Manuel J. Gayoso

Prenatal development of the mammalian vomeronasal organ

The vomeronasal organ (VNO) originates from the medial wall of the olfactory pit shortly after the middle of the embryonic period in mammals. The Anlage stage consists of a cellular bud that grows dorsally, caudally, and towards the midline leaving a groove. The following stage, Early Morphogenesis, includes the closure of the vomeronasal groove to form a parasagittal blind‐ended tube in the nasal septum, which opens into the nasal and/or oral cavities. The lumen adopts a crescent shape while the epithelial lining differentiates into an increasingly wider epithelium on the concave side and a gradually thinner epithelium on the convex side. The former goes on to occupy a medial position and develops neuroblasts among supporting and undifferentiated cells, with supporting cell nuclei tending to align in the upper rows. The lateral “non‐sensory” epithelium furrows, giving a kidney‐shaped appearance to the VNO cross section. The next stage, Late Morphogenesis is extended up to a difference in thickness between both epithelia becomes similar to the adult, generally by birth. An increasing number of ciliary generation complexes, larger and more abundant microvilli, and an evident glycocalyx are observed in the neuroepithelium at the luminal surface, while enzymatic activities become more intense. The non‐sensory epithelium appears quite mature save for its luminal surface, which is still devoid of cilia. Blood capillaries penetrate the most basal region of the neuroepithelium and vomeronasal glands are very few and immature. At birth, some neurons appear well developed to support certain functionality; however, persistence of architectural, histochemical, and ultrastructural signs of immaturity, suggests that full performance of the VNO does not occur in newborn mammals, but in prepubertal ages. Microsc. Res. Tech. 41:456–470, 1998.

Elastin-like recombinamers as substrates for retinal pigment epithelial cell growth

The aim of this study is to investigate the use of elastin-like recombinamers (ELRs) as a substrate that can maintain the growth, phenotype, and functional characteristics of retinal pigment epithelial (RPE) cells efficiently and as a suitable carrier for the transplantation of autologous RPE cells for treatment of age-related macular degeneration (AMD). ELR films containing a bioactive sequence, RGD (ELR-RGD), and one with no specific sequence (ELR-IK) as control, were obtained by solvent-casting onto glass and subsequent cross-linking. ARPE19 cells were seeded on sterilized ELR films as well as on the control surfaces. Cells were analysed after 4, 24, 72, and 120 h to study cell adhesion, proliferation, cell viability, morphology, and specificity by staining with Trypan blue, DAPI, Rhodamin-Phalloidin and RPE65, ZO-1 antibodies and observing under fluorescence as well as electron microscope. ARPE19 cells seeded on both ELR films and controls were 100% viable and maintained their morphology and set of characteristics at the different time points studied. Cell proliferation on ELR-RGD was significantly higher than that found on ELR-IK at all time points, although it was less than the growth rate on polystyrene. ARPE19 cells grow well on ELR-RGD maintaining their phenotype. These results should be extended to further studies with fresh human RPE cells and in vivo studies to determine whether this ELR-RGD matrix could be used as a Bruchs membrane prosthesis and carrier for transplantation of RPE cells in patients suffering with AMD.

Time course modifications in organotypic culture of human neuroretina

The purpose of this study was to characterize organ culture of human neuroretina and to establish survival and early degeneration patterns of neural and glial cells. Sixteen neuroretina explants were prepared from 2 postmortem eyes of 2 individuals. Four explants were used as fresh retina controls, and 12 were evaluated at 3, 6, and 9 days of culture. Neuroretina explants (5 × 5 mm) were cultured in Transwell(®) dishes with the photoreceptor layer facing the supporting membrane. Culture medium (Neurobasal A-based) was maintained in contact with the membrane beneath the explant. Cryostat and ultrathin sections were prepared for immunohistochemistry and electron microscopy. Neuroretinal modifications were evaluated after toluidine blue staining and after immunostaining for neuronal and glial cell markers. Ultrastructural changes were analyzed by electron microscopy. From 0 to 9 days in culture, there was progressive retinal degeneration, including early pyknosis of photoreceptor nuclei, cellular vacuolization in the ganglion cell layer, decrease of both plexiform layer thicknesses, disruption and truncation of photoreceptor outer segments (OS), and marked reduction in the number of nuclei at both nuclear layers where the cells were less densely packed. At 3 days there was swelling of cone OS with impairment of pedicles, loss of axons and dendrites of horizontal and rod bipolar cells that stained for calbindin (CB) and protein kinase C (PKC-α), respectively. After 9 days, horizontal cells were pyknotic and without terminal tips. There were similar degenerative processes in the outer plexiform layer for rod bipolar cells and loss of axon terminal lateral varicosities in the inner plexiform layer. Glial fibrillary acidic protein (GFAP) staining did not reveal a dramatic increase of gliosis in Müller cells. However, some Müller cells were CB immunoreactive at 6 days of culture. Over 9 days of culture, human neuroretina explants underwent morphological changes in photoreceptors, particularly the OS and axon terminals, and in postsynaptic horizontal and bipolar cells. These early changes, not previously described in cultured human samples, reproduce some celullar modifications after retinal damage. Thus, this model may be suitable to evaluate therapeutic agents during retinal degeneration processes.

