Maytê Bolean

Photodynamic Therapy in Planktonic and Biofilm Cultures of Aggregatibacter actinomycetemcomitans

OBJECTIVE To evaluate the inactivation of Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), responsible for causing aggressive periodontitis, using photodynamic therapy (PDT) by rose bengal (RB) as a model of a reactive oxygen species (ROS) generator, in planktonic and biofilm cultures. MATERIALS AND METHODS A. actinomycetemcomitans was grown in planktonic and biofilm cultures using tryptic soy broth medium. The sensibility (dark toxicity) to RB was determined, and its ideal concentration for PDT was established. Concentrations in the range from 0.01 to 50.0 micromol L(-1) RB, with different light potencies and incubation times, were used. An odontological resin photopolymerizer that emits the adequate wavelength for absorption of the RB dye was applied. Bacterial viability was determined by colony- forming units (CFU). RESULTS RB photosensitizer dye in concentrations up to 0.1 micromol L(-1) did not show toxicity per se toward A. actinomycetemcomitans cells. In a PDT study with photoirradiation (1 min) at 0.1 micromol L(-1), a 55% reduction of A. actinomycetemcomitans viability was obtained in planktonic cultures. Preincubation (30 min) of the bacteria with the dye resulted in a 90% reduction of its viability. It is important to note that, for dye concentrations up to 1 micromol L(-1), in the same experimental conditions, no death effect on gingival fibroblasts was observed. The A. actinomycetemcomitans biofilm was not affected by RB or light alone. After PDT, the reduction in the biofilm (about 45%) is significantly dependant on RB concentration and irradiation time when this dye was used as a ROS generator. CONCLUSION Photodynamic therapy-generated ROS inactivates A. actinomycetemcomitans both in planktonic and biofilm cultures, even in small concentrations of the photosensitizing agent, and it does not cause damage to fibroblast cells under the same conditions.

Proteoliposomes Harboring Alkaline Phosphatase and Nucleotide Pyrophosphatase as Matrix Vesicle Biomimetics

We have established a proteoliposome system as an osteoblast-derived matrix vesicle (MV) biomimetic to facilitate the study of the interplay of tissue-nonspecific alkaline phosphatase (TNAP) and NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) during catalysis of biomineralization substrates. First, we studied the incorporation of TNAP into liposomes of various lipid compositions (i.e. in pure dipalmitoyl phosphatidylcholine (DPPC), DPPC/dipalmitoyl phosphatidylserine (9:1 and 8:2), and DPPC/dioctadecyl-dimethylammonium bromide (9:1 and 8:2) mixtures. TNAP reconstitution proved virtually complete in DPPC liposomes. Next, proteoliposomes containing either recombinant TNAP, recombinant NPP1, or both together were reconstituted in DPPC, and the hydrolysis of ATP, ADP, AMP, pyridoxal-5′-phosphate (PLP), p-nitrophenyl phosphate, p-nitrophenylthymidine 5′-monophosphate, and PPi by these proteoliposomes was studied at physiological pH. p-Nitrophenylthymidine 5′-monophosphate and PLP were exclusively hydrolyzed by NPP1-containing and TNAP-containing proteoliposomes, respectively. In contrast, ATP, ADP, AMP, PLP, p-nitrophenyl phosphate, and PPi were hydrolyzed by TNAP-, NPP1-, and TNAP plus NPP1-containing proteoliposomes. NPP1 plus TNAP additively hydrolyzed ATP, but TNAP appeared more active in AMP formation than NPP1. Hydrolysis of PPi by TNAP-, and TNAP plus NPP1-containing proteoliposomes occurred with catalytic efficiencies and mild cooperativity, effects comparable with those manifested by murine osteoblast-derived MVs. The reconstitution of TNAP and NPP1 into proteoliposome membranes generates a phospholipid microenvironment that allows the kinetic study of phosphosubstrate catabolism in a manner that recapitulates the native MV microenvironment.

Catalytic Signature of a Heat-Stable, Chimeric Human Alkaline Phosphatase with Therapeutic Potential

Recombinant alkaline phosphatases are becoming promising protein therapeutics to prevent skeletal mineralization defects, inflammatory bowel diseases, and treat acute kidney injury. By substituting the flexible crown domain of human intestinal alkaline phosphatase (IAP) with that of the human placental isozyme (PLAP) we generated a chimeric enzyme (ChimAP) that retains the structural folding of IAP, but displays greatly increased stability, active site Zn2+ binding, increased transphosphorylation, a higher turnover number and narrower substrate specificity, with comparable selectivity for bacterial lipopolysaccharide (LPS), than the parent IAP isozyme. ChimAP shows promise as a protein therapeutic for indications such as inflammatory bowel diseases, gut dysbioses and acute kidney injury.

