The functional characteristics of a human apolipoprotein E variant (cysteine at residue 142) may explain its association with dominant expression of type III hyperlipoproteinemia.

Type III hyperlipoproteinemia typically is associated with homozygosity for apolipoprotein (apo) E2(Arg158----Cys). Dominant expression of type III hyperlipoproteinemia associated with apoE phenotype E3/3 is caused by heterozygosity for a human apoE variant, apoE3(Cys112----Arg, Arg142----Cys). However, this apoE3 variant was not separable from the normal apoE3 in these patients' plasma because the two proteins have identical amino acid composition, charge, and molecular weight. Therefore, to determine the functional characteristics of this protein, we used recombinant DNA techniques to produce this apoE variant in bacteria. We also produced a non-naturally occurring variant, apoE(Arg142----Cys), that had only the cysteine substituted at residue 142. These two apoE variants were purified from cell lysates of the transfected Escherichia coli by ultracentrifugal flotation in the presence of phospholipid, by gel filtration chromatography, and by heparin-Sepharose chromatography. Both Cys142 apoE variants bound to lipoprotein receptors on human fibroblasts with only about 20% of normal binding activity. Therefore, cysteine at residue 142, not arginine at residue 112, is responsible for the decreased receptor binding activity of the variants. Cysteamine treatment and removal of the carboxyl-terminal domain had little effect on the binding activity, whereas both modulate the receptor binding activity of apoE2(Arg158----Cys). The mutation at residue 142 decreased the binding activity of apoE to both heparin and the monoclonal antibody 1D7 (this antibody inhibits receptor binding of apoE), whereas apoE2(Arg158----Cys), which is associated with recessive expression of type III hyperlipoproteinemia, binds normally to both. The Arg112, Cys142 variant predominantes 3:1 over normal apoE3 in the very low density lipoproteins of plasma from an affected subject, as assessed by differential reactivity with the antibody 1D7. The unique combination of functional properties of the Arg112, Cys142 variant provides a possible explanation for its association with dominant expression of type III hyperlipoproteinemia.     

Effect of Light on Biomass and Community Structure of Estuarine Detrital Microbiota

Comparison of estuarine detrital microbiota grown with and without light in the absence of macroscopic grazing showed shifts in the community structure that enabled correlation between various biochemical measures. Analysis of these biochemical measures showed that growth in light induces the smallest increases in procaryotic attributes such as muramic acid; wall glucosamine; lipid phosphate; total extractable adenosine nucleotides; short-branched, cyclopropane, and cis-vaccenic fatty acids; lipid glucose and mannose; the incorporation of acetate into lipid; and the formation of deoxyribonucleic acid from thymidine. Measures of the microfauna such as lipid inositol and the γ-linolenic series of polyenoic fatty acids also increased minimally in the light-grown microbiota. Measures of sulfo-lipid synthesis, lipid glycerol, total extractable palmitate, 18-carbon polyenoic fatty acids, and total polyenoic fatty acids longer than 20 carbons increased 10- to 15-fold in algae and fungi. Chlorophyll a, lipid galactose, and the 16- and 20- carbon polyenoic fatty acids characteristic of diatoms increased maximally in the light. This increase of diatom measure correlated with the sheets of diatoms detected by scanning electron microscopy.     

Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice

Mature mouse sperm cells incubated in an isotonic buffer with cloned DNA capture DNA molecules over a 15 min period. Spermatozoa incubated with pSV2CAT plasmid in either circular or linear form were used to fertilize mouse eggs in vitro. Sequences complementary to pSV2CAT were identified in approximately 30% of 250 progeny by Southern blotting. A genomic library was constructed from the DNA of a positive mouse. Three positive clones were identified and two adjacent HincII restriction fragments of 240 and 370 bp showed identical sequences to the corresponding fragments of the pSV2CAT plasmid. F1 progeny showed paternal and maternal transmission of the transgenes from founders. CAT gene expression was detected on tissues of adult F1 individuals, preferentially on tails and muscle. We conclude that transgenic mice can be obtained using sperm cells as foreign DNA vectors.     

Tolerance,safety and efficacy of Hedera helix extract in inflammatory bronchial diseases under clinical practice conditions: A prospective,open, multicentre postmarketing study in 9657 patients

In this postmarketing study 9657 patients (5181 children) with bronchitis (acute or chronic bronchial inflammatory disease) were treated with a syrup containing dried ivy leaf extract. After 7 days of therapy, 95% of the patients showed improvement or healing of their symptoms. The safety of the therapy was very good with an overall incidence of adverse events of 2.1% (mainly gastrointestinal disorders with 1.5%). In those patients who got concomitant medication as well, it could be shown that the additional application of antibiotics had no benefit respective to efficacy but did increase the relative risk for the occurrence of side effects by 26%. In conclusion, it is to say that the dried ivy leaf extract is effective and well tolerated in patients with bronchitis. In view of the large population considered, future analyses should approach specific issues concerning therapy by age group, concomitant therapy and baseline conditions.     

