Efficacy of a polyphenolic extract from silver fir (Abies alba) bark on psoriasis: a randomised, double-blind, placebo-controlled trial.

Silver fir (Abies alba) bark extract contains a mixture of bioactive polyphenols. We tested their effectiveness in the treatment of psoriasis in order to further investigate the potential topical anti-inflammatory activity of polyphenols by means of a randomized, double-blind, placebo-controlled add-on clinical trial, after having examined their ability to downregulate the expression of IL-1ß cytokine in monocyte/macrophage primary cell culture. 61 patients with mild psoriasis met the inclusion criteria and were willing to comply with protocol requirements, were enrolled in the study. The severity of the disease was measured by psoriasis area severity index (PASI). Treatment efficacy was evaluated by assessing erythema (E, 0 to 4-point scale), desquamation (D, 0 to 4-point scale) and induration (I, 0 to 4-point scale) of lesions before and after the treatment. All patients enrolled in the study had symmetrical psoriasis plaques on the skin. All patients received O/V ointment with 2% of silver fir bark extract and/or placebo, respectively. We compared medications by right/left intra-patient comparison, so that the control group was always contralateral of the tested one. Location of the tested or control site was randomised, using a computer-generated randomisation schedule. Silver fir extract was well-tolerated. A superiority of active treatment above placebo, based on the clinical investigational PASI score system was observed by 15 % in all volunteers and in 40% regarding the improvement of psoriasis on elbows. However, statistical analysis showed no significant differences between placebo and active treatment with the extract from silver fir bark (p < 0.05).

A highly active PtCu3 intermetallic core-shell, multilayered Pt-skin, carbon embedded electrocatalyst produced by a scale-up sol-gel synthesis.

We present a novel, scaled-up sol-gel synthesis which enables one to produce 20 g batches of highly active and stable carbon supported PtCu3 nanoparticles as cathode materials for low temperature fuel cell application. We confirm the presence of an ordered intermetallic phase underneath a multilayered Pt-skin together with firm embedment of nanoparticles in the carbon matrix.

Surface with antimicrobial activity obtained through silane coating with covalently bound polymyxin B.

Surfaces exhibiting antimicrobial activity were prepared for potential medical application. A polycationic lipopeptide polymyxin B was selected as the bioactive agent for covalent immobilization onto the surface. First, by using sol-gel technology the inert glass substrate was functionalized by a silane coating with epoxide rings to which the peptide was coupled by means of a catalyst. Preparation of the coating and presence of the peptide on the surface were followed by FTIR, XPS and AFM analyses. The obtained material showed antimicrobial effect indicating that in spite of immobilization the peptide has retained its bioactivity. The coated surface was able to reduce bacterial cell counts of the Gram-negative bacterium Escherichia coli by more than five orders of magnitude in 24 h of incubation. It can be concluded that bioactive coatings with covalently bound polycationic peptides have potential for application on medical devices where leakage into the surrounding is not allowed in order to prevent bacterial growth and biofilm formation.

Janus nematic colloids driven by light.

We present an experimental analysis of different equilibrium orientations and light driven transformations of Janus particles in the nematic liquid crystal 5CB. Depending on the preparation technique of homeotropic (DMOAP) and planar (Au) hemispheres we have observed two types of director field configurations: dipolar-like in the case of Au/DMOAP capped colloids and boojum-like in the case of DMOAP/Au capped colloids. Using the manipulation of Au/DMOAP capped colloids with laser tweezers we report on light driven irreversible orientational transformations into Saturn-ring and a novel, boojum-ring configuration. On the contrary, boojum-like DMOAP/Au capped colloids can act as rotators when exposed to the laser filed. Observed rotation is continuous around an axis perpendicular to the laser beam axis, with the frequency increasing linearly with the laser power.

Expression of tight-junction proteins in the inflamed and clinically uninvolved skin in patients with venous leg ulcers.

