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.

Dermaseptin 01 as antimicrobial peptide with rich biotechnological potential: study of peptide interaction with membranes containing Leishmania amazonensis lipid-rich extract and membrane models

This article addresses the interactions of the synthetic antimicrobial peptide dermaseptin 01 (GLWSTIKQKGKEAAIAAA-KAAGQAALGAL-NH(2), DS 01) with phospholipid (PL) monolayers comprising (i) a lipid-rich extract of Leishmania amazonensis (LRE-La), (ii) zwitterionic PL (dipalmitoylphosphatidylcholine, DPPC), and (iii) negatively charged PL (dipalmitoylphosphatidylglycerol, DPPG). The degree of interaction of DS 01 with the different biomembrane models was quantified from equilibrium and dynamic liquid-air interface parameters. At low peptide concentrations, interactions between DS 01 and zwitterionic PL, as well as with the LRE-La monolayers were very weak, whereas with negatively charged PLs the interactions were stronger. For peptide concentrations above 1 mu g/ml, a considerable expansion of negatively charged monolayers occurred. In the case of DPPC, it was possible to return to the original lipid area in the condensed phase, suggesting that the peptide was expelled from the monolayer. However, in the case of DPPG, the average area per lipid molecule in the presence of DS 01 was higher than pure PLs even at high surface pressures, suggesting that at least part of DS 01 remained incorporated in the monolayer. For the LRE-La monolayers, DS 01 also remained in the monolayer. This is the first report on the antiparasitic activity of AMPs using Langmuir monolayers of a natural lipid extract from L. amazonensis. Copyright (C) 2011 European Peptide Society and John Wiley & Sons, Ltd.

Effect of mesoporosity of vanadium oxide prepared by sol-gel process as cathodic material evaluated by cyclability during Li(+) insertion/deinsertion

The effect of pore structure on the behavior of lithium intercalation into an electrode containing porous V(2)O(5) film has been investigated and compared with the electrode containing a non-porous V(2)O(5) film. X-ray diffraction patterns indicate a lamellar structure for both materials. Nitrogen adsorption isotherms, t-plot method, and Scanning Electronic Microscopy show that the route employed for the preparation of mesoporous V(2)O(5) was successful. The electrochemical performance of these matrices as lithium intercalation cathode materials was evaluated. The porous material reaches stability after several cycles more easily compared with the V(2)O(5) xerogel. Lithium intercalation into the porous V(2)O(5) film electrode is crucially influenced by pore surface and film surface irregularity, in contrast with the non-porous surface of the V(2)O(5) xerogel.

Shared structural determinants for the calcium-independent liposome membrane permeabilization and sarcolemma depolarization in Bothropstoxin-I, a LYS49-PLA(2) from the venom of Bothrops jararacussu

The structural determinants of myotoxicity of bothropstoxin-I (BthTX-I), a Lys49 phospholipase A(2) from Bothrops jararacussu venom, were studied by measuring the resting membrane potential in the mouse phrenic nerve-diaphragm preparation. This method proved to be around 100-fold more sensitive than the creatine kinase release assay, and was used to evaluate a total of 31 site-directed BthTX-I alanine scanning mutants. Mutants that reduced the resting membrane potential were located in a surface patch defined by residues in the C-terminal loop (residues 115-129), positions 37-39 in the membrane interfacial recognition surface (Y46 and K54), and residue K93. These results expand the known structural determinants of the biological activity as evaluated by previous creatine kinase release experiments. Furthermore, a strong correlation is observed between the structural determinants of sarcolemma depolarization and calcium-independent disruption of liposome membranes, suggesting that a common mechanism of action underlies the permeabilization of the biological and model membranes. (C) 2009 Elsevier Ltd. All rights reserved.

