Evidence for a Calcium/Calmodulin Involvement in Density-Dependent Melanogenesis in Murine B16 Melanoma Cells

Previous work from our laboratory has shown that both cyclic AMP and calcium/calmodulin appear to be involved in the regulation of melanogenesis in murine B16 melanoma cells. In these cells as in murine Cloudman S91 cells, melanogenic responsiveness to melanocyte-stimulating hormone (MSH) varies with cell density in culture. Our objective in this study was to learn more about the intracellular systems involved in the control of melanogenesis, particularly the role played by calcium. The melanogenic response to αMSH was compared to the response to drugs affecting intracellular free calcium and calmodulin over a range of cell densities in B16F1 cells. αMSH-stimulated melanin production was extremely density-dependent but αMSH-stimulated cyclic AMP production was independent of cell density. The melanogenic response to agents that increased intracellular calcium (A23187) or inhibited intracellular calmodulin varied with cell density. A drug (TMB8) that lowered intracellular free calcium, however, increased melanogenesis independently of cell density. At high cell density it was found that an elevation in calcium decreased melanogenesis, whereas agents that reduced calcium or inhibited calmodulin activity increased melanogenesis. At low cell density, however, the inhibitory response to A23187 was lost and in some experiments even stimulated melanogenesis. These data suggest that the calcium/calmodulin signalling system has an inhibitory influence on melanogenesis, and its expression, which depends upon cell density, may also modulate the response to stimulatory agents such as αMSH.     

A novel stress-acclimation response in Spirodela punctata (Lemnaceae): 2,4,6-trichlorophenol triggers an increase in the level of an extracellular peroxidase, capable of the oxidative dechlorination of this xenobiotic pollutant

Peroxidases are haem‐containing enzymes capable of oxidizing a wide range of substrates. This article describes the presence of peroxidase activity in the growth medium of axenic Spirodela punctata (Lemnaceae) cultures. It was found that the release of extracellular peroxidase activity is specifically enhanced by phytotoxic, halogenated phenols but not by other abiotic stress‐factors, elicitors or plant metabolites. Based on the concentration dependence of 2,4,6‐trichlorophenol (TCP)‐enhanced peroxidase release, it is concluded that release is not simply a consequence of physiological damage, but rather requires metabolically healthy fronds. In vitro studies (UV/VIS spectroscopy and liquid chromatography/mass spectrometry) show that the extracellular duckweed peroxidase (SpEx), which was partially purified from Spirodela growth medium, is capable of catalysing the oxidative dechlorination of TCP with hydrogen peroxide as the electron acceptor. It is proposed that the ability of S. punctata to specifically sense environmentally persistent phytotoxic chlorophenols, and to respond by increasing extracellular levels of a peroxidase capable of catalysing their oxidative dechlorination, is part of the protection strategy of this aquatic plant against xenobiotic stress.     

Identification and Partial Purification of a Stage-Specific 33 kDa Mitochondrial Protein as the Alternative Oxidase of the Trypanosoma brucei brucei Bloodstream Trypomastigotes

ABSTRACT. The glycerophosphate oxidase (GPO), the unique terminal oxidase of bloodstream trypanosome (TAO), appears to be functionally similar to the alternative oxidases of some plants and higher fungi. Immunoblotting of mitochondrial proteins of bloodstream trypomastigotes of Trypanosoma brucei brucei with monoclonal or polyclonal antibodies to Sauromatum guttatum (voodoo lily) and Symplocarpus foetidus (skunk cabbage) alternative oxidases respectively revealed two proteins of about 33 kDa (p33) and 68 kDa (p68). These proteins are not present in procyclic trypomastigotes. Electrophoresis under rigorous denaturing conditions indicated p68 to be the dimer of p33. Indirect immunofluorescent studies of bloodstream and procyclic trypomastigotes with monoclonal antibody to plant alternative oxidase also showed the localization of 33 kDa protein in the mitochondria of the bloodstream trypomastigotes. The functional TAO activity could be solubilized efficiently from the mitochondrial membrane of the bloodstream trypomastigotes by 1% NP-40 or 10 mM lauryl maltoside. When fractionated by Superose 12 gel filtration chromatography, p33 was co-purified with the TAO enzymatic activity. The apparent molecular size of the active enzyme complex was found to be 160 kDa. Gradual disappearance of the 33 kDa protein and the TAO enzymatic activity were well correlated during in vitro differentiation of the bloodstream to procyclic trypomastigotes. This study implies that the net biosynthesis of p33, an essential subunit of TAO, is decreased during differentiation from bloodstream to procyclic trypomastigotes.     

Rapid recycling of triose phosphates in oak stem tissue

We report the carbon-13 and oxygen-18 isotope ratios in cellulose from the early and late wood of pedunculate oak (Quercus robur L.). The δ¹³ C value of the early wood correlates best with that of the late wood of the previous year. The δ¹⁸O value of the early wood correlates best with that of the late wood of the same year. We suggest that a biochemical explanation of these data is that there is a rapid cycle between hexose monophosphates and triose phosphates in oak stem tissue during cellulose synthesis. Evidence in support of this explanation is provided by the intramolecular distribution of ¹⁴C in labelled fructose extracted from cores of wood that had been supplied with [1?¹⁴C]- and [6-¹⁴C]glucose.     

