Investigating Eukaryotic Elongation Factor 2 Kinase/Eukaryotic Translation Elongation Factor 2 Pathway Regulation and Its Role in Protein Synthesis Impairment during Disuse-Induced Skeletal Muscle Atrophy.

The principal mechanism underlying the reduced rate of protein synthesis in atrophied skeletal muscle is largely unknown. Eukaryotic elongation factor 2 kinase (eEF2k) impairs the ability of eukaryotic translation elongation factor 2 (eEF2) to bind to the ribosome via T56 phosphorylation. Perturbations in the eEF2k/eEF2 pathway during various stages of disuse muscle atrophy have been investigated utilizing a rat hind limb suspension (HS) model. Two distinct components of eEF2k/eEF2 pathway misregulation were demonstrated, observing a significant (P < 0.01) increase in eEF2k mRNA expression as early as 1-day HS and in eEF2k protein level after 3-day HS. We set out to determine whether eEF2k activation is a Ca2+-dependent process with involvement of Cav1.1. The ratio of T56-phosphorylated/total eEF2 was robustly elevated after 3-day HS, which was completely reversed by 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) and decreased by 1.7-fold (P < 0.05) by nifedipine. Transfection of C2C12 with cytomegalovirus promoter (pCMV)-eEF2k and administration with small molecules were used to modulate eEF2k and eEF2 activity. More importantly, pharmacologic enhancement of eEF2 phosphorylation induced phosphorylated ribosomal protein S6 kinase (T389) up-regulation and restoration of global protein synthesis in the HS rats. Taken together, the eEF2k/eEF2 pathway was up-regulated during disuse muscle atrophy involving calcium-dependent activation of eEF2k partly via Cav1.1. The study provides evidence, in vitro and in vivo, of the eEF2k/eEF2 pathway impact on ribosomal protein S6 kinase activity as well as protein expression of key atrophy biomarkers, muscle atrophy F-box/atrogin-1 and muscle RING finger-1.

P2Y1 and P2Y2 receptors differ in their role in the regulation of signaling pathways during unloading-induced rat soleus muscle atrophy.

The current study aimed to investigate the hypothesis that purinergic receptors P2Y1 and P2Y2 play a regulatory role in gene expression in unloaded muscle. ATP is released from cells through pannexin channels, and it interacts with P2Y1 and P2Y2 receptors, leading to the activation of markers of protein catabolism and a reduction in protein synthesis. To test this hypothesis thirty-two rats were randomly divided into four groups (8 per group): a non-treated control group (C), a group subjected to three days of hindlimb unloading with a placebo (HS), a group subjected to three days of hindlimb unloading treated with a P2Y1 receptor inhibitor, MRS2179 (HSM), and a group subjected to three days of hindlimb unloading treated with a P2Y2 receptor inhibitor, AR-C 118925XX (HSA). This study revealed several key findings following three days of soleus muscle unloading: 1: Inhibition of P2Y1 or P2Y2 receptors prevented the accumulation of ATP, the increase in IP3 receptor content, and the decrease in the phosphorylation of GSK-3beta. This inhibition also mitigated the reduction in the rate of protein synthesis. However, it had no significant effect on the markers of mTORC1-dependent signaling. 2: Blocking P2Y1 receptors prevented the unloading-induced upregulation of phosphorylated p38MAPK and partially reduced the increase in MuRF1mRNA expression. 3: Blocking P2Y2 receptors prevented muscle atrophy during unloading, partially maintained the levels of phosphorylated ERK1/2, reduced the increase in mRNA expression of MAFbx, ubiquitin, and IL-6 receptor, prevented the decrease in phosphorylated AMPK, and attenuated the increase in phosphorylated p70S6K. Taken together, these results suggest that the prevention of muscle atrophy during unloading, as achieved by the P2Y2 receptor inhibitor, is likely mediated through a reduction in catabolic processes and maintenance of energy homeostasis. In contrast, the P2Y1 receptor appears to play a relatively minor role in muscle atrophy during unloading.

Preparation, X-ray Characterization, and Reactivity of the Rod-like and Angular Germanium- and Titanium(IV)-Capped Iron(II) Bis-Clathrochelates and Their Mono- and Bis-Capped (Semi)clathrochelate Precursors.

