PubMed Health⌕ Search

Biomedical subjects

G M Villegas

Publications and source records attributed to G M Villegas.

At least 19 recordsLinked to original sources
PubMed1995

Neuronal differentiation of PC12 and chick embryo ganglion cells induced by a sciatic nerve conditioned medium: characterization of the neurotrophic activity.

The present work deals with the finding and characterization of a neurotrophic factor present in serum-free Dulbecco's modified Eagle's medium in which rat sciatic nerves previously cultured for 9 days were maintained for 24 h. This sciatic nerve conditioned medium (SNCM) produced neuronal differentiation and neurite outgrowth on PC12 cells, as well as survival and differentiation of eight-day old chick embryo dorsal root ganglion (E8-DRG) and ciliary ganglion (E8-CG) neurons. SNCM activity was decreased by dilution, heating and trypsin treatment; it was not inhibited by anti-NGF and anti-bFGF antibodies; and it was not mimicked by CNTF, laminin and fibronectin. By utilizing its neurite-promoting activity on PC12 cells, experiments oriented to purify the factor were carried out. Ultrafiltration, heparin-affinity chromatography and size-exclusion high pressure liquid chromatography (HPLC) were employed. The ability of SNCM to induce PC12 cell, E8-DRG and E8-CG neuronal differentiation, the heparin affinity of the active SNCM protein, and the size-exclusion HPLC elution characteristics of the active protein suggest that the active component of the SNCM is, in all probability, a novel sciatic nerve neurotrophic factor (SNTF).

Animals↗
PubMed1995

Order-disorder phenomena in myelinated nerve sheaths: V. Effects of temperature on rat sciatic and optic nerves, and structural differences between the two types of nerve.

We describe in this work X-ray scattering and electron microscope studies of rat sciatic and optic nerves as a function of temperature. The scattering experiments were analyzed as described in the previous papers of this series: a variety of parameters were determined, some of which characterize the lattice disorder, others the structure of the motif. The main results are the following. All the parameters determined by the X-ray scattering study vary with temperature and the temperature-dependence is specific for the type of nerve (sciatic or optic). Most of the disorder-related parameters display a minimum or a maximum in the vicinity of physiological temperature (38 degrees C in rat); this observation, strongly supported by the electron microscope study, shows that the degree of organization of myelin is highest near physiological temperature. The structure of the motif, as revealed by the electron density profile, is fairly different in the two types of nerves (in contrast with the assumption made by previous workers); the structure also varies with temperature and the temperature-induced alterations are nerve-type specific. In the two types of nerve the thickness of the lipid bilayer varies with temperature as expected for a lipid-containing system with hydrocarbon chains in the disordered conformation. In sciatic nerve the thickness of the (thinner) cytoplasmic polar layer, which is also the layer most affected by lattice disorder in this type of nerve, decreases dramatically with increasing temperature. In optic nerve, in which lattice disorder predominantly affects the extracellular layer, the thickness of both the cytoplasmic and the extracellular layer is barely affected by temperature.

Algorithms↗
PubMed1994

Expression of sodium channels with different saxitoxin affinity during rat forebrain development.

This work characterizes the development of the saxitoxin (STX)-sensitive Na+ channels from rat whole forebrain between embryonic day 15 (E15) and postnatal day 90 (P90), both with binding studies and with single channel studies. The Na+ channel total mRNA and the individual mRNAs encoding Na+ channels I, II and III were also determined. The total STX binding rose about 40-fold from E15 to reach a plateau at P30 and its temporal course correlated with the expression of Na+ channel total mRNA. Low affinity and high-affinity STX binding sites, predominant in embryonic and postnatal forebrains, respectively, were found. The single channel studies of batrachotoxin-modified channels also revealed two main populations. In E15 only low-affinity channels (KD = 32.7 nM; 200 mM NaCl) and in P30 only high affinity ones (KD = 1.6 nM) were present. At P0 channels with intermediate affinity (KD range 3-34 nM) were observed. The increase in affinity was due to a gradual increase in the STX association rate.

Animals↗
PubMed1992

Order-disorder phenomena in myelinated nerve sheaths. IV. The disordering effects of high levels of local anaesthetics on rat sciatic and optic nerves.

