Oxidative stress and delayed-onset muscle damage after exercise

Reactive oxygen species (ROS) produced during exercise may be involved in delayed-onset muscle damage related to inflammation. To investigate this hypothesis, we studied whether oxidative stress increases nuclear translocation of nuclear factor-kappaB and chemokine expression in skeletal muscle using myotube L6 cells. We also assessed whether prolonged acute exercise could increase these parameters in rats. In L6 cells, H(2)O(2) induced nuclear translocation of p65 and increased the expression of cytokine-induced neutrophil chemoattractant-1 (CINC-1) and monocyte chemoattractant protein-1 (MCP-1), whereas preincubation with alpha-tocopherol limited the increase in these proteins. Sprague Dawley rats were divided into the following groups: rested control, exercised, rested with a high alpha-tocopherol diet, and exercised with a high alpha-tocopherol diet. After 3 weeks of acclimation, both exercise groups ran on a treadmill at 25 m/min for 60 min. Exercise increased nuclear p65, CINC-1, and MCP-1 in gastrocnemius muscle cells, but these changes were ameliorated by the high alpha-tocopherol diet. Increases in myeloperoxidase and thiobarbituric acid-reactive substrates were ameliorated by a high alpha-tocopherol diet, as were the histological changes. Neutrophil activity was not altered by either exercise or a high alpha-tocopherol diet. These results indicate that delayed-onset muscle damage induced by prolonged exercise is partly related to inflammation via phagocyte infiltration caused by ROS and that alpha-tocopherol (an antioxidant) can attenuate such inflammatory changes.     

Determination of potato glycoalkaloids using high-pressure liquid chromatography-electrospray ionisation/mass spectrometry

A method for quantifying two toxic glycoalkaloids, alpha-solanine and alpha-chaconine, in potato (Solanum tuberosum) tuber tissue was developed using HPLC-electrospray ionisation (ESI)/MS. Potato samples were extracted with 5% aqueous acetic acid, and the extracts were subjected directly to HPLC-ESI/MS after filtration. By determining the intensities of the protonated molecules of alpha-solanine (m/z 868) and alpha-chaconine (m/z 852) using selected ion monitoring (positive ion mode), a sensitive assay was attained with detection limits of 38 and 14 ppb for the two glycoalkaloids, respectively. The high sensitivity and selectivity of MS detection effectively reduced the time of analysis thus enabling a high throughput assay of glycoalkaloids in potato tubers.     

Osteocyte network; a negative regulatory system for bone mass augmented by the induction of Rankl in osteoblasts and Sost in osteocytes at unloading

Reduced mechanical stress is a major cause of osteoporosis in the elderly, and the osteocyte network, which comprises a communication system through processes and canaliculi throughout bone, is thought to be a mechanosensor and mechanotransduction system; however, the functions of osteocytes are still controversial and remain to be clarified. Unexpectedly, we found that overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteoblast and osteoclast differentiation were unaffected by BCL2 transgene in vitro. However, the cortical bone mass increased due to enhanced osteoblast function and suppressed osteoclastogenesis at 4 months of age, when the frequency of TUNEL-positive lacunae reached 75%. In the unloaded condition, the trabecular bone mass decreased in both wild-type and BCL2 transgenic mice at 6 weeks of age, while it decreased due to impaired osteoblast function and enhanced osteoclastogenesis in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Rankl and Opg were highly expressed in osteocytes, but Rankl expression in osteoblasts but not in osteocytes was increased at unloading in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Sost was locally induced at unloading in wild-type mice but not in BCL2 transgenic mice, and the dissemination of Sost was severely interrupted in BCL2 transgenic mice, showing the severely impaired osteocyte network. These findings indicate that the osteocyte network is required for the upregulation of Rankl in osteoblasts and Sost in osteocytes in the unloaded condition. These findings suggest that the osteocyte network negatively regulate bone mass by inhibiting osteoblast function and activating osteoclastogenesis, and these functions are augmented in the unloaded condition at least partly through the upregulation of Rankl expression in osteoblasts and that of Sost in osteocytes, although it cannot be excluded that low BCL2 transgene expression in osteoblasts contributed to the enhanced osteoblast function.     

Genotoxicity of quinone pigments from pathogenic fungi

The genotoxicity and mutagenicity of several kinds of quinone pigments from pathogenic fungi were examined by means of the hepatocyte primary culture (HPC)/DNA repair test and of Ames test with TA98 and TA100. Clear genotoxicity of the two quinone chemicals, xanthomegnin and luteosporin were observed in the HPC/DNA repair test, though definite mutagenicity was not detected in the Salmonella microsome test. These two pigments are thus suspected to be genotoxic carcinogens.     

