A maurotoxin with constrained standard disulfide bridging: innovative strategy of chemical synthesis,pharmacology, and docking on K+ channels

Maurotoxin (MTX) is a 34-residue toxin that has been isolated initially from the venom of the scorpion Scorpio maurus palmatus. It presents a large number of pharmacological targets, including small conductance Ca2+-activated and voltage-gated K+ channels. Contrary to other toxins of the alpha-KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C1-C5, C2-C6, C3-C4, and C7-C8 (instead of the conventional C1-C5, C2-C6, C3-C7, and C4-C8, herein referred to as Pi1-like) that does not prevent its folding along the classic alpha/beta scaffold of scorpion toxins. Here, we developed an innovative strategy of chemical peptide synthesis to produce an MTX variant (MTXPi1) with a conventional pattern of disulfide bridging without any alteration of the toxin chemical structure. This strategy was used solely to address the impact of half-cystine pairings on MTX structural properties and pharmacology. The data indicate that MTXPi1 displays some marked changes in affinities toward the target K+ channels. Computed docking analyses using molecular models of both MTXPi1 and the various voltage-gated K+ channel subtypes (Shaker B, Kv1.2, and Kv1.3) were found to correlate with MTXPi1 pharmacology. A functional map detailing the interaction between MTXPi1 and Shaker B channel was generated in line with docking experiments.     

Thioredoxin motif of Caenorhabditis elegans PDI-3 provides Cys and His catalytic residues for transglutaminase activity

Previous reports have suggested that protein disulfide isomerases (PDIs) have transglutaminase (TGase) activity. The structural basis of this reaction has not been revealed. We demonstrate here that Caenorhabditis elegans PDI-3 can function as a Ca(2+)-dependent TGase in assays based on modification of protein- and peptide-bound glutamine residues. By site-directed mutagenesis the second cysteine residue of the -CysGlyHisCys- motif in the thioredoxin domain of the enzyme protein was found to be the active site of the transamidation reaction and chemical modification of histidine in their motif blocked TGase activity.     

Photoexcitation of the O-intermediate in bacteriorhodopsin mutant L93A

During the extended lifetime of the O-state in bacteriorhodopsin (bR) mutant L93A, two substates have been distinguished. The first O-intermediate (OI) is in rapid equilibrium with N and apparently still has a 13-cis chromophore. OI undergoes a photoreaction with a small absorbance change, positive charge transport in the pumping direction, and proton release and uptake. None of these effects was detected after photoexcitation of the late O (OII). The most likely interpretation of the effects seen is an accelerated return of the molecule from the OI- to the bR-state. However, with a lifetime approximately 140 ms, the reaction cannot account for the observed high pumping efficiency of the mutant under continuous illumination. We suggest that OII corresponds to the O-intermediate with a twisted all-trans chromophore seen in the photocycle of wild-type bR, where the 13-cis OI-intermediate under the usual conditions does not accumulate in easily detectable amounts and, therefore, has generally been overlooked. Both the OI- and OII-decays are apparently strongly inhibited in the mutant.     

Chiral detection of carotenoid assemblies

Carotenoid assemblies were produced by aqueous dilution of ethanolic solutions. UV/VIS and CD spectroscopy revealed the formation of J- and H-types of aggregates of both right- and left-handed kinds. Simulation of UV/VIS spectra of the aggregates showed characteristic differences between the two types. 6'-Epimers of capsanthol ((all-E,3R,3'S,5'R)-beta,kappa-carotene-3,3',6'-triols) formed assemblies with increased chirality in dilute solution. While the absorption of 6'R-capsanthol giving H-type aggregate does not depend on the concentration, 6'S-capsanthol yielding J-type assembly showed concentration-dependent absorption intensity. Dilute aggregate of 6'R-capsanthol is characterized by an extremely large A value of -6,600. The transformation of J- to H-type assembly was observed in the mixtures of the epimers producing an intermediate kind of aggregate. A hypothetical structure for H-type assemblies is proposed.     

Synthesis of amyloid β-peptides in solution employing chloroform-phenol mixed solvent for facile segment condensation of sparingly soluble protected peptides

Segment condensation reaction of sparingly soluble protected peptides proceeded smoothly in CHCl₃-phenol mixed solvent without danger of epimerization or of significant ester formationwith the carboxyl component when 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was employedin the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine(HOOBt). The optimal conditions for enhancement of peptide coupling mediated by EDC/HOOBt in CHCl₃-phenol were determined and successfully applied to the synthesis of amyloid -peptide (1-42), (1-43) and [Pyr³]-(3-42). These peptides of high homogeneity were used to examine the relation between structure and amyloidogenesis by means of CD spectra andfluorimetric assay.     

