Activation of RAT erythrocyte phosphofructokinase by AMP and by non-physiological concentrations of Cyclic AMP

Summary Cyclic AMP (300µ m) activates phosphofructokinase from dialyzed haemolysates of mature rat erythrocytes. The main conclusions are: a) Cyclic AMP, at pH 7.1 and low concentrations of fructose-6-phosphate, is able to reverse the inhibition produced by different amounts of ATP (up to 1.5mm). b) The cyclic nucleotide is a positive allosteric effector of the enzyme as shown by the displacement of sigmoidal fructose-6-phosphate saturation curve to hyperbolic kinetics in the presence of inhibitory concentrations (1.5mm) of ATP. c) Cyclic AMP has no significant influence as deinhibitor of phosphofructokinase either at pH 7.1 and non-inhibitory levels (0.25mm) of ATP or at pH 8.1 and inhibitory (1.5mm) of non-inhibitory (0.25mm) concentrations of ATP. Similar conclusions were obtained with 300µ m AMP but not at a lower concentration (3µ m) with both nucleotides.The comparison of cyclic AMP results with those obtained under similar concentrations of AMP suggest that cyclic AMP is really only an in vitro modulator of the enzyme from rat erythrocytes, presumably at an AMP regulatory site, since non-physiological concentrations are required to act as deinhibitor.     

Hydrogeochemistry of Lake Gallocanta (Aragón,NE Spain)

Lake Gallocanta has undergone drastic changes during the last thirteen years. Water level changed from a high level (Z_max = 2 m) to total dryness in 1985. From 1986 to 1988 slow refilling occurred. The water volume fluctuations have been studied in relation to climatic variations recorded for that period. Variations in the major dissolved ions were related to water volume fluctuations from data at two different stages, one corresponding to the drying phase and another to the refilling phase. Mineralogical composition of the salts precipitated at different stages was examined by X-ray diffraction. Interstitial water and mineralogical composition of recent sediments were also studied along a transect through the lake.The water column decrease from 1977 to 1985 is related to decreasing annual rainfall (500-250 mm respectively). The refilling in 1986–1988 is due to high annual rainfall (537 mm). In addition to these fluctuations, seasonal changes of the water level between 20 and 50 cm occurred every year.Gallocanta is a Na-Mg-Cl-(SO₄) type lake. During the drying period a typical salt enrichment occurs with linear relationships between TDS, Cl, Na and K. Alkalinity is linearly correlated with Ca at relatively low salinities. As salinity increases a linear relationship between Ca and SO₄ is observed. Minerals formed from the brine are halite, bischofite, epsomite, hexahydrite, mirabilite, gypsum, aragonite, calcite and dolomite. The molar ratio Mg/Ca of the interstitial water changes from 1.5 along the shorelines, where calcite and aragonite precipitate, to 40 in the center of the lake. Sediment cores from the central part of the lake show aragonite in the top layers, magnesian calcite and low proportions of quartz and illite, while at 20 cm depth a high proportion of gypsum is present. In contrast, cores from the shore of the lake are mainly composed of low magnesium calcite in the top layers and low magnesium calcite together high magnesium calcite and dolomite between 30 and 70 cm depth. Gypsum deposits only occur in significant proportions at 80–100 cm depth.The refilling process showed relationships between volume and salt concentration following the Langbein model. The salt mass in solution decreased about 50% from the drying to the refilling phase. However, Mg content decreased about 70 % for the same period, suggesting a contribution of this element to the dolomite formation.     

Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

The damaging effects of high plasma levels of cholesterol in the cardiovascular system are widely known, but little attention has been paid to direct effects on cardiomyocyte function. We therefore aimed at testing the hypothesis that Low Density Lipoprotein (LDL) cholesterol affects calcium dynamics and signal propagation in cultured atrial myocytes. For this purpose, mRNA and protein expression levels were determined by real time PCR and western blot analysis, respectively, and intracellular calcium was visualized in fluo-4 loaded atrial HL-1 myocyte cultures subjected to field stimulation. At low stimulation frequencies all cultures had uniform calcium transients at all tested LDL concentrations. However, 500 µg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.30±0.04 to 0.17±0.02 (p<0.05). Moreover, LDL-cholesterol dose-dependently increased the fraction of alternating and irregular beat-to-beat responses observed when the stimulation interval was shortened. This effect was linked to a concurrent reduction in SERCA2, RyR2, IP3RI and IP3RII mRNA levels. SERCA2 protein levels were also reduced by 43% at 200 µg LDL/mL (p<0.05) and SR calcium loading was reduced by 38±6% (p<0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDL-cholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.7±0.1 mm/s with 500 µg LDL/mL (p<0.05). This coincided with a reduction in Cx40 expression (by 44±3%; p<0.05 for mRNA and by 79±2%; p<0.05 for Cx40 protein at 200 µg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDL-cholesterol destabilizes calcium handling in cultured atrial myocytes subjected to rapid pacing by reducing SERCA2 and Cx40 expression and by slowing the conduction velocity of the calcium signal.     

Assessing the Environmental Impact of Water Consumption by Energy Crops Grown in Spain

The environmental impact of the water consumption of four typical crop rotations grown in Spain, including energy crops, was analyzed and compared against Spanish agricultural and natural reference situations. The life cycle assessment (LCA) methodology was used for the assessment of the potential environmental impact of blue water (withdrawal from water bodies) and green water (uptake of soil moisture) consumption. The latter has so far been disregarded in LCA. To account for green water, two approaches have been applied: the first accounts for the difference in green water demand of the crops and a reference situation. The second is a green water scarcity index, which measures the fraction of the soil‐water plant consumption to the available green water. Our results show that, if the aim is to minimize the environmental impacts of water consumption, the energy crop rotations assessed in this study were most suitable in basins in the northeast of Spain. In contrast, the energy crops grown in basins in the southeast of Spain were associated with the greatest environmental impacts. Further research into the integration of quantitative green water assessment in LCA is crucial in studies of systems with a high dependence on green water resources.     