Transmetalation of the bis{triethylantimony(V)}-capped iron(II) tris-α-dioximate with n-butylboronic acid afforded the mixed antimony, boron cross-linked clathrochelate with single reactive antimony(V)-based apical fragment. This macrobicyclic precursor easily underwent the transmetalation reactions with germanium and titanium(IV) alkoxides to give the rod-like and angular FeII2MIV-trinuclear bis-clathrochelates. Those of the aforementioned diantimony(V)-capped complex with 3- and 4-carboxyphenylboronic acids afforded the monoboron-capped iron(II) semiclathrochelates, undergoing a double-cyclization (macrobicyclization) with germanium- and titanium(IV)-based capping agents. The reactions in the low-temperature range unexpectedly gave the stable 2:1 associates, formed by the bridging of two carboxyl-terminated macrobicyclic molecules of the mixed carboxylboron, triethylantimony-capped iron(II) clathrochelate with a triethylantimony(V)-based linker fragment. The obtained complexes were characterized using elemental analysis, MALDI-TOF, 1H and 13C{1H} NMR and UV-vis spectra, and single-crystal XRD experiments. The encapsulated iron(II) ion in their 3D-molecules is situated almost in the center of its FeN6-coordination polyhedron possessing a truncated trigonal-pyramidal geometry. Fe-N distances fall in the range 1.887(7)-1.945(4) Å characteristic of the low-spin iron(II) complexes. The cross-linking titanium and germanium(IV) ions in the corresponding bis-clathrochelate molecules form the octahedral MIVO6-coordination polyhedra, the MIV-O distances of which vary from 1.946(2) to 1.964(2) Å and from 1.879(7) to 1.907(6) Å, respectively.

Metformin Pre-Treatment as a Means of Mitigating Disuse-Induced Rat Soleus Muscle Wasting.

Currently, no ideal treatment exists to combat skeletal muscle disuse-induced atrophy and loss of strength. Because the activity of AMP-activated protein kinase (AMPK) in rat soleus muscle is suppressed at the early stages of disuse, we hypothesized that pre-treatment of rats with metformin (an AMPK activator) would exert beneficial effects on skeletal muscle during disuse. Muscle disuse was performed via hindlimb suspension (HS). Wistar rats were divided into four groups: (1) control (C), (2) control + metformin for 10 days (C+Met), (3) HS for 7 days (HS), (4) metformin treatment for 7 days before HS and during the first 3 days of 1-week HS (HS+Met). Anabolic and catabolic markers were assessed using WB and RT-PCR. Treatment with metformin partly prevented an HS-induced decrease in rat soleus weight and size of slow-twitch fibers. Metformin prevented HS-related slow-to-fast fiber transformation. Absolute soleus muscle force in the HS+Met group was increased vs. the HS group. GSK-3ß (Ser9) phosphorylation was significantly increased in the HS+Met group vs. the HS group. Metformin pre-treatment partly prevented HS-induced decrease in 18S+28S rRNA content and attenuated upregulation of calpain-1 and ubiquitin. Thus, pre-treatment of rats with metformin can ameliorate disuse-induced reductions in soleus muscle weight, the diameter of slow-type fibers, and absolute muscle strength.

Changes in the Mechanical Properties of Fast and Slow Skeletal Muscle after 7 and 21 Days of Restricted Activity in Rats.