Some observations on the solitary male among Japanese monkeys

The social behavior pattern of a solitary male at Koshima was studied by means of radio-telemetry. The relationship between the solitary males and the troop was estimated from radio-tracking data of the former's location and movement, and by direct observation of the latter at each corresponding hour.For most of day, the solitary male stayed within a distance of about 20 to 150 m from the central part of the troop, occasionally approaching it. His movement also was synchronized with that of the troop. For two nights, the solitary male slept at places which were about 200 m from the sleeping sites of the troop and faced them across the beach. The relationship between the solitary male and the troop did not seem to be strongly antagonistic.It can be assumed that the solitary male was moving according to certain pre-determined relationships or social contacts with the troop. The example of this solitary male shows the existence of the solitary male that follows and maintains contact with the troop, even outside the copulatory season.This study was sponsored by Scientific Research Grant No. 91620 of the Ministry of Education to the Japan Monkey Centre.     

Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes.

Bacterial lipopolysaccharide (LPS) triggers innate immune responses through Toll-like receptor (TLR) 4, a member of the TLR family that participates in pathogen recognition. TLRs recruit a cytoplasmic protein, MyD88, upon pathogen recognition, mediating its function for immune responses. Two major pathways for LPS have been suggested in recent studies, which are referred to as MyD88-dependent and -independent pathways. We report in this study the characterization of the MyD88-independent pathway via TLR4. MyD88-deficient cells failed to produce inflammatory cytokines in response to LPS, whereas they responded to LPS by activating IFN-regulatory factor 3 as well as inducing the genes containing IFN-stimulated regulatory elements such as IP-10. In contrast, a lipopeptide that activates TLR2 had no ability to activate IFN-regulatory factor 3. The MyD88-independent pathway was also activated in cells lacking both MyD88 and TNFR-associated factor 6. Thus, TLR4 signaling is composed of at least two distinct pathways, a MyD88-dependent pathway that is critical to the induction of inflammatory cytokines and a MyD88/TNFR-associated factor 6-independent pathway that regulates induction of IP-10.     

An insect cell line discrimination method by RAPD-PCR

Summary RAPD-PCR with a tenmar single primer for discrimination of insect cell lines was devised. The base sequence of the primers used were TTCGAGCCAG, CCGCATCTAC, GAACGGACTC, and TGAGTGGGTG (GC contents were 60%). Genome DNA was extracted by modified Landry et al. (1993) method. The reaction mixture consisted of 10 μl buffer, 8 μl dNTP mixture (2.5 mM each), 4 μl primer (50 μM), Taq DNA polymerase (2.5 units), 1 μl template DNA; and the reaction was run at 94° C for 2 min (denaturation), followed by 31 cycles of 94° C for 1 min, 42° C for 1 min (annealing), and 72° C for 2 min (extension) and terminated with 72° C for 7 min. By developing the reaction products with agarose gel electrophoresis, it became evident that DNA fragments were amplified with all the primers used. Among four primers, the second primer was selected as a suitable primer for distinguishing cell lines. With this method, cell lines derived from different species were clearly distinguished.     

Inorganic ion compositions in the Ulvophyceae and the Rhodophyceae, with special reference to sulfuric acid ion accumulations

Cellular pH estimated from cell extract pH, and the ion compositions of major inorganic ions (Na⁺, NH₄⁺, K⁺, Mg²⁺, Ca²⁺, Cl^?, Br^?, NO₃^?, S0₄²‐) were studied by ion chromatography in 15 species of 4 orders (Cladophorales, Codiales, Siphonocladales and Ulvales) of Ulvophyceae and 49 species of 8 orders (Bangiales, Ceramiales, Corallinales, Cryptonemiales, Gelidiales, Gigartinales, Nemaliales and Rhodymeniales) of Rhodophyceae. Among the Rhodophyceae, relatively low intracellular pH (approximately 2.0 within cells) and high concentration of S0₄^2‐ was demonstrated in Plocamium telfairiae(Harvey) Harvey. Furthermore, five species of Hypnea Lamouroux were shown to contain high concentrations of S0₄^2‐ balanced by relatively high concentrations of Na⁺. Among the Ulvophyceae, Codium cylindricum Holmes and Ulva pertusa Kjellman contained high concentrations of S0₄^2‐ balanced by relatively high concentrations of Mg²⁺.