No abstract

Forkhead box O (Foxo) transcription factors induce muscle atrophy by upregulating the muscle-specific E3 ubiquitin ligases MuRF-1 and atrogin-1/MAFbx, but other than Akt, the upstream regulators of Foxos during muscle atrophy are largely unknown. To examine the involvement of the dystrophin glycoprotein complex (DGC) in regulation of Foxo activities and muscle atrophy, we analyzed the expression of DGC members during tail suspension, a model of unloading-induced muscle atrophy. Among several DGC members, only neuronal NOS (nNOS) quickly dislocated from the sarcolemma to the cytoplasm during tail suspension. Electron paramagnetic resonance spectrometry revealed production of NO in atrophying muscle. nNOS-null mice showed much milder muscle atrophy after tail suspension than did wild-type mice. Importantly, nuclear accumulation of dephosphorylated Foxo3a was not evident in nNOS-null muscle, and neither MuRF-1 nor atrogin-1/ MAFbx were upregulated during tail suspension. Furthermore, an nNOS-specific inhibitor, 7-nitroindazole, significantly prevented suspension-induced muscle atrophy. The NF-κB pathway was activated in both wildtype and nNOS-null muscle during tail suspension. We also show that nNOS was involved in the mechanism of denervation-induced atrophy. We conclude that nNOS/NO mediates muscle atrophy via regulation of Foxo transcription factors and is a new therapeutic target for disuse-induced muscle atrophy.

Lactobacillus reuteri is a heterofermentative lactic acid bacterium that naturally inhabits the gut of humans and other animals. The probiotic effects of L. reuteri have been proposed to be largely associated with the production of the broad-spectrum antimicrobial compound reuterin during anaerobic metabolism of glycerol. We determined the complete genome sequences of the reuterin-producing L. reuteri JCM 1112T and its closely related species Lactobacillus fermentum IFO 3956. Both are in the same phylogenetic group within the genus Lactobacillus. Comparative genome analysis revealed that L. reuteri JCM 1112T has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B12). The 58-gene cluster has a lower GC content and is apparently inserted into the conserved region, suggesting that the cluster represents a genomic island acquired from an anomalous source. Two-dimensional nuclear magnetic resonance (2D-NMR) with 13C3-glycerol demonstrated that L. reuteri JCM 1112T could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112T to produce reuterin in the intestine. Given that glycerol is shown to be naturally present in feces, the acquired ability to produce reuterin and cobalamin is an adaptive evolutionary response that likely contributes to the probiotic properties of L. reuteri.

The synthesis and characterization of a series of bis(cyanide)(meso-tetraalkylporphyrinatoiron(III)), [Fe(TRP)(CN) 2 ] -where R is H, Me, Et, and i Pr, are reported. The 1 H NMR spectrum of the unsubstituted [Fe(THP)(CN) 2 ] -shows a pyrrole signal at δ ) -23.19 ppm (-25 °C) in CD 2 Cl 2 , which is quite typical as a low spin ferric complex. As the bulkiness of the meso substituent increases, the pyrrole signal moves to lower magnetic field; 0.34, -2.26, and 11.94 ppm for [Fe(TMeP)(CN) 2 ] -, [Fe(TEtP)(CN) 2 ] -, and [Fe(T i PrP)(CN) 2 ] -, respectively. Corresponding to the pyrrole proton signal, the cyanide carbon signal also exhibits a large downfield shift. The difference in chemical shifts between [Fe(THP)(CN) 2 ] -and [Fe(T i PrP)(CN) 2 ] -reaches as much as 1443 ppm at -25 °C. The substituent dependent phenomena are also observed in EPR spectra taken in frozen CH 2 Cl 2 solution at 4.2 K. While the unsubstituted complex gives a so called large g max type signal at 3.65, the alkyl substituted complexes exhibit axial type spectra; the EPR parameters for [Fe(T i PrP)(CN) 2 ] -are g ⊥ ) 2.43 and g | ) 1.73. These results clearly indicate that the electronic ground state changes from the usual (d xy ) 2 (d xz , d yz ) 3 to the unusual (d xz , d yz ) 4 (d xy ) 1 as the substituent becomes bulkier. Analysis of the EPR g values reveals that the orbital of the unpaired electron has more than 90% d xy character in the alkyl substituted complexes. The unusual electron configuration is ascribed to the destabilization of d xy orbital and/or stabilization of d xz and d yz orbitals caused by the S 4 ruffled structure of the alkyl substituted porphyrin ring. Thus, in a strongly ruffled low spin complex such as [Fe(T i PrP)(L) 2 ] ( , electron configuration of iron is presented by (d xz , d yz ) 4 (d xy ) 1 regardless of the kind and basicity of the axial ligand (L). In fact, low spin bis(pyridine) complex [Fe(T i PrP)(Py) 2 ] + gives a pyrrole signal at quite a low field, δ ) +16.4 ppm at -87 °C, which is actually the lowest pyrrole signal ever reported for the low spin ferric porphyrin complexes. Correspondingly, the EPR spectrum taken at 77 K showed a clear axial type spectrum, g ⊥ ) 2.46 and g | ) 1.59. In every case examined, (d xz ,d yz ) 4 (d xy ) 1 ground state is more or less stabilized by the addition of methanol as exemplified by the further downfield shift of the pyrrole proton and cyanide carbon signals together with the smaller EPR g ⊥ values. The methanol effect is explained in terms of the stabilization of d xz and d yz relative to d xy due to the hydrogen bond formation between coordinated cyanide and methanol.

Nitric oxide (NO), a simple diatomic free radical, is known to play a critical physiological role in diverse organisms. An iron complex, with N-(dithiocarboxy)sarcosine (Fe-DTCS), has a high affinity for endogenous NO and can trap, stabilize, and accumulate it. The stable NO adduct thus formed is detectable at room temperature with electron paramagnetic resonance (EPR) spectrometry. We report in vivo EPR imaging of endogenous NO, trapped by an Fe-DTCS complex, in the abdomen of a live mouse. To our knowledge, this is the first report on EPR imaging of endogenous free radicals produced in vivo. This EPR imaging method will be useful for the noninvasive investigation of the spatial distribution of NO in pathologic organs or tissues.