Sphingosine kinase type 1 promotes estrogen-dependent tumorigenesis of breast cancer MCF-7 cells

The sphingolipid metabolite, sphingosine-1-phosphate (S1P), formed by phosphorylation of sphingosine, has been implicated in cell growth, suppression of apoptosis, and angiogenesis. In this study, we have examined the contribution of intracellular S1P to tumorigenesis of breast adenocarcinoma MCF-7 cells. Enforced expression of sphingosine kinase type 1 (SPHK1) increased S1P levels and blocked MCF-7 cell death induced by anti-cancer drugs, sphingosine, and TNF-alpha. SPHK1 also conferred a growth advantage, as determined by proliferation and growth in soft agar, which was estrogen dependent. While both ERK and Akt have been implicated in MCF-7 cell growth, SPHK1 stimulated ERK1/2 but had no effect on Akt. Surprisingly, parental growth of MCF-7 cells was only weakly stimulated by S1P or dihydro-S1P, ligands for the S1P receptors which usually mediate growth effects. When injected into mammary fat pads of ovariectomized nude mice implanted with estrogen pellets, MCF-7/SPHK1 cells formed more and larger tumors than vector transfectants with higher microvessel density in their periphery. Collectively, our results suggest that SPHK1 may play an important role in breast cancer progression by regulating tumor cell growth and survival.     

Purification and some catalytic properties of phosphoglucomutase from maize leaves

Phosphoglucomutase (EC 2.7.5.1, PGM) was purified to homogeneity from maize (Zea mays L.) leaves. The enzyme had specific activity 11. 7 U/mg protein and molecular mass (determined by gel-chromatography) of 133 +/- 4 kD. The molecular mass of PGM subunits determined by SDS-electrophoresis was 66 +/- 3 kD. The enzyme had Km for glucose-1-phosphate and glucose-1,6-diphosphate of 20.0 +/- 0.9 and 16.0 +/- 0.8 &mgr;M, respectively. Concentrations of glucose-1-phosphate and glucose-1,6-diphosphate above 3 and 0.4 mM, respectively, cause substrate inhibition. The enzyme activity was maximal at pH 8.0 and temperature 35 degreesC. Magnesium ions activate the enzyme and manganese ions inhibit it. 3-Phosphoglycerate is an uncompetitive inhibitor of the enzyme (Ki = 1.22 +/- 0.05 mM). Fructose-6-phosphate, 6-phosphogluconate, and ADP activate PGM, whereas ATP, UTP, and AMP inhibit the enzyme. Citrate was also a potent inhibitor, inhibitory effects of isocitrate and cis-aconitate being less pronounced.     

Urinary excretion of biologically active chorionic gonadotrophin by the pregnant marmoset (Callithrix jacchus jacchus).

No significant correlation exists between the amount of biologically active marmoset chorionic gonadotrophin (mCG) in urine and results obtained with an immunological pregnancy test. The pregnant marmoset excretes large amounts of oestrogenic steroids, which must be removed, to prevent the enhancement of the response of the bioassay for mCG. More than 99% of these unconjugated and conjugated urinary oestrogens can be removed by extraction with acetone and ether. mCG is excreted throughout pregnancy, maximum levels occurring between the 8th and 9th week of gestation. There is a considerable within- and between-animal variation in the amount of mCG excreted. However, the pattern of gonadotrophin excretion by the pregnant marmoset is similar to that of man and the apes but unlike that of baboons and macaques.     

The synthesis of S-adenosylmethionine by mutants with defects in S-adenosylmethionine synthetase

Summary Some metK mutants of Salmonella typhimurium with constitutive methionine biosynthesis have no detectable S-adenosylmethionine (SAM) synthetase, the enzyme which converts methionine to SAM, the postulated corepressor of the methionine pathway. However these mutants are not auxotrophic for SAM, an essential compound for many reactions. Here it is shown that these mutants have normal functioning of pathways involving SAM and do in fact produce SAM at as high levels as wild-type. Also, SAM synthetase is shown to be dispensible for growth but not for methionine regulation. These results indicate that there is another route of SAM synthesis independent of SAM synthetase. This route probably also uses methionine as substrate as metK mutants are shown to convert methionine to SAM as efficiently as analogous non-metK strains. The existence of a second route of SAM synthesis makes it necessary to postulate a compartmentalization of SAM made via the SAM synthetase reaction from SAM made in any other way to explain the reduced ability of metK mutants to repress methionine biosynthesis.     

