Two-step liquid-phase microextraction and high-performance liquid chromatography for the simultaneous analysis of the enantiomers of mefloquine and its main metabolite carboxymefloquine in plasma

A method for the simultaneous analysis of the enantiomers of mefloquine (MQ) and its main metabolite carboxymefloquine (CMQ) in plasma is described for the first time. The assay involves two-step liquid-phase micro-extraction (LPME) and enantioselective high-performance liquid chromatography. In the first LPME step, the enantiomers of MQ were extracted from an alkalinized sample through a thin layer of di-n-hexyl ether immobilized in the pores of the hollow fiber and into 0.01 M perchloric acid as acceptor solution. In the second LPME step, the same sample was acidified to enable the extraction of CMQ using the same organic solvent and 0.05 M sodium hydroxide as acceptor phase. The analytes were resolved on a Chirobiotic T column in the polar-organic mode of elution and detected at 285 nm. The recovery rates from 1 mL of plasma were in the range 35-38%. The method presented limits of quantification of 50 ng/mL for all analytes and was linear up to 1,500 and 3,000 ng/mL for the enantiomers of MQ and CMQ, respectively. The plasmatic concentrations of (+)-(RS)-MQ were higher than those of (-)-(SR)-MQ after oral administration of the racemic drug to rats.

Paracoccin from Paracoccidioides brasiliensis; purification through affinity with chitin and identification of N-acetyl-beta-D-glucosaminidase activity

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most frequent systemic mycosis in Latin America. Our group has been working with paracoccin, a P. brasiliensis lectin with MM 70 kDa. which is purified by affinity, with immobilized N-acetylglucosamine (GlcNAc). Paracoccin has been described to play a role in fungal adhesion to extracellular matrix components and to induce high and persistent levels or TNF alpha. and nitric oxide production by macrophages. In the cell wall, paracoccin colocalizes with the beta-1,4-homopolymer of GlcNAc into the budding sites of the P. brasiliensis yeast cell. In this paper we present a protocol for the chitin-affinity purification or paracoccin. This procedure provided higher yields than those achieved by means of the technique based oil the affinity of this lectin with GlcNAc and had an impact on downstream assays. SDS-PAGE and Western blot analysis revealed similarities between the N-acetylglucosamine- and chitin-bound fractions, confirmed by MALDI-TOF-MS of trypsinic peptides. Western blot of two-dimensional gel electrophoresis of the yeast extract showed a major spot with M(r) 70000 and pl approximately 5.63. Moreover, an N-acetyl-beta-D-glucosaminidase activity was reported for paracoccin, thereby providing new insights into the mechanisms that lead to cell wall remodelling and opening new perspectives for its structural characterization. Copyright (C) 2009 John Wiley & Sons. Ltd.

Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma

Phospholipases A(2) (PLA(2)s) are important components of Bothrops snake venoms, that can induce several effects on envenomations such as myotoxicity, inhibition or induction of platelet aggregation and edema. It is known that venomous and non-venomous snakes present PLA(2) inhibitory proteins (PLIs) in their blood plasma. An inhibitory protein that neutralizes the enzymatic and toxic activities of several PLA2s from Bothrops venoms was isolated from Bothrops alternatus snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on CNBr-activated Sepharose. Biochemical characterization of this inhibitory protein, denominated alpha BaltMIP, showed it to be a glycoprotein with Mr of similar to 24,000 for the monomeric subunit. CD spectra of the PLA(2)/inhibitor complexes are considerably different from those corresponding to the individual proteins and data deconvolution suggests that the complexes had a relative gain of helical structure elements in comparison to the individual protomers, which may indicate a more compact structure upon complexation. Theoretical and experimental structural studies performed in order to obtain insights into the structural features of aBaltMIP indicated that this molecule may potentially trimerize in solution, thus strengthening the hypothesis previously raised by other authors about snake PLIs oligomerization. (C) 2010 Elsevier Masson SAS. All rights reserved.