Sequences of 15 minute X-ray scattering spectra were recorded with rat sciatic and optic nerves, superfused with tetracaine-containing Ringer solutions. The spectra were analysed using the algorithm advocated in this series of papers. The main results, as a function of the time of exposure to tetracaine, were: the mean value of the repeat distance increases; its variance decreases; the average number of membrane pairs per coherent domain decreases; the fraction of isolated membrane pairs increases. Eventually, the spectra were observed to give way to the continuous intensity curve of a single, isolated membrane pair. At all stages of the experiment the continuous intensity curves were found to differ from one type of nerve to the other, and to be invariant, for each type of nerve, with respect to the tetracaine treatment. The X-ray scattering study clearly identified the nature of the structural differences between the two types of myelin sheaths: in that of native sciatic nerves, packing disorder preferentially affects the cytoplasmic space of the membrane pair, and tetracaine disrupts the packing in that space; in the myelin of optic nerves it is the external space that is preferentially affected by packing disorder and disrupted by tetracaine. The time-course of the structure parameters showed that, at any stage of the experiment, tetracaine acts preferentially on the more highly disordered regions of the structure and totally disrupts them. These results corroborate earlier conclusions reported in the previous papers of this series. An electron microscope study was also performed on tetracaine-treated nerves: the results, in close agreement with those of the X-ray scattering study, neatly confirm the conclusions given above. In a more general way, the remarkable agreement between the results of the analysis of the X-ray scattering spectra and the electron microscope observations strongly supports the validity of the physical model used in this series of papers and the correctness of the mathematical treatment that we advocate. Finally, the relations between this work and the work of others are discussed. It must be stressed that the present work bears on the toxic rather than on the anaesthetic effects of tetracaine.

Action Potentials↗
PubMed1991

Concomitant changes in endothelial cell junctions and extracellular matrix components in the chick embryo aorta.

According to previous studies, a process of endothelial activation seems to be occurring in the chick embryo between days 7 and 18. Also, endothelial cells respond to collagen as a substratum between 12 and 18 days, and this response diminishes until it almost disappears after birth. In the present study, aortas from chick embryos (days 7 to 21), and from chicks (14 days posthatching) were used. The results obtained by the freeze-fracturing technique, showed that between days 12 and 14 the intramembranous particles were aggregated into linear or clustered arrays in the fracture P-face of endothelial cells. This could signify that some kind of gap junction-like coupling may occur between adjacent endothelial cells. Our results also indicate that in advanced stages (21-day-old chick embryos and 14-day-old chicks) the growth of small aggregates into larger aggregates or plaques could occur. In addition to gap junctions, the presence of macular and linear tight junctions, reported as focal tight junctions (day 14 of development) macular and linear tight junctions with free-ending strands oriented parallel to one another (21 days) and smooth contoured ridges (14 days post-hatching) were observed. This sequence of changes may represent a development from linear to macular, to a more occluding arrangement, and may also reflect an endothelial cell polarization. Histochemical study of proteoglycans was done by using cuprolinic blue according to the critical electrolyte concentration method. Cuprolinic blue-positive granular, elongated and microfibrillar materials were found in the subendothelial region, forming a meshwork that occupies the extracellular space. Qualitative and quantitative changes were observed both in proteoglycans and in other extracellular matrix components throughout development, suggesting an increase in extracellular matrix complexity. These results lead us to suggest that the assembly of a more complex extracellular matrix, concomitantly with the formation of intercellular junctions during development, might influence the polarization of endothelium in the aorta of the chick embryo.

Animals↗
PubMed1991

Periaxonal ensheathment of lobster giant nerve fibres as revealed by freeze-fracture and lanthanum penetration.

Sheath structure and permeability have been studied in the nerve fibres of lobster (Panulirus argus) walking limbs, in particular the individually ensheathed larger giant fibres, 100-150 microns in diameter, of which there are five or six in a peripheral bundle. They are easily distinguished and can be separated from neighbouring fibre bundles in which smaller giant axons (65-80 microns diameter) and many axons of much smaller diameter (5-15 microns) are ensheathed together. Each of the larger giant axons is enveloped by a Schwann cell layer outside of which is a multilayered sheath consisting of one-cell thick belts of flattened cells and interleaved zones of collagen fibrils and extracellular matrix. The cells in each belt lack basal lamina and, after freeze-fracture, as well as in thin sections, exhibit intercellular gap junctions and incomplete, fascia type, tight junctions; their most striking aspect is an exceedingly large number of exo-endocytic profiles. Permeability to lanthanum chloride in the bathing medium studied before or during fixation both in intact nerves and in nerves with surgically breached (slit) epineurium showed penetration of lanthanum tracer between the cells around the giant fibres, but the electron-dense tracer was excluded from the Schwann cell layer and the periaxonal space unless the epineurium had been slit. The extent of lanthanum diffusion was evaluated by transmission electron microscopy of thin sections and confirmed by X-ray microanalysis (EDAX) of comparable selected areas in such sections. The results indicate structural similarities but distinct permeability differences between the multilayered sheath surrounding the lobster giant axons and the vertebrate nerve perineurium. Other ultrastructural details provided by the freeze-fracture replicas concern the distribution of intramembrane particles in the axolemma and the Schwann and sheath cell membranes.