Disuse muscle atrophy is usually accompanied by changes in skeletal muscle structure, signaling, and contractile potential. Different models of muscle unloading can provide valuable information, but the protocols of experiments with complete immobilization are not physiologically representative of a sedentary lifestyle, which is highly prevalent among humans now. In the current study, we investigated the potential effects of restricted activity on the mechanical characteristics of rat postural (soleus) and locomotor (extensor digitorum longus, EDL) muscles. The restricted-activity rats were kept in small Plexiglas cages (17.0 × 9.6 × 13.0 cm) for 7 and 21 days. After this, soleus and EDL muscles were collected for ex vivo mechanical measurements and biochemical analysis. We demonstrated that while a 21-day movement restriction affected the weight of both muscles, in soleus muscle we observed a greater decrease. The maximum isometric force and passive tension in both muscles also significantly changed after 21 days of movement restriction, along with a decrease in the level of collagen 1 and 3 mRNA expression. Furthermore, the collagen content itself changed only in soleus after 7 and 21 days of movement restriction. With regard to cytoskeletal proteins, in our experiment we observed a significant decrease in telethonin in soleus, and a similar decrease in desmin and telethonin in EDL. We also observed a shift towards fast-type myosin heavy chain expression in soleus, but not in EDL. In summary, in this study we showed that movement restriction leads to profound specific changes in the mechanical properties of fast and slow skeletal muscles. Future studies may include evaluation of signaling mechanisms regulating the synthesis, degradation, and mRNA expression of the extracellular matrix and scaffold proteins of myofibers.

Effect of enhanced muscle tone on the expression of atrogenes and cytoskeletal proteins during postural muscle unloading.

Skeletal muscle unloading leads to the decreased electrical activity and decline of muscle tone. AIMS: Current study evaluated the effect of muscle tone preservation achieved by tetanus toxin (TeNT) treatment on signaling pathways regulating atrophic processes during unloading. MAIN METHODS: Four groups of rats were used: non-treated control (C), control rats with TeNT administration (CT), 7 days of unloading/hindlimb suspension with placebo (HS), and 7 days of unloading with TeNT administration (HST). KEY FINDINGS: Absolute and relative force of tetanic contractions was decreased by 65% in soleus muscle of HS rats when compared with C. Treatment with TeNT significantly lessened force decline in soleus muscle of HST rats when compared with HS. TeNT administration increased myosin heavy chain I beta (MyHC Iß) expression in CT rats and prevented MyHC Iß loss in HST group when compared with C rats. Desmin content was lower by 31.4% (p < 0.05) in HS group when compared with HST. Calpain-1 expression was increased in HS group when compared with C, CT and HST. There was a decrease in p-p70S6K content (41%, p < 0,05) and an increase in p-eEF2 content (77%, p < 0,05) in HS group when compared with C, while there were no significant differences in the content of these proteins between HST, CT and C groups. SIGNIFICANCE: Treatment with TeNT significantly diminished unloading-induced decline of soleus muscle mass and mechanical properties and affected the regulation of MyHC Iß expression. These effects are mediated by signaling pathways regulating protein synthesis and degradation.

Metformin Attenuates Slow-to-Fast Fiber Shift and Proteolysis Markers Increase in Rat Soleus after 7 Days of Rat Hindlimb Unloading.

Muscle unloading leads to signaling alterations that cause muscle atrophy and weakness. The cellular energy sensor AMPK can regulate myofiber-type shift, calcium-dependent signaling and ubiquitin-proteasome system markers. We hypothesized that the prevention of p-AMPK downregulation during the first week of muscle unloading would impede atrophy development and the slow-to-fast shift of soleus muscle fibers, and the aim of the study was to test this hypothesis. Thirty-two male Wistar rats were randomly assigned to four groups: placebo control (C), control rats treated with metformin (C + M), 7 days of hindlimb suspension (HS) + placebo (7HS), and 7 days of HS + metformin administration (7HS + M). In the soleus of the 7HS rats, we detected a slow-to-fast fiber-type shift as well as a significant downregulation of MEF-2D and p300 in the nuclei. In the 7HS group, we also found decreases in p-ACC (AMPK target) protein level and in the expression of E3 ubiquitin ligases and p-CaMK II protein level vs. the C group. The 7-day metformin treatment for soleus muscle unloading (1) prevented slow-to-fast fiber-type shift; (2) counteracted changes in the p-ACC protein level; (3) hindered changes in the nuclear protein level of the slow myosin expression activators MEF-2D and p300, but did not affect NFATc1 signaling; and (4) attenuated the unloading-induced upregulation of MuRF-1, atrogin-1, ubiquitin and myostatin mRNA expression, but did not prevent soleus muscle atrophy. Thus, metformin treatment during muscle disuse could be useful to prevent the decrease in the percentage of slow-type fatigue-resistant muscle fibers.