Effect of total dissolved solids and irradiance on growth and toxin production by Nodularia spumigena

The objective of this work was to determine the influence of total dissolved solids/salinity (TDS mgL^-1) on growth and biomass specific rates of nodularin (hepatotoxin) production by Nodularia spumigena 001E isolated from Lake Alexandrina, South Australia. Maximum biomass yield (dry matter, chlorophyll a and particulate organic carbon/POC) at 80 μmol photon m^-2 s^-1 was recorded at 3300 mg TDS L^-1 and decreased at salinities above or below this value (p < 0.05). The maximum biomass yield (dry matter and chlorophyll a) at 30 μmol m^-2 s^-1 occurred at a higher salinity of 9900 mg TDS L^-1. Cultures grown at 80 μmol m^-2 s^-1, at a TDS> 6600 mg L^-1, had significantly (p < 0.05) lower nodularin content (ml^-1 medium) than cultures grown at the same salinities at 30 μmolm^-2 s^-1. The maximum total toxin concentration (mL^-1 medium) occurred at 9900 and 3300 mg TDS L^-1 at 30 μmol m^-2 s^-1and 80 μmol m^-2 s^-1 respectively. Toxin per unit biomass, expressed as dry matter, chlorophyll a and POC was similar for cultures grown at 30 μmol m^-2 s^-1 or 80 μmol m^-2s^-1 at salinities < 6600 mg TDS L^-1. At salinities > 9900 mg TDS L^-1 the toxin content per unit biomass decreased at both irradiances, however, cultures grown at 30 μmol m^-2s^-1 had a higher toxin content than those grown at 80 μmol m^-2 s^-1. The results indicate that not only do changes in irradiance and salinity directly influence growth and toxin production but that changes in irradiance affected the influence of salinity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ligand-linked stability of mutants of the C-domain of calmodulin

There is a necessary energetic linkage between ligand binding and stability in biological molecules. The critical glutamate in Site 4 was mutated to create two mutants of the C-domain of calmodulin yielding E140D and E140Q. These proteins were stably folded in the absence of calcium, but had dramatically impaired binding of calcium. We determined the stability of the mutant proteins in the absence and presence of calcium using urea-induced unfolding monitored by circular dichroism (CD) spectroscopy. These calcium-dependent unfolding curves were fit to models that allowed for linkage of stability to binding of a single calcium ion to the native and unfolded states. Simultaneous analysis of the unfolding profiles for each mutant yielded estimates for calcium-binding constants that were consistent with results from direct titrations monitored by fluorescence. Binding to the unfolded state was not an important energetic contributor to the ligand-linked stability of these mutants.     

Stable Isotope Analysis of Amphidromous Hawaiian Gobies Suggests Their Larvae Spend a Substantial Period of Time in Freshwater River Plumes

Synopsis We employed stable isotope analysis (δ¹³C, δ¹⁵N) to evaluate the sources of nutrients used by amphidromous gobiid fishes (Lentipes concolor, Sicyopterus stimpsoni, Awaous guamensis) caught migrating into and living in Hakalau Stream, Hawaii. Although considerable variation amongst the stable isotope values of stream items was noted across all 4 years of our study, the relationships between the fishes were relatively constant. Stable isotope values of recruiting gobies were consistently closer to those of both inshore plankton and freshwater adults than those of offshore plankton, suggesting that the larvae of these species derive much of their nutrition from inshore environments influenced by fresh water. Small differences between the stable values of these species further suggested that their larvae come from different inshore locations. After entering fresh water all species appear to swim rapidly upstream without feeding. Finally, once well upstream, adult L. concolor and A. guamensis appear to assume an omnivorous diet while adult S. stimpsoni rely upon autochthonous production within streams. We propose that freshwater food webs play an integral yet complex role in the lives of both larval and adult amphidromous Hawaiian fishes.     

Intrapopulation variation in gray wolf isotope (δ<Superscript>15</Superscript>N and δ<Superscript>13</Superscript>C) profiles: implications for the ecology of individuals

In many organisms reproductive success is strongly dependent on several breeding site characteristics, which often vary in space and time. Although we have a good understanding of how ovipositing organisms respond to single factors, we still have little information about how they respond under more complex natural conditions. We examined the oviposition behavior of a tree-hole breeding frog, Phrynobatrachus guineensis, with respect to abiotic and biotic oviposition site characteristics, including desiccation risk and the presence of conspecific offspring using both observation and experiments. Based on daily monitoring data, compiled from 69 natural oviposition sites during a complete reproductive season, we developed oviposition site-selection models. A model based on water presence, sediment depth and maximal possible water depth showed the best predictive performance and was transferable to the subsequent season. Field observations and experiments revealed that frogs could estimate water-holding capacity of sites and timed oviposition with respect to future water presence. Despite the negative effects on larval growth and the availability of sites without conspecifics, data suggest that ovipositing individuals are attracted to conspecific offspring because they serve as a cue for low predation risk. Our results imply that a sites potential for being used at least once for oviposition was determined by abiotic factors, whereas the relative use of breeding sites was determined by a response to conspecifics. Our study demonstrates the importance of including multiple biotic and abiotic factors in the analysis of oviposition site-selection.