Conformational study of new amphipathic alpha-helical peptide models of apoA-I as potential atheroprotective agents

Aiming at contributing to the development of potential atheroprotective agents, we report on the concept and design of two peptide models, which mimic the amphipathic helices of apoA-I and incorporate Met into their sequences to validate its role as oxidant scavenger: Ac-ESK(Palm)KELSKSW(10)SEM(13)LKEK(Palm)SKS-NH(2) (model 1 [W(10), M(13)]) and Ac-ESK(Palm)KELSKSM(10)SEW(13)LKEK(Palm)SKS-NH(2) (model 2 [M(10), W(13)]). Hydrophobic residues of both models cover about the half of the surface, while the positively and negatively charged residues constitute two separate clusters on the hydrophilic face. Palmitoyl groups were introduced into the Lys-N(epsilon)H(2) groups at positions 3 and 17 to contribute to the amphipathic character of the peptides and stabilize the nonpolar face of the helix. Conformational study by the combined application of 2D-NMR and molecular dynamics simulations, CD, FTIR, and fluorescence spectroscopy revealed that model 1 adopts helical conformation and Met is well exposed to the microenvironment. Model 2 that derives from model 1 by exchanging W(10) (model 1) with M(10) and M(13) (model 1) with W(13) also displays helical characteristics, while Met is rather shielded. Oxidation experiments indicated that model 1 exhibits a 2-fold more potent antioxidant activity towards LDL oxidation, compared to model 2, confirming the role of Met, when is devoid of steric hindrances, as oxidant scavenger for the protection of LDL.     

Development of an amperometric biosensing method for the determination of L-fucose in pretreated urine

The first amperometric biosensing method for the determination of L-fucose is described. L-Fucose is the objective of much current research, as it is considered as a potential marker for various pathologic disorders. Recombinant L-fucose dehydrogenase, having as cofactor beta-nicotinamide adenine dinucleotide phosphate (NAD+P), was cross-linked in a water-soluble photosensitive polymer matrix, that is, polyvinyl alcohol (PVA) modified with styrylpyridinium (SbQ), in the presence of BSA and glutaraldehyde. The resulting membrane was sandwiched between two polycarbonate membranes and was mounted in an amperometric cell. The oxidation of the enzymatically produced NADPH was monitored at a platinum anode at +0.25 V versus a silver pseudoreference electrode in the presence of ferricyanide. The system was fully optimized with respect to various analytical parameters. Regarding to the mechanical properties of the membrane and the storage stability of the immobilized enzyme, various parameters were also optimized. Several methods for the pretreatment of urine samples were investigated. Treatment of the samples with PbO2 found to eliminate the interference effect of various electroactive species exist in urine; optimum incubation time was determined since at prolonged incubation times L-fucose is also affected. Calibration curves for the direct and the mediated monitoring of NADPH were liner over the concentration ranges 0.04-1.0 mM (r2=0.9995) and 0.03-3.0 mM (r2=0.9997) fucose, respectively. The detection limits (S/N 3) were 2 and 1.5 microM fucose, respectively. The R.S.D. of the mediated biosensor is better than 1.5% (n=10, 0.5 mM fucose). The proposed biosensor correlates well with a reference enzymatic method and exhibits very good working and storage stability.     

Diterpenes from the brown algae Dictyota dichotoma and Dictyota linearis

Nineteen secondary metabolites of the brown alga Dictyota dichotoma (Huds.) Lam. and fifteen metabolites of the brown alga D. linearis (Ag.) Grev. were isolated and their chemical structures were elucidated on the basis of their NMR and mass spectral data. The diterpenes isopachydictyolal (1) from D. dichotoma and 4alpha-acetyldictyodial (2) from D. linearis are new natural products. The antiviral activity of metabolites isolated in adequate amounts was evaluated in laboratory assays against Herpes simplex virus I (HSV I) and Poliomyelitis Virus I, using Vero cells as hosts.     

Tendon conditioning: artefact or property?

