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International audience; Bistable molecules represent a potential miniaturization limit for high-density information technologies. However, molecules exhibit memory effect typically at very low temperatures. This is the case of spin-crossover (SCO) complexes, where the concept of a molecular memory has not been considered due to the fast spin-state interconversion of all previous systems. Breaking this principle, here we report a slow relaxation process found in an SCO iron-triazole polyanionic complex. Multiple experimental evidences confirm the opening of a thermal hysteresis upon solid dilution and even in liquid solution. Density functional theory (DFT) calculations reveal the origin of this unexpected phenomenon to the appearance of an energy barrier that slows down the spin-state relaxation processes at the molecular level. These results show how SCO molecules may store information at room temperature, opening unique opportunities for molecular data storage.

The search for new ways to suppress unwanted protein aggregation represents an important problem in modern biochemistry, bioengineering, and even medicine. Recently we succeeded in preventing the aggregation using synthetic polyelectrolytes. The present work describes a new approach to solubilizing pre-formed protein aggregates with sulfated or sulfonated polymers (polysulfoanions). For the first time it was shown that polysulfoanions are capable of solubilizing amorphous and amyloid protein inclusion bodies as well as thermal aggregates. Treatment of prion protein inclusion bodies with sulfonated polymers was shown to cause significant decrease in amyloid structure content, whereas in case of thermal aggregates of glyceraldehyde-3-phosphate dehydrogenase the observed solubilization was accompanied by a partial recovery of enzymatic activity. The suggested approach could be relevant in the task of extracting recombinant proteins from inclusion bodies and also useful in the development of amyloid disease therapy.

Recent studies have led us to suppose that synergistic action of multiple solute cofactors could play substantial roles in amyloid-type fibrillogenesis of pathogenic polypeptides. To support this view, we performed aggregation experiments of human islet amyloid polypeptide (IAPP) in media containing both polyanions and non-polar solvents. The results demonstrated that the fibrillation at sub-micromolar IAPP occurred only when polyanionic and non-polar solutes coexist. A simple sum of two independent cofactor’s effects could not account for the synergistic action. We propose that this synergy of polyanionic and nonpolar milieus could substantially modify the amyloidgenesis in the human body.

Bioartificial pancreas, in which the islets of Langerhans are enclosed in artificial membrane to be protected from the host immune system, is expected to be a promising medical device to treat patients who suffer from insulin-dependent diabetes. Our strategy for preparation of a bioartificial pancreas involves utilizing a membrane including polymeric materials that can inhibit the complement reaction. In this study, we examined the effects of poly(styrene sulfonic acid) (PSSa) on the alternative pathway of the serum complement system to identify the mechanism(s) involved. PSSa was dissolved in pooled normal human serum (NHS), and the mixtures were incubated at 37 degrees C for 30 min. Complement activities in sera were determined by hemolytic assays. Amounts of complement activation products released were determined by ELISA. Interactions of PSSa with complement components and fragments were examined with electrophoresis and immunoblotting. From these examinations, it appeared that the manner of PSSa effects on the alternative pathway (AP) highly depends on its concentration. PSSa seemingly acted as an activator when its concentration was 0.005 g/dl to 0.05 g/dl, while it acted as an inhibitor when its concentration was more than 0.1 g/dl. In terms of activation or inhibition of the AP, forming complex of PSSa with factor H induced activation, and that with factor D induced inhibition.

The transport of activated fatty acids across the mitochondrial outer membrane has not been fully addressed. A polyanion (Mn=22 kDa) inhibited the ADP-stimulated carnitine-dependent oxidation of both palmitoyl-CoA and palmitate plus CoA as well as mitochondrial hexokinase binding. In contrast, the oxidation of palmitoylcarnitine plus malate, as well as glutamate oxidation, was essentially unaffected. Mitochondrial carnitine palmitoyltransferase-1 was not inhibited by the polyanion. The data suggest an additional component in carnitine-dependent mitochondrial fatty acid oxidation, possibly porin.

