Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cells

Self-renewing tissues such as the intestine contain progenitor proliferating cells which subsequently differentiate. Cell proliferation and differentiation involve gene regulation processes which take place in the nucleus. A human intestinal epithelial cell line model (Caco2/TC7) which reproduces these dynamic processes has been used to perform proteomic studies on nuclear proteins. Nuclei from Caco2/TC7 cells at proliferative and differentiated stages were purified by subcellular fractionation. After two-dimensional gel electrophoresis separation and ruthenium staining, 400 protein spots were detected by image analysis. Eighty-five spots corresponding to 60 different proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry in nuclei from proliferative cells. Comparison of nuclear proteomes from proliferative or differentiated cells by differential display resulted in the identification of differentially expressed proteins such as nucleolin, hnRNP A2/B1 and hnRNP A1. By using Western blot analysis, we found that the expression and number of specific isoforms of these nuclear proteins decreased in differentiated cells. Immunocytochemistry experiments also showed that in proliferative cells nucleolin was distributed in nucleoli-like bodies. In contrast, hnRNPs A2/B1 and A1 were dispersed throughout the nucleus. This study of the nuclear proteome from intestinal epithelial cells represents the first step towards the establishment of a protein database which will be a valuable resource in future studies on the differential expression of nuclear proteins in response to physiological, pharmacological and pathological modulations.     

Genetic and molecular basis of grass cell wall biosynthesis and degradability. II. Lessons from brown-midrib mutants

The brown-midrib mutants of maize have a reddish-brown pigmentation of the leaf midrib and stalk pith, associated with lignified tissues. These mutants progressively became models for lignification genetics and biochemical studies in maize and grasses. Comparisons at silage maturity of bm1, bm2, bm3, bm4 plants highlighted their reduced lignin, but also illustrated the biochemical specificities of each mutant in p-coumarate, ferulate ester and etherified ferulate content, or syringyl/guaiacyl monomer ratio after thioacidolysis. Based on the current knowledge of the lignin pathway, and based on presently developed data and discussions, C3H and CCoAOMT activities are probably major hubs in controlling cell-wall lignification (and digestibility). It is also likely that ferulates arise via the CCoAOMT pathway.     

Helicoidal pattern in secondary cell walls and possible role of xylans in their construction

The helicoidal organization of secondary cell walls is overviewed from several examples. Both the plywood texture and the occurrence of characteristic defects strongly suggest that the wall ordering is relevant of a cholesteric liquid-crystal assembly that is rapidly and strongly consolidated by lignification. A preferential localization of glucuronoxylans, major matrix components, and in vitro re-association experiments emphasize their preeminent role: (1) during the construction of the composite as directing the cellulose microfibrils in a helicoidal array; (2) during the lignification of the composite as a host structure for lignin precursors.     

A xylan-degrading strain of<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis>: isolation and characterization of the xylanase activity

Two strains (O and X₂) of the hyperthermophilic crenarchaeon Sulfolobus solfataricus strain MT4 were selected and isolated for their ability to grow on xylan. O and X₂, grown on media containing oat spelt xylan and birchwood xylan as the sole nutrient source, respectively, produced the same thermostable xylanase that was demonstrated to be inducible in xylan cultures. In an oat spelt medium, S. solfataricus O underwent interesting morphological changes in the cell envelope, exhibiting mobile appendages not present in the typical coccal shape. The enzyme was prevalently membrane associated and showed a molecular mass of approximately 57.0 kDa. It was also highly thermostable, with a half-life of 47 min at 100°C, and exhibited an optimal temperature and pH of 90°C and 7.0, respectively. Xylo-oligosaccharides were the enzymatic products of xylan hydrolysis, and the smallest degradation product was xylobiose, thus indicating that the enzyme was an endoxylanase. The enzyme was able to bind weakly to crystalline cellulose (Avicel) and more strongly to insoluble xylan in a substrate amount-and temperature-dependent manner.Communicated by G. Antranikian     

Elemental and biochemical composition of<Emphasis Type="Italic"> Nephrops norvegicus</Emphasis> (Linnaeus 1758) larvae from the Mediterranean and Irish Seas

The Norway lobster, Nephrops norvegicus, is a commercially exploited decapod which is widely distributed throughout the north-eastern Atlantic and the Mediterranean Sea. Ovigerous females originating from the Mediterranean and the Irish Seas were held in the laboratory until larvae hatched. Biomass and biochemical composition, as well as digestive gland structure, were examined in newly hatched larvae from these two regions. In addition, previously published data from a North Sea population were included in our comparison. Elemental analyses showed that the absolute quantities of dry mass (DM), carbon (C), nitrogen (N) and hydrogen (H) (collectively referred to as CHN) per individual, and the C:N mass ratios, were significantly lower, while the relative CHN, protein and lipid values (in % of DM) were higher in samples from the Irish Sea compared to larvae originating from either the Mediterranean or the North Sea. As in CHN, the absolute level of protein per individual was higher in larvae from the Mediterranean, while no significant differences were observed in the individual lipid contents. Likewise, the digestive gland structure at hatching did not show any differences between study areas. Intraspecific variability in biomass and chemical composition of newly hatched larvae from different regions may be related to differential patterns of reproduction in regions with different climatic conditions. Lobster larvae hatch in the Mediterranean Sea predominantly in winter when both water temperature and planktonic food availability are at a minimum, while hatching in the Irish Sea occurs under more favourable conditions in spring. Hence, significantly higher wet mass, dry mass and protein values in Mediterranean larvae may represent adaptive traits allowing for early posthatching survival and development under food-limited conditions in an oligotrophic environment.Communicated by H.-D. Franke     

