Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.     

Molecular evolution of mammalian lactate dehydrogenase-A genes and pseudogenes: association of a mouse processed pseudogene with a B1 repetitive sequence

A mouse genomic clone containing a lactate dehydrogenase-A (LDH-A) processed pseudogene and a B1 repetitive element was isolated, and a nucleotide sequence of approximately 3 kb was determined. The pseudogene and B1 element are flanked by perfect 13-bp repeats, and the B1 sequence starts at 14 nucleotides 3' to the presumptive polyadenylation signal of the pseudogene. The nucleotide sequences of the LDH-A genes and processed pseudogenes from mouse, rat, and human were compared, and a phylogenetic tree was constructed. The rate and pattern of nucleotide substitutions in the LDH-A pseudogenes are similar to previously reported results (Li et al. 1984). The average rate of nucleotide substitutions in the LDH-A pseudogenes is 4.3 X 10(- 9)/site/year. The substitutions of C----T and G----A are most frequent, and A----G substitutions are relatively high. The rate of synonymous substitutions in the LDH-A genes is 5.3 X 10(-9), which is not significantly higher than the average rate of 4.7 X 10(-9) for 35 mammalian genes. The rate of nonsynonymous substitutions in the LDH-A genes is 0.20 X 10(-9), which is considerably lower than the average rate of 0.88 X 10(-9) for 35 mammalian genes. Thus, the mammalian LDH-A gene appears to be highly conserved in evolution.      

Genbank accession #: AF 124983

Plant Molecular Biology -     

Mechanisms of the antinociceptive action of (?) Epicatechin obtained from the hydroalcoholic fraction of Combretum leprosum Mart & Eic in rodents

ABSTRACT: BACKGROUND: The mechanisms of the antinociceptive activity of () epicatechin (EPI), a compound isolated from the hydroalcoholic fraction of Combreum leprosum Mart & Eicher. METHODS: were assessed in the model of chemical nociception induced by glutamate (20 mumol/paw). To evaluate the mechanisms involved, the animals , male Swiss mice (25-30 g), received EPI (50 mg/kg p.o.) after pretreatment with naloxone (2 mg/kg s.c. opioid antagonist), glibenclamide (2 mg/kg s.c. antagonist K + channels sensitive to ATP), ketanserin (0.3 mg/kg s.c. antagonist of receptor 5-HT2A), yoimbine (0.15 mg/kg s.c. alpha2 adrenergic receptor antagonist), pindolol (1 mg/kg s.c. 5-HT1a/1b receptor antagonist), atropine (0.1 mg/kg s.c. muscarinic antagonist) and caffeine (3 mg/kg s.c. adenosine receptor antagonist), ondansetron (0.5 mg/kg s.c. for 5-HT3 receptor) and L-arginine (600 mg/kg i.p.). RESULTS: The antinociceptive effect of EPI was reversed by pretreatment with naloxone and glibenclamide, ketanserin, yoimbine, atropine and pindolol, which demonstrates the involvement of opioid receptors and potassium channels sensitive to ATP, the serotoninergic (receptor 5HT1A and 5HT2A), adrenergic (receptor alpha 2) and cholinergic (muscarinic receptor) systems in the activities that were observed. The effects of EPI, however, were not reversed by pretreatment with caffeine, L-arginine or ondansetron, which shows that there is no involvement of 5HT3 receptors or the purinergic and nitrergic systems in the antinociceptive effect of EPI. In the Open Field and Rotarod test, EPI had no significant effect, which shows that there was no central nervous system depressant or muscle relaxant effect on the results. CONCLUSIONS: This study demonstrates that the antinociceptive activity of EPI in the glutamate model involves the participation of the opioid system, serotonin, adrenergic and cholinergic.     

Probing the Flexibility of Tropomyosin and Its Binding to Filamentous Actin Using Molecular Dynamics Simulations

Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments.     

Stress responses and metal tolerance of <Emphasis Type="Italic">Chlamydomonas acidophila</Emphasis> in metal-enriched lake water and artificial medium

Chlamydomonas acidophila faces high heavy-metal concentrations in acidic mining lakes, where it is a dominant phytoplankton species. To investigate the importance of metals to C. acidophila in these lakes, we examined the response of growth, photosynthesis, cell structure, heat-shock protein (Hsp) accumulation, and metal adsorption after incubation in metal-rich lake water and artificial growth medium enriched with metals (Fe, Zn). Incubation in both metal-rich lake water and medium caused large decreases in photosystem II function (though no differences among lakes), but no decrease in growth rate (except for medium + Fe). Concentrations of small Hsps were higher in algae incubated in metal-rich lake-water than in metal-enriched medium, whereas Hsp60 and Hsp70A were either less or equally expressed. Cellular Zn and Fe contents were lower, and metals adsorbed to the cell surface were higher, in lake-water-incubated algae than in medium-grown cells. The results indicate that high Zn or Fe levels are likely not the main or only contributor to the low primary production in mining lakes, and multiple adaptations of C. acidophila (e.g., high Hsp levels, decreased metal accumulation) increase its tolerance to metals and permit survival under such adverse environmental conditions. Supposedly, the main stress factor present in the lake water is an interaction between low P and high Fe concentrations.     

Seed after-ripening and dormancy determine adult life history independently of germination timing

? Seed dormancy can affect life history through its effects on germination time. Here, we investigate its influence on life history beyond the timing of germination. ? We used the response of Arabidopsis thaliana to chilling at the germination and flowering stages to test the following: how seed dormancy affects germination responses to the environment; whether variation in dormancy affects adult phenology independently of germination time; and whether environmental cues experienced by dormant seeds have an effect on adult life history. ? Dormancy conditioned the germination response to low temperatures, such that prolonged periods of chilling induced dormancy in nondormant seeds, but stimulated germination in dormant seeds. The alleviation of dormancy through after-ripening was associated with earlier flowering, independent of germination date. Experimental dormancy manipulations showed that prolonged chilling at the seed stage always induced earlier flowering, regardless of seed dormancy. Surprisingly, this effect of seed chilling on flowering time was observed even when low temperatures did not induce germination. ? In summary, seed dormancy influences flowering time and hence life history independent of its effects on germination timing. We conclude that the seed stage has a pronounced effect on life history, the influence of which goes well beyond the timing of germination.     

Dual role of nNOS in ischemic injury and preconditioning

Background  

Nitric oxide (NO) is cardioprotective and a mediator of ischemic preconditioning (IP). Endothelial nitric oxide synthase (eNOS) is protective against myocardial ischemic injury and a component of IP but the role and location of neuronal nitric oxide synthase (nNOS) remains unclear. Therefore, the aims of these studies were to: (i) investigate the role of nNOS in ischemia/reoxygenation-induced injury and IP, (ii) determine whether its effect is species-dependent, and (iii) elucidate the relationship of nNOS with mitoK_ATP channels and p38MAPK, two key components of IP transduction pathway.