Influence of community structure on the spatial distribution of critically endangered Dicerandra immaculata var. immaculata (Lamiaceae) at wild, introduced, and extirpated locations in Florida scrub

Community structure at local scales is a major factor controlling population and community dynamics of plant species. Dicerandra immaculata Lakela var. immaculata (Lamiaceae) is a critically endangered plant known only from a few locations in scrub habitat in Florida. Using seven sites where populations of D. immaculata were wild, introduced, and/or extirpated, we sought to answer the following questions: (1) how do habitat characteristics at locations supporting wild D. immaculata plants vary from random locations within the same habitat; (2) how do habitat characteristics differ between wild and extirpated populations; and (3) how do habitat characteristics differ between wild and introduced populations? At locations of wild D. immaculata, community structure had fewer woody stems, shorter understory vegetation, lower percent canopy coverage, and lower percent ground cover of detritus than random locations and locations with extirpated D. immaculata. In addition, bare ground decreased at extirpated locations because other plant species expanded their coverage, water saturation of the soil increased, diversity of shrubs decreased, and composition of the overstory changed compared to that of wild locations. Habitat characteristics associated with introduced plants were more similar to characteristics at randomly chosen locations than those with wild plants. However, introduced plants tended to occupy locations that had drier soil, a higher abundance of conspecifics, and a higher proportion of woody understory plants than that of random locations. Overall, gaps in the canopy and at ground level are likely essential for survival and recruitment of D. immaculata.     

Use of RT-Defective HIV Virions: New Tool to Evaluate Specific Response in Chronic Asymptomatic HIV-Infected Individuals

Background

Generation of new reagents that can be used to screen or monitor HIV-1-specific responses constituted an interesting field in the development of HIV vaccines to improve their efficacy.

Methods

We have evaluated the specific T cell response against different types of NL4-3 virions (including NL4-3 aldrithiol-2 treated, NL4-3/ΔRT and R5 envelopes: NL4-3/ΔRT/ΔEnv[AC10] and NL4-3/ΔRT/ΔEnv[Bal]) and against pools of overlapping peptides (15 mer) encompassing the HIV-1 Gag and Nef regions. Cryopreserved PBMC from a subset of 69 chronic asymptomatic HIV positive individuals have been employed using different techniques including IFN-γ ELISPOT assay, surface activation markers and intracellular cytokine staining (ICS) by flow cytometry.

Results

The differential response obtained against NL4-3 aldrithiol-2 treated and NL4-3/ΔRT virions (25% vs 55%, respectively) allow us to divide the population in three groups: “full-responders” (positive response against both viral particles), “partial-responders” (positive response only against NL4-3/ΔRT virions) and “non-responders” (negative responses). There was no difference between X4 and R5 envelopes. The magnitude of the total responses was higher against NL4-3/ΔRT and was positively correlated with gender and inverse correlated with viral load. On the contrary CD4+ T cell count was not associated with this response. In any case responses to the viruses tended to be lower in magnitude than those detected by the overlapping peptides tested. Finally we have found an increased frequency of HLA-B27 allele (23% vs 9%) and a significant reduction in some activation markers (CD69 and CD38) on T cells surface in responders vs non-responders individuals.

Conclusions

In summary these virions could be considered as alternative and useful reagents for screening HIV-1-specific T cell responses in HIV exposed uninfected people, HIV infected patients and to assess immunogenicity of new prototypes both in vitro and in vaccine trials, by a feasible, simply, effective and low cost assay.     

Seed size,number and strategies in annual plants: a comparative functional analysis and synthesis

Background and AimsPlants depend fundamentally on establishment from seed. However, protocols in trait-based ecology currently estimate seed size but not seed number. This can be rectified. For annuals, seed number should simply be a positive function of vegetative biomass and a negative function of seed size.MethodsUsing published values of comparative seed number as the ‘gold standard’ and a large functional database, comparative seed yield and number per plant and per m² were predicted by multiple regression. Subsequently, ecological variation in each was explored for English and Spanish habitats, newly calculated C-S-R strategies and changed abundance in the British flora.Key ResultsAs predicted, comparative seed mass yield per plant was consistently a positive function of plant size and competitive ability, and largely independent of seed size. Regressions estimating comparative seed number included, additionally, seed size as a negative function. Relationships differed numerically between regions, habitats and C-S-R strategies. Moreover, some species differed in life history over their geographical range. Comparative seed yield per m² was positively correlated with FAO crop yield, and increasing British annuals produced numerous seeds. Nevertheless, predicted values must be viewed as comparative rather than absolute: they varied according to the ‘gold standard’ predictor used. Moreover, regressions estimating comparative seed yield per m² achieved low precision.ConclusionsFor the first time, estimates of comparative seed yield and number for >800 annuals and their predictor equations have been produced and the ecological importance of these regenerative traits has been illustrated. ‘Regenerative trait-based ecology’ remains in its infancy, with work needed on determinate vs. indeterminate flowering (‘bet-hedging’), C-S-R methodologies, phylogeny, comparative seed yield per m² and changing life history. Nevertheless, this has been a positive start and readers are invited to use estimates for >800 annuals, in the Supplementary data, to help advance ‘regenerative trait-based ecology’ to the next level.