An alpha-type phospholipase A(2) inhibitor from Bothrops jararacussu snake plasma: Structural and functional characterization

An inhibitory protein that neutralizes the enzymatic, toxic and pharmacological activities of several phospholipases A(2) from Bothrops venoms was isolated from B. jararacussu snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on Sepharose gel. Biochemical characterization of this inhibitory protein, denominated alpha BjussuMIP, showed it to be an oligomeric glycoprotein with M-r of 24,000 for the monomeric subunit. Secondary structural analysis by circular dichroism revealed 44% alpha-helix, 18% beta-sheet, 10% beta-turn and 28% random coil structures. Circular dichroism spectroscopy indicated that no significant alterations in the secondary structure of either alpha BjussuMIP or the target protein occur following their interaction. The product from the reaction with reverse transcriptase produced a cDNA fragment of 432 bp that codifies for a mature protein of 144 amino acid residues. The first 21 amino acid residues from the N-terminal and five tryptic peptides were characterized by mass spectrometry of the mature protein and confirmed by the nucleotide sequence. Alignment of alpha BjussuMIP with other snake inhibitors showed a sequence similarity of 73-92% with these alpha PLIs. alpha BjussuMIP was relatively stable within the pH range of 6-12 and temperatures from 0 degrees C to 80 degrees C, even after deglycosylation. The results showed effects against Bothrops phospholipase A(2) activities (enzymatic, edema inducing, myotoxic, cytotoxic and bactericidal), suggesting that alpha BjussuMIP may prove useful in the treatment of snakebite envenomations. (C) 2008 Elsevier Masson SAS. All rights reserved.

Ironporphyrin immobilized onto montmorillonite as a bimimetical model for azo dye oxidation

In this work, we studied the oxidation of the azo dye Disperse orange 3 (DO3) by hydrogen peroxide, catalyzed by 5,10,15, 20-tetrakis(4-N-methylpyridyl)porphyrin iron(III) chloride immobilized onto montmorillonite K10, FeP-K10. Results showed that the FeP-K10/H2O2 system is efficient for discoloration of the DO3 dye, especially at pH 3.0. The catalyst was shown to be relatively stable and could be recycled many times, leading to good yields. DO3 oxidation products were analyzed by gas chromatography and mass spectrometry, being 4-nitroaniline the main product. Tert-butylhydroperoxide and iodosylbenzene were also used as oxidants, giving rise to 4-nitroaniline as product too. The studied system is a good biomimetic model of oxidative enzymes, being a promising discoloring agent for azo dyes. (C) 2007 Elsevier Ltd. All rights reserved.

Hyaluronidase Behavior at the Air/Liquid and Air/Lipid Interfaces and Improved Enzymatic Activity by Its Immobilization in a Biomembrane Model

Bovine testicular hyalurphidase (BT-HAase), a tetrameric enzyme responsible for randomly hyaluronic acid, catalytic hydrolysis, was successfully immobilized on Langmuir- Blodgett films prepared with the sodium salt of dihexadacylphosphoric acid, (DHP-Zn(II)) ending with dipalmitoylphosphatidylcholine, DPPC. Data of protein, adsorption at the air-liquid interface by means of pendant drop shipe analysis and interaction of the protein with Langmuir monolayers of DPPC, using a Langmuir trough, have provided information. about the conditions to be used in the protein immobilization. The dynamic surface pressure curves obtained from pendant drop experiments for the enzyme in buffer solutions indicate that, within the range of concentration investigated in this study, the enzyme exhibits the largest induction time at 5 mu g L(-1) attributed to diffusion processes. Nevertheless, it seems that, at this concentration, the most probable conformation should be the one which occupies the smallest area at pi -> 0. The surface pressure (pi) area curves obtained for BT-HAase and mixed DPPC- BT-HAase monolayers reveal the presence of the enzyme at the air-lipid interface up to 45 mN m(-1). Tests of enzymatic activity, using hyaluronic acid, HA, as the substrate, showed an increase of activity compared to the homogeneous medium. A simplified model of protein insertion into the lipid matrix is used to explain the obtained results.