Isolated tendons subjected to cyclic tensile loads higher than those experienced in the tendons' recent history exhibit 'conditioning', i.e. gradually increasing elongations upon loading and gradually increasing residual elongations after unloading in the first few loading-unloading cycles. The present study examines whether this behaviour is a measurement artefact or an actual time-dependent property. The gastrocnemius tendons of six men who refrained from rigorous physical activities prior to the experiment were loaded cyclically by 10 repeated isometric plantarflexion contractions at 80% of the moment generated during plantarflexion maximal voluntary contraction (MVC). In each contraction, the elongation of the gastrocnemius tendon at 80% of MVC and the residual tendon elongation after relaxation were obtained from the analysis of sonographs recorded during the test. The tendon elongation during activation and the residual tendon elongation after relaxation increased by ca. 5 mm from the first contraction to the tenth contraction, with no changes obtained after the fifth contraction. The behaviour of the tendon in the first five contractions indicates the presence of conditioning. It is therefore concluded that conditioning is a relevant property and not an artefact associated with in vitro testing. This has implications for joint kinematics and muscle excursion.     

Hydroxyl-platelet-activating factor exists in blood of healthy volunteers and periodontal patients

Periodontal diseases are localized chronic inflammatory conditions of the gingival and underlying bone and connective tissue. Platelet-activating factor (PAF), a potent inflammatory phospholipid mediator that has been previously detected in elevated levels in inflamed gingival tissues, in gingival crevicular fluid and in saliva, is implicated in periodontal disease. Our results from previous studies showed that the biologically active phospholipid detected in gingival crevicular fluid is a hydroxyl-PAF analogue. In this study, hydroxyl-PAF analogue was detected for the first time in human blood derived from patients with chronic periodontitis as well as from periodontally healthy volunteers. The hydroxyl-PAF analogue was purified by high-performance liquid chromatography, detected by biological assays and identified by electrospray analysis. In addition, the quantitative determination of PAF and hydroxyl-PAF analogue (expressed as PAF-like activity) showed a statistically significant increase in the ratio of hydroxyl-PAF analogue levels to PAF levels in periodontal patients, suggesting that this bioactive lipid may play a role in oral inflammation.     

Time-resolved resonance Raman and time-resolved step-scan FTIR studies of nitric oxide reductase from Paracoccus denitrificans: comparison of the heme b3-FeB site to that of the heme-CuB in oxidases

Time-resolved resonance Raman (TR(3)) and time-resolved step-scan (TRS(2)) FTIR spectroscopies have been used to probe the structural dynamics at the heme b(3) proximal and distal sites after carbon monoxide photolysis from fully reduced CO-bound nitric oxide reductase. The Raman spectra of the transient species exhibit structural differences relative to the equilibrium geometry of heme b(3). The most significant of these is a shift of 8 cm(-1) to higher frequency of the 207 cm(-1) mode, and a shift of 7 cm(-1) to lower frequency of the nu(4) mode. Our results indicate that the 207 cm(-1) mode observed in the equilibrium-reduced heme b(3) originates from nu(Fe-His). Its behavior in the photolytic transients indicates that the relaxed Fe-His state is not significantly populated. We suggest that relaxation along the tilt angle (theta) of the proximal histidine with respect to the heme plane and the out-of-plane displacement of the Fe (q) are coupled, and ligand binding and dissociation are accompanied by significant changes in the angular orientation of the His ligand. The results are compared to those obtained for the aa(3)-cytochrome c oxidase from Paracoccus denitrificans. The results are compared to those obtained for the aa(3)-cytochrome c oxidase from P. denitrificans. The TR(3) and TRS(2) FTIR data demonstrate significant alterations in the nature of the heme-protein dynamics between nitric oxide reductase and heme-copper oxidases resulting from specific structural differences in their respective hemepockets.     