Role of Pannexin 1 ATP-Permeable Channels in the Regulation of Signaling Pathways during Skeletal Muscle Unloading.

Skeletal muscle unloading results in atrophy. We hypothesized that pannexin 1 ATP-permeable channel (PANX1) is involved in the response of muscle to unloading. We tested this hypothesis by blocking PANX1, which regulates efflux of ATP from the cytoplasm. Rats were divided into six groups (eight rats each): non-treated control for 1 and 3 days of the experiments (1C and 3C, respectively), 1 and 3 days of hindlimb suspension (HS) with placebo (1H and 3H, respectively), and 1 and 3 days of HS with PANX1 inhibitor probenecid (PRB; 1HP and 3HP, respectively). When compared with 3C group there was a significant increase in ATP in soleus muscle of 3H and 3HP groups (32 and 51%, respectively, p < 0.05). When compared with 3H group, 3HP group had: (1) lower mRNA expression of E3 ligases MuRF1 and MAFbx (by 50 and 38% respectively, p < 0.05) and MYOG (by 34%, p < 0.05); (2) higher phosphorylation of p70S6k and p90RSK (by 51 and 35% respectively, p < 0.05); (3) lower levels of phosphorylated eEF2 (by 157%, p < 0.05); (4) higher level of phosphorylated GSK3ß (by 189%, p < 0.05). In conclusion, PANX1 ATP-permeable channels are involved in the regulation of muscle atrophic processes by modulating expression of E3 ligases, and protein translation and elongation processes during unloading.

Spectroelectrochemical Properties and Catalytic Activity in Cyclohexane Oxidation of the Hybrid Zr/Hf-Phthalocyaninate-Capped Nickel(II) and Iron(II) tris-Pyridineoximates and Their Precursors.

The in situ spectroelectrochemical cyclic voltammetric studies of the antimony-monocapped nickel(II) and iron(II) tris-pyridineoximates with a labile triethylantimony cross-linking group and Zr(IV)/Hf(IV) phthalocyaninate complexes were performed in order to understand the nature of the redox events in the molecules of heterodinuclear zirconium(IV) and hafnium(IV) phthalocyaninate-capped derivatives. Electronic structures of their 1e-oxidized and 1e-electron-reduced forms were experimentally studied by electron paramagnetic resonance (EPR) spectroscopy and UV-vis-near-IR spectroelectrochemical experiments and supported by density functional theory (DFT) calculations. The investigated hybrid molecular systems that combine a transition metal (pseudo)clathrochelate and a Zr/Hf-phthalocyaninate moiety exhibit quite rich redox activity both in the cathodic and in the anodic region. These binuclear compounds and their precursors were tested as potential catalysts in oxidation reactions of cyclohexane and the results are discussed.

Solvent-Induced Encapsulation of Cobalt(II) Ion by a Boron-Capped tris-Pyrazoloximate.

Boron-cross-linked cobalt(II) pseudoclathrochelate was obtained by the template reaction of 2-acetylpyrazoloxime, phenylboronic acid, and a new DMF cobalt(II) solvato complex with a decachloro-closo-decaborate dianion. As confirmed by single-crystal X-ray diffraction, this complex crystallizes with two symmetry-independent cobalt(II) pseudoclathrochelate cations, one decachloro-closo-decaborate dianion, one benzene, one dichloromethane solvent molecule, and two molecules of DMF. The latter act as pseudocapping fragments to the monocapped tris-pyrazoloximate ligands by forming N-H···O hydrogen bonds with their pyrazole groups. The CoIIN6-coordination polyhedra adopt a nearly ideal TP geometry with distortion angles φ equal to 1.22(16) and 2.58(17)° for two symmetry-independent pseudoclathrochelate cations, both containing the encapsulated cobalt(II) ion in its high-spin state (Co-N 2.115(4)-2.198(3) Å). Magnetic properties of this complex were studied both by dc-magnetometry and by solution-state NMR spectroscopy to reveal a high magnetic anisotropy, thus suggesting a large magnetic susceptibility tensor anisotropy (25.8 × 10-32 m3 at 298 K) and a large negative zero-field splitting energy (-85 cm-1). The results of magnetometry studies in the ac magnetic field suggest a single molecule magnet behavior of this TP complex with an effective magnetization reversal barrier of approximately 130 cm-1. Its pseudocapping DMF molecules that form H-bonds with tris-pyrazoloximate fragments are easy to substitute by strong H-bond acceptors, such as chloride ions and di- and tetramethylureas, thus affecting the magnetic properties of a whole pseudomacrobicyclic paramagnetic system.