Migration from plant to plant: an important factor controlling densities of the epiphytic cladoceran <Emphasis Type="Italic">Alona</Emphasis> (Chydoridae,Anomopoda) on lake vegetation

We investigated seasonal changes in the density of epiphytic cladocerans Alona spp. (Chydoridae, Anomopoda) in two habitats, emergent and submerged aquatic plants, in Lake Suwa, Japan, from April to August 1998 and from April to November 2000. Alona had a density peak in early June on reeds (emergent) and in late June on Potamogeton malaianus (submerged). In summer, Alona density remained low in both habitats. Although density was positively correlated with the abundance of epiphytic algae, the birth rate was constant and no correlation between algal abundance and clutch size was detected. In a field experiment using ropes as an artificial substrate covered with high and low densities of epiphytic algae as food, more Alona attached to the ropes with the high density of algae. These results suggest that Alona may select food-rich habitats and migrate seasonally, and that migration is an important factor in the population dynamics of epiphytic chydorid cladocerans such as Alona. In Lake Suwa, Alona may migrate from the reed zone to the submerged macrophyte zone in June.     

Diversity of the archaeal community in 44 anaerobic digesters as determined by single strand conformation polymorphism analysis and 16S rDNA sequencing

The diversity of Archaea in anaerobic digesters was characterized by strand conformation polymorphism (SSCP) analysis and the sequencing of 16S rDNA genes. The 44 digesters sampled, located in eight different countries, treated effluents from agriculture, the food processing and petro-chemical industries, pulp and paper plant, breweries, slaughterhouses and municipal waste. All the existing processes were represented among the samples (fixed-film, fluidized bed, stirred-tank, UASB, sequential batch reactor, lagoon). Single strand conformation polymorphism analysis targeting the V3 region of 16S rDNA revealed between four to six distinct archaeal peaks per digester. The diversity of dominant Archaea in the 44 digesters was estimated as 23 different 16S rDNA sequences. Cloning of archaeal 16S rRNA genes from 11 distinct total genomic DNA, screening of clones by SSCP and the sequencing of 170 of them made it possible to characterize these SSCP peaks. All the sequences retrieved were members of the Euryarchaeaota subdomain. Furthermore, most of the sequences retrieved were very close to already known and cultivated strains or to environmental clones. The most frequent archaeal sequences were close to Methanosaeta concilii and to a 16S rDNA clone vadinDC06 located in the Methanobacterium clade (84% and 73% of digesters respectively). The other sequences were members of the Methanobacteriales and the Methanomicrobiales families. Only one sequence was far from any sequence of the database and it could be grouped with several sequences of environmental clones. Each digester harboured between two to nine archaeal sequences with only one of them corresponding to a putative acetate-utilizing species. Furthermore, the process in the digesters appeared to play a part in the distribution of archaeal diversity.     

Large-scale exploration of growth inhibition caused by overexpression of genomic fragments in Saccharomyces cerevisiae

We have screened the genome of Saccharomyces cerevisiae for fragments that confer a growth-retardation phenotype when overexpressed in a multicopy plasmid with a tetracycline-regulatable (Tet-off) promoter. We selected 714 such fragments with a mean size of 700 base-pairs out of around 84,000 clones tested. These include 493 in-frame open reading frame fragments corresponding to 454 distinct genes (of which 91 are of unknown function), and 162 out-of-frame, antisense and intergenic genomic fragments, representing the largest collection of toxic inserts published so far in yeast.     

Production of tailor-made fructans in sugar beet by expression of onion fructosyltransferase genes

The consumption of fructans as a low caloric food ingredient or dietary fibre is rapidly increasing due to health benefits. Presently, the most important fructan source is chicory, but these fructans have a simple linear structure and are prone to degradation. Additional sources of high-quality tailor-made fructans would provide novel opportunities for their use as food ingredients. Sugar beet is a highly productive crop that does not normally synthesize fructans. We have introduced specific onion fructosyltransferases into sugar beet. This resulted in an efficient conversion of sucrose into complex, onion-type fructans, without the loss of storage carbohydrate content.     

Nucleotide diversity of the <Emphasis Type="Italic">ZmPox3</Emphasis> maize peroxidase gene: Relationships between a MITE insertion in exon 2 and variation in forage maize digestibility

Background  

Polymorphisms were investigated within the ZmPox3 maize peroxidase gene, possibly involved in lignin biosynthesis because of its colocalization with a cluster of QTL related to lignin content and cell wall digestibility. The purpose of this study was to identify, on the basis of 37 maize lines chosen for their varying degrees of cell wall digestibility and representative of temperate regions germplasm, ZmPox3 haplotypes or individual polymorphisms possibly associated with digestibility.