Skin around venous leg ulcers (VLUs) is often inflamed and prone to contact sensitization. Expression of tight-junction components (ZO-1, occludin, and claudins 1 and 4) was studied by immunofluorescence in inflamed and noninflamed lower leg skin (both uncovered skin and skin occluded under hydrocolloid dressings) in patients with VLUs. No major differences were found in the expression of occludin and claudin-4. ZO-1 protein had stronger and more wide-ranging expression in the inflamed epidermis. Expression of claudin-1 was lost from the basal layer of the inflamed skin and skin under the hydrocolloid dressing. The skin on the lower legs affected by VLU may have altered expression of ZO-1 and claudin-1, similar to that seen in psoriatic plaques.

Deletion analogues of transportan.

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.

Role of third intracellular loop of galanin receptor type 1 in signal transduction.

To determine the domains essential for G-protein coupling of the human galanin receptor type 1 (GalR1), we have used both GalR1 mutants and synthetic receptor-derived peptides in(125)I-galanin and [(35)S]-GTPgammaS binding studies. Replacement of potential phosphorylation sites by Leu in the third intracellular loop (IC3) of GalR1 did not affect K(D)values for the receptor. Peptides derived form the IC3 loop, and especially the N-terminal part of it were able to increase the rate of [(35)S]-GTPgammaS binding to the trimeric Gialpha1beta1gamma2, but not to Gsalphabeta1gamma2, whereas the peptides corresponding to the IC1 and IC2 loops had no such effect. IC3 loop peptides also inhibited the binding of(125)I-galanin to GalR1 in membranes from Rin m5F cells. Our results suggest that the IC3 loop of GalR1, especially its N-terminal part, defines the coupling of the receptor to the Gialpha1beta1gamma2 protein and consequently, to the signal transduction cascade.

Pluripotency of 17beta-hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus.

Cochliobolus lunatus 17beta-hydroxysteroid dehydrogenase (17beta-HSD) is pluripotent for several steroidal and nonsteroidal substrates. In the presence of NADPH the enzyme was found to reduce 3-keto groups of 4,5-dihydro steroids, 20-keto groups, and most efficiently, 17-keto groups of steroidal substrates. In addition, several quinones were accepted and found to be even better substrates as steroids due to their higher affinity for the enzyme-coenzyme complex and faster conversion of the enzyme-coenzyme-substrate complex into the corresponding products. As suggested by the competition studies quinones and 17-ketosteroids are converted by the same active center of the enzyme. For all tested substrates, the equilibrium ordered mechanism was established with NADPH binding first to the enzyme. According to our knowledge, the investigated 17beta-HSD is the first known fungal pluripotent enzyme of this type.

Effects of vasopressin-mastoparan chimeric peptides on insulin release and G-protein activity.

Two chimeric peptides, consisting of the linear vasopressin receptor V1 antagonist PhAc-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr, in the N-terminus and mastoparan in the C-terminus connected directly (M375) or via 6-aminohexanoic acid (M391), have been synthesised. At 10 microM concentration, these novel peptides increased insulin secretion from isolated rat pancreatic islet cells 18-26-fold at 3.3 mM glucose and 3.5-5-fold at 16.7 mM glucose. PTX pretreatment of the islets decreased the peptide-induced insulin release. M375 and M391 bind to V1a vasopressin receptors with affinities lower than the unmodified vasopressin antagonist, but with K(D) values of 3.76 nM and 9.02 nM, respectively, both chimeras are high affinity ligands. The GTPase activity and GTPgammaS binding in the presence of these peptides has been characterised in Rin m5F cells. Comparison of the influence of the peptides M375 and M391 on GTPase activity in native and pertussis toxin-treated cells suggests a selective activation of G alpha(i)/G alpha(o) subunits, combined with a suppression of other GTPases, primarily G alpha(s). However, the GTPgammaS binding data show that the peptides retain some of the activating property even in PTX-treated cell membranes. In conclusion, the conjugation of mastoparan with the V1a receptor antagonists produce peptides with properties different from the parent peptides that could be used to elucidate the role of different G proteins in insulin release.