Transport properties of mitochondrial porin were investigated on the basis of changes in the activity of hexokinase utilizing external ATP. Production of glucose: 6-phosphate is inhibited by polyanion both in intact brain mitochondria and in contact point vesicles. Hexokinase activity is restored by solubilization of the enzyme by high ionic strength or 0.5-1% Triton X-100. In very low concentrations (0.001–0.005%) Triton does not mobilize hexokinase from its binding sites but it is able to release polyanion-inhibition completely. This finding provides an explanation for the discrepancy observed in the transport properties of porin when studied ‘in situ’ or in artificial lipid membranes.

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and other proteins. It assembles actin filaments and microtubules, can bind actin filaments and SH3-domains to a membrane surface, and may be able to tether them to the oligodendrocyte membrane and participate in signal transduction in oligodendrocytes/myelin. In the present study, we have shown that the 18.5kDa MBP isoform can also bind microtubules to lipid vesicles in vitro. Phosphorylation of MBP at Thr94 and Thr97 (bovine sequence) by MAPK, and deimination of MBP (using a pseudo-deiminated recombinant form), had little detectable effect on its ability to polymerize and bundle microtubules, in contrast to the effect of these modifications on MBP-mediated assembly of actin. However, these modifications dramatically decreased the ability of MBP to tether microtubules to lipid vesicles. MBP and its phosphorylated and pseudo-deiminated variants were also able to bind microtubules to actin filaments. These results suggest that MBP may be able to tether microtubules to the cytoplasmic surface of the oligodendrocyte membrane, and that this binding can be regulated by post-translational modifications to MBP. We further show that MBP appears to be co-localized with actin filaments and microtubules in cultured oligodendrocytes, and also at the interface between actin filaments at the leading edge of membrane processes and microtubules behind them. Thus, MBP may also cross-link microtubules to actin filaments in vivo.

The treatment of 1,2-, 1,7- and 1,12-carbaborane lithiated isomers with [3,3′-Co-8-(CH2CH2O)2-(1,2-C2B9H10)-(1′,2′-C2B9H11)] (1) at molar ratios 1:1 or 1:2 at room temperature in THF leads generally to the formation of a series of orange double-cluster mono and dianions. These were characterized by NMR and MS methods as [1′′-X-1′′,2′′-closo-C2B10H11]−, [2]−; [1′′-X-1′′,7′′-closo-C2B10H11]−, [3]− and [1′′-X-1′′,12′′-closo-C2B10H11], [4]− for the monoanions, whereas [1′′,2′′-X2-1′′,2′′-closo-C2B10H10]2−, [2]2−; [1′′,7′′-X2-1′′,7′′-closo-C2B10H10]2−, [3]2−; and [1′′,12′′-X2-1′′,12′′-closo-C2B10H10]2−, [4]2− for the dianions (where X = 3,3′-Co-8-(CH2CH2O)2-(1,2-C2B9H10)-1′,2′-(C2B9H11)). Moreover, these borane-cage subunits can be easily modified via attaching variable substituents onto cage carbon and boron vertices, which makes these compounds structurally flexible potential candidates for BNCT of cancer and HIV-PR inhibition. Ministry of Education of the Czech Republic (Project LC523), Research Plan AV0Z40320502 from AS CR, MEC (Grant MAT2006-05339), CSIC (I3P grant to P.F.) and the Generalitat de Catalunya 2005/SGR/00709 peer-reviewed

The pharmacokinetics and metabolic fate of the intrinsically active (anti-HIV) drug carrier succinylated human serum albumin (Suc-HSA) was studied in rats. Suc-HSA was prepared by derivatizing HSA with 1,4-[C-14]-succinic anhydride, a modification by which all available epsilonNH2-groups in HSA were converted into carboxylic groups.After iv injections of 0.3, 1.0, 3.0 and 10.0 mg/kg in freely moving rats, Suc-HSA showed a dose dependent elimination pattern, indicating a saturable elimination pathway. The Michaelis-Menten parameters V-max and K-m were 98.7 mug.min(-1).kg(-1) and 8.5 mug.ml(-1) respectively. The kinetics of Suc-HSA was influenced by anaesthesia. In anaesthetised animals, V-max and K-m were found to be 26.9 mug.min(-1).kg(-1) and 0.26 mug.ml(-1), respectively. This implies an intrinsic clearance of 100 ml.min(-1).kg(-1), which is about 10-fold higher as compared to 12 ml.min(-1).kg(-1) in freely moving animals.Intravenous administration of a sub-saturable dose of 3.0 mg.kg(-1)1,4-[C-14]-Suc-HSA to freely moving rats resulted in a biphasic elimination with an initial t(1/2) of 20 min and a terminal t(1/2) of 40 hrs. Excretion of metabolites in urine and faeces lasted for at least 48 hours. About 70% of the radioactive dose was excreted in urine, whereas maximally 2% was detected in faeces. Suc-HSA was degraded to its individual amino acids including succinylated lysine (the only radioactive product formed). Succinylated lysine was not further metabolised and mainly excreted via the urine. Immunohistochemical staining showed that even after 48 hrs Suc-HSA could be detected in livers. Together with the urinary excretion patterns, this points to a gradual degradation of Suc-HSA.

