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A biochemical study of dental pulp of calves has been performed concerning:a) peroxydabilityb) A, E, C vitamins contentc) glutation (GSH) contentd) presence of paramagnetic compoundse) phosphorylation ratioThe dental pulp from incisives of 5 months old calves has been preserved. Immediately after decapitation the pulp was immersed in liquid nitrogen. Chromatographie (HPLC) and spectroscopic (NMR-ESR) techniques have been used.GSH in dental pulp are present and dosable (4.56 +/-0.08 n moles/mg prot.) and GSSG (1.05 +/-0.01 n moles/mg prot.).Because of blood traces in the extracted pulps, the AA. have determined the hemoglobin (Hb) dosage and GSH of erythrocytary derivation (Fig. 1).After deduction of GSH of erythrocytary derivation, the GSH really present in the pulp was 4.41 n moles/mg prot. and the GSSG was 0.90 n moles/mg prot.Peroxydability of the dental pulp has been evaluated with Lowry method with dental pulp homogenate and rat liver homogenate (see Table 1).The ESR spectre shows 4 resonances with the following values: g. 2.24 - 2.04 - 2.00 - 1.97; there are some free intermediary radicals (gr. - 2.00) (Fig.2).The NMR spectre shows the presence of ATP (0.22 n moles/g) of inorganic phosphate (16.58 n moles g) (Fig.3).The pulp seems to have a lot of antioxydant factors. The next researches will be to study E, A and C vitamins concentrations. This high presence of GSH and GSSG may be an embryonary peculiarity., Une étude biochimique de la pulpe dentaire a été entreprise pour étudier les propriétés antioxydantes et leurs interactions avec le métabolisme énergétique et les équilibres redox de la pulpe dentaire de veau. Le GSH réduit et oxydé (GSSG) a été mesuré et la spectroscopie avec ESR (Electron Spin Résonance) a été utilisée pour la recherche des substances paramagnétiques et la résonance magnétique nucléaire (NMR) pour la détermination des métabolites phosphorylés de petit poids moléculaire. Après la soustraction de la quantité de GSH érythrocytaire, le GSH présent dans la pulpe est resté identique 4,41 n moles/mg prot. (GSH) et 0.90 n moles/mg n moles/mg prot. (GSSG). La péroxydation lipidique de la pulpe dentaire a été étudiée. Le spectre ESR montre 4 résonances, respectivement de valeurs de G de 2.24, 2.04, 2.00, 1.97.L’analyse des résultats montre l’existence d’une petite quantité de radicaux libres intermédiaires (g-2.00) dépendant du métabolisme tissulaire. Le spectre NMR a montré la présence d’ATP (0.22 n moles/g) et de phosphate inorganique (16.50 n moles/g).

A biochemical study of dental pulp of calves has been performed concerning: a) peroxydability b) A, E, C vitamins content c) glutation (GSH) content d) presence of paramagnetic compounds e) phosphorylation ratio The dental pulp from incisors of 5-months-old calves has been preserved. Immediately after decapitation the pulp was immersed in liquid nitrogen. Chromatographic (HPLC) and spectroscopic (NMR-ESR) techniques have been used. GSH in dental pulp are present and dosable (4.56 +/- 0.08 n moles/mg prot.) and GSSG (1.05 +/- 0.01 n moles/mg prot.). Because of blood traces in the extracted pulps, the AA. have determined the hemoglobin (Hb) dosage and GSH of erythrocytic derivation (Fig. 1). After deduction of GSH of erythrocytic derivation, the GSH really present in the pulp was 4.41 n moles/mg prot. and the GSSG was 0.90 n moles/mg prot. Peroxydability of the dental pulp has been evaluated with Lowry method with dental pulp homogenate and rat liver homogenate (see Table 1). The ESR spectre shows 4 resonances with the following values: g. 2.24-2.04-2.00-1.97; there are some free intermediary radicals (gr.-2.00) (Fig.2). The NMR spectre shows the presence of ATP (0.22 n moles/g) of inorganic phosphate (16.58 n moles g) (Fig.3). The pulp seems to have a lot of antioxidant factors. The next researches will be to study E, A and C vitamins concentrations. This high presence of GSH and GSSG may be an embryonic peculiarity.

Morphological researches have been carried out through histological studies with fine and semifine sections at the electronic scan microscope on the dental pulp of calves. The general aim was to identify the antioxidant properties of pulp and study cellular density and microfibrillar architecture. The dental pulps of calves of 5-6 months taken immediately after slaughtering were used with immersions into the fixing liquid. The scan microscope showed fibroblasts in the deepest regions of the pulp in a stroma of collagenic fibres, not organised but scattered among the cells of the connective tissue. The odontoblasts presented well ordered one beside the others with well visible details such as the swollen basal portion, the nucleus and a very tight villosity. No clear interodontoblastic connections were evident. The electron transmission microscope revealed typical cells with histiocyte appearance with microfilaments evident in the cytoplasma (myofibroblasts) presenting a wide variety of cytoplasmatic interconnections with interdigitations.