Differences in the Role of HDACs 4 and 5 in the Modulation of Processes Regulating MAFbx and MuRF1 Expression during Muscle Unloading.

Unloading leads to skeletal muscle atrophy via the upregulation of MuRF-1 and MAFbx E3-ligases expression. Reportedly, histone deacetylases (HDACs) 4 and 5 may regulate the expression of MuRF1 and MAFbx. To examine the HDAC-dependent mechanisms involved in the control of E3-ubiquitin ligases expression at the early stages of muscle unloading we used HDACs 4 and 5 inhibitor LMK-235 and HDAC 4 inhibitor Tasqinimod (Tq). Male Wistar rats were divided into four groups (eight rats per group): nontreated control (C), three days of unloading/hindlimb suspension (HS) and three days HS with HDACs inhibitor LMK-235 (HSLMK) or Tq (HSTq). Treatment with LMK-235 diminished unloading-induced of MAFbx, myogenin (MYOG), ubiquitin and calpain-1 mRNA expression (p < 0.05). Tq administration had no effect on the expression of E3-ligases. The mRNA expression of MuRF1 and MAFbx was significantly increased in both HS and HSTq groups (1.5 and 4.0 folds, respectively; p < 0.05) when compared with the C group. It is concluded that during three days of muscle unloading: (1) the HDACs 4 and 5 participate in the regulation of MAFbx expression as well as the expression of MYOG, ubiquitin and calpain-1; (2) the inhibition of HDAC 4 has no effect on MAFbx expression. Therefore, HDAC 5 is perhaps more important for the regulation of MAFbx expression than HDAC 4.

P38α-MAPK Signaling Inhibition Attenuates Soleus Atrophy during Early Stages of Muscle Unloading.

To test the hypothesis that p38α-MAPK plays a critical role in the regulation of E3 ligase expression and skeletal muscle atrophy during unloading, we used VX-745, a selective p38α inhibitor. Three groups of rats were used: non-treated control (C), 3 days of unloading/hindlimb suspension (HS), and 3 days HS with VX-745 inhibitor (HSVX; 10 mg/kg/day). Total weight of soleus muscle in HS group was reduced compared to C (72.3 ± 2.5 vs 83.0 ± 3 mg, respectively), whereas muscle weight in the HSVX group was maintained (84.2 ± 5 mg). The expression of muscle RING-finger protein-1 (MuRF1) mRNA was significantly increased in the HS group (165%), but not in the HSVX group (127%), when compared with the C group. The expression of muscle-specific E3 ubiquitin ligases muscle atrophy F-box (MAFbx) mRNA was increased in both HS and HSVX groups (294% and 271%, respectively) when compared with C group. The expression of ubiquitin mRNA was significantly higher in the HS (423%) than in the C and HSVX (200%) groups. VX-745 treatment blocked unloading-induced upregulation of calpain-1 mRNA expression (HS: 120%; HSVX: 107%). These results indicate that p38α-MAPK signaling regulates MuRF1 but not MAFbx E3 ligase expression and inhibits skeletal muscle atrophy during early stages of unloading.

Cellular and molecular signatures of alcohol-induced myopathy in women.