Carbamazepine oxidation catalyzed by iron and manganese porphyrins supported on aminofunctionalized matrices

This work describes the catalytic activity of manganese and iron porphyrins, Mn and Fe(TFPP)Cl, covalently immobilized on the aminofunctionalized supports montmorillonite K-10 (MontX) and silica (SilX), where X= 1 or 2 represents the length of the organic chain (""arms"") binding the metalloporphyrin to the support. These systems were characterized by UV-vis and Electronic Paramagnetic Resonance (EPR), and they were used as catalysts in the oxidation of carbamazepine (CBZ) by the oxidants iodosylbenzene (PhIO) and hydrogen peroxide. The manganese porphyrin (MnP) catalysts proved to be efficient and selective for the epoxide, the main CBZ metabolite in natural systems. MnMont1 was an excellent catalyst when PhIO was used as oxidant, even better than the same MnP in homogeneous system. Supports bearing short ""arms"" led to the best yields. Although H2O2 is an environmentally friendly oxidant, low product yields were obtained when it was employed in CBZ oxidation. Fe(TFPP)CI immobilized on aminofunctionalized supports was not an efficient catalyst, probably due to the presence of Fe(H) species in the matrix, which led to the less reactive intermediate PFe(IV)(O). (c) 2007 Elsevier B.V. All rights reserved.

Jacobsen catalyst immobilized on chitosan membrane as interface catalyst in organic/aqueous system for alkene oxidation

The Jacobsen catalyst, Mn(salen), was immobilized in chitosan membrane. The obtained Mn(salen)-Chit was characterized by thermogravimetric analysis (TC), differential thermal analysis (DTA), differential scanning calorimetry (DSC), infrared spectroscopy (FT-IR), degree of N-acetylation by (1)H NMR, and UV-vis spectroscopy. The UV-vis absorption spectrum of the encapsulated catalyst displayed the typical bands of the Jacobsen catalyst, and the FT-IR presented an absorption band characteristic of the imines present in the Jacobsen catalyst. The chitosan membranes were available, in a biphasic system, as a catalytic barrier between two different phases: an organic substrate phase (cyclooctene or styrene) and an aqueous solution of either m-CPBA, t-BuOOH or H(2)O(2), and dismissing the need for phase transfer agents and leading to better product yields compared with the catalyst in homogeneous medium. This new catalyst did not leach from the support and was reused many times, leading to high turnover frequencies. (C) 2009 Elsevier B.V. All rights reserved.

Molecular View of the Interaction between iota-Carrageenan and a Phospholipid Film and Its Role in Enzyme Immobilization

Proteins incorporated into phospholipid Langmuir-Blodgett (LB) films are a good model system for biomembranes and enzyme immobilization studies. The specific fluidity of biomembranes, an important requisite for enzymatic activity, is naturally controlled by varying phospholipid compositions. In a model system, instead, LB film fluidity may be varied by covering the top layer with different substances able to interact simultaneously with the phospholipid and the protein to be immobilized. In this study, we immobilized a carbohydrate rich Neurospora crassa alkaline phosphatase (NCAP) in monolayers of the sodium salt of dihexadecylphosphoric acid (DHP), a synthetic phospholipid that provides very condensed Langmuir films. The binding of NCAP to DHP Langmuir-Blodgett (LB) films was mediated by the anionic polysaccharide iota-carrageenan (iota-car). Combining results from surface isotherms and the quartz crystal microbalance technique, we concluded that the polysaccharide was essential to promote the interaction between DHP and NCAP and also to increase the fluidity of the film. An estimate of DHP:iota-car ratio within the film also revealed that the polysaccharide binds to DHP LB film in an extended conformation. Furthermore, the investigation of the polysaccharide conformation at molecular level, using sum-frequency vibrational spectroscopy (SFG), indicated a preferential conformation of the carrageenan molecules with the sulfate groups oriented toward the phospholipid monolayer, and both the hydroxyl and ether groups interacting preferentially with the protein. These results demonstrate how interfacial electric fields can reorient and induce conformational changes in macromolecules, which may significantly affect intermolecular interactions at interfaces. This detailed knowledge of the interaction mechanism between the enzyme and the LB film is relevant to design strategies for enzyme immobilization when orientation and fluidity properties of the film provided by the matrix are important to improve enzymatic activity.

