Romildo de Albuquerque Nogueira

A Novel Approach to Study the Geometry of the Water Lumen of Ion Channels: Colicin Ia Channels in Planar Lipid Bilayers

Abstract. This paper describes a new approach to evaluate the inner structure (including a main constriction and its localization) of the water lumen of an ion channel. The method is based on the determination of channel filling by different nonelectrolyte molecules through each side of an ion channel. The method has two characteristic features that make its use attractive: (i) the possibility to ascertain the existence, localization and size of a narrow part inside an ion channel water lumen and (ii) the chances to determine the maximal size of both entrances of an ion channel and to obtain additional information about the geometry of its water lumen at the same time. Determinations were made on colicin Ia ion channels inserted into planar lipid bilayers. This channel was chosen because there is an apparent contradiction between its low single channel conductance and the large diameter of its water lumen. Our results show that the water lumen of the colicin Ia channel has a funnel-like structure with a small trans-entrance, with a diameter of about 1.0 nm, and a large cis-entrance, with a diameter of approximately 1.8 nm. A constriction with a diameter of approximately 0.7 nm is shown to be located close to the trans-entrance of the channel. The method can also be applied to patch clamp studies of single ion channels.

Pore-forming properties of proteolytically nicked staphylococcal ⋅-toxin: the ion channel in planar lipid bilayer membranes

Abstract Staphylococcal α-toxin is a single-chain protein with a molecular mass of 33.2 kDa, which can form large water-filled pores both in lipid bilayers and in erythrocyte membranes. Limited proteolysis of the purified toxin with proteinase K led to time-dependent changes of all the functional features of the channels formed by the toxin. Single-channel conductance in planar bilayers was decreased about threefold. The anion selectivity of the channel was replaced with cation selectivity and the asymmetry in the current-voltage relationship of the channel became more pronounced. At the same time the nicked toxin kept its full ability to form ion channels in lipid bilayers, although it lost a considerable part of its hemolytic activity. In planar bilayers and in erythrocyte membranes, the proteolytically nicked toxin actually formed channels with a slightly smaller diameter (∼1.2 times) than that formed by the native toxin. This decrease was not marked enough to explain changes in the biological effects of the nicked toxin. The change in channel selectivity induced by the cleavage is considered to be the major determinant of the changes in the biological effects of the nicked toxin.

The hinge portion of the S. aureus α-toxin crosses the lipid bilayer and is part of the trans-mouth of the channel

This paper compares the functional properties of ion channels formed in planar lipid membranes by the wild and mutant Staphylococcus aureus alpha-toxin. It was shown that replacement of the amino acid Gly at position 130 by Cys in the primary structure of the toxin decreases the single-channel conductance with a concomitant decrease in the pH at which the channel becomes unable to discriminate between Cl- and K+ ions. The mutation also induced an increase in the asymmetry in the current-voltage relationship of the channel. The results of our experiments suggest that the trans-mouth of the channel is responsible for all the observed changes in channel properties. It was assumed that this entrance is built by the glycine-rich hinge portion of the toxin and is situated close to the surface of monolayer facing the trans-compartment.

Fractal analysis of extra-embryonic vessels of chick embryos under the effect of glucosamine and chondroitin sulfates.

Like heparan sulfate proteoglycans, some monosaccharides and glycosaminoglycans, such as sulfated glucosamine (GS) and chondroitin (CS), integrate the vascular extracellular matrix and may influence vascular endothelial cell growth. To assess the effects of these substances on blood vessel formation, we used the chick yolk sac membrane (YSM) model and fractal geometry quantification, which provided an objective in vivo method for testing potential agents that promote vasculogenesis and angiogenesis. An image processing method was developed to evaluate YSM capillary vessels after they were implanted in a methylcellulose disk of GS or CS at a concentration between 0.001-0.1mg/disk (performed on 2-day old embryos). This method resulted in a binary image of the microvascular network (white vessels on a black background). Fractal box-counting (DBC) and information (DINF) dimensions were used to quantify the activity of GS and CS in vasculogenesis and angiogenesis. YSM treated with GS (0.001-0.1mg) and CS (0.03-0.1mg) showed an increase in fractal dimensions that corresponded to vitelline vessel growth compared to the control group (vehicle), with GS displaying higher fractal dimension values.

Non stochastic distribution of single channels in planar lipid bilayers

The selectivity of the planar lipid bilayers modified by two channel-forming proteins (alpha-toxin S. aureus and colicin Ia) was examined. It was established that in all cases the value of zero current potential depended on the amount of open ion channels and increased with the number of channels (from one to about 5-7). These facts point out both the interactions among ion channels and their non stochastic distribution on the membrane surface.