Alcoholic myopathy is characterized by the reduction in cross-sectional area (CSA) of muscle fibers and impaired anabolic signaling. The goal of the current study was to investigate the causes and compare the changes in CSA and fiber type composition with the modifications of anabolic and catabolic signaling pathways at the early stages of chronic alcohol consumption in women. Skeletal muscle samples from 5 female patients with alcohol abuse (AL; 43 ± 5 yr old; alcohol abuse duration 5,6 ± 0,6 yr) were compared with the muscle from the control group of 8 healthy women (C; 35 ± 4 yr old). The average daily dose of alcohol consumption was 110 ± 10 ml of pure ethanol. In women patients, a significant decrease in CSA of type I and II muscle fibers, titin and nebulin content, plasma IGF-1 level and total IRS-1, p-Akt and p-4E-BP1 in vastus lateralis was found in comparison with the control group. The p-AMPK level was found to be increased versus the control group. In women patients with chronic alcoholic myopathy 1) both fast and slow muscle fibers are subjected to atrophy; 2) impairments in IGF-I-dependent signaling and pathways controlling translation initiation (AMPK/mTOR/4E-BP1), but not translation elongation, are observed; 3) the level of calpain-1 and ubiquitinated proteins increases, unlike E3 ligases content.

Elevated p70S6K phosphorylation in rat soleus muscle during the early stage of unloading: Causes and consequences.

Currently, there is a lack of investigation into the initial signaling events underlying the development of disuse muscle atrophy. The study was aimed to (i) identify an assumed relationship between AMPK dephosphorylation and p70S6K hyperphosphorylation in the initial period of hindlimb unloading (HS), and (ii) assess the signaling consequences of p70S6K hyperphosphorylation following 24-h HS. For experiment 1, rats were treated with AMPK activator (AICAR) for 6 d before HS as well as during 24-h HS. For experiment 2, rats were treated with mTORC1 inhibitor rapamycin during 24-h HS. The key signaling markers implicated in protein turnover were assessed using WB and RT-PCR. One-day HS resulted in a significant upregulation of MuRF-1 and MAFbx expression, increase in p70S6K (Thr389) and IRS-1 (Ser639) phosphorylation and a significant decrease in phosphorylated AMPK, AKT, FOXO3, total IRS-1 content, and HDAC5 nuclear content. AMPK and p70S6K phosphorylation did not differ from control in AICAR-treated unloaded rats. Rapamycin treatment during unloading abolished p70S6K and E3 ligases upregulation and increased HDAC5 nuclear accumulation. The results of the study suggest that mTORC-1/p70S6K signaling pathway in rat soleus muscle is activated following 24-h mechanical unloading. This activation is facilitated by a decrease in AMPK phosphorylation. Increased p70S6K activity at the initial stage of hindlimb unloading could lead to the upregulation of E3 ligases MAFbx/atrogin-1 and MuRF-1 via nuclear export of HDAC5.

Edaphobacter lichenicola sp. nov., a member of the family Acidobacteriaceae from lichen-dominated forested tundra.

An isolate of aerobic, Gram-stain-negative, rod-shaped, non-motile and light-pink pigmented bacteria, designated SBC68T, was obtained from slightly decomposed thalli of the lichen Cladonia sp. collected from the forested tundra of north-western Siberia. Cells of this isolate occurred singly, in pairs or in rosettes. These bacteria were acidophilic (optimum growth at pH 4.3-5.6) and mesophilic (optimum growth at 20-30 °C) but were also capable of growth at low temperatures, down to 7 °C. The preferred growth substrates were sugars, some organic acids and lichenan. The major fatty acids were iso-C15 : 0, C16 : 1ω7c, C16 : 0, C16 : 1ω7t, and 13,16-dimethyl octacosanedioic acid. The only quinone was MK-8, and the G+C content of the DNA was 54.7 mol%. SBC68T represented a member of the family Acidobactericeae; the closest taxonomically described relatives were Edaphobacter dinghuensis DHF9T and Granulicella aggregans TPB6028T (97.2 and 97.1 % 16S rRNA gene sequence similarity, respectively). In 16S rRNA gene-based trees, SBC68T clustered together with species of the genus Edaphobacter. However, this isolate differed from all previously described species of the genus Edaphobacter with respect to the pink pigmentation, formation of cell rosettes and substrate utilization pattern. On the basis of these data, strain SBC68T should be considered to represent a novel species of acidobacteria, for which the name Edaphobacter lichenicola sp. nov. is proposed. The type strain is SBC68T (=DSM 104462T=VKM B-3208T).

Effect of Chronic Alcohol Abuse on Anabolic and Catabolic Signaling Pathways in Human Skeletal Muscle.

