Short-time infusion of fish oil-based lipid emulsions, approved for parenteral nutrition, reduces monocyte proinflammatory cytokine generation and adhesive interaction with endothelium in humans

Potential impact of omega-3 fatty acids, as contained in fish oil, on immunological function has been suggested because observations of reduced inflammatory diseases in Greenland Inuit were published. A fish oil-based lipid emulsion has recently been approved for parenteral nutrition in many countries. We investigated the influence of a short infusion course of fish oil-based (omega-3) vs conventional (omega-6) lipid emulsion on monocyte function. In a randomized design, twelve healthy volunteers received omega-3 or omega-6 lipid infusion for 48 h, with cross-over repetition of the infusion course after 3 mo. Fatty acid profiles, monocyte cytokine release and adhesive monocyte-endothelium interaction were investigated. Resultant omega-6 lipid emulsion increased plasma-free fatty acids including arachidonic acid, whereas the omega-3/omega-6 fatty acid ratio in monocyte membranes remained largely unchanged. It also caused a tendency toward enhanced monocyte proinflammatory cytokine release and adhesive monocyte-endothelium interaction. In contrast, omega-3 lipid emulsion significantly increased the omega-3/omega-6 fatty acid ratio in the plasma-free fatty acid fraction and in monocyte membrane lipid pool, markedly suppressing monocyte generation of TNF-alpha, IL-1, IL-6, and IL-8 in response to endotoxin. In addition, it also significantly inhibited both monocyte-endothelium adhesion and transendothelial monocyte migration, although monocyte surface expression of relevant adhesive molecules (CD11b, CD18, CD49 days, CCR2) was unchanged. Although isocaloric, omega-3 and omega-6 lipid emulsions exert differential impact on immunological processes in humans. In addition to its nutritional value, fish oil-based omega-3 lipid emulsion significantly suppresses monocyte proinflammatory cytokine generation and features of monocyte recruitment.     

Clonal expansions of mitochondrial genomes: implications for in vivo mutational spectra

It is often assumed mutant frequencies, as measured in a DNA sample, faithfully represent basic mutation rates associated with these mutations. This paradigm was extremely helpful for in vitro studies of the mechanisms of mutagenesis/repair and causes of mutations. However, in vivo, mutant fractions appear to vary dramatically and randomly from sample to sample. It's unlikely that basic mutational rates vary so much. Such variations are probably caused by clonal expansions of mutants within tissue. Whether a particular tissue sample includes an expansion or not, is a matter of chance, which explains the observed random fluctuations of mutant fractions. Well-known examples of clonal expansions involve pathological conditions such as cancer or mitochondrial disease. It is less appreciated that even in normal tissue, expansions of somatic mutants create local deviations from the "expected" mutant frequencies. The sizes of clonal expansions appear to span a wide range and thus, may affect samples of various sizes, from individual cells to individuals. In conclusion, human body appears to be a sort of a "gambling ground" for clonally expanding mutants. We speculate that expansion of early mutants rather than de novo mutation at old age may be the major source of at least some aging-specific mutants in our bodies.     

High stability of Discosoma DsRed as compared to Aequorea EGFP

Comparative analysis of conformational stabilities was performed for two widely used genetic reporters, EGFP and DsRed, proteins exhibiting similar beta-can folds, but possessing different oligomeric organization and chromophore structures. Two factors affecting protein stability in vitro, such as elevated temperatures and a chaotropic agent guanidine hydrochloride, were studied. In vivo tolerance of the fluorescence proteins to proteasomal-based degradation was studied in insect and mammalian cells, and in Xenopus embryos. The apparent rate constants of thermal and GdmCl-induced denaturation were several orders of magnitude lower for DsRed than for EGFP. DsRed lifetimes severalfold longer than those of EGFP were observed in cultured cells and in embryos. The remarkable fluorescence stability of DsRed under the all conditions that have been studied is attributed to a significant extent to its tetrameric organization. Therefore, DsRed can be used as a genetic reporter and advanced population marker with a significantly extended intracellular lifespan.     

Omega-3 fatty acids suppress monocyte adhesion to human endothelial cells: role of endothelial PAF generation

Monocyte-endothelium interaction is a fundamental process in many acute and chronic inflammatory diseases. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are fish oil-derived alternative (omega-3) precursor fatty acids implicated in the suppression of inflammatory events. We investigated their influence on rolling and adhesion of monocytes to human umbilical vein endothelial cells (HUVEC) under laminar flow conditions in vitro. Exposure of HUVEC to tumor necrosis factor (TNF-alpha) strongly increased 1) surface expression of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and E-selectin, 2) platelet-activating factor (PAF) synthesis as assessed by thrombin challenge, and 3) rate of rolling and adhesion of monocytes. Preincubation of HUVEC with EPA or DHA markedly suppressed PAF synthesis, monocyte rolling, and adherence, whereas expression of endothelial adhesion molecules was unchanged. Also, PAF receptor antagonists markedly suppressed the adhesion rate of monocytes, and EPA or DHA revealed no additional inhibitory capacity. In contrast, arachidonic acid partially reversed the effect of the antagonist. We conclude that omega-3 fatty acids suppress rolling and adherence of monocytes on activated endothelial cells in vitro by affecting endothelial PAF generation.     

Long-term circulation of vaccine-derived poliovirus that causes paralytic disease

Successful implementation of the global poliomyelitis eradication program raises the problem of vaccination against poliomyelitis in the posteradication era. One of the options under consideration envisions completely stopping worldwide the use of the Sabin vaccine. This strategy is based on the assumption that the natural circulation of attenuated strains and their derivatives is strictly limited. Here, we report the characterization of a highly evolved derivative of the Sabin vaccine strain isolated in a case of paralytic poliomyelitis from a 7-month-old immunocompetent baby in an apparently adequately immunized population. Analysis of the genome of this isolate showed that it is a double (type 1-type 2-type 1) vaccine-derived recombinant. The number of mutations accumulated in both the type 1-derived and type 2-derived portions of the recombinant genome suggests that both had diverged from their vaccine predecessors approximately 2 years before the onset of the illness. This fact, along with other recent observations, points to the possibility of long-term circulation of Sabin vaccine strain derivatives associated with an increase in their neurovirulence. Comparison of genomic sequences of this and other evolved vaccine-derived isolates reveals some general features of natural poliovirus evolution. They include a very high preponderance and nonrandom distribution of synonymous substitutions, conservation of secondary structures of important cis-acting elements of the genome, and an apparently adaptive character of most of the amino acid mutations, with only a few of them occurring in the antigenic determinants. Another interesting feature is a frequent occurrence of tripartite intertypic recombinants with either type 1 or type 3 homotypic genomic ends.     

Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme

A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of His106 has been directly observed by improved TROSY-NMR techniques in the 44 kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of ¹⁵N-¹⁵N and ¹H-¹⁵N trans-hydrogen bond scalar couplings, ^2h J _NN and ^1h J _HN, and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic models.