Olfactory sensory neurons (OSNs) of fish belong to three main types: ciliated olfactory sensory neurons (cOSNs), microvillous olfactory sensory neurons (mOSNs), and crypt cells. Mercury is a toxic metal harmful for olfaction. We exposed the olfactory epithelium of zebrafish to three sublethal Hg2+ concentrations. Molecular markers specific for the different types of OSNs were immunohistochemically detected. Image analysis of treated sections enabled counting of marked cells and measurement of staining optical density indicative of the response of OSNs to Hg2+ exposure. The three types of OSNs reacted to mercury in a different way. Image analysis revealed that mOSNs are more susceptible to Hg2+ exposure than cOSNs and crypt cell density decreases. Moreover, while the ratio between sensory/nonsensory epithelium areas is unchanged, epithelium thickness drops, and dividing cells increase in the basal layer of the olfactory epithelium. Cell death but also reduction of apical processes and marker expression could account for changes in OSN immunostaining. Also, the differential results between dorsal and ventral halves of the olfactory rosette could derive from different water flows inside the olfactory chamber or different subpopulations in OSNs.

Vasa, PIWI and TDRKH are conserved components of germ granules that in metazoans are involved in germline specification and differentiation, as documented by mutational experiments in some model animals. So far, investigations on PIWI during spermatogenesis of fish has been limited to a few species, and no information is available for TDRKH, another protein involved in the piRNA pathway. In this study, the immunolocalization of these three germline determinants was analyzed in male gonads of the teleost fish Poecilia reticulata to document their localization pattern in the different stages of germ cell differentiation. To analyze their distribution pattern during the different stages of spermatogenesis we performed immunohistochemistry (IHC) and immunofluorescence (IF) assays using primary polyclonal antibodies after testing their specificity with Western Blot. Moreover, sections of testis stained with haematoxylin and eosin clarified the structural organization of P. reticulata testis, while the use of the confocal microscope and the nuclear staining clarified the different stages of germ cell differentiation during spermatogenesis. The results showed that Vasa, PIWI and TDRKH were specifically immunolocalized in the germ cells of P. reticulata, with no specific signal detected in Sertoli cells and in other somatic cells of the gonad. These markers were detected in all stages of differentiation from early spermatogonia to advanced spermatids. Vasa staining was the strongest in spermatogonia, and then decreases throughout differentiation. Instead, both PIWI and TDRKH staining increases during differentiation, and their distribution pattern, similar to what observed in the mouse, suggests their concerted participation in the piRNA pathway also in this fish.

The germline is a key feature of sexual animals and the ways in which it separates from the soma differ widely across Metazoa. However, at least at some point during germline differentiation, some cytoplasmic supramolecular structures (collectively called germ plasm-related structures) are present and involved in its specification and/or differentiation. The factors involved in the assembly of these granular structures are various and non-ubiquitous among animals, even if some functional patterns and the presence of certain domains appear to be shared among some. For instance, the LOTUS domain is shared by Oskar, the Holometabola germ plasm master regulator, and some Tudor-family proteins assessed as being involved in the proper assembly of germ granules of different animals. Here, we looked for the presence of LOTUS-containing proteins in the transcriptome of Ruditapes philippinarum (Bivalvia). Such species is of particular interest because it displays annual renewal of gonads, sided by the renewal of germline differentiation pathways. Moreover, previous works have identified in its early germ cells cytoplasmic granules containing germline determinants. We selected the orthologue of TDRD7 as a candidate involved in the early steps of germline differentiation through bioinformatic predictions and immunohistological patterning (immunohistochemistry and immunofluorescence). We observed the expression of the protein in putative precursors of germline cells, upstream to the germline marker Vasa. This, added to the fact that orthologues of this protein are involved in the assembly of germ granules in mouse, zebrafish, and fly, makes it a worthy study unit for investigations on the formation of such structures in bivalves. Supplementary Information The online version contains supplementary material available at 10.1007/s00418-021-01983-0.

Background Kanamycin, mainly used in the treatment of drug-resistant-tuberculosis, is known to cause irreversible hearing loss. Using the xeno-transplant model, we compared both in vitro and in vivo characteristics of human mesenchymal stromal cells (MSCs) derived from adult tissues, bone marrow (BM-MSCs) and adipose tissue (ADSCs). These tissues were selected for their availability, in vitro multipotency and regenerative potential in vivo in kanamycin-deafened nod-scid mice. Methods MSCs were isolated from informed donors and expanded ex vivo. We evaluated their proliferation capacity in vitro using the hexosaminidase assay, the phenotypic profile using flow-cytometry of a panel of surface antigens, the osteogenic potential using alkaline phosphatase activity and the adipogenic potential using oil-red-O staining. MSCs were intravenously injected in deafened mice and cochleae, liver, spleen and kidney were sampled 7 and 30 days after transplantation. The dissected organs were analyzed using lectin histochemistry, immunohistochemistry, polymerase chain reaction (PCR) and dual color fluorescence in situ hybridization (DC-FISH). Results MSCs showed similar in vitro characteristics, but ADSCs appeared to be more efficient after prolonged expansion. Both cell types engrafted in the cochlea of damaged mice, inducing regeneration of the damaged sensory structures. Several hybrid cells were detected in engrafted tissues. Discussion BM-MSCs and ADSCs showed in vitro characteristics suitable for tissue regeneration and fused with resident cells in engrafted tissues. The data suggest that paracrine effect is the prevalent mechanism inducing tissue recovery. Overall, BM-MSCs and ADSCs appear to be valuable tools in regenerative medicine for hearing loss recovery.

