Lipid polymorphism and hydrocarbon order

The use of 2H nuclear magnetic resonance for the characterization of the polymorphic behavior of lipids is illustrated. Different lipid phase preferences may be expected to influence the orientational order and its variation along the acyl chains. Several results are presented to support that view. An increase of motional freedom and a redistribution of the order along the acyl chains are observed during the lamellar-to-hexagonal phase transition, showing that the order profile is sensitive to the lipid phase symmetry. In addition, if the preferences for nonlamellar phases are not expressed explicitly, the presence of "nonbilayer" lipids constrained in bilayer environment induces increased hydrocarbon order. This suggests that order parameters of the acyl chains and lipid polymorphic tendencies are intimately related.     

Monoclonal antibodies against histone H5. Epitope mapping and binding to chromatin

Monoclonal antibodies against chicken erythrocyte histone H5 were produced. Nine hybridomas of different clonal origin were selected, and the antibodies were purified by affinity chromatography. Typing of the antibodies indicated that all but one (IgM) belong to the IgG1 class and contain kappa light chains. Indirect immunoprecipitation, solid-phase radioimmunoassay, and competitive inhibition assays using various H5 fragments revealed that the antigen-binding sites were localized on the central region of H5 (GH5, residues 22-100). Results of immunoblots from gels containing different denaturing agents indicate that some of the antibodies recognize related continuous epitopes localized at the junction of the GH5 with the rest of the molecule. Competition experiments between pairs of the eight different IgGs suggest that they recognize at least seven distinct sites on GH5. The epitopes appear to represent different regions of GH5 although some of them overlap. In general, the antibodies recognize epitopes which are not too accessible to the environment in the native conformation of the histone. All of the antibodies examined, except one of them (5H10), react with nuclei and chromatin from the erythroid cells but not from other cell lines. The site recognized by 5H10 is likely to be one of the regions where GH5 interacts with the nucleosome. No cross-reactivity of the antibodies with other histones including H1, H2A, H2B, H3, H4, and rat liver histone H1(0) was observed.     

Repair of damage in double-stranded phi X174 (RF) DNA due to radiation-induced water radicals

Experiments in which the yields of radiation-induced OH and H radicals were varied, showed that both types of water radicals inactivate phi X174 RF DNA to about the same extent as measured by transfection of the (irradiated) DNA to E. coli wild-type spheroplasts. On the other hand, using spheroplasts prepared from E. coli strains, deficient in one of the proteins involved in excision DNA repair (uvrA- or uvrC-) or in post-replication repair (recA-), clear differences between damage originating from OH or H radical attack were found. Part of the radiation damage due to H radicals appeared to be repairable by an uvrA-gene-dependent repair mechanism, whereas this repair pathway does not play an important role in the case of OH radical damage. The reverse applies to uvrC-gene-dependent repair, which only affects OH radical damage (obtained under anoxic conditions), but has no influence on damage due to H radicals. Irradiation of double-stranded phi X174 (RF) DNA in the presence of oxygen however, yields damage--due to OH radicals only--which appeared not to be sensitive to either uvrC- or uvrA-gene-dependent repair. Furthermore, post-replication repair (recA) has only very little effect on the amount of inactivation by H or OH radicals, when irradiation is carried out under anoxic conditions. We did not find significant inactivation due to hydrated electrons, whether the biological activity was determined by use of wild-type spheroplasts or of strains deficient in excision or post-replication repair proteins.     

Modulation of the orientational order profile of the lipid acyl chain in the Lα phase

The orientational order profile along the lipid acyl chain has been characterized under several different conditions of polar headgroup composition, temperature, and cholesterol content. Despite the different nature of these factors, the variation of the order is governed by two common trends. First, the relative change of order induced by the variation of these factors is always more pronounced towards the end of the chain than for the methylene groups near the interface. Second, there is, to a first approximation, a distinct correlation between the magnitude of the order parameters and the shape of the order profile. For example when the chain is highly ordered, the relative width of the order distribution is narrow indicating that the plateau region is longer. These conclusions suggest that the orientational order profile depends on only a small number of parameters and demonstrate clearly that the correlation length for changes in orientational order is much greater than one C-C bond length. Our results also show that the reduced temperature is not related in simple terms to orientational order and probably has little theoretical significance. The orientational order profiles of POPC and POPE bilayers are significantly different even when expressed in terms of reduced temperature. The behavior of POPC/cholesterol systems also indicates that the orientational order of the lipid chain and the gel-to-liquid crystalline phase transition temperature are not related in a straightforward manner.Abbreviations POPC 1-palmitoyl-2-oleoyl-phosphatidylcholine - POPE 1-palmitoyl-2-oleoyl-phosphatidylethanolamine - PC phosphatidylcholine - PE phosphatidylethanolamine - NMR nuclear magnetic resonance - EDTA ethylenediaminetetraacetic acid Offprint requests to: M. Bloom     

Osmotic and pH transmembrane gradients control the lytic power of melittin.