Organization of the outer plexiform layer of the diurnal and nocturnal bird retinae

Cajal (1892) described the outer plexiform layer of the gallinaceous retina as composed of three distinct strata or “concentric plexuses”: the external plexus formed by the basal rod filaments and the dendritic expansions of some bipolar cells; the intermediate plexus formed by the vitreal end or synaptic bodies of the “straight” cones and the ascending dendrites of other bipolar cell types; the internal plexus formed by the basal filaments of the “oblique” cones and the dendritic expansions of the bipolar cells. No further information about the structure of the outer plexiform layer in other bird species is found in Cajal’s work. The presence of rods, single cones and double cones in the birds retina was noted by Schultze (1866, 1867) who observed in the cones red, yellow and colorless droplets. A comprehensive review on the visual cells of several bird species was made with light microscopy by Rochon-Duvigneaud (1943). Nevertheless later studies (Meyer and Cooper, 1966; Morris and Shorey, 1967; and Morris, 1970) on bird visual cells using phase contrast and electron microscopic techniques, have contributed largely to the current knowledge on this subject. Morris (1970) describes different types of visual cells in the chicken retina. namely, rods with no oil droplets, double cones whose “chief member possesses a large yellow oil droplet and whose “accessory” one has a small yellow-green droplet and three types of single cones. All types of single cones have oil droplets and can be differentiated by their affinity for lead staining and their electron density. According to Morris and Shorey (1967) the synaptic bodies of the chicken photoreceptors are located at two different levels in the outer plexiform layer: at the external or sclerad level lie the rod and double cone synaptic bodies; at the internal or v&read level lie the synaptic bodies of the single cones. In the present investigation, the retinae of three diurnal birds, GalLs domesticus, Hieratrrs fasciurus and Mihs mihs and of two nocturnal birds, Carinae noctua and Asiojammetrs were used. Histological processing started immediately after sacrificing the animals. In several instances portions of the retinas were studied in fresh or just after formaldehyde fixation. with the light microscope using Nomarsky optics. For regular light microscopy the techniques used were as follows: the Gallego technique (1953) of silver impregnation of the entire retina. the classical Golgi procedure and its Colonnier modification. Routinely all the retinas were prepared as flat-mounts and studied in toto. Samples of the retinas were selected either for embedding in paraffin and sectioning at different orientations or for plastic embedding, ultrathin sectioning and examination by the electron microscope. The electron microscope is necessary to identify the photoreceptor types and to decide upon the organization of the synaptic bodies of the photoreceptors in the outer plexiform layer.

Postnatal development of striatal connections in the rat: a transport study with wheat germ agglutinin-horseradish peroxidase.

This paper deals with the postnatal development of afferent and efferent connections of the rat striatum as revealed by the transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP). Tracer was injected weekly from birth to the end of the first postnatal month in the head of the caudate nucleus. To control for transport from cortical areas contaminated by the micropipette, injections in newborn rats were made by either vertical or lateral penetrations. In addition some newborn and 14-day-old animals were injected only in the cortex. The results showed that at birth there was retrograde transport to the thalamus, substantia nigra and raphe nuclei. Labelling in the cortex was seen at birth but was probably due to cortical contamination. Transport from the striatum was clearly established on day 7, when a few labelled neurons were observed on both the ipsi and contralateral sides. These neurons increased in number and were distributed through layers III to VI by day 14. At this time labelled cell bodies were observed in the claustrum and lateral amygdaloid nucleus as well as in the globus pallidus and entopeduncular nucleus. On day 21 the contralateral labelling of the lateral amygdaloid nucleus was apparent. The anterograde transport from the striatum to globus pallidus, entopeduncular nucleus and substantia nigra was already visible at birth although its intensity increased during the first postnatal month.

Elderberries: A Source of Ribosome-Inactivating Proteins with Lectin Activity

Sambucus (Adoxaceae) species have been used for both food and medicine purposes. Among these, Sambucus nigra L. (black elder), Sambucus ebulus L. (dwarf elder), and Sambucus sieboldiana L. are the most relevant species studied. Their use has been somewhat restricted due to the presence of bioactive proteins or/and low molecular weight compounds whose ingestion could trigger deleterious effects. Over the last few years, the chemical and pharmacological characteristics of Sambucus species have been investigated. Among the proteins present in Sambucus species both type 1, and type 2 ribosome-inactivating proteins (RIPs), and hololectins have been reported. The biological role played by these proteins remains unknown, although they are conjectured to be involved in defending plants against insect predators and viruses. These proteins might have an important impact on the nutritional characteristics and food safety of elderberries. Type 2 RIPs are able to interact with gut cells of insects and mammals triggering a number of specific and mostly unknown cell signals in the gut mucosa that could significantly affect animal physiology. In this paper, we describe all known RIPs that have been isolated to date from Sambucus species, and comment on their antiviral and entomotoxic effects, as well as their potential uses.