Multifractal dimension and lacunarity of yolk sac vasculature after exposure to magnetic field

Several studies have reported about the effects of magnetic fields (MFs) on vascular tissue. Extremely low frequency magnetic fields (ELF-MFs) can promote either inhibition or stimulation of vasculogenesis and angiogenesis, depending upon the intensity and time of exposure to the MF. To investigate the possible effects of ELF-MF on vascular processes, it is necessary to employ methods that allow parameterization of the vascular network. Vascular network is a structure with fractal geometry; therefore, fractal methods have been used to evaluate its morphometric complexity. Here, we used the lacunarity parameter (complementary method of fractal analysis) and multifractal analyses to investigate angiogenesis and vasculogenesis in the embryonic yolk sac membrane (YSM) of Japanese quails (Coturnix japonica) with and without exposure to an external MF of 1 mT and 60 Hz. Lacunarity results showed that the vascular density was lower for the group exposed to the magnetic field for 9 h/day. In addition, multifractal analysis showed reduced vascularization in the experimental groups (6 h/day and 9 h/day of exposure to MF). Furthermore, multifractal analysis showed difference between the groups exposed for 12 and 24 h/day. Using multifractal methods (generalized dimensions and singularity spectrum), it was possible to characterize the vascular network of the quail embryo YSM as a multifractal object, therefore proving this method to be a more appropriate application than the traditional monofractal methods.

Effect of diet with omega-3 in basal brain electrical activity and during status epilepticus in rats

Western diets are high in saturated fat and low in omega-3. Certain animals cannot produce omega-3 from their own lipids, making it necessary for it to be acquired from the diet. However, omega-3s are important components of the plasma membrane, and altering their proportions can promote physical and chemical alterations in the membranes, which may modify neuronal excitability. These alterations occur in healthy individuals, as well as in patients with epilepsy who are more sensitive to changes in brain electrical activity. This study evaluated the effect of a diet supplemented with omega-3 on the basal brain electrical activity both before and during status epilepticus in rats. To evaluate the brain electrical activity, we recorded electrocorticograms (ECoG) of animals both with and without omega-3 supplementation before and during status epilepticus induced by pilocarpine. Calculation of the average brain wave power by a power spectrum revealed that omega-3 supplementation reduced the average power of the delta wave by 20% and increased the average power of the beta wave by 45%. These effects were exacerbated when status epilepticus was induced in the animals supplemented with omega-3. The animals with and without omega-3 supplementation exhibited increases in basal brain electrical activities during status epilepticus. The two groups showed hyperactivity, but no significant difference between them was noted. Even though the brain activity levels observed during status epilepticus were similar between the two groups, neuron damage to the animals supplemented with omega-3 was more slight, revealing the neuroprotective effect of the omega-3.

Pro-angiogenic Activity Assay of Chondroitin Sulfate and Glucosamine Sulfate on Vascular Network of Mouse and of Chick Embryo Chorioallantoic Membrane

Objective: Target of this study was to test the capacity of chondroitin sulfate (CS) and glucosamine sulfate (GS) to induce in vivo angiogenesis. Methods: The proangiogenic activity of these compounds was analyzed through the assays in chorioallantoic membrane (CAM) of chick embryo and dorsal skin vascularization in mice, but before was realized a cell viability assay with human umbilical veins endothelial cells (HUVEC). Results: In the viability assay, concentrations tested between 30 and 3000 μg/ml showed a reduction of viable HUVEC number. In the CAM assay, CS and GS in an amount 2.0 mg/implant increased the vessels number as compared to control (phosphate buffered saline-PBS). In the assay of the dorsal skin vascularization of adult Swiss mice, the groups treated with CS (2 mg/implant; Gelfoam plug) exhibited an increase in the vessels number into plugs (0.52 ± 0.08 g/dl; measured as plug-hemoglobin content), a similar effect to that promoted by Fibroblast growth factor-2 (FGF-2; 50 ng/implant) (0.53 ± 0.1 g/dl). However the group treated with GS did not exhibit significant effect on mice skin vascularization. Conclusion: CS was capable to promote angiogenesis on CAM and dorsal skin vascularization, but GS only had pro-angiogenic activity in CAM vascular network.

Analysis of the Height of Lingual Vallates Papillae in Brazilian Individuals

Las papilas valadas linguales estan localizadas en la superficie dorsal de la lengua, en un area denominada V lingual y presentan celulas gustativas acumuladas en botones gustativos. Se evaluo la altura papilar en relacion con la superficie de la lengua de 519 papilas valadas linguales. Fueron seleccionadas 45 lenguas humanas, de individuos de ambos sexos, de 45 a 50 anos de edad, obtenidas de autopsias del Servicio de Verificacion de Obitos de la Universidade Federal ed Pernambuco /Secretaria de Salud del Estado de Pernambuco, Recife, Brasil. Las muestras fueron clasificadas de acuerdo a los niveles de altura en relacion con la superficie de la lengua, no estableciendose la altura exacta, donde hubo predominio de papilas ubicadas sobre la superficie lingual