Experimental chronic low‐frequency resistance training produces skeletal muscle hypertrophy in the absence of muscle damage and metabolic stress markers

Volitional animal resistance training constitutes an important approach to modeling human resistance training. However, the lack of standardization protocol poses a frequent impediment to the production of skeletal muscle hypertrophy and the study of related physiological variables (i.e., cellular damage/inflammation or metabolic stress). Therefore, the purposes of the present study were: (1) to test whether a long‐term and low frequency experimental resistance training program is capable of producing absolute increases in muscle mass; (2) to examine whether cellular damage/inflammation or metabolic stress is involved in the process of hypertrophy. In order to test this hypothesis, animals were assigned to a sedentary control (C, n = 8) or a resistance trained group (RT, n = 7). Trained rats performed 2 exercise sessions per week (16 repetitions per day) during 12 weeks. Our results demonstrated that the resistance training strategy employed was capable of producing absolute mass gain in both soleus and plantaris muscles (12%, p < 0.05). Furthermore, muscle tumor necrosis factor (TNF‐α) protein expression (soleus muscle) was reduced by 24% (p < 0.01) in trained group when compared to sedentary one. Finally, serum creatine kinase (CK) activity and serum lactate concentrations were not affected in either group. Such information may have practical applications if reproduced in situations where skeletal muscle hypertrophy is desired but high mechanical stimuli of skeletal muscle and inflammation are not. Copyright © 2010 John Wiley & Sons, Ltd.     

Testosterone suppresses oxidative stress in human neutrophils

The in vitro effect of testosterone on human neutrophil function was investigated. Blood neutrophils from healthy male subjects were isolated and treated with 10 nM, 0.1 and 10 µM testosterone for 24 h. As compared with untreated cells, the testosterone treatment produced a significant decrease of superoxide production as indicated by the measurement of extra‐ and intracellular superoxide content. An increment in the production of nitric oxide was observed at 0.1 and 10 µM testosterone concentrations, whereas no effect was found for 10 nM. Intracellular calcium mobilization was significantly increased at 10 nM, whereas it was reduced at 10 µM testosterone. There was an increase in phagocytic capacity at 10 nM and a decrease of microbicidal activity in neutrophils treated with testosterone at 10 µM. Glutathione reductase activity was increased by testosterone treatment, whereas no effect was observed in other antioxidant enzyme activities. An increase in the content of thiol groups was observed at all testosterone concentrations. Lipid peroxidation in neutrophils evaluated by levels of TBARS was decreased at 10 nM and 0.1 µM testosterone. These results indicate the antioxidant properties of testosterone in neutrophils as suggested by reduction of superoxide anion production, and lipid peroxidation, and by the increase in nitric oxide production, glutathione reductase activity and the content of thiol groups. Therefore, the plasma levels of testosterone are important regulators of neutrophil function and so of the inflammatory response. Copyright © 2010 John Wiley & Sons, Ltd.     

Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents.

Colyophilization or codrying of subtilisin Carlsberg with the crown ethers 18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activity in THF, acetonitrile, and 1,4-dioxane in the transesterification reactions of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (+/-)-1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V(0), ranged from less than 10-fold to more than 100-fold. All crown ethers activated subtilisin substantially, which excludes a specific macrocyclic effect from being responsible. The secondary structure of subtilisin was studied by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-crown-5 led to a more nativelike structure of subtilisin in the organic solvents employed when compared with that of the dehydrated enzyme obtained from buffer alone. However, the high level of activation with 12-crown-4 where this effect was not observed excluded overall structural preservation from being the primary cause of the observed enzyme activation. The conformational mobility of subtilisin was investigated by performing thermal denaturation experiments in 1,4-dioxane. Although only a small effect of temperature on subtilisin structure was observed for the samples prepared with or without 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature at quite low temperatures (38 degrees C and 56 degrees C, respectively). No relationship between this property and V(0) was evident, but increased conformational mobility of the protein decreased its storage stability. The possibility of a "molecular imprinting" effect was also tested by removing 18-crown-6 from the subtilisin-18-crown-6 colyophilizate by washing. V(0) was only halved as a result of this procedure, an effect insignificant compared with the ca. 80-fold rate enhancement observed prior to washing in THF. This suggests that molecular imprinting is likely the primary cause of subtilisin activation by crown ethers, as recently suggested.     

