy-coumarin showed a similar dependence on the proteoliposomal concentration. The results were in accordance with models for kinetic or equilibrium processes in biphasic systems containing membrane-bound catalytic or acceptor sites, in which a linear solute partition in the bilayer membrane is postulated. The methyl, ethyl and n-propyl ether were readily dealkylated. However, the O-dealkylation rate of 7-n-butoxycoumarin was low and became very small for longer alkyl ethers. Both the effective dissociation constants and effective Michaelis constants decreased with elongation of the alkyl side chain of the coumarins. From plots of the apparent dissociation constants and apparent Michaelis constants against the lipid volume fraction of the proteoliposomes, the membrane partition coefficients for several homologues were calculated. When protein-free liposomes were added to 7-n-alkoxycoumarin solutions, the fluorescence intensity of the coumarins decreased and eventually became negligible in the presence of an excess of liposomal material. On the assumption that the overall fluorescence can be ascribed exclusively to the fraction of 7-n-alkoxycoumarin molecules present in the aqueous phase, partition coefficients for liposomal accumulation of the test compounds could be determined directly. For several coumarin ethers, comparable values were derived for the membrane partition coefficients from binding, kinetic and fluorescence intensity measurements. The change in free energy per methylene group of the 7-n-alkoxycoumarins for partitioning between n-octanol and buffer was significantly different from the value for liposome partitioning.