Manuel Garrosa

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.

The "Big-Bang" for modern glial biology: Translation and comments on Pío del Río-Hortega 1919 series of papers on microglia.

The word “glia” was coined in the mid‐19th century and defined as “the nerve glue”. For decades, it was assumed to be a uniform matrix, until cell theorists raised the “neuron doctrine” which stipulated that nervous tissue was composed of individual cells. The term “astrocytes” was introduced in the late 19th century as a synonym for glial cells, but it was Santiago Ramón y Cajal who defined a “third element” distinct from glial cells (astrocytes) and neurons. It was not until 1919 when Pío del Río‐Hortega, an alumnus of the Cajal School, introduced the modern terms we use today, and thoroughly described both “oligodendrocytes” and “microglia” to clearly distinguish them from astrocytes. In a series of four papers published that year in Spanish, Río‐Hortega described the distribution and morphological phenotype of microglia. He also noted that these cells were the origin of the rod cells described earlier in pathologic tissue, and recognized that resting microglia transformed into an ameboid phenotype in different types of brain diseases and pathologies. He also noted the mesodermal origin of these cells and recognized their phagocytic capacity. We here provide the first English translation of these landmark series of papers, which paved the way for modern glial research. To heighten the value and accessibility of these classic papers and their original figures, an introduction to this critical period of neuroscience is provided, along with unpublished photographs. By adding comments to the translated text, we provide sufficient context so that contemporary scientists may fully appreciate it. GLIA 2016;64:1801–1840

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.

FGF signalling controls expression of vomeronasal receptors during embryogenesis

Fibroblast growth factors (FGFs) have been shown to control formation and differentiation of multiple organ systems in the developing vertebrate embryo. The analysis of differential gene expression during embryogenesis is, therefore, a potent tool to identify novel target genes regulated by FGF signalling. Here, we have applied microarray analysis to identify differentially regulated genes in FGF mutant mouse embryos. Surprisingly, transcripts corresponding to vomeronasal receptors (VRs), which so far have been only detected in the vomeronasal organ (VNO), were found to be downregulated in FGF mutant embryos. VR expression was detected in the developing olfactory pit and the anlage of the VNO. Interestingly, several FGFs can be detected in the developing olfactory pit during mouse embryogenesis [Bachler, M., Neubuser, A. 2001. Expression of members of the Fgf family and their receptors during midfacial development. Mech. Dev. 100, 313-316]. FGF signalling may thus control expression of VRs in the olfactory pit and formation of the VNO. Moreover, VR expression was detected in unexpected locations within the developing embryo including retina, dorsal root ganglia and neural tube. The relevance of VR expression in these structures and for formation of the VNO is discussed.

Toxicity of the Anti-ribosomal Lectin Ebulin f in Lungs and Intestines in Elderly Mice

All parts of dwarf elder (Sambucus ebulus L.) studied so far contain a ribosome-inactivating protein with lectin activity (ribosome-inactivating lectin; RIL), known as ebulin. Green fruits contain ebulin f, the toxicity of which has been studied in six-week-old mice, where it was found that the intestines were primary targets for it when administered intraperitoneally (i.p.). We performed experiments to assess whether ebulin f administration to six- and 12-month-old mice would trigger higher toxicity than that displayed in six-week-old mice. In the present report, we present evidence indicating that the toxicological effects of ebulin f after its i.p. administration to elderly mice are exerted on the lungs and intestines by an increased rate of apoptosis. We hypothesize that the ebulin f apoptosis-promoting action together with the age-dependent high rate of apoptosis result in an increase in the lectin’s toxicity, leading to a higher lethality level.

Paneth cells are also target of the ribotoxic lectin nigrin b

Ribosome-inactivating lectins (RILs) are A-B type toxins like ricin whose molecular target is the large rRNA of eukaryotic ribosome. Administration of lethal doses of the RIL nigrin b isolated from elderberry (Sambucus nigra L.) bark triggers specific intestinal derangement. The aim of the present research was to explore the early effects of a lethal dose of nigrin b (16 mg/kg body weight) on the small intestine using light and electron microscopy to ascertain intestinal epithelium changes. 6 h after nigrin administration, the small intestine crypts began to show signs of damage with cells appearing at different stages of apoptosis. 16 h after injection crypts appeared more impaired, including the derangement of Paneth cells. The novelty of our results is that the Paneth cells in the small intestine in addition to stem cells are the early cellular targets for nigrin b.

Lectin Digestibility and Stability of Elderberry Antioxidants to Heat Treatment In Vitro

Elderberry contains healthy low molecular weight nutraceuticals and lectins which are sequence-related to the elderberry allergen Sam n1. Some of these lectins are type II ribosome-inactivating proteins. The sensitivity of native lectins present in elderberry fruits and bark to the proteolysis triggered by in vitro simulated gastric and duodenal fluids has been investigated. It was found that these lectins are refractory to proteolysis. Nonetheless, incubation for 5–10 min in a boiling water bath completely sensitized them to the hydrolytic enzymes in vitro. Under these conditions neither total Folin-Ciocalteau’s reagent reactive compounds, total anthocyanins and the mixture of cyanidin-3-glucoside plus cyanidin-3-sambubioside, nor antioxidant and free-radical scavenging activities were affected by more than 10% for incubations of up to 20 min. Therefore, short-time heat treatment reduces potential allergy-related risks deriving from elderberry consumption without seriously affecting its properties as an antioxidant and free-radical scavenging food.

Endotoxins from a Pharmacopoeial Point of View

A pyrogen is a substance that causes fever after intravenous administration or inhalation. Gram negative endotoxins are the most important pyrogens to pharmaceutical laboratories. In the International, United States, Japanese and European Pharmacopoeias, there are two official methods to evaluate pyrogenicity—that is, the bacterial endotoxin test, and the pyrogen test. The main objective of this review is to compare the monographs of each test among the different Pharmacopeias, to detect similarities and differences. The former can be considered fully harmonized, and only non-significant differences were detected. The latter, which is the only available assay for some products and formulations to demonstrate apyrogenicity, shows large differences, which should be considered.

The ligamentum teres femoris in orangutans

OBJECTIVES It is widely viewed that orangutans lack a ligamentum teres femoris (LTF) inserting on the femoral head because orangutans lack a distinct fovea capitis. Orangutans employ acrobatic quadrumanous clambering that requires a high level of hip joint mobility, and the absence of an LTF is believed to be an adaptation to increase hip mobility. However, there are conflicting reports in the literature about whether there may be a different LTF configuration in orangutans, perhaps with a ligament inserting on the femoral neck instead. Here we perform a dissection-based study of orangutan hip joints, assess the soft tissue and hard tissue correlates of the orangutan LTF, and histologically examination the LTF to evaluate whether it is homologous to that found in other hominoids. MATERIALS AND METHODS The hip joints from six orangutans were dissected. In the two orangutans with an LTF passing to the femoral head, the LTF was assessed histologically. Skeletonized femora (n=56) in osteological repositories were examined for evidence of a foveal pit. RESULTS We observed an LTF in two of the three infant orangutans but not in the sub-adult or adult specimens. Histological examination of the infant LTF shows a distinct artery coursing through the LTF to the head of the femur. One percent of orangutan femora present with a foveal scar, but no pit, on the femoral head. DISCUSSION Despite being absent in adults, the LTF is present in at least some orangutans during infancy. We suggest that the LTF maintains a role in blood supply to the femoral head early in life. Because the LTF can limit hip mobility, this may explain why the LTF may be lost as an orangutan ages and gains locomotor independence. These findings enhance our understanding of orangutan hip morphology and underscore the need for future soft tissue investigations.