Polyglutamylation of nucleosome assembly proteins

Polyglutamylation is an original posttranslational modification, discovered on tubulin, consisting in side chains composed of several glutamyl units and leading to a very unusual protein structure. A monoclonal antibody directed against glutamylated tubulin (GT335) was found to react with other proteins present in HeLa cells. After immunopurification on a GT335 affinity column, two prominent proteins of approximately 50 kDa were observed. They were identified by microsequencing and mass spectrometry as NAP-1 and NAP-2, two members of the nucleosome assembly protein family that are implicated in the deposition of core histone complexes onto chromatin. Strikingly, NAP-1 and NAP-2 were found to be substrates of an ATP-dependent glutamylation enzyme co-purifying on the same column. We took advantage of this property to specifically label and purify the polyglutamylated peptides. NAP-1 and NAP-2 are modified in their C-terminal domain by the addition of up to 9 and 10 glutamyl units, respectively. Two putative glutamylation sites were localized for NAP-1 at Glu-356 and Glu-357 and, for NAP-2, at Glu-347 and Glu-348. These results demonstrate for the first time that proteins other than tubulin are polyglutamylated and open new perspectives for studying NAP function.     

Posttranslational modifications of the lutropin receptor: mass spectrometric analysis

Our present knowledge of the lutropin (LH/hCG) receptor structure derives from deductions made from its amino acid sequence as established by studying the cDNA. To obtain direct experimental information, luteinizing hormone (LH) receptor expressed in L cells was immunopurified in sufficient amounts to warrant analysis by mass spectrometry and microsequencing. The mature receptor, complexed to human chorionic gonadotropin (hCG), was purified by using monoclonal antibodies recognizing the hormone, whereas the mannose-rich non-hormone-binding precursor was purified by use of antireceptor antibodies. Determination of the N-terminus showed that (2)/(3) of protein molecules started at Thr24 whereas (1)/(3) started at Ala28. All these molecules bound hCG, suggesting that the most N-terminal region of the receptor does not participate in hormone binding. Six N-glycosylation sites have been predicted from the amino acid sequence. One of them (Asn299) was found to be nonglycosylated in both the precursor and the mature protein. The most heavily glycosylated residue was Asn291, followed by Asn195 and Asn99. These three sites accounted for 82% and 97% of carbohydrate moieties in the mature receptor and in the mannose-rich precursor, respectively. The presence of some receptor molecules nonglycosylated at sites 99, 174, and 195 in hormone-receptor complexes dismisses a direct role of these glycosylation sites in hormone binding or in the correct folding of the protein. The mature carbohydrate chains were homogeneous at position 174, 195, and 313 (absence of Golgi mannosidase II activity at positions 174 and 313, absence of GlcNAc tranferases III and IV activity at position 195). Heterologous carbohydrates were present at sites 99 and 291. The latter, which is highly variable in carbohydrate chains, is unlikely to participate in a direct interaction with hormone. Site 313 thus remains as the main candidate for a role in hormone binding.     

Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells

We have previously reported that 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAc alpha-O-bn), an inhibitor of glycosylation, perturbed apical biosynthetic trafficking in polarized HT-29 cells suggesting an involvement of a lectin-based mechanism. Here, we have identified galectin-4 as one of the major components of detergent-resistant membranes (DRMs) isolated from HT-29 5M12 cells. Galectin-4 was also found in post-Golgi carrier vesicles. The functional role of galectin-4 in polarized trafficking in HT-29 5M12 cells was studied by using a retrovirus-mediated RNA interference. In galectin-4-depleted HT-29 5M12 cells apical membrane markers accumulated intracellularly. In contrast, basolateral membrane markers were not affected. Moreover, galectin-4 depletion altered the DRM association characteristics of apical proteins. Sulfatides with long chain-hydroxylated fatty acids, which were also enriched in DRMs, were identified as high-affinity ligands for galectin-4. Together, our data propose that interaction between galectin-4 and sulfatides plays a functional role in the clustering of lipid rafts for apical delivery.     

Specific secretion of gel-forming mucins and TFF peptides in HT-29 cells of mucin-secreting phenotype

Trefoil factor family (TFF) peptides are typical secretory products of mucin-producing cells, e.g. of the gastrointestinal tract. Here, the expression and secretion of mucins and TFF peptides was studied in the HT-29 cell line throughout cellular growth and differentiation in relation to a mucin-secreting (HT-29 MTX) or an enterocyte-like (HT-29 G(-)) phenotype. mRNAs of several MUC and TFF genes were expressed in both cell subpopulations. However, for most MUC and TFF genes, the expression appeared strongly induced with the differentiation into the mucin-secreting phenotype. On the other hand, TFF2 was specifically expressed in the mucin-secreting HT-29 MTX cells. The differentiation of HT-29 MTX cells into the mucin-secreting phenotype was characterised by secretion of the gel-forming mucins MUC2, MUC5AC, and MUC5B, however, according to a different pattern in the course of differentiation. A significant amount of TFF1 and TFF3 was secreted after differentiation, also according to a different pattern, whereas TFF2 was only faintly detected. Secretagogues, known to induce the secretion of mucus, increased the secretion of all three TFF peptides. In contrast, neither a secretory mucin nor a TFF peptide was found in the culture medium of HT-29 G(-) cells. Overlay assays indicated that HT-29 MTX mucins bound to secretory peptides of HT-29 MTX cells with relative molecular mass similar to TFF peptides. TFF1 and TFF3 were specifically localised in the mucus layer of HT-29 MTX cells by confocal microscopy. Finally, the secretion of TFF peptides and mucins appears as a co-ordinated process which only occurs after differentiation into goblet cell-like phenotype.     