Transmembrane osmotic gradients applied on large unilamellar 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles were used to modulate the potency of melittin to induce leakage. Melittin, an amphipathic peptide, changes the permeability of vesicles, as studied using the release of entrapped calcein, a fluorescent marker. A promotion of the ability of melittin to induce leakage was observed when a hyposomotic gradient (i.e., internal salt concentration higher than the external one) was imposed on the vesicles. It is proposed that structural perturbations caused by the osmotic pressure loosen the compactness of the outer leaflet, which facilitates the melittin-induced change in membrane permeability. Additionally, we have shown that this phenomenon is not due to enhanced binding of melittin to the vesicles using intrinsic fluorescence of the melittin tryptophan. Furthermore, we investigated the possibility of using a transmembrane pH gradient to control the lytic activity of melittin. The potency of melittin in inducing release is known to be inhibited by increased negative surface charge density. A transmembrane pH gradient causing an asymmetric distribution of unprotonated palmitic acid in the bilayer is shown to be an efficient way to modulate the lytic activity of melittin, without changing the overall lipid composition of the membrane. We demonstrate that the protective effect of negatively charged lipids is preserved for asymmetric membranes.     

Correlation between lipid plane curvature and lipid chain order.

The 1-palmitoyl-2-oleoyl-phosphatidylethanolamine: 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE:POPC) system has been investigated by measuring, in the inverted hexagonal (HII) phase, the intercylinder spacings (using x-ray diffraction) and orientational order of the acyl chains (using 2H nuclear magnetic resonance). The presence of 20 wt% dodecane leads to the formation of a HII phase for the composition range from 0 to 39 mol% of POPC in POPE, as ascertained by x-ray diffraction and 2H nuclear magnetic resonance. The addition of the alkane induces a small decrease in chain order, consistent with less stretched chains. An increase in temperature or in POPE proportion leads to a reduction in the intercylinder spacing, primarily due to a decrease in the water core radius. A temperature increase also leads to a reduction in the orientational order of the lipid acyl chains, whereas the POPE proportion has little effect on chain order. A correlation is proposed to relate the radius of curvature of the cylinders in the inverted hexagonal phase to the chain order of the lipids adopting the HII phase. A simple geometrical model is proposed, taking into account the area occupied by the polar headgroup at the interface and the orientational order of the acyl chains reflecting the contribution of the apolar core. From these parameters, intercylinder spacings are calculated that agree well with the values determined experimentally by x-ray diffraction, for the variations of both temperature and POPE:POPC proportion. This model suggests that temperature increases the curvature of lipid layers, mainly by increasing the area subtended by the hydrophobic core through chain conformation disorder, whereas POPC content affects primarily the headgroup interface contribution. The frustration of lipid layer curvature is also shown to be reflected in the acyl chain order measured in the L alpha phase, in the absence of dodecane; for a given temperature, increased order is observed when the curling tendencies of the lipid plane are more pronounced.     

Pan-Arctic soil moisture control on tundra carbon sequestration and plant productivity

Long-term atmospheric CO₂ concentration records have suggested a reduction in the positive effect of warming on high-latitude carbon uptake since the 1990s. A variety of mechanisms have been proposed to explain the reduced net carbon sink of northern ecosystems with increased air temperature, including water stress on vegetation and increased respiration over recent decades. However, the lack of consistent long-term carbon flux and in situ soil moisture data has severely limited our ability to identify the mechanisms responsible for the recent reduced carbon sink strength. In this study, we used a record of nearly 100 site-years of eddy covariance data from 11 continuous permafrost tundra sites distributed across the circumpolar Arctic to test the temperature (expressed as growing degree days, GDD) responses of gross primary production (GPP), net ecosystem exchange (NEE), and ecosystem respiration (ER) at different periods of the summer (early, peak, and late summer) including dominant tundra vegetation classes (graminoids and mosses, and shrubs). We further tested GPP, NEE, and ER relationships with soil moisture and vapor pressure deficit to identify potential moisture limitations on plant productivity and net carbon exchange. Our results show a decrease in GPP with rising GDD during the peak summer (July) for both vegetation classes, and a significant relationship between the peak summer GPP and soil moisture after statistically controlling for GDD in a partial correlation analysis. These results suggest that tundra ecosystems might not benefit from increased temperature as much as suggested by several terrestrial biosphere models, if decreased soil moisture limits the peak summer plant productivity, reducing the ability of these ecosystems to sequester carbon during the summer.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Influence of anoxic sensitizers on the radiation damage in biologically active DNA in aqueous solution

The competition between biologically active single-stranded phiX174 DNA and the anoxic radiosensitizers metronidazole, misonidazole, paranitroacetophenone or nifuroxime for OH radicals is studied. The results are compared with experiments in which the protection of the DNA by t-butanol is determined. Also the effects of the sensitizers on the chemical nature of the damage (immediate and potential break, immediate and potential base damage) is studied. It is found that in diluted aqueous solutions of DNA these radiosensitizers do not sensitize with respect to the biological inactivation. The only effect observed is a shift from potential to immediate breaks with misonidazole and also nifuroxime.     

Inosine synthesis and phosphoribosylpyrophosphate availability in rat liver cells in the presence of allopurinol

In the presence of allopurinol, apparent phosphoribosylpyrophosphate (PP-ribose-P) availability as measured by adenine incorporation into ribonucleotides was decreased in rat liver cells, hypoxanthine incorporation into ribonucleotides was increased, and there was a large synthesis of inosine from hypoxanthine. Inosine was formed directly by the reversal of the purine nucleoside phosphorylase reaction which was very rapid in liver cells. We tested the hypothesis that utilization of ribose 1-phosphate for inosine synthesis could decrease PP-ribose-P availability. Our results indicate that the apparent decrease of PP-ribose-P availability in the presence of allopurinol was due to competition between adenine and hypoxanthine salvage pathways into nucleotides, and not to the synthesis of inosine.