Animals↗
PubMed1989

Peripheral nerve regeneration in quail-chick spinal cord chimeras treated with oral ciclosporin.

Regeneration of chimeric wing nerves in 2 quail-chick spinal cord chimeras is reported. The large DNA-containing quail nucleolar marker could not be distinguished in flattened cells, and in particular, in perineurial cell nuclei of chimeric nerves. With oral ciclosporin treatment, rejection of graft-derived nervous system tissues was acute and florid in one chimera, but followed a chronic, remitting course in the other.

Administration, Oral↗
PubMed1988

The lobster nerve sodium channel: solubilization and purification of the tetrodotoxin receptor protein.

Solubilization and purification of the tetrodotoxin (TTX) binding protein of the lobster walking-leg nerve Na+ channel were carried out utilizing [3H]tetrodotoxin [( 3H]tetrodotoxin) as a marker. The nerve membrane was solubilized with Lubrol-PX and the Na+ channel protein was purified with diethylaminoethyl Bio-Gel A, Bio-Gel hydroxylapatite powder and two Sepharose 6B columns. Care was taken to keep the temperature of the Na+ channel preparation as close to 1 degrees C as possible and to use solutions (pH 7.5) that contain Na channel protectors, i.e., egg phosphatidylcholine/Lubrol-PX mixture, TTX, EDTA, EGTA, phenylmethylsulfonyl fluoride, pepstatin A, iodoacetamide, antipain, phosphoramidon, soybean trypsin inhibitor, leupeptin and bacitracin. From an initial specific binding of 20.1 pmol of [3H]TTX/mg protein for the solubilized membrane, the binding increased to 1241 pmol/mg protein for the most active fraction of the last Sepharose 6B column. The [3H]TTX specific binding of the Sepharose 6B fractions correlated with a large peptide of Mr 260,000 (240-280K), although other peptides were also present in lesser amounts.

Adsorption↗
PubMed1988

[Electron microscopy of giant nerve fibers].

The present paper is a brief review on the ultrastructural details of giant nerve fibers and their possible correlation to some physiological findings which have been reported in the squid giant axon. In both, thin sections and freeze-fracture replicas, the most striking feature is the exceeding amount of paired membranes populating the Schwann cell layer. These membranes represent permeable intercellular clefts connecting the axon surface to the endoneurial extracellular space, thus leaving the axolemma as the only continuous barrier between the axoplasm and the neuron exterior. Close apposition of the axon and Schwann cell at the level of structural complexes involving both cells plasma membranes are observed in sections and replicas. These zones could represent the morphologic expression of the functional coupling reported in the same preparation. Besides, the Schwann cell appears to be very active according to the amount of exo-endocytotic profiles seen in all its fracture faces. Finally, the endoneurial cells are different in the various giant fibers studied: in the squid they appear as spongy cells, whereas in the lobster they exhibit an extraordinary amount of exo-endocytosis mixed with some gap and a few incomplete tight junctions, and in the crayfish they present the same features as the adaxonal glia.

Animals↗
PubMed1987

Anemone toxin II receptor site of the lobster nerve sodium channel. Studies in membrane vesicles and in proteoliposomes.