Aminoacyl-tRNA formation in the extreme thermophile Thermus thermophilus

Thermophilic organisms must be capable of accurate translation at temperatures in which the individual components of the translation machinery and also specific amino acids are particularly sensitive. Thermus thermophilus is a good model organism for studies of thermophilic translation because many of the components in this process have undergone structural and biochemical characterization. We have focused on the pathways of aminoacyl-tRNA synthesis for glutamine, asparagine, proline, and cysteine. We show that the T. thermophilus prolyl-tRNA synthetase (ProRS) exhibits cysteinyl-tRNA synthetase (CysRS) activity although the organism also encodes a canonical CysRS. The ProRS requires tRNA for cysteine activation, as is known for the characterized archaeal prolyl-cysteinyl-tRNA synthetase (ProCysRS) enzymes. The heterotrimeric T. thermophilus aspartyl-tRNA(Asn) amidotransferase can form Gln-tRNA in addition to Asn-tRNA: however, a 13-amino-acid C-terminal truncation of the holoenzyme A subunit is deficient in both activities when assayed with homologous substrates. A survey of codon usage in completed prokaryotic genomes identified a higher Glu:Gln ratio in proteins of thermophiles compared to mesophiles.     

Unmasking the anti-La/SSB response in sera from patients with Sjogren's syndrome by specific blocking of anti-idiotypic antibodies to La/SSB antigenic determinants

BACKGROUND: Autoantigen La/SSB is molecular target of humoral autoimmunity in patients with primary Sjogren's Syndrome (pSS) and systemic lupus erythematosus (SLE). In this study, we investigated the existence and possible influence of anti-idiotypic response to anti-La/SSB antibodies. MATERIALS AND METHODS: Synthetic peptide analogs (pep) of the major antigenic determinants of La/SSB (289-308 aa and 349-364 aa) were prepared. Based on "molecular recognition" theory, complementary peptides (cpep), derived by anti-parallel readings of the noncoding strand of La/SSB DNA encoding for its antigenic determinants, were constructed. Sera from 150 patients with anti-La/SSB antibodies, 30 patients without anti-La/SSB antibodies, and 42 normal individuals were tested against all four peptides. F(ab')(2) fragments from anti-peptide IgG were prepared and F(ab')(2) - IgG interactions were evaluated using a specific anti-idiotypic ELISA. RESULTS: All four peptides were recognized by anti-La positive sera (83% and 51% for pep and cpep 349-364 and 51% and 28% for pep and cpep289-308, respectively). Anti-cpep F(ab')(2 )bound to a common idiotype (Id) located within or spatially close to the antigen combining site of anti La/SSB (anti-pep) antibodies. Homologous and cross-inhibition experiments further confirmed this relation. The anti-idiotypic antibodies inhibited the anti-La/SSB antibody binding to recombinant La/SSB by 91%. To overcome the anti-idiotypic interference in anti-La/SSB detection, a specific assay was developed. Sera were heated for dissociation of Id-anti-Id complexes, anti-Id antibodies blocked with cpep, and anti-La/SSB reactivity was recovered. Application of this method to anti-Ro positive-anti-La/SSB "negative" sera showed that all anti-Ro/SSA positive autoimmune sera also possess anti-La/SSB antibodies. This reaction was not observed in 14 anti-Ro negative- anti-Sm/RNP positive sera from patients with SLE. CONCLUSIONS: Autoimmune sera from patients with pSS and SLE contain anti-idiotypic antibodies targeting a common anti-La/SSB idiotype. These antibodies can be detected using complementary peptides of La/SSB epitopes. The antiidiotypic antibodies mask the anti-La/SSB response. Hidden anti-La/SSB antibodies can be released and detected using complementary epitope analogs.     

The dorsal reverse adipofascial flap for fingertip reconstruction

Nine patients who presented with fingertip amputations were treated with the dorsal reverse adipofascial flap. The mean age of the patients was 41.3 years and the mean follow-up was 18 months. The flap described here was used only for amputations at the level of the nail fold, from approximately the lunula to the proximal nail matrix. This flap is based on the dorsal arterial branches that originate from the volar digital arteries just distal to the distal interphalangeal joint. The flap uses only the adipofascial tissue over the middle phalanx of the injured finger; it is turned over to cover the fingertip defect and then covered with a split-thickness skin graft. All flaps survived completely, and the patients continue to use their fingertips as before the amputation injury. This one-step operation is easily performed (even in the emergency department), makes no use of the adjacent digits, and provides a pleasing and stable cover for the fingertips.