BACKGROUND: Animal studies showed that alcoholic myopathy is characterized by the reduction in myofiber cross-sectional area (CSA) and by impaired anabolic signaling. The goal of this study was to compare changes in CSA and fiber type composition with modifications in anabolic and catabolic signaling pathways at the early stages of alcohol misuse in humans. METHODS: Skeletal muscle samples from 7 male patients with chronic alcohol abuse (AL; 47.7 ± 2.0 years old; alcohol misuse duration 7.7 ± 0.6 years) were compared with muscle from a control group of 7 healthy men (C; 39.7 ± 5.0 years old). Biopsies from vastus lateralis muscles were taken and analyzed for the changes in fiber type composition, fiber CSA, and for the alterations in anabolic and catabolic signaling pathways. RESULTS: AL patients did not have detectable clinical myopathy symptoms or muscle fiber atrophy, but the relative proportion of fast fibers was increased. There was a significant decrease in IGF-1 in plasma and IRS-1 protein content in muscle of AL group. Levels of total and phosphorylated p70S6K1, GSK3ß, and p90RSK1 were not different between AL and C groups. Muscle of AL patients had increased mRNA expression of HSP70 and HSP90. A marker of anabolic pathway p-4E-BP1 was decreased, while catabolic markers (MuRF-1, MAFbx, ubiquitinated proteins) were increased in AL patients when compared with C group. CONCLUSIONS: At the early stages of alcohol misuse in humans, changes in the regulation of anabolic and catabolic signaling pathways precede the development of skeletal muscle atrophy and manifestation of clinical symptoms of alcoholic myopathy.

Prevalence of house dust mites in the homes of atopic dogs in Finland.

BACKGROUND: The house dust mites (HDM) Dermatophagoides farinae and D. pteronyssinus are important environmental allergens implicated in the pathogenesis of human and canine atopic dermatitis. Sensitization to HDM measured by allergen-specific IgE is common in Finnish atopic dogs. Studies on HDM prevalence in Finland are few but suggest that HDM are scarce. OBJECTIVE: The aim of the present study was to evaluate the prevalence of HDM in the home environments of atopic dogs in Finland. METHODS: Dust samples were obtained from the homes of 50 atopic dogs. Samples were collected by vacuuming the owners' mattresses and each dog's bed. In each case, an area of 21 × 30 cm was vacuumed for 2 min. Samples weighing 100 mg or more were considered sufficient for determination of HDM allergen concentrations (Der f 1 and Der p 1) using standardized ELISA. Samples sufficient for further analysis were also examined by direct microscopy for the presence of mites and by multiplex PCR for HDM DNA. RESULTS: Eighty one samples were sufficient for analysis by ELISA, 59 by PCR and 29 by direct microscopy. A single sample was analysed from four homes in which the dog shared the owner's bed. Der f 1 was detected in three samples (3.7%). Der p 1 was not detected in any sample. No mites were identified on microscopy. Five samples were positive for HDM on multiplex PCR (8.4%). CONCLUSION: House dust mites seem to be uncommon in the home environment of atopic dogs in Finland despite reported frequent allergen-specific IgE antibodies.

Methylovulum psychrotolerans sp. nov., a cold-adapted methanotroph from low-temperature terrestrial environments, and emended description of the genus Methylovulum.

