Effects of arachidonic acid and its major prostaglandin derivatives on bovine myoblast proliferation, differentiation, and fusion.

Many studies have shown positive effects of prostaglandins (PGs) on various steps of skeletal muscle formation such as myoblast proliferation and myotube hypertrophy. In animals, PGs are synthesized through the action of the rate-limiting enzymes cyclooxygenase (COX) -1 and COX-2 from arachidonic acid (AA), a conditionally essential fatty acid. As a step toward exploring the possibility of using dietary supplementation of AA to improve skeletal muscle growth in cattle, which are major meat-producing animals, we determined the effects of AA and its major PG derivatives PGE2, PGF2α, and PGI2 on proliferation, differentiation, and fusion of primary bovine myoblasts in vitro. In the proliferation experiment, myoblasts were cultured in a growth medium to which was added 10 µM AA, 1 µM PGE2, 1 µM PGF2α, 1 µM PGI2, or vehicle control for 24 h, and the proliferating cells were identified by 5-ethynyl-2'-deoxyuridine (EdU) labeling. This experiment revealed that AA, PGE2, PGF2α, and PGI2 each increased the number of proliferating cells by 13%, 24%, 16%, and 16%, respectively, compared to the control (n = 7, P < 0.05). In the differentiation and fusion test, myoblasts were induced to differentiate and fuse into myotubes in the presence of the aforementioned treatments for 0, 24, 48, and 72 h. Based on quantitative reverse transcription PCR analyses of mRNAs of myoblast differentiation and fusion markers (myogenin; myosin heavy chain 3; creatine kinase, muscle; myomaker) at 0, 24, and 48 h of differentiation, AA, PGE2, and PGF2α promoted myoblast differentiation (n = 6, P < 0.05). Based on Giemsa staining and counting the number of myotubes at 72 h of differentiation, PGE2 enhanced the number of formed myotubes by 14% (P < 0.05) compared to the control. Treating the myoblasts with AA and either the COX-1 and COX-2 common inhibitor indomethacin or the COX-2-specific inhibitor NS-398 reversed the stimulatory effect of AA on myoblast proliferation (n = 4, P < 0.05). Overall, this study demonstrates that exogenous AA stimulates bovine myoblast proliferation and differentiation in culture. The results of this study suggest that AA stimulates myoblast proliferation through its metabolites PGE2, PGF2α, or PGI2, and that AA stimulates myoblast differentiation through PGE2.

Cloning and functional expression of the human glucagon-like peptide-1 (GLP-1) receptor.

Truncated forms of glucagon-like peptide-1 are the most potent endogenous stimuli of insulin secretion and have powerful antidiabetogenic effects. To determine the structure and coupling mechanisms of the human GLP-1 receptor we have isolated two pancreatic islet cDNAs, encoding the 463 amino acid receptor and differing mainly in their 3' untranslated regions. The deduced amino acid sequence is 90% homologous with the rat GLP-1 receptor. Northern blot analysis shows expression of a single 2.7 kb transcript in pancreatic tissue. When expressed in COS-7 cells the recombinant receptor conferred specific, high affinity GLP-1(7-37) binding. GLP-1(7-37) increased intracellular cAMP in a concentration dependent manner and caused an increase in the free cytosolic calcium ([Ca2+]i) from an intracellular pool, characteristic of phospholipase C (PLC) activation. Thus, like the structurally related glucagon and parathyroid hormone receptors, the human GLP-1 receptor can activate multiple intracellular signaling pathways including adenylyl cyclase and PLC. Knowledge of the GLP-1 receptor structure will facilitate the development of receptor agonists and elucidation of the important role of GLP-1 in normal physiology and disease states.

Reversal of epileptic state by patterned electrical stimulation suggested by trion model calculations.