uPA/plasmin system-mediated MMP-9 activation is implicated in bronchial epithelial cell migration

To examine the effects of the uPA/plasmin system on cell migration in relation to the activation of MMP-9, we used ex vivo and in vitro wound-repair models of human bronchial epithelial cells and videomicroscopy techniques that make possible cell tracking and quantification of cell migration speeds. We observed that uPA was only detected in migrating cells at the wound edges and located at crucial sites for cell/extracellular matrix interactions. The implication of uPA in human bronchial epithelial cell migration was studied by incubating cultures with a monoclonal antibody raised against uPA and these experiments led to a 70% reduction in cell velocity. To examine the effects of the plasmin system on cell migration, we incubated cultures with increasing concentrations of plasmin or activated MMP-9. We observed a significant dose-dependent increase in cell migration velocity with plasmin (P < 0.001) and MMP-9 (P < 0.001). Moreover, addition of exogenous plasmin led to a twofold increase of activated MMP-9 in migrating cells. We also demonstrated that the addition of anti-uPA IgG led to an inhibition of 43% of activated MMP-9. In conclusion, these results show that uPA is involved in human bronchial epithelial cells migration. This action is mediated by the generation of plasmin, which in turn activates MMP-9, thus making possible cell migration.     

高等植物中的磷酸烯醇式丙酮酸羧激酶

简要介绍了近年来有关高等植物中磷酸烯醇式丙酮酸羧激酶（ＰＥＰＣＫ）的研究进展,并讨论了此酶的结构、功能和调节等方面的问题。     

Differences in need for antihypertensive drugs among those aware and unaware of their hypertensive status: a cross sectional survey

Peripheral arterial disease (PAD) is a common, progressive manifestation of atherothrombotic vascular disease, which should be managed no different to cardiac disease. Indeed, there is growing evidence that PAD patients are a high risk group, although still relatively under-detected and under treated. This is despite the fact that PAD patients are an increased mortality rate comparable to those with pre-existing or established cardiovascular disease [myocardial infarction, stroke]. With a holistic approach to atherothrombotic vascular disease, our management of PAD can only get better.     

The Spatial Distribution of Mustelidae in France

We estimated the spatial distribution of 6 Mustelidae species in France using the data collected by the French national hunting and wildlife agency under the “small carnivorous species logbooks” program. The 1500 national wildlife protection officers working for this agency spend 80% of their working time traveling in the spatial area in which they have authority. During their travels, they occasionally detect dead or living small and medium size carnivorous animals. Between 2002 and 2005, each car operated by this agency was equipped with a logbook in which officers recorded information about the detected animals (species, location, dead or alive, date). Thus, more than 30000 dead or living animals were detected during the study period. Because a large number of detected animals in a region could have been the result of a high sampling pressure there, we modeled the number of detected animals as a function of the sampling effort to allow for unbiased estimation of the species density. For dead animals -- mostly roadkill -- we supposed that the effort in a given region was proportional to the distance traveled by the officers. For living animals, we had no way to measure the sampling effort. We demonstrated that it was possible to use the whole dataset (dead and living animals) to estimate the following: (i) the relative density -- i.e., the density multiplied by an unknown constant -- of each species of interest across the different French agricultural regions, (ii) the sampling effort for living animals for each region, and (iii) the relative detection probability for various species of interest.     

Purification and amino acid sequence of chicken liver cathepsin L

Cathepsin L was purified from chicken liver lysosomes by a two-step procedure. Cathepsin L exhibited a single band of Mr 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, presented a high affinity for the substrate Z-Phe-Arg-NMec, was very unstable at neutral pH, and was inhibited by Z-Phe-Phe-CHN2. The complete amino acid sequence of cathepsin L has been determined and consists of 215 residues. The sequence was deduced from analysis of peptides generated by enzymatic digestions and by chemical cleavage at methionyl bonds. Comparison of the amino acid sequence of cathepsin L with those of rat liver cathepsins B and H and papain demonstrates a striking homology among their primary structures.     

Biochemical aspect of photoassimilated carbon partitioning at late kernel fill in maize under climatic stress

The aim of this work was to describe the incorporation of 14CO2 into maize at the late kernel fill under chilling and the subsequent movement of the photoassimilated 14C out the fed ear leaf. Cool temperatures were observed to decrease the photosynthetic rate and to alter the operation of the carbon assimilation pathway with 14C accumulation in alpha-alanine. They were shown also to affect the rate of photoassimilated carbon out of the fed area, and especially by delaying the seed import processes.