Lipid composition modulates ATP hydrolysis and calcium phosphate mineral propagation by TNAP-harboring proteoliposomes.

Bone biomineralization is mediated by a special class of extracellular vesicles, named matrix vesicles (MVs), released by osteogenic cells. The MV membrane is enriched in sphingomyelin (SM), cholesterol (Chol) and tissue non-specific alkaline phosphatase (TNAP) compared with the parent cells' plasma membrane. TNAP is an ATP phosphohydrolase bound to cell and MV membranes via a glycosylphosphatidylinositol (GPI) anchor. Previous studies have shown that the lipid microenvironment influences the catalytic activity of enzymes incorporated into lipid bilayers. However, there is a lack of information about how the lipid microenvironment controls the ability of MV membrane-bound enzymes to induce mineral precipitation. Herein, we used TNAP-harboring proteoliposomes made of either pure dimyristoylphosphatidylcholine (DMPC) or DMPC mixed with either Chol, SM or both of them as MV biomimetic systems to evaluate how the composition modulates the lipid microenvironment and, in turn, TNAP incorporation into the lipid bilayer by means of calorimetry. These results were correlated with the proteoliposomes' catalytic activity and ability to induce the precipitation of amorphous calcium phosphate (ACP) in vitro. DMPC:SM proteoliposomes displayed the highest efficiency of mineral propagation, apparent affinity for ATP and substrate hydrolysis efficiency, which correlated with their highest degree of membrane organization (highest ΔH), among the tested proteoliposomes. Results obtained from turbidimetry and Fourier transformed infrared (FTIR) spectroscopy showed that the tested proteoliposomes induced ACP precipitation with the order DMPC:SM>DMPC:Chol:SM≈DMPC:Chol>DMPC which correlated with the lipid organization and the presence of SM in the proteoliposome membrane. Our study arises important insights regarding the physical properties and role of lipid organization in MV-mediated mineralization.

The yeast GRASP Grh1 displays a high polypeptide backbone mobility along with an amyloidogenic behavior.

GRASPs are proteins involved in cell processes that seem paradoxical: responsible for shaping the Golgi cisternae and involved in unconventional secretion mechanisms that bypass the Golgi. Despite its physiological relevance, there is still a considerable lack of studies on full-length GRASPs. Our group has previously reported an unexpected behavior of the full-length GRASP from the fungus C. neoformans: its intrinsically-disordered characteristic. Here, we generalize this finding by showing that it is also observed in the GRASP from S. cerevisae (Grh1), which strongly suggests it might be a general property within the GRASP family. Furthermore, Grh1 is also able to form amyloid-like fibrils either upon heating or when submitted to changes in the dielectric constant of its surroundings, a condition that is experienced by the protein when in close contact with membranes of cell compartments, such as the Golgi apparatus. Intrinsic disorder and fibril formation can thus be two structural properties exploited by GRASP during its functional cycle.

Antimicrobial evaluation of new metallic complexes with xylitol active against P. aeruginosa and C. albicans: MIC determination, post-agent effect and Zn-uptake.

Xylitol (xylH5) is metabolized via the pentose pathway in humans, but it is unsuitable as an energy source for many microorganisms where it produces a xylitol-induced growth inhibition and disturbance in protein synthesis. For this reason, xylitol is used in the prophylaxis of several infections. In the search of better antimicrobial agents, new copper and zinc complexes with xylitol were synthesized and characterized by analytical and spectrosco pic methods: Na2[Cu3(xylH−4)2]·NaCl·4.5H2O (Cu-xyl) and [Zn4(xylH−4)2(H2O)2]·NaCl·3H2O (Zn-xyl). Both copper and zinc complexes presented higher MIC against Pseudomona aeruginosa than the free xylitol while two different behaviors were found against Candida albicans depending on the complex. The growth curves showed that Cu-xyl presented lower activity than the free ligand during all the studied period. In the case of Znxyl the growth curves showed that the inhibition of the microorganism growth in the first stage was equivalent to that of xylitol but in the second stage (after 18 h) Zn-xyl inhibited more. Besides, the PAE (post agent effect)obtained for Zn-xyl and xyl showed that the recovery from the damage of microbial cells had a delay of 14 and 13 h respectively. This behavior could be useful in prophylaxis treatments for infectious diseases where it is important that the antimicrobial effect lasts longer. With the aim to understand the microbiological activities the analysis of the particle size, lipophilicity and Zn uptake was performed.

Effects of GPI-anchored TNAP on the dynamic structure of model membranes.

Tissue-nonspecific alkaline phosphatase (TNAP) plays a crucial role during skeletal mineralization, and TNAP deficiency leads to the soft bone disease hypophosphatasia. TNAP is anchored to the external surface of the plasma membranes by means of a GPI (glycosylphosphatidylinositol) anchor. Membrane-anchored and solubilized TNAP displays different kinetic properties against physiological substrates, indicating that membrane anchoring influences the enzyme function. Here, we used Electron Spin Resonance (ESR) measurements along with spin labeled phospholipids to probe the possible dynamic changes prompted by the interaction of GPI-anchored TNAP with model membranes. The goal was to systematically analyze the ESR data in terms of line shape changes and of alterations in parameters such as rotational diffusion rates and order parameters obtained from non-linear least-squares simulations of the ESR spectra of probes incorporated into DPPC liposomes and proteoliposomes. Overall, the presence of TNAP increased the dynamics and decreased the ordering in the three distinct regions probed by the spin labeled lipids DOPTC (headgroup), and 5- and 16-PCSL (acyl chains). The largest change was observed for 16-PCSL, thus suggesting that GPI-anchored TNAP can give rise to long reaching modifications that could influence membrane processes halfway through the bilayer.

