Nutrient signalling in the regulation of human muscle protein synthesis

The mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are important nutrient- and energy-sensing and signalling proteins in skeletal muscle. AMPK activation decreases muscle protein synthesis by inhibiting mTOR signalling to regulatory proteins associated with translation initiation and elongation. On the other hand, essential amino acids (leucine in particular) and insulin stimulate mTOR signalling and protein synthesis. We hypothesized that anabolic nutrients would be sensed by both AMPK and mTOR, resulting in an acute and potent stimulation of human skeletal muscle protein synthesis via enhanced translation initiation and elongation.
We measured muscle protein synthesis and mTOR-associated upstream and downstream signalling proteins in young male subjects ( n = 14) using stable isotopic and immunoblotting techniques. Following a first muscle biopsy, subjects in the 'Nutrition' group ingested a leucine-enriched essential amino acid–carbohydrate mixture (EAC). Subjects in the Control group did not consume nutrients. A second biopsy was obtained 1 h later. Ingestion of EAC significantly increased muscle protein synthesis, modestly reduced AMPK phosphorylation, and increased Akt/PKB (protein kinase B) and mTOR phosphorylation ( P < 0.05). mTOR signalling to its downstream effectors (S6 kinase 1 (S6K1) and 4E-binding protein 1 (4E-BP1) phosphorylation status) was also increased ( P < 0.05). In addition, eukaryotic elongation factor 2 (eEF2) phosphorylation was significantly reduced ( P < 0.05). Protein synthesis and cell signalling (phosphorylation status) was unchanged in the control group ( P > 0.05).
We conclude that anabolic nutrients alter the phosphorylation status of both AMPK- and mTOR-associated signalling proteins in human muscle, in association with an increase in protein synthesis not only via enhanced translation initiation but also through signalling promoting translation elongation.     

Short half-lives of ataxia-associated aprataxin proteins in neuronal cells

Early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH)/ataxia with oculomotor apraxia type 1 (AOA1) is caused by mutations in the gene encoding aprataxin (APTX). Although several in vitro findings proposed that impaired enzymatic activities of APTX are responsible for EAOH/AOA1, potential instability of mutant proteins has also been suggested as the pathogenesis based on in vivo finding that mutant proteins are almost undetectable in EAOH/AOA1 tissues or cells. The present study aimed to experimentally prove instability of mutant proteins in neuronal cells, the cell type preferentially affected by this disease. Results of pulse-chase experiments demonstrated that all of the disease-associated mutants had extremely shorter half-lives than the WT. We further found that mutants were targeted for rapid proteasome-mediated degradation. These results help establish pathogenic and physiological protein characteristics of APTX in neuronal cells.     

Comparative chromosome mapping of sex-linked genes and identification of sex chromosomal rearrangements in the Japanese wrinkled frog (Rana rugosa, Ranidae) with ZW and XY sex chromosome systems

There are regional variations of sex chromosome morphologies in the Japanese wrinkled frog, Rana rugosa (2n = 26): heterogametic ZZ/ZW-type and XX/XY-type sex chromosomes, and two different types of homomorphic sex chromosomes. To search for homology between the ZW and XY sex chromosomes and the chromosome rearrangements that have occurred during sex chromosomal differentiation in R. rugosa, we performed chromosome mapping of sexual differentiation genes for R. rugosa by FISH. Three genes, AR, SF-1/Ad4BP and Sox3, were localized to both the ZW and XY chromosomes, and their locations were all different between the Z and W and between the X and Y. AR and SF-1/Ad4BP were located on the short arms of the W and X and the long arms of Z and Y, and Sox3 was mapped to the different locations on the long arms between the Z and W and between the X and Y, probably as a result of multiple rearrangements that occurred during the process of sex chromosome differentiation. However, the chromosomal locations of three genes were almost consistent between the Z and Y and between the W and X, indicating that the Z and Y chromosomes and the W and X chromosomes were respectively derived from the same origins. Dmrt1, which is located on avian sex chromosomes, was localized to autosomes in R. rugosa with both the ZW and XY sex chromosomes, suggesting that Dmrt1 might not be related to sex determination in this species.     

