Chemical characterization of cell-wall polysaccharides from tank-cultivated and wild plants ofDelesseria sanguinea (Hudson) Lamouroux (Ceramiales,delesseriaceae): culture patterns and potent anticoagulant activity

Tank cultivation ofDelesseria sanguinea was investigated in order to manipulate conditions for vegetative growth and to provide biomass for the analysis of cell wall polysaccharides. Seasonality is subject to short-day photoperiodic control. Night-break or long-day conditions prevented fertility in tetrasporophytes and gametophytes and triggered outgrowth of new blades. Long-day illuminations allowed a 1% daily growth rate. Seawater temperature below 13 °C was necessary for inducing formation of new blades. Both wild and cultivated ofD. sanguinea plants contained a non gelling sulfated heteropolysaccharide composed of a galactosyl backbone branched with xylosyl residues. The hot water extract at neutral pH displayed the highest anticoagulant activity (5 μg ml^-1 polysaccharide concentration in APTT clotting assay). No obvious differences were found in polysaccharide chemical composition and properties between gametophytes and sporophytes or between cultivated and wild plants.     

Morphogenesis in tobacco subepidermal cells: Putrescine as marker of root differentiation

We have used the tobacco thin cell layer in vitro system to evaluate changes in polyamine titers as correlated with root differentiation and with variations in external pH during culture. We show that root differentiation in this system depends on both a rise in putrescine titers and a drop of pH, each of these two factors acting independently. With respect to polyamine titers, the most dramatic changes occur in the levels of putrescine liberated from perchloric acid-soluble conjugates. These titers increase from day 0 to day 7 of culture, reaching almost 2000 nmol g^-1 fresh weight. Inhibition of putrescine biosynthesis prevents root initiation, while exogenous putrescine supply reverses this effect. We conclude that putrescine is a good marker for root differentiation.     

Distinct Effects of Imipramine on 5-Hydroxytryptamine Uptake Mediated by the Recombinant Rat Serotonin Transporter SERT1

Abstract: Tricyclic and nontricyclic serotonin [5-hydroxytryptamine (5-HT)] uptake inhibitors are widely used for the treatment of depression. Here, we show that both the tricyclic antidepressant imipramine and the nontricyclic antidepressant citalopram competitively inhibit 5-HT transport mediated by the recombinant rat 5-HT transporter SERT1. For citalopram, the concentration producing half-maximal transport inhibition was in the same order of magnitude as its K _D value determined by equilibrium binding. In contrast, the inhibitory potency of imipramine was more than one order of magnitude lower than its K _D value. Our data are consistent with low-affinity imipramine binding occurring at or close to the substrate recognition site, which also binds citalopram. Occupation of the high-affinity imipramine binding site on SERT1 did not affect 5-HT transport but allosterically displaced citalopram from the substrate recognition site. Consequently, low concentrations of imipramine partially protected 5-HT transport from citalopram inhibition. This protection was only observed in the presence of Na⁺ because high-affinity imipramine binding is strictly sodium-dependent. Thus, depending on which of its binding sites on SERT1 is occupied, imipramine may exert distinct effects on 5-HT uptake mediated by the recombinant rat 5-HT transporter.     

Flow cytometry is a rapid and reliable method for evaluating heat shock protein 70 expression in human monocytes

The increasing interest in stress/heat shock proteins (Hsps) as markers of exposure to environmental stress or disease requires an easily applicable method for Hsp determination in peripheral blood cells. Of these cells, monocytes preferentially express Hsps upon stress. An appropriate fixation/permeabilization procedure was developed, combined with immunofluorescence staining and flow cytometry for the detection of the inducible, cytosolic, 72 kDa Hsp (Hsp70) in human monocytes. Higher relative fluorescence intensity was observed in cells exposed to heat shock (HS), reflecting a higher expression of Hsp70 in these cells as compared with cells kept at 37°C. The heat-inducible increased Hsp70 expression was temperature- and time-dependent. Expression of Hsp70 was not uniform within the monocyte population, indicating the presence of subpopulations expressing variable levels of Hsp70 in response to HS. Simultaneous measurements of intracellular Hsp70 and membrane CD14 expression revealed that the higher Hsp70 inducibility coincided with the higher CD14 expression. Comparisons performed with biometabolic labelling, Western blotting, immunofluorescence and immunoperoxidase microscopic analysis, showed a high concordance between these different methods; however, cytometry was more sensitive for Hsp70 detection than Western blotting. Flow cytometric detection of intracellular Hsp70 is a rapid, easy and quantitative method, particularly suited for the determination of protein levels in individual cells from an heterogeneous population such as peripheral mononuclear blood cells, and applicable to cohort studies.     

Apo-Hsp90 coexists in two open conformational states in solution

Background information. Hsp90 (90 kDa heat‐shock protein) plays a key role in the folding and activation of many client proteins involved in signal transduction and cell cycle control. The cycle of Hsp90 has been intimately associated with large conformational rearrangements, which are nucleotide‐binding‐dependent. However, up to now, our understanding of Hsp90 conformational changes derives from structural information, which refers to the crystal states of either recombinant Hsp90 constructs or the prokaryotic homologue HtpG (Hsp90 prokaryotic homologue). Results and discussion. Here, we present the first nucleotide‐free structures of the entire eukaryotic Hsp90 (apo‐Hsp90) obtained by small‐angle X‐ray scattering and single‐particle cryo‐EM (cryo‐electron microscopy). We show that, in solution, apo‐Hsp90 is in a conformational equilibrium between two open states that have never been described previously. By comparing our cryo‐EM maps with HtpG and known Hsp90 structures, we establish that the structural changes involved in switching between the two Hsp90 apo‐forms require large movements of the NTD (N‐terminal domain) and MD (middle domain) around two flexible hinge regions. Conclusions. The present study shows, for the first time, the structure of the entire eukaryotic apo‐Hsp90, along with its intrinsic flexibility. Although large structural rearrangements, leading to partial closure of the Hsp90 dimer, were previously attributed to the binding of nucleotides, our results reveal that they are in fact mainly due to the intrinsic flexibility of Hsp90 dimer. Taking into account the preponderant role of the dynamic nature of the structure of Hsp90, we reconsider the Hsp90 ATPase cycle.     

Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii

Toxoplasma gondii is a unicellular parasite characterized by unique extracellular and intracellular membrane compartments. The lipid composition of subcellular membranes has not been determined, limiting our understanding of lipid homeostasis, control, and trafficking, a series of processes involved in pathogenesis. In addition to a mitochondrion, Toxoplasma contains a plastid called the apicoplast. The occurrence of a plastid raised the question of the presence of chloroplast galactolipids. Using three independent rabbit and rat antibodies against digalactosyldiacylglycerol (DGDG) from plant chloroplasts, we detected a class of Toxoplasma lipids harboring a digalactolipid-like epitope (DGLE). Immunolabeling characterization supports the notion that the DGLE polar head is similar to that of DGDG. Mass spectrometry analyses indicated that dihexosyl lipids having various hydrophobic moieties (ceramide, diacylglycerol, and acylalkylglycerol) might react with anti-DGDG, but we cannot exclude the possibility that more complex dihexosyl-terminated lipids might also be immunolabeled. DGLE localization was analyzed by immunofluorescence and immunoelectron microscopy and confirmed by subcellular fractionation. No immunolabeling of the apicoplast could be observed. DGLE was scattered in pellicle membrane domains in extracellular tachyzoites and was relocalized to the anterior tip of the cell upon invasion in an actin-dependent manner, providing insights on a possible role in pathogenetic processes. DGLE was detected in other Apicomplexa (i.e., Neospora, Plasmodium, Babesia, and Cryptosporidium).     

Effects of docosahexaenoic acid on some megakaryocytic cell gene expression of some enzymes controlling prostanoid synthesis

Beneficial effects of docosahexaenoic acid (DHA) intake in the prevention of cardiovascular diseases are known, and platelets play a crucial role in cardiovascular complications. However, high doses of DHA may increase lipid peroxidation and induce deleterious effects, notably in platelets. This led us to investigate the effect of DHA on gene expression of some enzymes controlling redox status and prostanoid formation in human megakaryoblastic cells (MEG-01 cell line). MEG-01 cells were incubated in presence of DHA (10 and 100 μmol/L) for 6 h. DHA enrichment up-regulated glutathione peroxidase-1 and thromboxane synthase mRNA. DHA increased gene catalase expression and up-regulated PPAR β/δ and PPAR γ mRNA in presence of high concentration of DHA. In conclusion, our results support an antioxidant mechanism of DHA. The effects of DHA on cellular redox status could, with others, provide an explanation for the beneficial influence of low consumption of DHA on cardiovascular events.     

Genetic Clustering of Hepatitis C Virus Strains and Severity of Recurrent Hepatitis after Liver Transplantation

The influence of viral factors on the severity of hepatitis C virus (HCV)-related liver disease is controversial. We studied 68 liver transplant patients with recurrent hepatitis C, of whom 53 were infected by genotype 1 strains. Relationships between core sequences, serum HCV RNA levels, and fibrosis scores for each patient were analyzed in pairwise fashion 5 years after transplantation. We used Mantel's test, a matrix correlation method, to evaluate the correspondence between measured genetic distances and observed phenotypic differences. No clear relationship was found when all 68 patients were analyzed. In contrast, when the 53 patients infected by genotype 1 strains were analyzed, a strong positive relationship was found between genetic distance and differences in 5-year fibrosis scores (P = 0.001) and differences in virus load (P = 0.009). In other words, the smaller the genetic distance between two patients' viral core sequences, the smaller the difference between the two patients' fibrosis scores and viral replication levels. No relationship was found between genetic distance and differences in age, sex, or immunosuppression. In multivariate analysis, the degree of fibrosis was negatively related to the virus load (r = -0.68; P = 0.003). In the particular setting of liver transplantation, and among strains with closely related phylogenetic backgrounds (genotype 1), this study points to a correlation between the HCV genetic sequence and the variability of disease expression.     

Depletion of SMN by RNA interference in HeLa cells induces defects in Cajal body formation

Neuronal degeneration in spinal muscular atrophy (SMA) is caused by reduced expression of the survival of motor neuron (SMN) protein. The SMN protein is ubiquitously expressed and is present both in the cytoplasm and in the nucleus where it localizes in Cajal bodies. The SMN complex plays an essential role for the biogenesis of spliceosomal U-snRNPs. In this article, we have used an RNA interference approach in order to analyse the effects of SMN depletion on snRNP assembly in HeLa cells. Although snRNP profiles are not perturbed in SMN-depleted cells, we found that SMN depletion gives rise to cytoplasmic accumulation of a GFP-SmB reporter protein. We also demonstrate that the SMN protein depletion induces defects in Cajal body formation with coilin being localized in multiple nuclear foci and in nucleolus instead of canonical Cajal bodies. Interestingly, the coilin containing foci do not contain snRNPs but appear to co-localize with U85 scaRNA. Because Cajal bodies represent the location in which snRNPs undergo 2′-O-methylation and pseudouridylation, our results raise the possibility that SMN depletion might give rise to a defect in the snRNA modification process.