Electron spin-relaxation via vibronic level of nickel (I) and nickel (III) cyanide complexes in NaCl single crystals.

Electron spin-lattice relaxation rates for the low spin [Ni(CN)(4)](1-) and [Ni(CN)(4)](3-) complexes in NaCl host lattice were measured by the inversion recovery technique in the temperature range 7-50K. The data for both paramagnetic species fit very well to a relaxation process involving localized anharmonic vibration modes, also responsible for the g-tensor temperature dependence.

Electron spin resonance in studies of membranes and proteins.

We provide a review of current electron spin resonance (ESR) techniques for studying basic molecular mechanisms in membranes and proteins by using nitroxide spin labels. In particular, nitroxide spin label studies with high-field/high-frequency ESR and two-dimensional Fourier transform ESR enable one to accurately determine distances in biomolecules, unravel the details of the complex dynamics in proteins, characterize the dynamic structure of membrane domains, and discriminate between bulk lipids and boundary lipids that coat transmembrane peptides or proteins; these studies can also provide time resolution to studies of functional dynamics of proteins. We illustrate these capabilities with recent examples.

Sulfated glycosaminoglycans from ovary of Rhodnius prolixus.

We have characterized sulfated glycosaminoglycans from ovaries of the blood-sucking insect Rhodnius prolixus, and determined parameters of their synthesis and distribution within this organ by biochemical and histochemical procedures. The major sulfated glycosaminoglycan is heparan sulfate while chondroitin 4-sulfate is a minor component. These glycosaminoglycans are concentrated in the ovarian tissue and are not found inside the oocytes. Besides this, we detected the presence of a sulfated compound distinguished from sulfated glycosaminoglycans and possibly derived from sulfated proteins. Conversely to the compartmental location of sulfated glycosaminoglycans, the unidentified sulfated compound is located in the ovarian tissue as well as inside the oocytes. Based on these and other findings, the possible roles of ovarian sulfated glycosaminoglycans on the process of oogenesis in these insects are discussed.

Influence of diameter variation of Gates-Glidden drills on torsion resistance.

The aim of this study was to determine the variation of the shaft diameter close to the active cutting region and close to the radius of concordance of 28- and 32-mm Gates-Glidden drills and to analyze the diameter influence on the torsion fracture resistance. Seventy-two stainless steel Maillefer drills were studied. The drill diameter varied along the shaft, the smallest diameter occurring close to the radius of concordance. This variation causes stress concentration which leads to complete fracture of the instrument.

Agentes inalatórios halogenados empregados pelo método quantitativo de anestesia / Inhalatory halogenated agents employed by the quantitative method of anesthesia

Neste ensaio clínico, o enflurano, o isoflurano e o halotano foram usados com baixos fluxos de gases e absorçäo total do dióxido de carbono. Participaram do estudo, 90 pacientes adultos (30 em cada grupo), estado físico I ou II sem doença cardiopulmonar e escalados para diferentes tipos de cirurgias. Diazepan foi usado como medicaçäo pré-anestésica em todos os casos, por via oral, na dose de 0,2 a 0,4 mg.kg-1. A induçäo da anestesia, em todos os pacientes, foi realizada com pancurônio 1 mg. tiopental 4 a 5 mg.kg-1 e succinilcolina 1,5 mg. kg-1, para facilitar a intubaçäo orotraqueal (IOT). O teste "t" de Student foi usado na análise estatística de todos os dados. Foi observada uma predominância de pacientes do sexo feminino nos três grupos. A idade média dos enfermos foi de 38,8 + ou - 12,4 anos no grupo do enflurano, 39,0 + ou - 16,7 anos no grupo do isoflurano e 40,1 + ou - 14,1 anos de idade no grupo do halotano. A duraçäo média das cirurgias foi de 190,7 + ou - 51,7 minutos no grupo do enflurano, 205,4 + ou - 70,7 minutos no grupo do isoflurano e 206,4 + ou - 76,0 minutos no grupo do halotano. A dose média de tiopental para a induçäo da anestesia foi de 340,8 + ou - 79,4 mg no grupo do enflurano, 303,4 + ou - 81,2 mg no grupo do isoflurano e 295,7 + ou - 65,7 mg no grupo do halotano. O fluxo de O2 usado foi de 249,0 + ou - 42,2 ml no grupo do enflurano, 225,0 + ou - 38,8 ml no grupo do isoflurano e 265,0 + ou - 63,9 ml no grupo do halotano. O volume de anestésico injetado foi menor que o previsto em todos os grupos, com diferenças estatisticamente significantes apenas nos grupos do enflurano e isoflurano. O volume total de enflurano, isoflurano e halotano foi de 421,5 ml, 327,7 ml e 335,4 ml gastos em cada grupo. Conclui-se o método quantitativo de anestesia diminui dramaticamente o consumo de anestésicos inalatórios e que proporcionalmente, parece que o enflurano é o mais econômico dos anestésicos halogenados estudados

Aneurisma da arteria esplenica. / Aneurysm of splenic artery

Apresentam os autores a experiencia de dois casos de aneurismas de arteria esplenica agudos no servico de cirurgia do HECC. Fazem revisao da literatura enfatizando o exame fisico e radiologico simples do abdomen dados fundamentais para o diagnostico em que o sinal do "anel de sinete" devera ser sempre pesquisado. Referem ainda os magnificos resultados obtidos com a aneurismectomia e esplenectomia