Biomechanical evaluation on tendon reinsertion by comparing trans-osseous suture and suture anchor at different stages of healing: Experimental study on rabbits

Background: Through an experimental biomechanical study on rabbits, tendon reinsertion by means of trans-osseous suture on a spongy bone bed and suture anchor were evaluated comparatively at different phases of healing. Methods: Twenty-four New Zealand White rabbits were used: 2 as pilots, 4 as the control group, and 18 as the experimental group. These 18 animals underwent sectioning and reinsertion of the Achilles tendon bilaterally, using the technique of trans-osseous suture on 1 side and suture anchor on the other. All the pelvic limbs that underwent the procedure were then immobilized for 3 weeks. The experimental group was divided into 3 groups that were sacrificed, respectively, 3, 6, and 12 weeks later. The tendon-bone complex was subjected to biomechanical tests to evaluate the parameters of maximum strength, stiffness, and yield strength. Results: There was no statistically significant difference between the suture anchor group and the trans-osseous suture group, in relation to yield strength (3 weeks, P = .222; 6 weeks, P = .465; and 12 weeks, P = .200) or maximum strength (3 weeks, P = .222; 6 weeks, P = .076; and 12 weeks, P = .078). In relation to stiffness, the suture anchor group showed a statistically significant difference only at 3 weeks of healing ( P = .032) over the trans-osseous suture group. Conclusion: The technique of suturing with an anchor was shown to be similar to the technique of trans-osseous suture for the studied parameters. Level of evidence: Basic Science Study, In-Vitro Biomechanics Study. (C) 2010 Journal of Shoulder and Elbow Surgery Board of Trustees.

Quartz Crystal Microbalance monitoring the real-time binding of lectin with carbohydrate with high and low molecular mass

Quartz Crystal Microbalance (QCM) was used to monitor the mass changes on a quartz crystal surface containing immobilized lectins that interacted with carbohydrates. The strategy for lectin immobilization was developed on the basis of a multilayer system composed of Au-cystamine-glutaraldehyde-lectin. Each step of the immobilization procedure was confirmed by FTIR analysis. The system was used to study the interactions of Concanavalin A (ConA) with maltose and Jacalin with Fetuin. The real-time binding of different concentrations of carbohydrate to the immobilized lectin was monitored by means of QCM measurements and the data obtained allowed for the construction of Langmuir isotherm curves. The association constants determined for the specific interactions analyzed here were (6.4 +/- 0.2) X 10(4) M-1 for Jacalin-Fetuin and (4.5 +/- 0.1) x 10(2) M-1 for ConA-maltose. These results indicate that the QCM constitutes a suitable method for the analysis of lectin-carbohydrate interactions, even when assaying low molecular mass ligands such as disaccharides. Published by Elsevier B.V.

Metalloproteinase inhibition ameliorates hypertension and prevents vascular dysfunction and remodeling in renovascular hypertensive rats

Altered activity of matrix metalloproteinases (MMPs) is implicated in the vascular remodeling of hypertension. We examined whether increased MMP-2 expression/activity plays a role in the vascular remodeling and dysfunction found in the two-kidney, one-clip (2K-1C) hypertension. Sham operated or 2K-1C hypertension rats were treated with doxycycline 30 mg/(kg day) (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes, collagen, and elastin contents in the aortic wall were studied in hematoxylin/eosin, Sirius Red, and Orceine stained aortic sections, respectively. Aortic MMP-2 levels were determined by gelatin zymography and aortic MMP-2 proteolytic activity was measured using DQ gelatin as the substrate after MMP-2 was captured by a specific antibody and immobilized on a microplate. Aortic MMP-2/tissue inhibitor of metalloprotemases (TIMP)-2 mRNA levels were determined by real time RT-PCR. Doxycycline attenuated 2K-1C hypertension (215 +/- 8 mmHg versus 167 +/- 13 mmHg in 2K-1C rats and 2K-1C + doxy rats, respectively; P < 0.01) and prevented the 35% reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Doxycycline prevented the increases in media thickness, and was associated with lower media/lumen and cross-sectional areas (all P<0.01). Doxycycline also prevented excessive collagen and elastin deposition in the vascular wall. Increased MMP-2 and Pro-MMP-2 levels and MMP-2 activity were found in the aortas of 2K-1C rats (all P<0.05). A 21-fold increase (P<0.001) in the ratio of MMP-2/TIMP-2 mRNA expression was found in the 2K-1C group, whereas this ratio remained unaltered in 2K-1C+doxy rats. Our results suggest that MMP-2 plays a role in 2K-1C hypertension and its structural and functional vascular changes, which were attenuated by doxycycline. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Cloning, expression and purification of a glycosylated form of the DNA-binding protein Dps from Salmonella enterica Typhimurium