In the present study, we described the interaction of succinylated human serum albumin (Suc-HSA), a negatively charged anti-HIV-1 active protein, with HIV-1 gp120 and in detail with the third variable domain of gp120 (V3 loop). To this end, different assay formats were tested in which gp120- and V3-related peptides were presented in various configurations in order to investigate the effect of the conformational structure of the V3 loop on the interaction with negatively charged albumins. When gp120 presented via a lectin was used, it was observed that Suc-HSA bound to native gp120. The binding site appeared to be located at or near the thrombin digestion site (GPGRAF sequence) in the V3 loop of gp120, since the cleavage of the loop resulted in decreased binding of Suc-HSA. In addition, Suc-HSA was able to protect the V3 region of gp120 from cleaving with thrombin. In contrast, significant binding of Suc-HSA to V3 loop or gp120 peptides was not observed when both were presented in a fluid phase system, suggesting the involvement of a monovalent low affinity binding of Suc-HSA. Using overlapping peptides delineating the whole V3 loop immobilized to CNBr-Sepharose, we noticed that the interaction of the V3 loop with Suc-HSA was predominantly induced by electrostatic interactions between positively charged linearized peptide fragments and Suc-HSA and was positively influenced by the presence of hydrophobic amino in the V3 loop fragments as well. Moreover, the highest affinity site was located at sites near the GPGRAF sequence These observations add to the evidence, collected earlier, that Suc-HSA interferes at the level of virus entry, independent of interaction with the CD4 receptor. Since the recently discovered chemokine receptors are negatively charged, we can hypothesize that Suc-HSA is able to prevent the positively charged V3 loop from interacting with these types of receptors, thereby inhibiting virus entry. BIOCHEM PHARMACOL 57;8:889-898, 1999. (C) 1999 Elsevier Science Inc.

The HIV-1 transactivating factor (Tat) acts as an extracellular cytokine on target cells, including endothelium. Here, we report about the Tat-antagonist capacity of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. O-sulfated K5 with high sulfation degree (K5-OS(H)) and N,O-sulfated K5 with high (K5-N,OS(H)) or low (K5-N,OS(L)) sulfation degree, but not unmodified K5, N-sulfated K5, and O-sulfated K5 with low sulfation degree, bind to Tat preventing its interaction with cell surface heparan sulfate proteoglycans, cell internalization, and consequent HIV-LTR-transactivation. Also, K5-OS(H) and K5-N,OS(H) prevent the interaction of Tat to the vascular endothelial growth factor receptor-2 on endothelial cell (EC) surface. Finally, K5-OS(H) inhibits alphav beta3 integrin/Tat interaction and EC adhesion to immobilized Tat. Consequently, K5-OS(H) and K5-N,OS(H) inhibit the angiogenic activity of Tat in vivo. In conclusion, K5 derivatives with distinct sulfation patterns bind extracellular Tat and modulate its interaction with cell surface receptors and affect its biological activities. These findings provide the basis for the design of novel extracellular Tat antagonists with possible implications in anti-AIDS therapies.

To determine the regulatory mechanism for human tyrosine hydroxylase, we examined modulations of the activity of the enzyme from human pheochromocytoma by cyclic AMP-dependent protein kinase, calmodulin-dependent protein kinase II and polyanion. The most remarkable activation was observed when the enzyme was assayed at physiological pH (pH 7) after being subjected to phosphorylation by cyclic AMP-dependent protein kinase. Calmodulin-dependent protein kinase II and polyanion also modulated the enzyme activity. The results suggest that tyrosine hydroxylase may be regulated similarly in both human and rat.