Two isolates of aerobic methanotrophic bacteria, strains Sph1T and Sph2, were obtained from cold methane seeps in a floodplain of the river Mukhrinskaya, Irtysh basin, West Siberia. Another morphologically and phenotypically similar methanotroph, strain OZ2, was isolated from a sediment of a subarctic freshwater lake, Archangelsk region, northern Russia. Cells of these three strains were Gram-stain-negative, light-pink-pigmented, non-motile, encapsulated, large cocci that contained an intracytoplasmic membrane system typical of type I methanotrophs. They possessed a particulate methane monooxygenase enzyme and utilized only methane and methanol. Strains Sph1T, Sph2 and OZ2 were able to grow at a pH range of 4.0-8.9 (optimum at pH 6.0-7.0) and at temperatures between 2 and 36 °C. Although their temperature optimum was at 20-25 °C, these methanotrophs grew well at lower temperatures, down to 4 °C. The major cellular fatty acids were C16 : 1ω5c, C16 : 1ω6c, C16 : 1ω7c, C16 : 1ω8c, C16 : 0 and C14 : 0; the DNA G+C content was 51.4-51.9 mol%. Strains Sph1T, Sph2 and OZ2 displayed nearly identical (99.1-99.7 % similarity) 16S rRNA gene sequences and belonged to the family Methylococcaceae of the class Gammaproteobacteria. The most closely related organism was Methylovulum miyakonense HT12T (96.0-96.5 % 16S rRNA gene sequence similarity and 90 % pmoA sequence similarity). The novel isolates, however, differed from Methylovulum miyakonense HT12T by cell morphology, pigmentation, absence of soluble methane monooxygenase, more active growth at low temperatures, growth over a broader pH range and higher DNA G+C content. On the basis of these differences, we propose a novel species, Methylovulum psychrotolerans sp. nov., to accommodate these methanotrophs. Strain Sph1T (=LMG 29227T=VKM B-3018T) is the type strain.

Calpain-dependent regulation of the skeletal muscle atrophy following unloading.

Unloading causes rapid skeletal muscle atrophy due to increased protein degradation via activation of calpains and decreased protein synthesis. Our study elucidated role of calpain-1 in the regulation of ubiquitin proteasome pathway (UPP) and anabolic processes mediated by Akt-mTOR-p70S6K and MAPK-Erk (p90RSK) signaling. We hypothesized that blocking calpain will inhibit activation of UPP and decrease protein degradation resulting in reduction of unloading-induced skeletal muscle atrophy. Rats were divided into three groups: non-treated control (C), three day hindlimb suspension with (HSPD) or without (HS) treatment with calpain inhibitor PD150606. When compared with control PD150606 treatment during unloading: 1) attenuated loss of muscle mass, 2) prevented accumulation of calpain-1 (1.8-fold in HS vs 1.3-fold in HSPD) and ubiquitin (2.3-fold in HS vs 0.7-fold in HSPD) mRNA and ubiquitinated proteins (1.6-fold in HS vs 0.8-fold in HSPD), 3) prevented decrease in the pAkt (0.4-fold in HS vs 1-fold in HSPD) and pFOXO3 (0.2-fold in HS vs 1.2-fold in HSPD) levels, 4) prevented increase in MAFbx (3.8-fold in HS vs 1.3-fold in HSPD) and eEF2k (1.8-fold in HS vs 0.6-fold in HSPD) mRNA. Our study indicates that blocking of calpain during unloading decreases skeletal muscle atrophy by inhibiting UPP activation and preserving anabolic signaling.

Methylocapsa palsarum sp. nov., a methanotroph isolated from a subArctic discontinuous permafrost ecosystem.

An aerobic methanotrophic bacterium was isolated from a collapsed palsa soil in northern Norway and designated strain NE2T. Cells of this strain were Gram-stain-negative, non-motile, non-pigmented, slightly curved thick rods that multiplied by normal cell division. The cells possessed a particulate methane monooxygenase enzyme (pMMO) and utilized methane and methanol. Strain NE2T grew in a wide pH range of 4.1­8.0 (optimum pH 5.2­6.5) at temperatures between 6 and 32 °C (optimum 18­25 °C), and was capable of atmospheric nitrogen fixation under reduced oxygen tension. The major cellular fatty acids were C18 : 1ω7c, C16 : 0 and C16 : 1ω7c, and the DNA G+C content was 61.7 mol%. The isolate belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and was most closely related to the facultative methanotroph Methylocapsa aurea KYGT (98.3 % 16S rRNA gene sequence similarity and 84 % PmoA sequence identity). However, strain NE2T differed from Methylocapsa aurea KYGT by cell morphology, the absence of pigmentation, inability to grow on acetate, broader pH growth range, and higher tolerance to NaCl. Therefore, strain NE2T represents a novel species of the genus Methylocapsa, for which we propose the name Methylocapsa palsarum sp. nov. The type strain is NE2T ( = LMG 28715T = VKM B-2945T).