Peptitergent PD1 affects the GTPase activity of rat brain cortical membranes.

Peptitergent PD1 shows complex effects on GTPase activity of rat brain cortical membranes: inhibition in the presence of lower concentrations of GTP and activation at a higher concentration, above 0.5 microM, of GTP. Its effect is dose dependent and is characterized by an EC50 of 1.8 +/- 0.2 microM and a Hill coefficient of 1.6 +/- 0.3, and it increases both Km and Vmax of the GTP hydrolysis. PD1 that was unable to solubilize G-proteins from the membranes probably acts on them by direct binding near the C-terminal alpha-helical region of the Galpha subunit, similarly to mastoparan.

Regulation of GTPase and adenylate cyclase activity by amyloid beta-peptide and its fragments in rat brain tissue.

Modulation of GTPase and adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) activity by Alzheimer's disease related amyloid beta-peptide, A beta (1-42), and its shorter fragments, A beta (12-28), A beta (25-35), were studied in isolated membranes from rat ventral hippocampus and frontal cortex. In both tissues, the activity of GTPase and adenylate cyclase was upregulated by A beta (25-35), whereas A beta (12-28) did not have any significant effect on the GTPase activity and only weakly influenced adenylate cyclase. A beta (1-42), similar to A beta (25-35), stimulated the GTPase activity in both tissues and adenylate cyclase activity in ventral hippocampal membranes. Surprisingly, A beta (1-42) did not have a significant effect on adenylate cyclase activity in the cortical membranes. At high concentrations of A beta (25-35) and A beta (1-42), decreased or no activation of adenylate cyclase was observed. The activation of GTPase at high concentrations of A beta (25-35) was pertussis toxin sensitive, suggesting that this effect is mediated by Gi/G(o) proteins. Addition of glutathione and N-acetyl-L-cysteine, two well-known antioxidants, at 1.5 and 0.5 mM, respectively, decreased A beta (25-35) stimulated adenylate cyclase activity in both tissues. Lys-A beta (16-20), a hexapeptide shown previously to bind to the same sequence in A beta-peptide, and prevent fibril formation, decreased stimulation of adenylate cyclase activity by A beta (25-35), however, NMR diffusion measurements with the two peptides showed that this effect was not due to interactions between the two and that A beta (25-35) was active in a monomeric form. Our data strongly suggest that A beta and its fragments may affect G-protein coupled signal transduction systems, although the mechanism of this interaction is not fully understood.

Differential regulation of GTPase activity by mastoparan and galparan.

The chimeric peptide galparan, composed of galanin (1-13) in the N-terminus and mastoparan in the C-terminus, was recently designed and synthesized. The effect of galparan on GTPase activity of rat brain cortical membranes was studied in comparison with the effect of mastoparan and galanin. GTPase was activated by mastoparan but it was noncompetitively inhibited by galparan, while no effect of galanin and galanin (1-13) was found in this tissue. EC50 of 12.1 +/- 2.1 microM and Hill coefficient of 2.1 +/- 0.6 was calculated for galparan from a dose-response curve and Ki of 19.1 +/- 0.3 microM was obtained by fitting the experimental data to the Michaelis-Menten equation valid in the presence of noncompetitive inhibitor. Mastoparan reversed the effect of galparan in a fully competitive manner while benzalkonium chloride did not prevent the inhibition of GTPase activity by galparan. Pertussis-toxin-catalyzed ribosylation of G proteins from rat brain cortical membranes resulted in 15% lower basal GTPase activity of our preparation but did not alter the parameters of the dose-response curve for galparan inhibition. The rate of GTP gamma S binding to G proteins from rat brain cortical membranes was not influenced by galparan. CD spectra revealed predominantly antiparallel beta-structure and unordered secondary structure of galparan in the buffer solution, while in the presence of lipid vesicles it adopted a higher amount of alpha-helix. Critical micelle concentration of galparan in buffer solution of 22 microM was determined. It is suggested that the reversal of GTPase activation by mastoparan to inhibition by galparan is due to different loci of action of these two peptides on G proteins.

