J. Martins-Silva

Multidisciplinary utilization of dimethyl sulfoxide: pharmacological, cellular, and molecular aspects

DMSO is an amphipathic molecule with a highly polar domain and two apolar methyl groups, making it soluble in both aqueous and organic media. It is one of the most common solvents for the in vivo administration of several water-insoluble substances. Despite being frequently used as a solvent in biological studies and as a vehicle for drug therapy, the side-effects of DMSO (undesirable for these purposes) are apparent from its utilization in the laboratory (both in vivo and in vitro) and in clinical settings. DMSO is a hydrogen-bound disrupter, cell-differentiating agent, hydroxyl radical scavenger, intercellular electrical uncoupler, intracellular low-density lipoprotein-derived cholesterol mobilizing agent, cryoprotectant, solubilizing agent used in sample preparation for electron microscopy, antidote to the extravasation of vesicant anticancer agents, and topical analgesic. Additionally, it is used in the treatment of brain edema, amyloidosis, interstitial cystitis, and schizophrenia. Several systemic side-effects from the use of DMSO have been reported, namely nausea, vomiting, diarrhea, hemolysis, rashes, renal failure, hypertension, bradycardia, heart block, pulmonary edema, cardiac arrest, and bronchospasm. Looking at the multitude of effects of DMSO brought to light by these studies, it is easily understood how many researchers working with DMSO (or studying one of its specific effects) might not be fully aware of the experiences of other groups who are working with it but in a different context.

Evaluation of Lipopolysaccharide Aggregation by Light Scattering Spectroscopy

Lipopolysaccharides (LPS) are cell wall components of Gram‐negative bacteria. These molecules behave as bacterial endotoxins and their release into the bloodstream is a determinant of the development of a wide range of pathologies. These amphipathic molecules can self‐aggregate into supramolecular structures with different shapes and sizes. The formation of these structures occurs when the LPS concentration is higher than the apparent critical micelle concentration (CMCa). Light scattering spectroscopy (both static and dynamic) was used to directly characterize the aggregation process of LPS from Escherichia coli serotype 026:B6. The results point to a CMCa value of 14 μg mL−1 and the existence of premicelle LPS oligomers below this concentration. Both structures were characterized in terms of molecular weight (5.5×106 and 16×106 g mol−1 below and above the CMCa, respectively), interaction with the aqueous environment, gyration radius (56 and 105 nm), hydrodynamic radius, (60 and 95 nm) and geometry of the supramolecular structures (nearly spherical). Our data indicates that future in vitro experiments should be carried out both below and above the CMCa. The search for drugs that interact with the aggregates, and thus change the CMCa and condition LPS interactions in the bloodstream, could be a new way to prevent certain bacterial‐endotoxin‐related pathologies.

Biochemical characterization of human umbilical vein endothelial cell membrane bound acetylcholinesterase.

Acetylcholinesterase is an enzyme whose best‐known function is to hydrolyze the neurotransmitter acetylcholine. Acetylcholinesterase is expressed in several noncholinergic tissues. Accordingly, we report for the first time the identification of acetylcholinesterase in human umbilical cord vein endothelial cells. Here we further performed an electrophoretic and biochemical characterization of this enzyme, using protein extracts obtained by solubilization of human endothelial cell membranes with Triton X‐100. These extracts were analyzed under polyacrylamide gel electrophoresis in the presence of Triton X‐100 and under nondenaturing conditions, followed by specific staining for cholinesterase or acetylcholinesterase activity. The gels revealed one enzymatically active acetylcholinesterase band in the extracts that disappeared when staining was performed in the presence of eserine (an acetylcholinesterase inhibitor). Performing western blotting with the C‐terminal anti‐acetylcholinesterase IgG, we identified a single protein band of approximately 70 kDa, the molecular mass characteristic of the human monomeric form of acetylcholinesterase. The western blotting with the N‐terminal anti‐acetylcholinesterase IgG antibody revealed a double band around 66–70 kDa. Using the Ellmans method to measure the cholinesterase activity in human umbilical vein endothelial cells, regarding its substrate specificity, we confirmed the existence of an acetylcholinesterase enzyme. Our studies revealed a predominance of acetylcholinesterase over other cholinesterases in human endothelial cells. In conclusion, we have demonstrated the existence of a membrane‐bound acetylcholinesterase in human endothelial cells. In future studies, we will investigate the role of this protein in the endothelial vascular system.

Fluorescent probes DPH, TMA-DPH and C17-HC induce erythrocyte exovesiculation.

An experimental approach has been developed to study human erythrocyte vesiculation, using the fluorescent probes diphenylhexatriene (DPH), trimethylamino-diphenylhexatriene (TMA-DPH) and heptadecyl-hydroxycoumarin (C17-HC). Acetylcholinesterase (AChE) enzyme activity measurements confirmed the presence of exovesicles released from erythrocyte membranes labeled with DPH, TMA-DPH or C17-HC. The fluorescence intensity and anisotropy values obtained showed that the amphiphilic probes TMA-DPH and C17-HC are preferentially incorporated in the exovesicles (when compared with DPH). There is a significant decrease of the cholesterol content of the exovesicle suspensions with time, independently of the fluorescence probe used, reaching undetectable cholesterol levels for the samples incubated for 48 hr. The ratios between the concentration of cholesterol released in the exovesicles after 1 hr incubation with DPH, TMA-DPH or C17-HC and the probe concentration used in the incubation were 84.7, 3.82 and 0.074, respectively. The size of the released vesicles was evaluated by dynamic light scattering spectroscopy. Some hypotheses are proposed that could explain the resemblance and differences between the results obtained for erythrocytes labeled with each probe, considering the present knowledge of membrane vesiculation mechanisms, lipid microdomains (rafts), erythrocyte membrane phospholipid asymmetry and AChE inhibition by TMA-DPH and C17-HC. This work demonstrates that the fluorescent probes DPH, TMA-DPH and C17-HC induce rapid erythrocyte exovesiculation; their use can lead to new methodologies for the study of this still poorly understood mechanism.

Gramicidin D and dithiothreitol effects on erythrocyte exovesiculation

The use of either diphenylhexatriene, trimethylamino-diphenylhexatriene, or heptadecyl-hydroxy coumarin (C17-HC) allows, simultaneously and with the same molecule, the induction of erythrocyte exovesiculation and labeling of the released vesicles with the fluorescent probe. This method was used to evaluate gramicidin D (a channel-forming peptide) and dithiothreitol (a reducing agent) effects on the human erythrocytes vesiculation process. The release of cholesterol and phospholipids in exovesicles at longer incubation times was only detectable in the presence of gramicidin or dithiothreitol lead to a drastic decrease on the [phospholipids]/[cholesterol] ratio. However, in the samples with dithio-threitol, this variation did not result in the expectable decrease of membrane fluidity. These effects can be related with the presence of lipid rafts, the transbilayer lipids reorientation induced by gramicidin or dithiothreitol, and the cholesterol-dependent gramicidin channels inactivation.

A Colorimetric Process to Visualize Erythrocyte Exovesicles Aggregates.

A biochemistry laboratory class protocol is described in order to create an opportunity for students to apply by doing the theoretical concepts underlying biomolecules and vesicles properties, together with the principles of centrifugation and colorimetric methodologies. Through simple procedures the students will i) observe the segregation of the vesicles suspensions into two separate phases (phtalate esters gradient and vesicle aggregates); ii) visualize the vesicle aggregates by protein, enzyme, and phospholipids coloration processes; and iii) discuss and explain the visualized colors by proper biochemical reactions. The aims, objectives, methodology of teaching/learning, and assessment for this laboratory class are indicated.