Ebulin from dwarf elder (Sambucus ebulus L.): a mini-review.

Sambucus ebulus L. (dwarf elder) is a medicinal plant, the usefulness of which also as food is restricted due to its toxicity. In the last few years, both the chemistry and pharmacology of Sambucus ebulus L. have been investigated. Among the structural and functional proteins present in the plant, sugar-binding proteins (lectins) with or without anti-ribosomal activity and single chain ribosome-inactivating proteins (RIPs) have been isolated. RIPs are enzymes (E.C. 3.2.2.22) that display N-glycosidase activity on the 28S rRNA subunit, leading to the inhibition of protein synthesis by arresting the step of polypeptide chain elongation. The biological role of all these proteins is as yet unknown. The evidence suggests that they could be involved in the defense of the plant against predators and viruses or/and a nitrogen store, with an impact on the nutritional characteristics and food safety. In this mini-review we describe all the isoforms of ebulin that have to date been isolated from dwarf elder, as well as their functional characteristics and potential uses, whilst highlighting concern regarding ebulin toxicity.

Sialic acid-binding dwarf elder four-chain lectin displays nucleic acid N-glycosidase activity

Sialic acid-binding dwarf elder agglutinin (SEA) present only in rhizomes of the medicinal plant Sambucus ebulus L., was found to be a tetrameric glycoprotein consisting of two covalently-associated dimers of an enzymic A chain with rRNA N-glycosidase activity (EC 3.2.2.22) linked to a B chain with agglutinin properties. The lectin inhibited protein synthesis by a cell-free system and depurinated ribosomes. Cloning of the corresponding gene and molecular modeling of the deduced amino acid sequence demonstrated that SEA has a three-dimensional structure which resembles that reported for other two tetrameric type 2 RIPs from Sambucus (SNAI and SSA). The lectin agglutinated red blood cells and displayed sugar affinity for sialic acid residues apart from d-galactose, binding to the mucin-producing gut goblet cells. Since sialic acid is present in animal cells, especially in epithelial lining gut cells, but not in plants, SEA could play a role in the defense against insect attack. The nucleotide sequence reported in this paper has been submitted to the GenBank nucleotide database under accession number AM981401.

Horizontal cells of the avian retina

Consistent with his own general description of the retinal structure of vertebrates, Cajal (1892, 1904, 1933) described two types of horizontal cells in the avian retina: “stellate” and “brush” horizontal cells. Cajal’s distinction was based on the different location of each cell type with regard to the outer plexiform layer and on some of their morphological features. Recently, a great deal of work has been done on the structure and classification of the photoreceptor cells. as well as on the efferent centrifugal fiber system in the avian retina. However, much less information is available concerning the organization of the outer plexiform layer or the horizontal cells morphology and their relationship with the photoreceptor cells. A previous paper (Gallego, Baron and Gayoso, 1975) shows that in the diurnal bird retina the synaptic bodies of the photoreceptors are found at three different strata in the outer plexiform layer. whereas in the nocturnal species they are found at a single stratum. The present investigation attempts to establish the organization of the outer plexiform layer which includes the horizontal cell types found in the retinae of both diurnal and nocturnal birds. The retinae of three diurnal bird species, Gallus domesticus. Hieratus fasciatus and Milcus milaus, and those of two nocturnal ones, Carinae noctua and Asio jlammeus, were studied. The retinae were obtained inmediately after sacrificing the animal. In the study of the horizontal cells the most suitable procedure was found to be the Golgi silver impregnation and its Colonnier and Lasansky modifications. All retinas were prepared as flat-mounts and observed in toto. Samples of the retinas showing horizontal cells were selected and embedded in paraffin. These samples were then sectioned at different orientations. Further samples of the cell soma and/or the axonic terminal were prepared for thin sectioning and analysis by the electron microscope.

Toxicity in mice of lectin ebulin f present in dwarf Elderberry (Sambucus ebulus L.)

Dwarf elder fruits (Sambucus ebulus) contain the ribosome-inactivating lectin ebulin f structurally related to ricin. We investigated intraperitoneal toxicity of ebulin f in mice and found that it triggers specific derangement of the intestines. Ebulin f was much less toxic than ricin to mice when administered intraperitoneally. The targets were cells of the intestinal crypts, which underwent apoptosis. Small intestine crypts were more sensitive than large intestine crypts.