Salt tolerance in Eucalyptus spp.: identity and response of putative osmolytes

In four species of salt-tolerant eucalypts (Eucalyptus raveretiana, E. spathulata, E. sargentii and E. loxophleba), we found substantial concentrations of quercitol – a cyclitol known for its accumulation in seeds of Quercus. Quercitol was absent in old foliage of E. globulus, a species noted for greater susceptibility to salinity, and also absent in the moderately tolerant E. camaldulensis, but, relative to other species, both had higher foliar concentrations of inositol. Simple sugars and cyclitols accumulated to osmotically significant concentrations in all species. The osmotic potential of expressed sap was always less than that of the external ‘soil’ solution and increasing salinity produced predictable reductions in growth and increases in ion concentrations in foliage of saplings of four eucalypt species. The more salt-tolerant species, E. spathulata, E. loxophleba and E. sargentii, were able to maintain well-regulated leaf Na⁺ concentrations even at 300 mol m⁻³ NaCl. These more salt-tolerant species also showed an apparent increase in net selectivity for K⁺ over Na⁺ as salinity increased, irrespective of the Na⁺ : Ca²⁺ ratio of the external medium (range 25 : 1 to 75 : 1; Ca²⁺ always ≥ 4.0 mol m⁻³). By contrast, E. globulus was unable to exclude Na⁺ when exposed to higher NaCl concentrations (e.g. 200 and 300 mol m⁻³). Carbon isotope signatures of foliage reflected imposed salinity but were not strongly enough correlated with growth to support previous suggestions that isotope discrimination be a means of evaluating salt tolerance. On the other hand, patterns of sugar and cyclitol accumulation should be further explored in eucalypts as traits contributing to salt tolerance, and with potential use as markers in breeding programmes.     

Sinapine Leakage from Non-viable Cabbage Seeds

Seeds leak many compounds during the early phases of germinationand seed viability may be associated with differential leakageof specific compounds. Leakage of a fluorescent compound fromnon-viable cabbage (Brassica oleracca var. capitata L.) wasdocumented and studies were performed to identify the fluorescentcompound. Imbibed samples of both heat-killed (HK) and viablecabbage seeds were submerged in a viscous colloidal gel. After2 h to 4 h, a fluorescent halo was observed under U.V. lightaround the heat-killed seed but not around the viable seed.Viable and HK seeds were imbibed in water for 8 h and the pHof the leachate was adjusted to either 7 or 10. The absorptionspectra of leakage from HK seeds revealed peak values at 322nm and 388 nm at pH 7 and 10, respectively. This pattern wasnot observed from viable seed leakage. Two-dimensional paperchromatography was conducted on the HK seed leachate. Four fluorescentspots were observed after development first with BAW (n-butanol:acetic acid: water, 4: 1: 5 by vol.) followed by 6% acetic acid.One of the fluorescent spots (spot 3) was studied further dueto its observed intensity. Sinapine thiocyanate was preparedfrom rapeseed oilmeal and used as a reference sample. Absorptionspectra of spot 3 and sinapine thiocyanate were similar at bothpH 7 and 10. Spot 3 had identical R_F values using three solventsystems and identical colour reactions in five tests comparedto sinapine thiocyanate. It was concluded that sinapine wasthe major compound responsible for the fluorescent leakage fromHK cabbage seeds. Key words: Leachate, viability, Brassica     

Seed Viability Determinations in Cabbage Utilizing Sinapine Leakage and Electrical Conductivity Measurements

A need exists for the development of rapid seed quality teststhat determine viability (defined as the ability to germinate)without the necessity for completion of germination. In cabbage(Brassica oleracea var. capitata L.) the compound sinapine,a choline ester of sinapic acid, has been shown to leak fromheat-killed (HK.) but not from viable seeds. Sinapine leakagewas studied as a more accurate method for identifying viableseeds than the conductivity test. After an 8 h soak, a 26-folddifference in sinapine leakage was detected between HK and viableseeds (as measured by the absorbance at 388 nm at pH 12) comparedto a 4-fold difference measured by electrolyte leakage. Theremainder of the studies were conducted on two seedlots of thesame cultivar. Viability was predicted on the same seed by assessingleakage from individual seeds into the soak water using threemethods; soak water colour, absorbance at 388 nm, and electricalconductivity. This information was compared with the actualgermination of each seed for the two seedlots. For both seedlots,the presence of sinapine in seed soak water (detected eitherby a yellow solution colour at high pH or by high absorbance),identified more seeds that were non-viable than the partitioncoefficient calculated from conductivity measurements (76%,72% and 28% of non-viable seeds were identified, respectively).It is proposed that leakage of sinapine is a better predictorof cabbage seed viability than electrical conductivity. Key words: Seed deterioration, seed leakage, germination prediction