The Effect of Divalent Cations on Aggregation of Dictyostelium discoideum

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20–40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl₂ accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 × 10⁻⁵ M progesterone considerably delays it These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg²⁺ or the ionophore and greatly decreased accumulation in the case of the steroid.
Treatment with EDTA and Mg²⁺ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn²⁺ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg²⁺ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca²⁺ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca²⁺.
These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.     

Influence of C18 long chain fatty acids on hydrogen metabolism

During anaerobic treatment, several microorganisms mediate a series of reactions to convert reduced compounds (electron donors) into methane. Inhibitors such as long chain fatty acids (LCFAs) can affect several anaerobic microbial populations and decrease the treatment efficiency. The effects of three C18 LCFAs on hydrogenotrophic methanogens in a flocculated mixed anaerobic culture were assessed in this study. The reaction half-life and the hydrogen versus time profiles were used to characterize the inhibition process. The half-life values and profiles were similar for controls and cultures exposed to LCFAs for 1 h. The hydrogen inhibition was a function of the exposure time and the LCFA concentration except for cultures exposed to stearic acid (SA). A statistical analysis of the reaction half-life for cultures incubated with 1,500 and 2,000 mg L(-1) LCFAs for 48 h, revealed the following inhibition trend: linoleic acid (LA) > oleic acid (OA) > SA. After 48 h of exposure, no clear inhibition trend was observed for cultures inoculated with LCFA mixtures; however, at levels of 1,500 and 2,000 mg L(-1), the reaction half-life values were less than that observed for cultures fed with only LA. Based on the reaction half-life data, all of the LCFAs except SA at threshold levels of approximately 1,500 mg L(-1) inhibited hydrogen metabolism. The greatest inhibition and, hence, the largest amount of accumulated hydrogen was observed in cultures fed with 2,000 mg L(-1) LA and incubated for 48 h.     

Sequence variations in small-subunit ribosomal RNAs of Hartmannella vermiformis and their phylogenetic implications

Evidence of associations between free-living amoebas and human disease has been increasing in recent years. Knowledge about phylogenetic relationships that may be important for the understanding of pathogenicity in the genera involved is very limited at present. Consequently, we have begun to study these relationships and report here on the phylogeny of Hartmannella vermiformis, a free-living amoeba that can harbor the etiologic agent of Legionnaires' disease. Our analysis is based on studies of small-subunit ribosomal RNA genes (srDNA). Nucleotide sequences were determined for nuclear srDNA from three strains of H. vermiformis isolated from the United Kingdom, Germany, and the United States. These sequences then were compared with a sequence previously obtained for a North American isolate by J. H. Gunderson and M. L. Sogin. The four genes are 1,840 bp long, with an average GC content of 49.6%. Sequence differences among the strains range are 0.38%-0.76%. Variation occurs at 19 positions and includes 2 single-base indels plus 14 monotypic and 3 ditypic single-base substitutions. Variation is limited to eight helix/loop structures according to a current model for srRNA secondary structure. Parsimony, distance, and bootstrap analyses used to examine phylogenetic relationships between the srDNA sequences of H. vermiformis and other eukaryotes indicated that Hartmannella sequences were most closely related to those of Acanthamoeba and the alga Cryptomonas. All ditypic sites were consistent with a separation between European and North American strains of Hartmannella, but results of other tests of this relationship were statistically inconclusive.      

Manipulating the folding of membrane proteins: using the bilayer to our advantage

The folding mechanisms of integral membrane proteins have largely eluded detailed study. This is owing to the inherent difficulties in folding these hydrophobic proteins in vitro, which, in turn, reflects the often apparently insurmountable problem of mimicking the natural membrane bilayer with lipid or detergent mixtures. There is, however, a large body of information on lipid properties and, in particular, on phosphatidylcholine and phosphatidylethanolamine lipids, which are common to many biological membranes. We have exploited this knowledge to develop efficient in vitro lipid-bilayer folding systems for the membrane protein, bacteriorhodopsin. Furthermore, we have shown that a rate-limiting apoprotein folding step and the overall folding efficiency appear to be controlled by particular properties of the lipid bilayer. The properties of interest are the stored curvature elastic energy within the bilayer, and the lateral pressure that the lipid chains exert on the their neighbouring folding proteins. These are generic properties of the bilayer that can be achieved with simple mixtures of biological lipids, and are not specific to the lipids studied here. These bilayer properties also seem to be important in modulating the function of several membrane proteins, as well as the function of membranes in vivo. Thus, it seems likely that careful manipulations of lipid properties will shed light on the forces that drive membrane protein folding, and will aid the development of bilayer folding systems for other membrane proteins.     

Pitfalls in Portacath location using the landmark technique: case report

A 34 year old woman diagnosed with breast cancer and liver metastases underwent a left subclavian Portacath insertion. During the procedure, the clinical features and the findings of intra-operative investigations provided conflicting evidence of the catheter position. This report highlights the potential difficulties in establishing long-term central venous access, the limitations of common investigations and safety issues relating to the process of subclavian line insertion.     

Nitrogen Addition Increases Carbon Storage in Soils, But Not in Trees, in an Eastern U.S. Deciduous Forest

Forest ecosystems in most industrialized and agricultural regions receive elevated rates of atmospheric nitrogen (N) deposition from air pollution. To evaluate the effects of excess N deposition on carbon (C) and N cycling, we experimentally added N (as NH₄NO₃) to naturally-occurring, single-species plots of five different tree species that are common in the Northern Hardwood forests of northeastern North America: sugar maple (Acer saccharum Marsh), American beech (Fagus grandifolia Ehrh.), yellow birch (Betula alleghaniensis Britton), eastern hemlock (Tsuga canadensis (L.) Carr), and northern red oak (Quercus rubra L.). The experiment was performed in the Catskill Mountains of southeastern New York State, USA, and used a paired-plot design with six replicate plots per species. After 6 years of treatment, most species showed increases in foliar N concentrations in N-treated plots, but only for maple and birch were those increases statistically significant. No significant effects of the N treatment were observed on woody biomass increment or aboveground net primary production (ANPP) for any species. In the oak plots, the N treatment increased acorn production in mast years. In the soils, the N treatment was associated with a significant decline in potential N mineralization and nitrification rates in the mineral horizon but not in the forest floor, and in the mineral horizon the effect of the N treatment varied among species. The N treatment caused a significant increase in C stock, N stock and C:N ratio in the forest floor, with the largest effect in the hemlock plots. Nitrate leaching increased significantly in treated plots compared to controls. Dissolved organic carbon (DOC) in soil solution was unaffected by the N treatment, but the variation in DOC across plots was correlated with the C stock in the forest floor. These results suggest that the ANPP of these forests is not limited by N availability, but that excess N may cause accumulations of C in the forest floor, particularly in hemlock stands, perhaps through inhibition of decomposition rates or by altering phenolic chemistry of the litter. The magnitude, and sometimes the direction of the N treatment responses varied among species, suggesting that predictions of forest responses to elevated N deposition should take into account spatial and temporal variation in tree species composition.