Evaluation of endocrine disrupting activity of plasticizers in polyvinyl chloride tubes by estrogen receptor alpha binding assay

Polyvinyl chloride (PVC) tubing is an indispensable medical material for extracorporeal circulation therapy. However, di(2-ethylhexyl)phthalate (DEHP), a suspected endocrine disruptor, can be eluted from PVC, suggesting that an alternative material that does not contain DEHP is needed for clinical applications. First, we evaluated the endocrine disrupting risks of the plasticizers contained in PVC tubes by investigating their binding affinities for the human estrogen receptor alpha (ERα). Our results revealed that, while DEHP has some binding affinity for ERα, neither epoxidized soybean oil nor tris(2-ethylhexyl)trimellitate (an alternative to DEHP) has any affinity for ERα. Second, we evaluated the endocrine disrupting risks of a tube made of newly developed plasticizer-free (PF) materials. We confirmed the presence of DEHP and detected several unidentified substances in plasma stored within the PVC tube. This plasma's competitive binding affinity for ERα was significantly higher than that of control plasma (P < 0.01). In contrast, the profile of plasma stored in the PF tube was similar to that of the control, both in terms of high-performance liquid chromatography chromatograms and competitive binding capacity for ERα, suggesting that the PF tube is biocompatible and is useful for reducing the elution of substances capable of binding to ERα. Presented in part at the 42nd Congress of the Japanese Society for Artificial Organs, October 5–7, 2004, Tokyo, Japan     

Interferon-{beta} inhibits bleomycin-induced lung fibrosis by decreasing transforming growth factor-{beta} and thrombospondin

Pulmonary fibrosis is the result of abnormal processes of repair that occur after lung injury. Transforming growth factor (TGF)-beta is a key molecule in the progression of pulmonary fibrosis. Although clinical use of interferon (IFN)-beta did not improve survival in patients with idiopathic pulmonary fibrosis, because some preclinical studies have suggested that IFN-beta is a potent inhibitor of fibrogenesis, beneficial effects of IFN-beta have been expected. We therefore attempted to determine effects of IFN-beta and investigated the mechanism of action of IFN-beta in bleomycin-induced pulmonary fibrosis. Bleomycin at Day 0 and IFN-beta for 4 wk were administered intravenously to ICR mice. At 28 d after bleomycin injection, histologic and chemical analysis was performed for evaluation of effects of IFN-beta. Tissue distribution and amounts of TGF-beta1 and thrombospondin (TSP)-1/2 were analyzed. IFN-beta attenuated prolylhydroxylase activity, resulting in inhibition of pulmonary fibrosis. Bleomycin-induced increase in TGF-beta1 in epithelial cells and extracellular matrix was attenuated by IFN-beta. TSP-1/2 was limited in platelets of control mice, but was present in foamy cells in fibrotic regions induced by bleomycin. These findings suggest that the antifibrotic effect of IFN-beta is inhibition of TGF-beta and its activation via decrease in TSP-1/2 in lung tissue and change in location of TSP-1/2 from platelets to foamy cells.     

Detrimental effects after dobutamine infusion on rat left ventricular function: mechanical work and energetics

We have previously reported that continuous infusion of dobutamine into the coronary artery induces positive inotropic effects but induces no detrimental effects in cross-circulated, excised normal rat hearts and even in Ca2+ overload-induced contractile failing rat hearts. However, we hypothesized that some detrimental effects on left ventricular (LV) function are induced after continuous dobutamine infusion and the following clearance of blood dobutamine, as is the case after beta-adrenergic receptor stimulation. To test this hypothesis, we investigated LV mechanical work and energetics in the same type of preparations that underwent continuous dobutamine infusion and clearance of blood dobutamine. We found that both mean end-systolic pressure and systolic pressure-volume area (PVA; a measure of total mechanical energy per beat) at midrange LV volume were significantly (P < 0.01) decreased. The mean myocardial oxygen consumption per beat intercept, which is composed of for the total Ca2+ handling in excitation-contraction coupling and basal metabolism, of the and PVA linear relation was also significantly (P < 0.05) decreased (n=8). The mean slope of the linear relation was unchanged in such hearts. Post-dobutamine basal metabolism was unchanged (n = 5 of the 8 hearts). The moderate proteolysis of a cytoskeleton protein, alpha-fodrin was identified (n = 7 of the 8 hearts with the decreased intercept), after clearance of blood dobutamine. In agreement with our hypothesis, the detrimental effect of the post-beta-adrenergic receptor stimulation was induced by a moderate concentration of dobutamine; we found systolic dysfunction due to the impairment of Ca2+ handling in excitation-contraction coupling in the rat LV and proteolysis of a cytoskeleton protein, alpha-fodrin.     