The kinetic mechanism of the glutamate-aspartate carrier in rat intestinal brush-border membrane vesicles: the role of potassium

The sodium dependent transport system for L-glutamate and L-aspartate localized in the apical part of rat enterocytes has previously been kinetically characterized (Prezioso, G., and Scalera, V. (1996). Biochim. Biophys. Acta 1279, 144–148). In this paper the mechanism by which the potassium cation specifically activates the L-glutamate–sodium cotransport process is investigated. Potassium has been found to act as an activator when it is present inside the membrane vesicles, while its presence outside is ineffective, and the effect is saturable. The kinetic parameters with respect to sodium and glutamate have been compared in the presence and in the absence of the activator. The results indicate that the ordered sodium–sodium glutamate mechanism is not altered by potassium, and that the activation is probably exerted on both the rate determining steps of the transport process. It is proposed that (1) a specific binding site for potassium is present on the inside hydrophilic part of the membrane carrier, (2) the binding of the effector accelerates the intramembrane rearrangement steps of both the disodium glutamate–carrier complex and the free carrier, (3) the affinity of the carrier is lowered with respect to sodium whereas it is increased for glutamate, and (4) K⁺ antiport is not performed by this carrier.     

Treatment options in extravasation injury: an experimental study in rats

Local skin necrosis after extravasation of doxorubicin hydrochloride (Adriamycin), a widely used chemotherapeutic agent, is a common problem in cancer patients. Even though several treatment options have been proposed for extravasation injury, there is still controversy regarding the management of such lesions. The aim of this study was to compare the efficacy of saline infiltration, vitamin C infiltration, suction technique, and early surgical excision as a treatment in a rat extravasation model. The authors planned their study in two stages. In stage 1, the lowest effective dose of doxorubicin at which a homogeneous skin necrosis was formed and the method of administration were investigated. Intradermal and subpannicular injections were made for six rats, using six different concentrations of doxorubicin (0.33, 0.5, 0.66, 1.0, 1.33, and 1.5 mg/ml). In stage 1, the intradermal injection produced homogeneous and uniform tissue necrosis. In stage 2, the efficacy of saline infiltration (group 1), vitamin C infiltration (group 2), suction (group 3), suction and saline washout (group 4), suction and vitamin C washout (group 5), and early surgical excision (group 6) was compared. The treatment options were applied 2 hours after doxorubicin injection. At the end of the seventh day, the presence and size of ulcers at the injection site were calculated. Fourteen days after injection, a histopathologic examination was performed for each treatment and control group. In groups 1 and 3, there was no statistically significant difference in the size of necrosis compared with the control groups. In groups 2, 4, and 5, the size of necrosis was smaller compared with the control groups, and this was statistically significant. Furthermore, in group 4 (suction and saline washout) and group 5 (suction and vitamin C washout), the calculated area of necrosis was smaller compared with other treatment groups, and this was statistically significant. The findings supported the assertion that suction and saline or vitamin C washout reduce necrotic tissue size in extravasation injury.     

Vegetation dynamics of south-western Italy in the last 28 kyr inferred from pollen analysis of a Tyrrhenian Sea core

A continuous pollen record covering the last 28 kyr was obtained from core C106 collected in the Bay of Salerno in the southern Tyrrhenian Basin, seven radiocarbon dates and the recognition of two tephra layers (Y3 and Pompeii Pumice) providing good chronological constraints. The clear climatic signal given by the pollen spectra integrated by isotopic data, combined with comparisons with other Mediterranean sites, allowed the Last Glacial, Late Glacial and Holocene periods to be distinguished in the core. In particular, the Last Glacial period is characterised by large quantities of herbaceous and steppe elements such as Artemisia. The beginning of the Late Glacial has been correlated with the first increase of deciduous Quercus and the reduction of steppe and herbaceous elements. The Younger Dryas event is recorded only by oxygen isotopes while the vegetation does not seem to change, as in other Mediterranean sites. The Holocene corresponds to rich deciduous and evergreen forests. The first features which could be interpreted as signs of human presence are represented by a few grains of Juglans, Castanea and cereal-type while intensive olive cultivation and deforestation seem to fall within the Middle Ages. Received October 10, 2001 / Accepted June 20, 2002 Correspondence to: Elda Russo Ermolli