Cell penetration by transportan.

Transportan is a 27 amino acid-long peptide containing 12 functional amino acids from the amino terminus of the neuropeptide galanin and mastoparan in the carboxyl terminus, connected via a lysine. Transportan is a cell-penetrating peptide as judged by indirect immunofluorescence using N epsilon13-biotinyl-transportan. The internalization of biotinyl-transportan is energy independent and takes place efficiently at 37 degrees, 4 degrees, and 0 degrees C. Cellular uptake of transportan is probably not mediated by endocytosis, since it cannot be blocked by treating the cells with phenylarsine oxide or hyperosmolar sucrose solution and is nonsaturable. The kinetics of internalization was studied with the aid of the 125I-labeled peptide. At 37 degrees C, the maximal intracellular concentration is reached in about 20 min. The internalized transportan is protected from trypsin. The cell-penetrating ability of transportan is not restricted by cell type, but seems to be a general feature of this peptide. In Bowes' melanoma cells, transportan first localizes in the outer membrane and cytoplasmatic membrane structures. This is followed by a redistribution into the nuclear membrane and uptake into the nuclei where transportan concentrates in distinct substructures, probably the nucleoli.

Kinetic characterization of all steps of the interaction between acetylcholinesterase and eserine.

The three-step carbamylenzyme mechanism of the action of eserine on acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) has been known for a long time, but its complete kinetic characterization has never been done. Some of our investigations indicated that the determination of missing kinetic parameters should include the inspection of the enzyme-eserine interaction in a very wide range of eserine concentrations. Therefore, the activity of acetylcholinesterase as a function of time in the presence of low concentrations of eserine comparable to the enzyme concentration was followed. The reaction mechanism was analysed by fitting numerically integrated differential equations that describe the time dependences of all reactants and reaction intermediates to these data. Additionally, the progress curve measurements at higher eserine concentrations were carried out on a stopped-flow apparatus. The corresponding progress curve equations were derived and the kinetic parameters evaluated by non-linear regression treatment. The complex analysis confirmed the three-step mechanism. The values of the constants showed that the very high affinity of eserine for binding into the active centre of the enzyme is not so much a consequence of the fast initial complex formation but rather a consequence of its slow dissociation. The subsequent covalent bonding of eserine is also slow, but faster than the dissociation of the initial complex. In this manner, the decarbamoylation is the only process responsible for the reactivation of acetylcholinesterase after removal of eserine.

Purification and characterization of 17beta-hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus.

17beta-Hydroxysteroid dehydrogenase (17beta-HSD) from the filamentous fungus Cochliobolus lunatus was purified in three steps, yielding a protein of an apparent molecular mass of 28 kDa. According to the obtained experimental data, the native form of the enzyme could be a dimer (60 kDa) and/or a tetramer (120 kDa). The enzyme was found to catalyse preferentially the reduction of steroid substrates using NADPH as an electron donor. Both androgens and estrogens are substrates for 17beta-HSD. Kinetic studies revealed the equilibrium ordered kinetic mechanism with NADPH as the first ligand to be bound to the enzyme followed by the addition of the substrate androstenedione. The purification and characterization of 17beta-HSD from Cochliobolus lunatus represents a step towards the elucidation of the role of this enzyme in fungal metabolism.

Rupture of cornual ectopic pregnancy after dilatation and curretage.

A case of a cornual ectopic pregnancy missed at attempted pregnancy termination and with subsequent rupture is presented. The positive pregnancy test at the time of presentation to the emergency department was mistakenly thought to be due to continued detectable blood levels of human chorionic gonadotrophin from the aborted pregnancy. This caused a delay in surgical intervention and subsequent rupture of the ectopic pregnancy with hypovolemic shock.