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.     

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.     

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.     

Light and electron microscopic analyses of immediate and late tissue damage caused by radiofrequency ablation in porcine liver

Radiofrequency ablation (RFA) is an effective procedure for localized hepatocellular carcinoma. Contrast-enhanced CT depicts the ablated area as a hypoattenuated area without hepatic blood flow; however, light microscopy does not show obvious necrosis in the ablated area. We evaluated liver tissue changes after RFA by light microscopy and electron microscopy. The normal livers of three anesthetized pigs were coagulated using RFA after laparotomy. The liver was examined immediately, and 1 week after operation by light and electron microscopy. After RFA, the liver parenchyma surrounding the needle electrode was brown in color and surrounded by a red marginal zone separate from the normal liver parenchyma. Hematoxylin-eosin staining of the central area did not show cell necrosis, and the structures of liver sinusoids, liver cell cord and the nuclei of hepatocytes were preserved. However, electron microscopic examination of tissue immediately after RFA showed destruction of mitochondria of hepatocytes and fixation of sinusoidal cells. One week later, there was a large quantity of debris in the enlarged sinusoids, in addition to irreversible destruction of hepatocyte organelles. RFA of the porcine liver causes hepatocyte damage. This damage was not evident by light microscopy but clearly identified by electron microscopy.     

Peroxyl radical scavenging capability of fish sauces measured by the chemiluminescence method

Oxygen-related free radicals have been suggested as a cause of aging and various diseases, for example, various cancers and rheumatoid arthritis. A radical scavenger as an antioxidant has been sought in foods. Fish sauces are traditional Asian fermented seasonings. Using the luminol chemiluminescence method, the peroxyl radical scavenging capability of fish sauces was examined. From the IC50 values, many fish sauces have been shown to have a strong scavenging capability as well as soy sauces. A scavenging mechanism is also proposed.     

Estrogen receptor-associated expression of keratinocyte growth factor and its possible role in the inhibition of apoptosis in human breast cancer

Although estrogen is known to play a crucial role in the pathogenesis of breast cancer, the molecular mechanisms underlying the action of estrogen remain elusive. In the present study, we focused on keratinocyte growth factor (KGF) and its receptor (KGFR) in the pathogenesis of breast cancer, as a growth factor mediating estrogen action, since significant roles of KGF were demonstrated in various steroid hormone-dependent tissues. First, using paraffin-embedded specimens from 42 breast cancer patients, we examined expression patterns of KGF and KGFR by both immunohistochemistry using newly generated antibodies and nonradioactive in situ hybridization with T-T dimerized synthetic oligonucleotide probes. We next compared the results with the expression of estrogen receptor (ER) alpha and beta, proliferative activity and apoptotic frequency (TUNEL staining). Also, the similar approaches were taken to analyze the expression and role of KGF in ER-positive (MCF7, ZR-75-1) and ER-negative (SK-BR-3, MDA-MB-231) human breast cancer cell lines in vitro. In the surgical specimens, KGF was expressed in cancer cells as well as stromal cells in 19/42 cases (45%), while KGFR was found in cancer cells in 24/42 cases (57%). The distribution of protein and mRNA in the analysis of both KGF and KGFR expression generally coincided. Moreover, KGF expression was closely associated with the expression of ER alpha, and the coexpression of KGF and KGFR significantly correlated with lower TUNEL index, but not with proliferative activity. In accordance with the in vivo findings, KGF expression was detected only in ER alpha-positive MCF7 and ZR-75-1 cells in vitro. And more importantly, we found the inhibitory effect of KGF upon the induction of apoptosis by anticancer drugs in MCF7 cells. Collectively, our results indicate that ER alpha may be involved in KGF expression, and that KGF may play antiapoptotic roles, rather than mitogenic, in human breast cancer.     

Generators of visual evoked potentials for faces and eyes in the human brain as determined by dipole localization

Human visual evoked potentials were recorded during presentation of photos of human and animal faces and various face features. Negative waves with approximate peak latencies of 165 msec (N170) were bilaterally recorded from the occipito-temporal regions. Mean peak latencies of the N170 were shorter for faces than eyes only. Analyses of amplitudes of evoked potentials indicated that the N170 elicited by faces reflected activity of a specific neural system which was insensitive to detailed differences among individual faces regardless of species, and consequently suggest that this system might function to detect existence of faces in general. On the other hand, the mean amplitude of the N170 elicited by human eyes was significantly larger than those by animal eyes. These differences in response latencies and amplitudes of the N170 suggest existence of at least 2 different visual evoked potentials with similar latencies (i.e., N170) which are sensitive to faces in general and human eyes, respectively. Dipole source localization analysis indicated that dipoles for the N170 elicited by eyes were located in the posterior inferior temporal gyrus, and those for faces, located initially in the same region, but moved toward the fusiform and lingual gyri at the late phase of the N170. The results indicated that information processing of faces and eyes separated at least as early as the latency of the N170 at the posterior inferior temporal gyrus as well as the fusiform and lingual gyri, and might provide neurophysiological and anatomical bases to an initial structural encoding stage of human faces.     

A new selective agent for poly(tetrahydrofuran) degradation as a tool for the determination of the crystallite core size

The method formerly used to determine the crystallite core size of polyethylene was applied to poly(tetrahydrofuran) (PTHF). This method is based on the different diffusivity of a chaincleaving agent in crystalline and amorphous parts of the polymer. Butyllithium was used as a cleaving agent of PTHF. The degree of polymerization of the undecomposed part of the polymer (PTHF crystallites) was determined by gel permeation chromatography. This value agrees well with the value to be expected from the size of the crystallites determined small-angle X-ray scattering.     

Enhancement of an inhibitory input to the feeding central pattern generator in Lymnaea stagnalis during conditioned taste-aversion learning

To study the neuronal mechanism of a conditioned taste-aversion (CTA) learning in the pond snail Lymnaea stagnalis, we examined the synaptic connection between the neuron 1 medial (N1M) cell and the cerebral giant cell (CGC), the former is an interneuron in central pattern generator for the feeding response and the latter is a regulatory neuron to the central pattern generator. Inhibitory postsynaptic potential (IPSP) which was evoked in the N1M cell by activation of the CGC was larger and lasted longer in the conditioned animal than that in the control animal. The electrical properties of the cell body of CGC and the responses of the CGC to the chemosensory inputs were not changed during the CTA learning. These results, together with the previous report indicating the existence of excitatory projection from the N1M cell to the feeding motoneuron, suggest that enhanced IPSP in the N1M cell may underlie the suppression of feeding responses in the Lymnaea CTA learning.