Lipid microspheres loaded with antigenic membrane proteins of the Leishmania amazonensis as a potential biotechnology application

Lipid microspheres (LM) are excellent drug delivery or vaccines adjuvant systems and are relatively stable. The aim of this work is to develop and characterize a system that is able to encapsulate and present antigenic membrane proteins from Leishmania amazonensis. Membrane proteins are important for vaccine`s formulation because these proteins come in contact with the host cell first, triggering the cell mediated immune response. This is a useful tool to avoid or inactivate the parasite invasion. The LM are constituted by soybean oil (SO), dipalmitoylphosphatidilcholine (DPPC), cholesterol and solubilized protein extract (SPE). The particles formed presented an average diameter of 200 run, low polydispersion and good stability for a period of 30 days, according to dynamic light scattering assays. Isopycnic density gradient centrifugation of LM-protein showed that proteins and lipids floated in the sucrose gradient (5-50%w/v) suggesting that the LM-protein preparation was homogeneous and that the proteins are interacting with the system. The results show that 85% of SPE proteins were encapsulated in the LM. Studies of cellular viability of murine peritoneal macrophages show that our system does not present cytotoxic effect for the macrophages and still stimulates their NO production (which makes its application as a vaccine adjuvant possible). LM-protein loaded with antigenic membrane proteins from L. amazonensis seems to be a promising vaccine system for immunization against leishmaniasis. (C) 2009 Elsevier Inc. 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.

Insertion/deletion polymorphism of the angiotensin converting enzyme gene and loss of the insertion allele in aldosterone-producing adenoma

The genetic mechanisms responsible for the formation of adrenocortical adenomas which autonomously produce aldosterone are largely unknown, The adrenal renin-angiotensin system has been implicated in the pathophysiology of these tumours, Angiotensin-converting enzyme (ACE) catalyses the generation of angiotensin II, and the insertion/deletion (I/D) polymorphism of the ACE gene regulates up to 50% of plasma and cellular ACE variability in humans. We therefore examined the genotypic and allelic frequency distributions of the ACE gene I/D polymorphism in 55 patients with aldosterone-producing adenoma, APA, (angiotensin-unresponsive APA n = 28, angiotensin-responsive APA n = 27), and 80 control subjects with no family history of hypertension, We also compared the ACE gene I/D polymorphism allelic pattern in matched tumour and peripheral blood DNA in the 55 patients with APA, The frequency of the D allele was 0.518 and 0.512 and the I allele was 0.482 and 0.488 in the APA and control subjects respectively, Genotypic and allelic frequency analysis found no significant differences between the groups, Examination of the matched tumour and peripheral blood DNA samples revealed the loss of the insertion allele in four of the 25 patients who were heterozygous for the ACE I/D genotype. The I/D polymorphism of the ACE gene does not appear to contribute to the biochemical and phenotypic characteristics of APA, however, the deletion of the insertion allele of the ACE gene I/D polymorphism in 16% of aldosterone-producing adenomas may represent the loss of a tumour suppressor gene/s or other genes on chromosome 17q which may contribute to tumorigenesis in APA.

Elevated plasma membrane and sarcoplasmic reticulum Ca2+ pump mRNA levels in cultured aortic smooth muscle cells from spontaneously hypertensive rats

We have observed previously that Ca2+ pump-mediated Ca2+ efflux is elevated in cultured aortic smooth muscle cells from spontaneously hypertensive rats compared to those from Wistar-Kyoto rat controls. The objective of this work was to determine if these strains differ in mRNA levels for the PMCA1 isoform of the plasma membrane Ca2+-ATPase and the SERCA2 isoform of the sarcoplasmic reticulum Ca2+-ATPase. mRNA levels were compared in cultured aortic smooth muscle cells from 10-week-old male rats. PMCA1 and SERCA2 mRNA levels were elevated in SHR compared to WKY. Angiotensin II increased the level of PMCA1 and SERCA2 mRNA in both strains. These studies provide further evidence for alterered Ca2+ homeostasis in hypertension at the level of Ca2+ transporting ATPases in the spontaneously hypertensive rat model. These data are also consistent with the hypothesis that the expression of these two Ca2+ pumps may be linked. (C) 1997 Academic Press