Muramylpeptide shedding modulates cell sensing of Shigella flexneri

Bacterial infections trigger the activation of innate immunity through the interaction of pathogen-associated molecular patterns (PAMPs) with pattern recognition molecules (PRMs). The nucleotide-binding oligomerization domain (Nod) proteins are intracellular PRMs that recognize muramylpeptides contained in peptidoglycan (PGN) of bacteria. It is still unclear how Nod1 physically interacts with PGN, a structure internal to the Gram-negative bacterial envelope. To contribute to the understanding of this process, we demonstrate that, like Escherichia coli , Bordetella pertussis and Neisseria gonorrheae , the Gram-negative pathogen Shigella spontaneously releases PGN fragments and that this process can be increased by inactivating either ampG or mppA , genes involved in PGN recycling. Both Shigella mutants, but especially the strain carrying the mppA deletion, trigger Nod1-mediated NF-κB activation to a greater extent than the wild-type strain. Likewise, muramylpeptides spontaneously shed by Shigella are able per se to trigger a Nod1-mediated response consistent with the relative amount. Finally, we found that qualitative changes in muramylpeptide shedding can alter in vivo host responses to Shigella infection. Our findings support the idea that muramylpeptides released by pathogens during infection could modulate the immune response through Nod proteins and thereby influence the outcome of disease.     

Chronobiologic blood pressure assessment: maturation of the daily rhythm in newborn foals

Circadian rhythmicity of physiological processes in animals has been described for some variables. In order to investigate the daily rhythmicity of blood pressure, seven foals (Equus caballus) were used for 40 days after birth. Measurements of blood pressure were done by means of an oscillometric apparatus (Argus TM-7, Schiller, Barr Switzerland) with the foals in a standing position and the cuff placed around the tail. Blood pressure was recorded twice a day, 1 hour before dawn and 1 hour before dusk for the first 10 days of life, every 2 days from the 11th to the 32nd day, on the 36th and on the 40th day. Two-way repeated measures analysis of variance (ANOVA) were used to determine statistically significant differences between the mean values recorded at dawn and dusk during the study. Analysis of variance showed a significant effect of time (p<0.0001), during the first 40 days of life. This study confirms a strong correlation between age and blood pressure in horse and the absence of blood pressure maturation in foals 40 days old. The results on the maturation of the daily rhythm of blood pressure in foals is a contribution to the study of the development of circadian rhythms in mammals. These results are also useful for the chronophysiological assessment of blood pressure in the horse.     

The recycling of apolipoprotein E in macrophages: influence of HDL and apolipoprotein A-I

The ability of apolipoprotein E (apoE) to be spared degradation in lysosomes and to recycle to the cell surface has been demonstrated by our group and others, but its physiologic relevance is unknown. In this study, we characterized apoE recycling in primary murine macrophages and probed the effects of HDL and apoA-I on this process. In cells pulsed with (125)I.apoE bound to VLDL, intact apoE was found in the chase medium for up to 24 h after the pulse. Approximately 27 +/- 5% of the apoE internalized during the pulse was recycled after 4 h of chase. Addition of apoA-I and HDL increased apoE recycling to 45 +/- 3% and 46 +/- 3%, respectively, similar to the amount of apoE recycled after pulsing the cells with (125)I.apoE.HDL. In addition, apoA-I-producing macrophages from transgenic mice showed increased apoE recycling at 4 h (38 +/- 3%). Increased ABCA1 expression potentiated apoE recycling, suggesting that recycling occurs via ABCA1. Finally, in the presence of apoA-I, recycled apoE exited the cells on HDL-like particles. These results suggest that apoE recycling in macrophages may be part of a larger signaling loop activated by HDL and directed at maximizing cholesterol losses from the cell.     

Comparative analysis of response to phenotypic and marker-assisted selection for multiple lateral branching in cucumber (Cucumis sativus L.)

Yield increase in processing cucumber ( Cucumis sativus L.) is positively correlated with an increase in number of fruit-bearing branches. Multiple lateral branching (MLB) is a metric trait controlled by at least five effective factors. Breeding efficacy might be improved through marker-assisted selection (MAS) for MLB. Experiments were designed to independently confirm previously determined linkage of molecular markers (L18-2-H19A SNP, CSWTAAA01 SSR, CSWCT13 SSR, W7-2 RAPD and BC-551 RAPD) to MLB, and to determine their utility in MAS. These markers were present in significantly higher frequency than expected (1, presence:3, absence; p < 0.001) in BC(2) plants selected based on a high MLB phenotype (BC(2)PHE). However, markers that were considered selectively neutral fit the expected segregation of donor parent DNA in BC(2) progeny. Markers linked to MLB were used in MAS of BC(1) and BC(2) plants to produce BC(2)MAS, and BC(3)MAS progeny. Means for MLB in MAS populations were compared with backcross populations developed through phenotypic selection (BC(2)PHE, BC(3)PHE) and by random mating where no selection had been applied (BC(2)RND, BC(3)RND). Statistical analysis showed no significant differences ( p < 0.001) between means of phenotypic (BC(2)PHE = 3.02, BC(3)PHE = 3.29) and marker-aided selection (BC(2)MAS = 3.12, BC(3)MAS = 3.11) for MLB. However, both phenotypic and MAS population means were significantly higher than the random control (BC(2)RND = 2.27, BC(3)RND = 2.41) for MLB. Thus, given the observed response to selection and the rapid life-cycle of cucumber (4 months), markers linked to MLB when used in MAS will most likely be effective tools in cucumber improvement.