Network identification and flux quantification in the central metabolism of Saccharomyces cerevisiae under different conditions of glucose repression

The network structure and the metabolic fluxes in central carbon metabolism were characterized in aerobically grown cells of Saccharomyces cerevisiae. The cells were grown under both high and low glucose concentrations, i.e., either in a chemostat at steady state with a specific growth rate of 0.1 h(-1) or in a batch culture with a specific growth rate of 0.37 h(-1). Experiments were carried out using [1-(13)C]glucose as the limiting substrate, and the resulting summed fractional labelings of intracellular metabolites were measured by gas chromatography coupled to mass spectrometry. The data were used as inputs to a flux estimation routine that involved appropriate mathematical modelling of the central carbon metabolism of S. cerevisiae. The results showed that the analysis is very robust, and it was possible to quantify the fluxes in the central carbon metabolism under both growth conditions. In the batch culture, 16.2 of every 100 molecules of glucose consumed by the cells entered the pentose-phosphate pathway, whereas the same relative flux was 44.2 per 100 molecules in the chemostat. The tricarboxylic acid cycle does not operate as a cycle in batch-growing cells, in contrast to the chemostat condition. Quantitative evidence was also found for threonine aldolase and malic enzyme activities, in accordance with published data. Disruption of the MIG1 gene did not cause changes in the metabolic network structure or in the flux pattern.     

NMR characterization of a DNA duplex containing the major acrolein-derived deoxyguanosine adduct gamma -OH-1,-N2-propano-2'-deoxyguanosine

The environmental and endogenous mutagen acrolein reacts with cellular DNA to produce several isomeric 1,N(2)-propanodeoxyguanosine adducts. High resolution NMR spectroscopy was used to establish the structural features of the major acrolein-derived adduct, gamma-OH-1,N(2)-propano-2'-deoxyguanosine. In aqueous solution, this adduct was shown to assume a ring-closed form. In contrast, when gamma-OH-1,N(2)-propano-2'-deoxyguanosine pairs with dC at the center of an 11-mer oligodeoxynucleotide duplex, the exocyclic ring opens, enabling the modified base to participate in a standard Watson-Crick base pairing alignment. Analysis of the duplex spectra reveals a regular right-handed helical structure with all residues adopting an anti orientation around the glycosidic torsion angle and Watson-Crick alignments for all base pairs. We conclude from this study that formation of duplex DNA triggers the hydrolytic conversion of gamma-OH-1,N(2)-propano-2'-deoxyguanosine to an open chain form, a structure that facilitates pairing with dC during DNA replication and accounts for the surprising lack of mutagenicity associated with this DNA adduct.     

Mechanistic implications of mutations to the active site lysine of porphobilinogen synthase

Porphobilinogen synthase (PBGS) is a homo-octameric protein that catalyzes the complex asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). The only characterized intermediate in the PBGS-catalyzed reaction is a Schiff base that forms between the first ALA that binds and a conserved lysine, which in Escherichia coli PBGS is Lys-246 and in human PBGS is Lys-252. In this study, E. coli PBGS mutants K246H, K246M, K246W, K246N, and K246G and human PBGS mutant K252G were characterized. Alterations to this lysine result in a disabled but not totally inactive protein suggesting an alternate mechanism in which proximity and orientation are major catalytic devices. (13)C NMR studies of [3,5-(13)C]porphobilinogen bound at the active sites of the E. coli PBGS and the mutants show only minor chemical shift differences, i.e. environmental alterations. Mammalian PBGS is established to have four functional active sites, whereas the crystal structure of E. coli PBGS shows eight spatially distinct and structurally equivalent subunits. Biochemical data for E. coli PBGS have been interpreted to support both four and eight active sites. A unifying hypothesis is that formation of the Schiff base between this lysine and ALA triggers a conformational change that results in asymmetry. Product binding studies with wild-type E. coli PBGS and K246G demonstrate that both bind porphobilinogen at four per octamer although the latter cannot form the Schiff base from substrate. Thus, formation of the lysine to ALA Schiff base is not required to initiate the asymmetry that results in half-site reactivity.