Dps, found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress and/or nutrient-limited environment. Dps has been shown to accumulate during the stationary phase, to bind to DNA non-specifically, and to form a crystalline structure that compacts and protects the chromosome. Our previous results have indicated that Dps is glycosylated at least for a certain period of the bacterial cell physiology and this glycosylation is thought to be orchestrated by some factors not yet understood, explaining our difficulties in standardizing the Dps purification process. In the present work, the open reading frame of the dps gene, together with all the upstream regulatory elements, were cloned into a PCR cloning vector. As a result, the expression of dps was also controlled by the plasmid system introduced in the bacterial cell. The gene was then over-expressed regardless of the growth phase of the culture and a glycosylated fraction was purified to homogeneity by lectin-immobilized chromatography assay. Unlike the high level expression of Dps in Salmonella cells, less than 1% of the recombinant protein was purified by affinity chromatography using jacalin column. Sequencing and mass spectrometry data confirmed the identity of the dps gene and the protein, respectively. In spite of the low level of purification of the jacalin-binding Dps, this work shall aid further investigations into the mechanism of Dps glycosylation. (C) 2008 Elsevier Inc. All rights reserved.

Gingival and bone necrosis caused by accidental sodium hypochlorite injection instead of anaesthetic solution

Aim To report clinical complications (pain, necrotic gingival tissue and bone sequestration) resulting from accidental injection of sodium hypochlorite. Summary Root canal treatment is a routine clinical procedure with few reported complications. Sodium hypochlorite (NaOCl) is commonly used as an irrigant during the procedure because of its tissue-dissolving, antibacterial and lubricating properties. This paper presents a case in which accidental injection of sodium hypochlorite into the lingual gingiva of a female patient caused gingival and bone necrosis. Surgical intervention was required. Key learning points Sodium hypochlorite is dangerous if injected into the tissues. The presentation of sodium hypochlorite in glass, anaesthetic type cartridges is potentially dangerous, and should be condemned. All healthcare workers should check carefully the contents of any syringe before injecting into patients.

Activation of Leishmania (Leishmania) amazonensis arginase at low temperature by binuclear Mn(2+) center formation of the immobilized enzyme on a Ni(2+) resin

In Leishmania, arginase is responsible for the production of ornithine, a precursor of polyamines required for proliferation of the parasite. In this work, the activation kinetics of immobilized arginase enzyme from L. (L.) amazonensis were studied by varying the concentration of Mn(2+) applied to the nickel column at 23 degrees C. The intensity of the binding of the enzyme to the Ni(2+) resin was directly proportional to the concentration of Mn(2+). Conformational changes of the enzyme may occur when the enzyme interacts with immobilized Ni(2+), allowing the following to occur: (1) entrance of Mn(2+) and formation of the metal bridge; (2) stabilization and activation of the enzyme at 23 degrees C; and (3) an increase in the affinity of the enzyme to Ni(2+) after the Mn(2+) activation step. The conformational alterations can be summarized as follows: the interaction with the Ni(2+) simulates thermal heating in the artificial activation by opening a channel for Mn(2+) to enter. (C) 2010 Elsevier Inc. All rights reserved.