Effect of Desipramine on a Glycoprotein Sialyltransferase Activity in C6 Cultured Glioma Cells

The tricyclic antidepressant desipramine, when added to culture medium, gave rise in C6 rat glioma cells to a decrease of the activity of the enzyme asialofetuin sialyltransferase. The inhibition was dose and time dependent and was observed in both multiplying cells and cells blocked with 2 mM thymidine or depletion of amino acids. This inhibition was rather specific to the sialyltransferase, as under the conditions where this enzyme was inhibited up to 70%, other enzymes such as dolichol phosphate mannose synthetase, glutamine synthetase, and glycerol phosphate dehydrogenase remained unaffected. This inhibition was not reversed after removal of desipramine from the medium and was not observed by direct addition of desipramine to the sialyltransferase incubation assay. Under the same conditions, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], which is known to be a potent calmodulin antagonist and an inhibitor of calmodulin-dependent kinases, gave the same concentration-dependent inhibition profile of sialyltransferase as desipramine, whereas H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], which is an inhibitor of protein kinase C and cyclic nucleotide-dependent kinases, had no effect. So, it is suggested that desipramine inhibits the sialyltransferase activity in C6 glioma cells through a calmodulin-dependent system.     

Preparative separation of small molecular weight peptides from casein hydrolysate using gel filtration and immobilized metal ion affinity chromatography

A two step method consisting of a gel filtration step, followed by a Immobilized Metal Affinity Chromatography (IMAC) step using a IDA-Cu coupled Sephadex G-25 column, on a preparative scale is described for the group separation of peptides from a casein hydrolysate. The 48 groups of peptides thus separated are further characterised by RP-HPLC and amino acid analysis. Some peptides after the analytical RP-HPLC step are further characterised by sequencing. An insight into the mechanism of retention on IMAC of the peptides is attempted. In such complex mixtures as casein hydrolysate, the peptide-peptide interaction can mask the potential sites of interactions in a single peptide. The results obtained using volatile buffers as eluents show the possibility of using IMAC step as an alternative to obtain gram quantities of group of peptides free of salts from complex protein hydrolysates.     

Carbon-Flux Distribution within Streptomyces coelicolor Metabolism: A Comparison between the Actinorhodin-Producing Strain M145 and Its Non-Producing Derivative M1146

Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two Streptomyces coelicolor A3 (2) strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and ¹³C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest.     

Optimization of trans-Resveratrol bioavailability for human therapy

We have developed an innovative soluble galenic form to overcome the low absorption of trans-Resveratrol (t-Res) as a dry powder. We present here data on pharmacokinetics, bioavailability, and toxicity of t-Res in human volunteers treated with this soluble form, plus additional data on biological effects in rodents. Fifteen healthy volunteers of both sexes received 40 mg of t-Res in two forms, the soluble formulation (caplets) and the original powder (capsules), in a crossover design. Blood samples were collected at 15 min, 30 min, and every hour for 5 h. Plasma concentrations of t-Res and its metabolites were analyzed by liquid chromatography and mass spectrometry. The single dose (40 mg) of the soluble t-Res was well absorbed and elicited biologically efficient blood levels (0.1–6 μM) for several hours, despite metabolization into glucuronide and sulfate conjugates coupled to renal elimination. In contrast, t-Res administered as a dry powder barely elicited efficient blood levels for a short duration. The new formulation led to 8.8-fold higher t-Res levels in plasma versus the powder. t-Res metabolism was not modified and neither intolerance nor toxicity were observed during the study and the following week. The soluble formulation elicited a robust anti-inflammatory effect in various tissues of mice fed a high-fat diet, while dry powder t-Res was almost inactive. Our data suggest that significant improvements in t-Res bioavailability and efficiency can be obtained by this soluble galenic form, also allowing lower doses. The use of t-Res in human therapy is thus greatly facilitated and the toxicity risk is reduced.     

Evidence for new C-terminally truncated variants of α- and β-tubulins

Cellular α-tubulin can bear various carboxy-terminal sequences: full-length tubulin arising from gene neosynthesis is tyrosinated, and two truncated variants, corresponding to detyrosinated and Δ2 α‑tubulin, result from the sequential cleavage of one or two C-terminal residues, respectively. Here, by using a novel antibody named 3EG that is highly specific to the –EEEG C-terminal sequence, we demonstrate the occurrence in neuronal tissues of a new αΔ3‑tubulin variant corresponding to α1A/B‑tubulin deleted of its last three residues (EEY). αΔ3‑tubulin has a specific distribution pattern: its quantity in the brain is similar to that of αΔ2-tubulin around birth but is much lower in adult tissue. This truncated α1A/B-tubulin variant can be generated from αΔ2-tubulin by the deglutamylases CCP1, CCP4, CCP5, and CCP6 but not by CCP2 and CCP3. Moreover, using 3EG antibody, we identify a C‑terminally truncated β-tubulin form with the same –EEEG C-terminal sequence. Using mass spectrometry, we demonstrate that β2A/B-tubulin is modified by truncation of the four C-terminal residues (EDEA). We show that this newly identified βΔ4-tubulin is ubiquitously present in cells and tissues and that its level is constant throughout the cell cycle. These new C-terminally truncated α- and β-tubulin variants, both ending with –EEEG sequence, are expected to regulate microtubule physiology. Of interest, the αΔ3-tubulin seems to be related to dynamic microtubules, resembling tyrosinated-tubulin rather than the other truncated variants, and may have critical function(s) in neuronal development.     