EXPRESSION OF MEMBRANE TYPE 1 MATRIX METALLOPROTEINASE IN MEDULLARY THYROID CARCINOMA: PROGNOSTIC IMPLICATIONS

Background. Clinical and pathologic examinations cannot always provide a prognosis for patients with medullary thyroid carcinoma. Membrane type 1 matrix metalloproteinase (MT1-MMP) can act directly on carcinogenesis and takes part in 1 of the processes of metalloproteinase 2 activation, an enzyme related to prognostic impairment of patients with such tumor. Methods. Thirty-five patients who were submitted to surgery were followed up for an average of 74 months, Postoperative and final medical conditions were characterized for comparison with MT1-MMP immunostainings, performed in surgical paraffin blocks. A value of p < .05 was considered statistically significant. Results. Proposed index (association of proportion and intensity of immunostaining) and proportion of immunostained cells in primary specimens were correlated with cure or persistence after initial operations (p = .0216 and p = .0098, respectively). Conclusion. MT1-MMP immunostaining in primary tumor specimens is a new and complementary prognostic predictor in patients with medullary thyroid carcinomas. (C) 2009 Wiley Periodicals, Inc. Head Neck 32: 58-67, 2010

Muscle Transplants for Facial Reanimation Rationale and Results of Insertion Technique Using the Palmaris Longus Tendon

Thirty-one patients with unilateral long-standing facial palsy underwent I-stage reanimation with free gracilis muscle transplant innervated by the masseteric branch of the trigeminal nerve. They were divided into 2 nonrandomized groups according to insertion technique: group 1 (9 patients), interrupted suture between the free flap and the orbicularis oris of the upper and lower lip on the paralyzed side; group It (22 patients), palmaris longus tendon graft placed between the gracilis free flap and the orbicularis oris of the upper and lower lip on the nonparalyzed side. Qualitative evaluation of the smile demonstrated better results in patients from group II. Comparing the position Of the Cupid`s bow at rest, pre- and postoperatively in each patient, we observed significant improvement of facial symmetry in both groups. During smile, however, there was a significantly higher rate of centralization of the Cupid`s bow in patients submitted to reanimation with the use of the palmaris longus tendon (group II).

The Role of Prostate Specific Membrane Antigen and Pepsinogen C Tissue Expression as an Adjunctive Method to Prostate Cancer Diagnosis

Purpose: The diagnosis of prostate cancer in men with persistently increased prostate specific antigen after a negative prostate biopsy has become a great challenge for urologists and pathologists. We analyzed the diagnostic value of 6 genes in the tissue of patients with prostate cancer. Materials and Methods: The study was comprised of 50 patients with localized disease who underwent radical prostatectomy. Gene selection was based on a previous microarray analysis. Among 4,147 genes with different expressions between 2 pools of patients 6 genes (PSMA, TMEFF2, GREB1, TH1L, IgH3 and PGC) were selected. These genes were tested for diagnostic value using the quantitative reverse transcription polymerase chain reaction method. Initially malignant tissue samples from 33 patients were analyzed and in the second part of the study we analyzed benign tissue samples from the other 17 patients with prostate cancer. The control group was comprised of tissue samples of patients with benign prostatic hyperplasia. Results: Analysis of malignant prostatic tissue demonstrated that prostate specific membrane antigen was over expressed (mean 9 times) and pepsinogen C was under expressed (mean 1.3 X 10(-4) times) in all cases compared to benign prostatic hyperplasia. The other 4 tested genes showed a variable expression pattern not allowing for differentiation between benign and malignant cases. When we tested these results in the benign prostate tissues from patients with cancer, pepsinogen C maintained the expression pattern. In terms of prostate specific membrane antigen, despite over expression in most cases (mean 12 times), 2 cases (12%) presented with under expression. Conclusions: Pepsinogen C tissue expression may constitute a powerful adjunctive method to prostate biopsy in the diagnosis of prostate cancer cases.

Pirellulosomes: A new type of membrane-bounded cell compartment in planctomycete bacteria of the genus Pirellula

A distinct type of cellular organization was found in two species of the planctomycete genus Pirellula, Pirellula marina and Pirellula staleyi. Both species possess two distinct regions within the cell which are separated by a single membrane. The major region of the cell, the pirellulosome, contains the fibrillar condensed nucleoid. The other area, the polar cap region, forms a continuous layer surrounding the entire pirellulosome and displays a cap of asymmetrically distributed material at one cell pole. Immuno- and cytochemical-labelling of P. marina demonstrated that DNA is located exclusively within the pirellulosome; cell RNA is concentrated in the pirellulosome, with some RNA also located in the polar cap region.

