Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.     

Molecular evolution of a multigene family in group A streptococci

The emm genes are members of a gene family in group A streptococci (GAS) that encode for antiphagocytic cell-surface proteins and/or immunoglobulin-binding proteins. Previously sequenced genes in this family have been named "emm," "fcrA," "enn," "arp," "protH," and "mrp"; herein they will be referred to as the "emm gene family." The genes in the emm family are located in a cluster occupying 3-6 kb between the genes mry and scpA on the chromosome of Streptococcus pyogenes. Most GAS strains contain one to three tandemly arranged copies of emm-family genes in the cluster, but the alleles within the cluster vary among different strains. Phylogenetic analysis of the conserved sequences at the 3' end of these genes differentiates all known members of this family into four evolutionarily distinct emm subfamilies. As a starting point to analyze how the different subfamilies are related evolutionarily, the structure of the emm chromosomal region was mapped in a number of diverse GAS strains by using subfamily-specific primers in the polymerase chain reaction. Nine distinct chromosomal patterns of the genes in the emm gene cluster were found. These nine chromosomal patterns support a model for the evolution of the emm gene family in which gene duplication followed by sequence divergence resulted in the generation of four major-gene subfamilies in this locus.      

RNA editing in eag potassium channels: Biophysical consequences of editing a conserved S6 residue

RNA editing at four sites in eag, a Drosophila voltage-gated potassium channel, results in the substitution of amino acids into the final protein product that are not encoded by the genome. These sites and the editing alterations introduced are K467R (Site 1, top of the S6 segment), Y548C, N567D and K699R (sites 2–4, within the cytoplasmic C-terminal domain). We mutated these residues individually and expressed the channels in Xenopus oocytes. A fully edited construct (all four sites) has the slowest activation kinetics and a paucity of inactivation, whereas the fully unedited channel exhibits the fastest activation and most dramatic inactivation. Editing Site 1 inhibits steady-state inactivation. Mutating Site 1 to the neutral residues resulted in intermediate inactivation phenotypes and a leftward shift of the peak current-voltage relationship. Activation kinetics display a Cole-Moore shift that is enhanced by RNA editing. Normalized open probability relationships for 467Q, 467R and 467K are superimposable, indicating little effect of the mutations on steady-state activation. 467Q and 467R enhance instantaneous inward rectification, indicating a role of this residue in ion permeation. Intracellular tetrabutylammonium blocks 467K significantly better than 467R. Block by intracellular, but not extracellular, tetraethylammonium interferes with inactivation. The fraction of inactivated current is reduced at higher extracellular Mg⁺² concentrations, and channels edited at Site 1 are more sensitive to changes in extracellular Mg⁺² than unedited channels. These results show that even a minor change in amino acid side-chain chemistry and size can have a dramatic impact on channel biophysics, and that RNA editing is important for fine-tuning the channel’s function.     

Relationships between lake ecology and morphological characters in Icelandic Arctic charr,Salvelinus alpinus

The common occurrence of parallel phenotypic patterns suggests that a strong relationship exists between ecological dynamics and micro‐evolution. Comparative studies from a large number of populations under varying sets of ecological drivers could contribute to a better understanding of this relationship. We used data on morphology of arctic charr (Salvelinus alpinus) and ecological factors from 35 Icelandic lakes to test the hypothesis that morphological patterns among monomorphic charr populations from different lakes are related to interlake variation in ecological characteristics. There is extensive phenotypic diversity among populations of Icelandic charr, and populations are easily distinguished based on overall body morphology. The results obtained in the present study showed that the morphological diversity of charr was related to large‐scale diversity in lake ecology. Variation in charr morphology was related to water origin (e.g. spring fed versus run‐off), bedrock age, and fish community structure. The present study shows how various ecological factors can shape the biological diversity that we observe. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 761–771.     

Differentiation of SH-SY5Y cells to a neuronal phenotype changes cellular bioenergetics and the response to oxidative stress

Cell differentiation is associated with changes in metabolism and function. Understanding these changes during differentiation is important in the context of stem cell research, cancer, and neurodegenerative diseases. An early event in neurodegenerative diseases is the alteration of mitochondrial function and increased oxidative stress. Studies using both undifferentiated and differentiated SH-SY5Y neuroblastoma cells have shown distinct responses to cellular stressors; however, the mechanisms remain unclear. We hypothesized that because the regulation of glycolysis and oxidative phosphorylation is modulated during cellular differentiation, this would change bioenergetic function and the response to oxidative stress. To test this, we used retinoic acid (RA) to induce differentiation of SH-SY5Y cells and assessed changes in cellular bioenergetics using extracellular flux analysis. After exposure to RA, the SH-SY5Y cells had an increased mitochondrial membrane potential, without changing mitochondrial number. Differentiated cells exhibited greater stimulation of mitochondrial respiration with uncoupling and an increased bioenergetic reserve capacity. The increased reserve capacity in the differentiated cells was suppressed by the inhibitor of glycolysis 2-deoxy-d-glucose. Furthermore, we found that differentiated cells were substantially more resistant to cytotoxicity and mitochondrial dysfunction induced by the reactive lipid species 4-hydroxynonenal or the reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone. We then analyzed the levels of selected mitochondrial proteins and found an increase in complex IV subunits, which we propose contributes to the increase in reserve capacity in the differentiated cells. Furthermore, we found an increase in MnSOD that could, at least in part, account for the increased resistance to oxidative stress. Our findings suggest that profound changes in mitochondrial metabolism and antioxidant defenses occur upon differentiation of neuroblastoma cells to a neuron-like phenotype.     

Nine new polymorphic microsatellite loci for the amplification of archived otolith DNA of Atlantic cod,Gadus morhua L.

Nine out of 22 microsatellite primers tested were successfully amplified on three samples of cod Gadus morhua L. (two contemporary and one archived otolith samples). All loci were polymorphic (5–23 alleles/locus). The average observed heterozygosity across loci and samples was 0.625, ranging from 0.294 to 0.895 at each locus. All loci were under Hardy–Weinberg equilibrium, except PGmo56 that showed significant excess of heterozygotes in all studied samples. The isolated loci were suitable for degraded DNA and therefore useful for conducting a long‐term temporal study with DNA obtained from archived otoliths of cod.