Evidence of a balance between phosphorylation and O-GlcNAc glycosylation of Tau proteins--a role in nuclear localization

Both phosphorylation and O-GlcNAc glycosylation posttranslationally modify microtubule-associated Tau proteins. Whereas the hyperphosphorylation of these proteins that occurs in Alzheimer's disease is well characterized, little is known about the O-GlcNAc glycosylation. The present study demonstrates that a balance exists between phosphorylation and O-GlcNAc glycosylation of Tau proteins, and furthermore that a dysfunction of this balance correlates with reduced nuclear localization.The affinity of Tau proteins for WGA lectin, together with evidence from [3H]-galactose transfer and analysis of beta-eliminated products, demonstrated the presence of O-GlcNAc residues on both cytosolic and nuclear Tau proteins. In addition, our data indicated the existence of a balance between phosphorylation and O-GlcNAc glycosylation events. Indeed, as demonstrated by 2D-electrophoresis and Western blotting, O-GlcNAc residues were mainly located on the less phosphorylated Tau 441 variants, whereas the more phosphorylated forms were devoid of O-GlcNAc residues. Furthermore, the Tau protein hyperphosphorylation induced by cellular okadaic acid treatment was correlated with reduced incorporation of O-GlcNAc residues into Tau proteins and with diminished Tau transfer into the nucleus. Hence, this paper establishes a direct relationship between O-GlcNAc glycosylation, phosphorylation and cellular localization of Tau proteins.     

Relationship between expression of matrix metalloproteinases and migration of epidermal and in vitro generated Langerhans cells

Langerhans cells (LC) are dendritic cells that capture foreign antigens and migrate with them to the regional lymph nodes where they are presented to naive T cells. The possible role of matrix metalloproteinase-9 (MMP-9) in migration was suggested following experiments in a mouse model and in human skin explants. Using in vitro generated LC (iLC) derived from CD34+ cord blood cells and epidermal LC (eLC), we investigated the correlation between MMP-9 and other MMPs production and cell migration. Cells were activated by Bandrowski's base (BB), a chemical allergen, or by recombinant birch pollen allergen 1 (rBetv 1). Contact with allergens triggered migration of these cells, with a maximum rate being reached after 24 h. Migration was preceded by production of MMP-2 and MMP-9; part of the molecules were recovered as pro-MMPs in cell culture supernatant and part were associated with cell membrane proteins. At the cellular level, membrane-type 1 (MT1) and MT3-MMP were also identified. Addition of tumor necrosis factor-alpha (TNF-alpha) initiated pro-MMP-2 and pro-MMP-9 production followed by cell migration in a dose-dependent manner. These data imply that TNF-alpha is a key molecule for MMP production and cell migration. Furthermore, activation of iLC with BB or rBet v 1 induced synthesis of TNF-a and expression of TNF RII on the cell membrane, suggesting an autocrine loop. In conclusion, membrane-associated MMP-2 and-9 rather than soluble MMPs appear to be involved in cell migration.     

Gene expression profiling of 3T3-L1 adipocytes exposed to phloretin

Adipocyte dysfunction plays a major role in the outcome of obesity, insulin resistance and related cardiovascular complications. Thus, considerable efforts are underway in the pharmaceutical industry to find molecules that target the now well-documented pleiotropic functions of adipocyte. We previously reported that the dietary flavonoid phloretin enhances 3T3-L1 adipocyte differentiation and adiponectin expression at least in part through PPARγ activation. The present study was designed to further characterize the molecular mechanisms underlying the phloretin-mediated effects on 3T3-L1 adipocytes using microarray technology. We show that phloretin positively regulates the expression of numerous genes involved in lipogenesis and triglyceride storage, including GLUT4, ACSL1, PEPCK1, lipin-1 and perilipin (more than twofold). The expression of several genes encoding adipokines, in addition to adiponectin and its receptor, is positively or negatively regulated in a way that suggests a possible reduction in systemic insulin resistance and obesity-associated inflammation. Improvement of insulin sensitivity is also suggested by the overexpression of genes associated with insulin signal transduction, such as CAP, PDK1 and Akt2. Many of these genes are PPARγ targets, confirming the involvement of PPARγ pathway in the phloretin effects on adipocytes. In light of these microarray data, it is reasonable to assume that phloretin may be beneficial for reducing insulin resistance, in a similar way to the thiazolidinedione class of antidiabetic drugs.