Cross-linking studies and membrane localization and assembly of radiolabelled large mechanosensitive ion channel (MscL) of Escherichia coli

The gene encoding the large conductance mechanosensitive ion channel (MscL) of Escherichia coli and several deletion mutants of mscL were cloned under the control of the T7 RNA polymerase promoter. Transformation of these constructs into an E. coli strain carrying an inducible T7 RNA polymerase gene allowed the specific production and labelling of MscL with [S-35]methionine. Preparation of membrane fractions of E. coli cells by sucrose gradient centrifugation indicated that the radiolabelled MscL was present in the inner cytoplasmic membrane in agreement with results of several studies. However, treatment of the labelled cells and cell membrane vesicles with various cross-linkers resulted in the majority of labelled protein migrating as a monomer with a small proportion of molecules (approximate to 25%) migrating as dimers and higher order multimers. This result is in contrast with a finding of a study suggesting that the channel exclusively forms hexamers in the cell membrane off. coli (1) and therefore may have profound implication for the activation and/or ''multimerization'' of the channel by mechanical stress exerted to the membrane. In addition, from the specific activity of the radiolabelled protein and the amount of protein in the cytoplasmic membrane fraction we estimated the number of MscL ion channels expressed under these conditions to be approximately 50 channels per single bacterium. (C) 1997 Academic Press.

Platelet protease-activated receptor 1 and membrane expression of P-selectin in pulmonary arterial hypertension

Introduction: Pulmonary arterial hypertension (PAH) is frequently associated with thrombotic events, particularly involving the pulmonary microcirculation at sites of vascular injury. We therefore decided to analyse protease-activated receptor 1 (PAR1), a key element in the activation of human platelets by thrombin, in PAH patients in stable clinical condition. Methods: Using flow cytometry, we analyzed platelet PAR1 density, PAR1-mediated exposure of P-selectin and the formation of platelet-leukocyte aggregates in 30 PAH patients aged 11 to 78 years (median 50.5 years). The control group consisted of 25 healthy subjects with the same age range as patients. Results: In patients, total platelet PAR1 density and uncleaved PAR1 density correlated negatively with platelet count (r(2) = 0.33 and r(2) = 0.34 respectively, p < 0.0015). In patients with a low platelet count (<150 x 10(9) platelets/L), both densities were increased relative to controls (82% and 33% respectively, p < 0.05). Thrombin peptide-induced platelet exposure of P-selectin was directly related to total and uncleaved PAR1 density (respectively, r(2) = 0.33 and r(2) = 0.29, p < 0.0025) and increased in subjects with low platelet count (46% versus those with normal platelet count, p < 0.05). Patients with low platelet count had decreased in vitro thrombin-induced formation of platelet-leukocyte aggregates (57% decrease versus controls, p < 0.05). Conclusions: There seems to be a subpopulation of PAH patients with increased propensity to thrombotic events as suggested by increased platelet PAR1 expression and PAR-mediated surface exposure of P-selectin associated with decreased platelet count. (C) 2009 Elsevier Ltd. All rights reserved.

Histology of dart tag insertion sites in the epaulette shark

Samples of dermal and epidermal tissues of epaulette sharks Hemiscyllium ocellatum were examined histologically to assess damage caused by tagging. Tissues from around tag sites were removed at time intervals ranging from 100 min to 284 days post-tagging. These samples showed acute and chronic responses to tagging. Acute responses consisted of localized tissue breakdown and haemorrhaging, and occurred within the first few hours after tag insertion. At 10 h post-tagging, an intermediate response was apparent. This phase was characterized by further haemorrhaging and red and white blood cell movement into the wound area. The chronic response observed in the 10-284-day post-tagging samples was characterized by fibrous tissue formation to sequester the tag. This tissue presumably protects the adjacent musculature from further trauma produced by movement of the tag and provides a continuous barrier between the internal and external milieu. Tissue repair appeared to progress consistently in all specimens and no secondary infections at the tag site were seen. Tagging produced only localized tissue disruption and did not appear to be detrimental to the long term health of individual sharks. Our findings show that spaghetti style dart tagging is an acceptable method for marking individuals (40-75+ cm total length) of this species. (C) 1997 The Fisheries Society of the British Isles.