The effect of cholesterol on the reconstitution of alkaline phosphatase into liposomes

Tissue-nonspecific alkaline phosphatase (TNAP), present on the surface of chondrocyte- and osteoblast-derived matrix vesicles (MVs), plays key enzymatic functions during endochondral ossification. Many studies have shown that MVs are enriched in TNAP and also in cholesterol compared to the plasma membrane. Here we have studied the influence of cholesterol on the reconstitution of TNAP into dipalmitoylphosphatidylcholine (DPPC)-liposomes, monitoring the changes in lipid critical transition temperature (T(c)) and enthalpy variation (∆H) using differential scanning calorimetry (DSC). DPPC-liposomes revealed a T(c) of 41.5 °C and ∆H of 7.63 Kcal mol(-1). The gradual increase in cholesterol concentration decrease ∆H values, reaching a ∆H of 0.87Kcalmol(-1) for DPPC:cholesterol system with 36mol% of cholesterol. An increase in T(c), up to 47 °C for the DPPC:cholesterol liposomes (36 mol% of Chol), resulted from the increase in the area per molecule in the gel phase. TNAP (0.02 mg/mL) reconstitution was done with protein:lipid 1:10,000 (molar ratio), resulting in 85% of the added enzyme being incorporated. The presence of cholesterol reduced the incorporation of TNAP to 42% of the added enzyme when a lipid composition of 36 mol% of Chol was used. Furthermore, the presence of TNAP in proteoliposomes resulted in a reduction in ∆H. The gradual proportional increase of cholesterol in liposomes results in broadening of the phase transition peak and eventually eliminates the cooperative gel-to-liquid-crystalline phase transition of phospholipids bilayers. Thus, the formation of microdomains may facilitate the clustering of enzymes and transporters known to be functional in MVs during endochondral ossification.

Photodynamic Therapy with Rose Bengal Induces GroEL Expression in Streptococcus mutans

UNLABELLED Heat-shock proteins (HSPs) are indicative of stressing conditions that may affect cell viability. In Streptococcus mutans, acid stress induces high levels of GroEL, an HSP, in addition to metabolic alterations, as shown by proteomic analysis. OBJECTIVE We tested whether the expression of GroEL by S. mutans was enhanced after photodynamic therapy (PDT) with rose bengal. METHODS S. mutans was grown in complete medium supplemented with 50 mmol/L glucose. The test conditions used were as follows: Rose bengal (0.1 micromol/L) with and without light treatment (500 mJ/cm(2)), light treatment alone, and 1 mol/L NaCl (as a stress condition). The extracellular pH of bacteria was monitored; HSP expression was assayed with Western blot, and possible DNA damage analyzed. RESULTS Higher HSP expression was detected in bacteria after PDT treatment as compared with light or dye alone (negative controls). The expression of HSP after PDT was similar to that induced by osmotic stress. No DNA degradation was observed after PDT of S. mutans. CONCLUSIONS PDT may cause effects similar to those of other stressing conditions in S. mutans, and cell death induced by this treatment reflects its incapacity to protect itself sufficiently against the deleterious effects of PDT with Rose bengal.

Proteoliposomes in nanobiotechnology

Proteoliposomes are systems that mimic lipid membranes (liposomes) to which a protein has been incorporated or inserted. During the last decade, these systems have gained prominence as tools for biophysical studies on lipid–protein interactions as well as for their biotechnological applications. Proteoliposomes have a major advantage when compared with natural membrane systems, since they can be obtained with a smaller number of lipidic (and protein) components, facilitating the design and interpretation of certain experiments. However, they have the disadvantage of requiring methodological standardization for incorporation of each specific protein, and the need to verify that the reconstitution procedure has yielded the correct orientation of the protein in the proteoliposome system with recovery of its functional activity. In this review, we chose two proteins under study in our laboratory to exemplify the steps necessary for the standardization of the reconstitution of membrane proteins in liposome systems: (1) alkaline phosphatase, a protein with a glycosylphosphatidylinositol anchor, and (2) Na,K-ATPase, an integral membrane protein. In these examples, we focus on the production of the specific proteoliposomes, as well as on their biochemical and biophysical characterization, with emphasis on studies of lipid–protein interactions. We conclude the chapter by highlighting current prospects of this technology for biotechnological applications, including the construction of nanosensors and of a multi-protein nanovesicular biomimetic to study the processes of initiation of skeletal mineralization.

Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80% and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 microM) and oligomycin (4 microg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.

Liposomal systems as carriers for bioactive compounds

Since the revolutionary discovery that phospholipids can form closed bilayered structures in aqueous systems, the study of liposomes has become a very interesting area of research. The versatility and amazing biocompatibility of liposomes has resulted in their wide-spread use in many scientific fields, and many of their applications, especially in medicine, have yielded breakthroughs in recent decades. Specifically, their easy preparation and various structural aspects have given rise to broadly usable methodologies to internalize different compounds, with either lipophilic or hydrophilic properties. The study of compounds with potential biotechnological application(s) is generally related to evaluation and risk assessment of the possible cytotoxic or therapeutic effects of the compound under study. In most cases, undesirable side-effects are associated with an interaction of the liposome with the cell membrane and/or its absorption and subsequent interaction with a cellular biomolecule. Liposomal carrier systems have an unprecedented potential for delivering bioactive substances to specific molecular targets due to their biocompatibility, biodegradability and low toxicity. Liposomes are therefore considered to be an invaluable asset in applied biotechnology studies due to their potential for interaction with both hydrophilic and lipophilic compounds.

Biophysical aspects of biomineralization

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix (ECM) by promoting the synthesis of hydroxyapatite (HA) seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Several lipid and proteins present in the membrane of the MVs mediate the interactions of MVs with the ECM and regulate the initial mineral deposition and posterior propagation. Among the proteins of MV membranes, ion transporters control the availability of phosphate and calcium needed for initial HA deposition. Phosphatases (orphan phosphatase 1, ectonucleotide pyrophosphatase/phosphodiesterase 1 and tissue-nonspecific alkaline phosphatase) play a crucial role in controlling the inorganic pyrophosphate/inorganic phosphate ratio that allows MV-mediated initiation of mineralization. The lipidic microenvironment can help in the nucleation process of first crystals and also plays a crucial physiological role in the function of MV-associated enzymes and transporters (type III sodium-dependent phosphate transporters, annexins and Na+/K+ ATPase). The whole process is mediated and regulated by the action of several molecules and steps, which make the process complex and highly regulated. Liposomes and proteoliposomes, as models of biological membranes, facilitate the understanding of lipid–protein interactions with emphasis on the properties of physicochemical and biochemical processes. In this review, we discuss the use of proteoliposomes as multiple protein carrier systems intended to mimic the various functions of MVs during the initiation and propagation of mineral growth in the course of biomineralization. We focus on studies applying biophysical tools to characterize the biomimetic models in order to gain an understanding of the importance of lipid–protein and lipid–lipid interfaces throughout the process.

Matrix vesicles from chondrocytes and osteoblasts: Their biogenesis, properties, functions and biomimetic models

BACKGROUND Matrix vesicles (MVs) are released from hypertrophic chondrocytes and from mature osteoblasts, the cells responsible for endochondral and membranous ossification. Under pathological conditions, they can also be released from cells of non-skeletal tissues such as vascular smooth muscle cells. MVs are extracellular vesicles of approximately 100-300nm diameter harboring the biochemical machinery needed to induce mineralization. SCOPE OF THE REVIEW The review comprehensively delineates our current knowledge of MV biology and highlights open questions aiming to stimulate further research. The review is constructed as a series of questions addressing issues of MVs ranging from their biogenesis and functions, to biomimetic models. It critically evaluates experimental data including their isolation and characterization methods, like lipidomics, proteomics, transmission electron microscopy, atomic force microscopy and proteoliposome models mimicking MVs. MAJOR CONCLUSIONS MVs have a relatively well-defined function as initiators of mineralization. They bind to collagen and their composition reflects the composition of lipid rafts. We call attention to the as yet unclear mechanisms leading to the biogenesis of MVs, and how minerals form and when they are formed. We discuss the prospects of employing upcoming experimental models to deepen our understanding of MV-mediated mineralization and mineralization disorders such as the use of reconstituted lipid vesicles, proteoliposomes and, native sample preparations and high-resolution technologies. GENERAL SIGNIFICANCE MVs have been extensively investigated owing to their roles in skeletal and ectopic mineralization. MVs serve as a model system for lipid raft structures, and for the mechanisms of genesis and release of extracellular vesicles.