The receptor-site for the sea anemone toxin II from Anemonia sulcata (ATX) and its functional relationship with the Na+ channel were studied in plasma membrane preparations from lobster walking leg nerves. The modification of the 22Na influx by ATX was determined in membrane vesicles and in proteoliposomes prepared by reconstitution of detergent-extracted, unfractionated membrane particles into soybean liposomes. The effects of two other toxins, veratridine (VER) and tetrodotoxin (TTX), which bind to Na+ channel receptor-sites other than that for polypeptide toxins, were also studied, ATX and VER stimulated 22Na flux into membrane vesicles with K0.5 values in the order of 10(-7) and 10(-5) M, respectively. Positive cooperativity among these toxins was also seen; ATX displaces the K0.5 for VER towards lower VER concentrations. TTX abolishes the 22Na influx increment caused by ATX and/or VER with a K0.5 in the order of 10(-8) M. In proteoliposomes, in contrast, ATX modified the 22Na influx only at high concentrations (greater than 1 microM) and in the presence of VER. VER stimulation and TTX inhibition of the VER and the VER plus ATX modified fluxes, had the same characteristics as in the vesicle preparations. Measurable ATX and VER toxin effects were only seen in the presence of an outwardly directed K+ gradient for both vesicles and proteoliposomes. Detergent treatment and the reconstitution procedure seem to affect the functional properties of the ATX receptor site whereas the VER and the TTX sites remain unaltered.

Animals↗
PubMed1987

Freeze-fracture studies on the giant axon and ensheathing Schwann cells of the squid.

The giant axons and encompassing sheaths from the stellar nerves of the squids Sepioteuthis sepioidea and Loligo forbesi have been analysed by freeze-fracture. The axolemma exhibits many intramembranous particles (IMPs) that fracture onto the cytoplasmic membrane half-leaflet (P-face); the larger IMPs may be aggregated into clusters. Axoplasmic subsurface cisternae are found beneath this membrane. Clustered or aligned arrays of P-face IMPs are also found on the membranes of the Schwann cells that intimately encapsulate the giant axons as well as 'capitate' projections of Schwann cells into the axons. When adjacent Schwann cells abut directly against one another, aligned E-face IMPs are found along the fracture plane of the upturning membranes. These E-face alignments of IMPs have complementary furrows on the Schwann cell membranes which exhibit no complementary structure on the axolemma as they represent the clefts between adjacent glial cells. The other Schwann cell membranes exhibit P-face dimples and E-face (extracellular membrane half-leaflet) protuberances which may reflect endo- or exocytotic activity; alternatively they may represent caveolae. Comparable structures are occasionally observed at axo-glial interfaces. However, those in the Schwann cell membrane could be part of the transverse tubular lattice system which also exists in adaxonal glia. Beyond the Schwann cells, layers of endoneurial cells (fibrocytes) are interleaved by collagen-filled spaces. These cells exhibit extensive cross-fractured intracellular invaginations as well as inpushings of the extracellular matrix material. Their membranes exhibit a large number of IMPs.

Animals↗
PubMed1983

Mouse sciatic nerve regeneration through semipermeable tubes: a quantitative model.

The regeneration of transected mouse sciatic nerves using semipermeable acrylic copolymer tubes to enclose both stumps has been qualitatively assessed from 1 to 30 weeks post-operative. Quantitative morphometric analysis of electron micrograph montages of complete transverse sections of the segment regenerated between stumps has permitted determinations of the percents of total area occupied by the various tissue constituents--blood vessels, epineurium, perineurium, endoneurium, myelinated axon/Schwann cell units, and unmyelinated axon/Schwann cell units. Significant differences were found in the total cross-sectional area of segments regenerated through tubes of 1.0 mm versus 0.5 mm internal diameters. Segments regenerated with the distal stump inserted in the tube contained significantly greater percentages of neural units and were significantly larger at 8 weeks post-operative compared to segments regenerated for 9-10 weeks with the distal stump avulsed. The morphometric method permits rapid quantitation of sizeable electron micrograph montages which at 1300 X permit all types of tissue components, including the unmyelinated axons, to be visualized.

Acrylic Resins↗
PubMed1981

Reconstitution of the sodium channel with partially solubilized lobster nerve membrane.

Reconstitution experiments were carried out with particles obtained from lobster nerve plasma membrane preparations by detergent treatment, differential centrifugation and ammonium sulfate fractionation. The NA channel activity of the three fractions obtained, which have different amounts of the same peptides present in the original membrane, appears related to their content in a large component which does not enter the 9% polyacrylamide gel and in peptides with 220,000 and 110,000 apparent molecular weight. Other reconstitution experiments made with two fractions obtained by detergent treatment, differential centrifugation and gel exclusion chromatography, revealed that the Na channel active fraction contains the material which does not enter the gel in addition to the 220,000 and 110,000 molecular weight peptides. The other fraction was inactive and does not contain those components. The 220,000 dalton peptide has a molecular weight similar to those determined for the tetrodotoxin-saxitoxin receptor and the scorpion toxin receptor of the Na channel. Whether any of the other peptides is a Na channel constituent is unknown at present.

Animals↗