Vipera lebetina venom contains all types of snake venom metalloproteases

Snake venoms contain four classes of metalloproteases that all have a typical zinc-chelating sequence (HEXXHGXXH). N-terminal sequences and internal sequences of different purified metalloproteases were determined using Edman sequencing and LC MS/MS technique. Oligonucleotides were designed and used as primers for cDNA cloning from Vipera lebetina venom gland cDNA library. We found that isoforms of fibrinolytic enzyme lebetase Le-4 and Le-3 are synthesized in different way: Le-4 is synthesized as P-I type metalloprotease, Le-3 is synthesized with disintegrin-like domain as P-II type protease and processed post-translationally. An endothelial cell apoptosis-inducing heterodimeric glycosylated metalloprotease, V. lebetina apoptosis-inducing protease (VLAIP), belongs to P-III type containing metalloprotease, disintegrin-like and cysteine-rich domains. All these enzymes hydrolyze the Aalpha-chain and more slowly the Bbeta-chain of fibrinogen. Treatment of HUVEC cells with VLAIP induces changes in the attachment of cells to the substrate and causes apoptosis. V. lebetina venom contains also P-IV type-specific coagulant factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein composed of a heavy chain and two C-type lectin-like light chains linked by disulfide bonds. The heavy and light chains of VLFXA are synthesized from different genes.     

Comparing non-hierarchical models: Application to non-linear mixed effects modeling

There is no method available to compare the fit of two non-hierarchical non-linear mixed effects models, although the common practice is to select the model with the lower objective function. Bootstrapping the log-likelihood differences (LLDs) of non-hierarchical models and constructing a bootstrap confidence interval on the LLDs is proposed for comparing the goodness-of-fit of such models. This is illustrated with different parameterizations of clearance models for an anti-infective agent in a longitudinal pharmacokinetic study which are compared. Additive and exponential models of creatinine clearance as a predictor of clearance are used as examples.     

A novel metalloprotease from Vipera lebetina venom induces human endothelial cell apoptosis.

A novel endothelial cell apoptosis inducing metalloprotease (VLAIP) was found in the snake venom of Vipera lebetina. This metalloprotease is a heterodimeric glycoprotein with molecular mass of about 106 kDa. The protease hydrolyzes azocasein, fibrinogen and oxidized insulin B-chain. The enzyme readily hydrolyzes the Aalpha-chain and more slowly Bbeta-chain of fibrinogen. VLAIP does not cleave fibrin. The complete amino acid sequences of the two different monomers of VLAIP are deduced from the nucleotide sequences of cDNAs encoding these proteins. The full-length cDNA sequences of the VLAIP-A and VLAIP-B encode open reading frames of 616 and 614 amino acids that include signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. VLAIP belongs to the metalloprotease/disintegrin family of reprolysins and has high identity with the proteins that induce apoptosis of endothelial cells. Treatment of HUVEC cells with VLAIP induces changes in the attachment of cells to the substrate and causes cell death. We demonstrated that VLAIP inhibits endothelial cell adhesion to extracellular matrix proteins: fibrinogen, fibronectin, vitronectin, collagen I, and collagen IV. The induction of apoptosis by VLAIP was shown by means of a typical DNA fragmentation pattern of apoptotic cells as well as by monitoring phosphatidylserine externalization using annexin V-FITC staining and flow cytometric analysis.     

Evaluation of Photon Interaction Parameters of Some Antioxidants for Food Irradiation Applications

This study aimed to investigate the interaction parameters of antioxidant molecules in some spices and vegetables with gamma radiation. At first, mass attenuation coefficients (MACs, cm²/g) of gingerol, rosmarinic acid, quercetin, curcumin, eugenol, piperine, allicin, and capsaicin molecules were determined at the photon energies (13–1332 keV) emitted from the radioactive isotopes Am-241, Ba-133, Co-60, and Cs-137 with the help of the EpiXS and WinXCOM programs. The smallest and largest MAC values were found as 1.20 and 8.48 cm²/g at 13 keV and 0.059 and 0.058 cm²/g at 1332 keV for eugenol and allicin, respectively. It was observed that both results support each other. Using the MAC values, the effective atomic number and electron density (Z_eff and N_eff) values of the molecules were derived. The Z_eff values for gingerol and allicin were obtained in the range of 5.79–3.40 and 13.85–4.53, respectively. The variation of the buildup factors of antioxidants in the range of 0.015–15 MeV depending on the chemical composition and penetration depth were also examined. It was noticed that the photon accumulation was the lowest in allicin and the highest in gingerol and eugenol. The results obtained from this study will make an essential contribution to dose calculations in food irradiation studies.     

Systemic absorption of ketoconazole from vaginal pessaries.

Eight healthy female subjects were each given (a) ketoconazole 400 mg orally, (b) ketoconazole 400 mg as a single vaginal pessary, (c) ketoconazole 800 mg as two vaginal pessaries, and (d) ketoconazole 1200 mg as three vaginal pessaries. The area under the plasma concentration time curve (AUC) after the oral dose was 51.41 +/- 10.99 mg l-1 h (mean +/- s.d.) and the half-life of ketoconazole was 2.98 +/- 1.41 h. The AUCs after vaginal administration were 0.27 +/- 0.14, 0.52 +/- 0.25, and 0.43 +/- 0.22 mg-1 l h following the 400, 800 and 1200 mg pessaries respectively. Systemic absorption of single doses of vaginally administered ketoconazole appears to be negligible in the absence of vaginal infection. There were no local or systemic side effects related to ketoconazole in these healthy volunteers.     

Collagen-Carboxymethylcellulose Biocomposite Wound-Dressings with Antimicrobial Activity

Microbial infections associated with skin diseases are frequently investigated since they impact on the progress of pathology and healing. The present work proposes the development of freeze-dried, glutaraldehyde cross-linked, and non-cross-linked biocomposite dressings with a porous structure, which may assist the reepithelization process through the presence of collagen and carboxymethylcellulose, along with a therapeutic antimicrobial effect, due to silver nanoparticles (AgNPs) addition. Phisyco-chemical characterization revealed the porous morphology of the obtained freeze-dried composites, the presence of high crystalline silver nanoparticles with truncated triangular and polyhedral morphologies, as well as the characteristic absorption bands of collagen, silver, and carboxymethylcellulose. In vitro tests also assessed the stability, functionality, and the degradability rate of the obtained wound-dressings. Antimicrobial assay performed on Gram-negative (Escherichia coli), Gram-positive (Staphyloccocus aureus) bacteria, and yeast (Candida albicans) models demonstrated that composite wound dressings based on collagen, carboxymethylcellulose, and AgNPs are suitable for skin lesions because they prevent the risk of infection and have prospective wound healing capacity. Moreover, the cell toxicity studies proved that the obtained materials can be used in long time treatments, with no cytotoxic effects.