Search

A proteomic approach was employed to elucidate possible differential expression of native and oxidized glycoproteins using pooled plasma samples derived from ten patients with sporadic Alzheimer's disease (AD) and pooled plasma samples from nine normal elderly control (NEC) subjects. The plasma samples were fractionated by sequential affinity chromatography on heparin-agarose (HepA) and concanavalin A-agarose (ConA) columns followed by separation on one-dimensional and two-dimensional polyacrylamide gels. Carbonylation (oxidation) of proteins was monitored by in-strip derivatization with 2,4-dinitrophenylhydrazine (DNP) and anti-DNP immunoblotting. Nine spots representing glycoproteins which showed enrichment or high specific oxidation indices in AD HepA-ConA 2-D gels relative to NEC samples were analyzed by matrix-assisted laser desorption-time of flight-mass spectrometry and identified with high probability (p < 0.001) as isoforms of human transferrin (Tf), hemopexin (Hpx) and alpha-1-antitrypsin (alpha-1-AT). These glycoproteins were concentrated, respectively, 5-, 6.5- and 107-fold in HepA-ConA eluates derived from AD plasma relative to the NEC samples. Specific oxidation indices of the identified Tf and Hpx isoforms in AD plasma were respectively, 7.4 and 2.8 relative to NEC. Our findings provide further evidence for systemic derangements in heme/iron/redox homeostasis and activation of the acute phase response in sporadic AD. Moreover, the data implicate isoforms of Tf, Hpx and alpha-1-AT as potential biological markers of this condition.

Productions de TNF, IL-6, IL-12 et IFN-γ chez des cellules immunocompetentes traitees avec des exopolysaccharides du Lactobacillus rhamnosus RW-9595M. Difference entre les reponses de cellules de sang peripherique humain et de splenocytes de souris. Les exopolysaccharides (EPS) du Lactobacillus rhamnosus RW- 9595M ont ete prepares a partir de cultures bacteriennes, isoles, concentres, fractionnes et testes in vitro quant a leur potentiel immunomodulateur sur des splenocytes de souris C57B1/6 et BALB/c, sur la lignee macrophagique murine RAW.264.7 et sur des cellules mononuclees du sang humain provenant de 14 donneurs. Nous avons fractionne ces EPS en fonction des tailles moleculaires, avec des membranes filtrantes de differentes porosites. Les fractions correspondent aux tailles suivantes: F1: > 1000 kg.mol -1 , F2: 1000-100 kg.mol -1 , F3: 100-10 kg.mol -1 et F4: < 10 kg.mol -1 . Les EPS concentres, non fractionnes, ainsi que F1, se sont averes legerement mitogenes sur des splenocytes de la souris, ou des cellules sanguines humaines, cultivees 2-3 jours. Dans le dernier cas, F3 manifeste egalement cette propriete. Les EPS non fractionnes, ainsi que F1, sont capables de provoquer la production de TNF, IL-6, IL-12p40 apres 6 ou 24 h de culture, l'origine des cellules etant importante pour l'intensite de la reponse. Apres 24, 48 et 72 h de culture, les EPS non fractionnes, ainsi que F1, ont provoque la production d'interferon gamma par des splenocytes de souris C57B1/6 et BALB/c. Au contraire, dans le cas des cellules sanguines humaines, aucune production d'interferon gamma n'a pu etre observee, en depit d'une bonne reponse en IL-12p40. Ces resultats suggerent que les EPS de bacteries lactiques peuvent stimuler certaines fonctions immunitaires chez des sujets aptes a repondre.

In vitro translation products from two tomato cultivars, Lycopersicon esculentum cv. ‘Early Cherry’ (‘EC’) and L. esculentum x L. pimpinellifolium cv. ‘New York 280’ (‘NY’), differing in chilling tolerance, were compared before and after 16 days of chilling at 4 °C, and after return of the fruit to 20 °C for one and five days. Ripening parameters, color and texture, were monitored during the experiments. Color development showed normal ripening of chill-tolerant ‘NY’ after the fruit were returned to 20 °C, but failure of chillsensitive ‘EC’ to ripen. Before chilling, slight differences in the patterns of translation products resolved by 2D-PAGE were observed between the two cvs. probably reflecting their different genetic background. Chilling induced changes in gene expression that were for a large part similar in fruit of both cvs. However, among responses that differed in the two cvs., changes in the levels of two low-molecular-weight basic translation products identified as ‘A’ (ca. 18 kDa, pH 8.0) and ‘B’ (ca. 14 kDa, pH 8.2) were most noticeable. Translation product ‘A’, faintly visible in ‘NY’ before chilling, was strongly expressed after 16 days of chilling and 24 h after transfer to 20 °C, but had disappeared five days after transfer to 20 °C. Translation product ‘A’ also increased in abundance in ‘EC’ at low temperature and was retained at appreciable levels five days after the fruit were returned to 20 °C. Translation product ‘B’ highly expressed in chilling-tolerant ‘NY’ before chilling, was sharply reduced in chilled fruit. This change was readily reversible upon transfer to 20 °C. By contrast, the level of polypeptide ‘B’ remained low in ‘EC’ under all treatments.