The trion model is based on the Mountcastle columnar organizational principle of cortex. A trion represents an idealized minicolumn with three levels of firing activity and is highly structured in time and in spatial connections. A network of trions has a large repertoire of quasi-stable, periodic spatial-temporal firing patterns, MPs, which can be excited and each MP can be readily enhanced by a Hebb learning rule. A particular MP present in most repertoires has all trions firing together in synchrony, which we identify as the 'Epileptic' MP (EMP). In trion model simulations, the EMP can be enhanced via the Hebb rule after electrical stimulation so that an epileptic focus with after discharge (about 3-6 Hz) is formed and spontaneous firing of the EMP occurs (as in kindling). Following this, by using a small array of closely-spaced stimulating electrodes out of phase, other MPs are enhanced via the Hebb rule eliminating the dominance of the EMP. We strongly urge that these predictions be tested in the animal models for possible clinical relevance.

A double-blind trial of oral immunotherapy for Artemisia pollen asthma with evaluation of bronchial response to the pollen allergen and serum-specific IgE antibody.

Eighteen asymptomatic Artemisia pollen asthma patients with normal pulmonary function were selected for a double-blind trial of oral immunotherapy. Each patient had a positive skin test to Artemisia pollen extract and also a positive bronchial challenge response to the same extract. The patients were randomly assigned to active treatment or placebo group and received intensive oral administration of Artemisia pollen extract over a 50-day course. The nine patients who received the active treatment ingested a cumulative dose of 396,652 PNU and showed a significant decrease in serum-specific IgE antibodies (P less than .05) and a significant reduction in bronchial sensitivity to the same extract (P less than .01). The changes in these two variables correlated well (r = .8787, P less than .01). The nine patients who received the placebo showed no significant changes in serum-specific IgE or bronchial sensitivity to Artemisia pollen extract. Follow-up of two cases with the same extract showed that the reductions in serum-specific IgE as well as bronchial sensitivity induced by oral immunotherapy were maintained for 3 months.

An investigation on in vivo allergenicity of Artemisia annua leaves and stems.

Pollen of Artemisia annua is considered to be one of the most important allergens in autumnal hay fever in China, just as ragweed is in North America. In order to clarify the allergenicity of non-pollen containing components of the plant, Artemisia annua leaves and stems were collected and extracted before pollination time. The extracts of these pollen-free plant components were studied for allergenic activities using skin prick tests, intradermal tests, intranasal challenge and bronchial provocation tests. In 52 subjects sensitive to Artemisia pollen, 92.3% gave positive responses in skin prick tests, 100% gave positive responses in intradermal tests, 66.7% gave positive responses in intranasal challenge and 59.3% gave positive responses in bronchial provocation tests. Negative results to skin prick tests, intradermal tests and bronchial provocation tests were revealed in 30 seasonal asthmatics who gave negative responses to the pollen skin tests. Strict placebo control showed all negative tests in non-atopic adult human volunteers. We concluded that pollen-free plant extracts did have in vivo allergenic activities. Analysis of the plant allergenic components in vitro will be the subject of further studies.

One year observation of immunotherapy for Artemisia hay fever in China: a clinical and immunological study.

A controlled trial of one year immunotherapy was conducted in 50 Artemisia-sensitive hay fever patients (treatment group). From October 1985 to July 1986, all of the treatment group patients received one year regular injection of Artemisia pollen allergen extract totalling 30,000 protein nitrogen units (PNU). For these patients, symptom score indices of the posttreatment 1986 pollination season were compared with those from the pretreatment 1985 season and also with the scores of a similar group of 30 Artemisia-sensitive patients treated only with symptomatic medications during the 1986 season (control group). The 1986 symptom scores to the treatment group were significantly improved and the effective rate was 78%. Immunological study with the Human Basophil Degranulation Test (HBDT) showed a significant decrease in degranulation reactions after immunotherapy. Moreover, The decline of the HBDT positive rate in the treatment group was significantly greater in patients with improved symptoms than patients with unchanged symptoms. No difference was observed in basophil degranulation in those patients tested with a pollen-free plant extract, which was not applied in immunotherapy. The results suggested that immunotherapy could induce desensitization of basophils and that the induction might be allergen specific. Basophil desensitization may play an important role in the mechanism of immunotherapy.