Low-grade fibromyxoid sarcoma arising in the big toe

Low-grade fibromyxoid sarcoma (LGFMS) is a rare tumor. Reported herein is a case of LGFMS arising in the big toe. The patient was a 58-year-old man who underwent excision of the tumor. The tumor was well-demarcated. Histologically, there were proliferating spindle-shaped tumor cells arranged in a whorled growth pattern, and the stroma showed hyalinized collagen bundles and a myxoid matrix. Nuclear mitotic figures were conspicuous in part. A large rosette-like structure with hyalinized stroma was found, which is characteristic of LGFMS. The differential diagnosis included tumor occurrence in adults; tending to arise in distal extremities; and having bland fibromyxoid histological features, such as fibroma of tendon sheath, low-grade myxofibrosarcoma and acral myxoinflammatory fibroblastic sarcoma. It was not possible to detect the FUS/CREB3L2 and FUS/CREB3L1 fusion genes from the formalin-fixed and paraffin-embedded tissue, although the histological features of the present case were typical of LGFMS. LGFMS may become more common with time, and unique cases may accumulate.     

Cutaneous inputs can activate the ipsilateral primary motor cortex during bimanual sensory-driven movements in humans

Using transcranial magnetic stimulation (TMS), we examined whether sensory input from a finger affects activity of the ipsilateral primary motor cortex (M1) when human subjects hold a virtual object bimanually and whether this ipsilateral activation varies under different contexts. Subjects used both index fingers to hold two plates, which were subjected to unpredictable pulling loads from torque motors. Loads were delivered in a random sequence to either plate or concurrently to both, although the latter occurred most frequently. Finger forces vertical to the plates and surface electromyographs from the first dorsal interosseous muscles were recorded bilaterally during the task. TMS was sometimes applied over the finger area of the left M1 at variable times relative to load onset to examine cortical excitability. Strength of TMS was set around the active motor threshold of the right finger muscle while subjects waited for loading to the handheld plates. When one plate was singly loaded, the M1 contralateral to the loaded finger was activated, causing automatic force increases in the finger. In addition, the ipsilateral M1 was activated during such loading, associated with transient force increases in the contralateral nonloaded finger. Activations in the ipsilateral M1 were also observed during concurrent loading, when activations were stronger than those following single loading of the contralateral plate. Ipsilateral activations weakened when concurrent loading was less frequent. These results suggest interactions between bilateral sensorimotor cortices during bimanual coordinated movements, with strength varying by context.     

Comparison of the inhibitory effects of glucocorticoids on the expression of eotaxin in airway epithelial cell line BEAS-2B]

OBJECTIVE: Inhaled corticosteroids play a pivotal role in the treatment of asthma. To observe the mechanisms of glucocorticoids, we focused our study on the comparison of several glucocorticoids' effects on eotaxin expression in the airway epithelial cells. METHODS: Airway epithelial cell line BEAS-2B was cultured in vitro. Cells were preincubated with or without glucocorticoids (becromethasone dipropionate; BDP, budesonide; BUD, fluticasone propionate; FP) and stimulated with TNFalpha and/or IL-4. Protein levels of eotaxin in the supernatants of the cultured cells were determined by ELISA. RESULTS AND CONCLUSIONS: TNFalpha and IL-4 increased the levels of eotaxin in BEAS-2B cells. Combination of these cytokines synergistically upregulated the eotaxin expression as reported previously. Each glucocorticoid significantly inhibited the expression of eotaxin protein induced with TNFalpha and IL-4 and the compared efficacy was in order of FP>BUD>BDP. FP seemed most potent and the inhibitory effect was also observed with relatively low concentration such as 10 (-10)M. Taken together, the comparison of the potency of each glucocorticoid using airway epithelial cells may reflect the efficacy of these drugs in asthmatics.