Summary Pericarp lipids of mature-green tomato fruits of three cultivars of Lycopersicon esculentum Mill., and one of L. esculentum × pimpinellifolium were analyzed to clarify the relationship between membrane lipid composition and chilling sensitivity. Only the hybrid cultivar, ( ), ripened normally at 20 °C after 16 d of chilling at 4 °C. At low temperature phospholipid content was maintained, while galactolipid content markedly declined in the four cultivars. Phosphatidylcholine content and the 18:3 content of phosphatidylcholine decreased slightly in while they increased slightly in sensitive ( ) during chilling. Before chilling, the molar ratio of di- to monogalactosyldiacylglycerol of chilling-tolerant was double that of the sensitive cultivars. This ratio increased only in during chilling. Diacylglycerol content decreased at 4 °C, while free fatty acid content and the % of 16:0, 18:0 and 18:1 in the free fatty acid fraction increased. The low degree of unsaturation of diacylglycerol and free fatty acids suggests that they were not the products of galactolipid catabolism during chilling. Phosphatidyl-glycerol fatty acid composition was similar in the four cultivars under all treatments. Taken together, our data indicate that phosphatidylglycerol is not a factor differentiating fruit-chilling sensitivity in tomato cultivars. Galactolipid biosynthesis may be impaired at chilling temperature; sensitive steps may be the acylation of glycerol-3-phosphate and the transfer of phosphatidylcholine diglyceride moieties to monogalactosyldiacylglycerol.

We examined the relationship between reduced galactolipid content in tomato fruit at 4C and chilling injury. Galactolipid biosynthesis from 14C-acetate was compared in pericarp discs of cold-tolerant `New York 280' (`NY') and -sensitive `Early Cherry' (`EC') at 4C and 20C. Labeled lipids were separated by 2D-TLC. Labeled monogalactosyldiglyceride (MGDG) molecular species were hydrolyzed using a position-specific lipase; the fatty acids released were hydrogenated and separated according to chain length by reverse-phase TLC. At 4C, the relative amount of radioactivity was reduced in MGDG and enhanced in phosphatidylcholine (PC) in both cultivars, in comparison with labeling at 20C. In discs from fruit chilled for 6 h, labeling was similar in `NY' and `EC'. In fruit held at 4C for 8 days, labeling of MGDG was reduced and that in PC was enhanced to a greater extent in chilling-sensitive `EC' than in `NY'. The proportion of the MGDG label in eukaryotic species (i.e., the ratio in C18/C16 fatty acids in position sn-2), was less in `EC' at 4C than at 20C, even for fruit held at 4C for only 6 h. The ratio was little affected in `NY'. The data indicate that biosynthesis of eukaryotic MGDG was inhibited in tomato fruit at chilling injury-inducing temperatures.

Translatable mRNAs from two tomato (Lycopersicon esculentum Mill.) cultivars differing in chilling tolerance were compared after 16 days of chilling at 4C and after return to 20C for 1 and 5 days. Before chilling, the translation products, resolved by 2D NEPHGE, showed significant differences between more tolerant `New York 280' (NY) and less tolerant `Early Cherry' (EC). In NY, chilling reduced the level of five to 10 mRNAs and enhanced or induced that of several other mRNAs. After transfer to 20C, the trend was progressively reversed. Changes in the levels of two low-molecular-weight basic peptides were most noticeable. One, absent in NY before chilling, was strongly expressed after chilling and 24 h after transfer to 20C, but disappeared 5 days after transfer. The level of this peptide increased slightly in EC at low temperature and was maintained after transfer to 20C. The level of the other, high in NY before chilling, was sharply reduced after chilling. In contrast, the level of this polypeptide was low in EC under all treatments.