Heteroplasmy is the presence of more than one type of mitochondrial genome within an individual, a condition commonly reported as unfavorable and affecting mitonuclear interactions. So far, no study has investigated heteroplasmy at protein level, and whether it occurs within tissues, cells, or even organelles. The only known evolutionarily stable and natural heteroplasmic system in Metazoa is the Doubly Uniparental Inheritance (DUI)—reported so far in ∼100 bivalve species—in which two mitochondrial lineages are present: one transmitted through eggs (F-type) and the other through sperm (M-type). Because of such segregation, mitochondrial oxidative phosphorylation proteins reach a high amino acid sequence divergence (up to 52%) between the two lineages in the same species. Natural heteroplasmy coupled with high sequence divergence between F- and M-type proteins provides a unique opportunity to study their expression and assess the level and extent of heteroplasmy. Here, for the first time, we immunolocalized F- and M-type variants of three mitochondrially-encoded proteins in the DUI species Ruditapes philippinarum, in germline and somatic tissues at different developmental stages. We found heteroplasmy at organelle level in undifferentiated germ cells of both sexes, and in male soma, whereas gametes were homoplasmic: eggs for the F-type and sperm for the M-type. Thus, during gametogenesis, only the sex-specific mitochondrial variant is maintained, likely due to a process of meiotic drive. We examine the implications of our results for DUI proposing a revised model, and we discuss interactions of mitochondria with germ plasm and their role in germline development. Molecular and phylogenetic evidence suggests that DUI evolved from the common Strictly Maternal Inheritance, so the two systems likely share the same underlying molecular mechanism, making DUI a useful system for studying mitochondrial biology.

Among vertebrates, the physiologically uremic Chondrichthyes are the only class which are not presenting the ciliated olfactory receptor neurons in the olfactory neuroepithelium. The only sequenced genome for this class revealed only three olfactory receptor genes and the immunohistochemical detection of G protein alpha subunit typically coupled to the olfactory receptors (Gα(olf)) failed in different species. Chronic renal disease can represent a cause of olfactory impairment in human. In this context, our present study focused on investigating potential effects of high urea concentration on the olfactory epithelium of vertebrates. Larvae of the teleost fish Danio rerio were exposed to urea in order to assess the effects on the olfactory signal transduction; in particular on both the olfactory receptors and the Gα(olf). The endocytosis of neutral red dye in the olfactory mucosa was detected in control and urea-exposed larvae. The amount of neutral red dye uptake was used as a marker of binding and internalization of the Gα(olf). The neutral red dye endocytosis was not affected by urea exposure, hence suggesting that the presence of the Gα(olf) and their binding to the odorants are not affected by urea treatment, either. The presence and distribution of Gα(olf) were investigated in the olfactory epithelium of control and urea-exposed larvae, using a commercial antibody. The immunoreactivity was increased after urea treatment, suggesting an effect of urea on the expression or degradation of this G protein alpha subunit.

Continuous lifelong neurogenesis is typical of the vertebrate olfactory system. The regenerative ability of olfactory receptor neurons is dependent on the glial cell type specific to the olfactory pathway, designated 'olfactory ensheathing cells'. Several studies to date have focused on mammalian olfactory ensheathing cells, owing to their potential roles in cell-based therapy for spinal cord injury repair. However, limited information is available regarding this glial cell type in non-mammalian vertebrates, particularly anamniotes. In the current immunocytochemical study, we analysed the features of olfactory ensheathing cells in the zebrafish, Danio rerio. Fish provide a good model for studying glial cells associated with the olfactory pathway of non-mammalian vertebrates. In particular, zebrafish has numerous valuable features that enable its use as a prime model organism for genetic, neurobiological and developmental studies, as well as toxicology and genomics research. Paraffin sections from decalcified heads of zebrafish were processed immunocytochemically to detect proteins used in the research on mammalian olfactory ensheathing cells, including glial fibrillary acid protein (GFAP), S100, neural cell adhesion molecule (NCAM), polysialylated NCAM (PSA-NCAM), vimentin (VIM), p75NTR and galactin (Gal)-1. Notably, GFAP, S100, NCAM and Gal-1 were clearly observed, whereas no vimentin staining was detected. Weak immunostaining for PSA-NCAM and p75NTR was evident. Moreover the degree of marker expression was not uniform in various tracts of the zebrafish olfactory pathway. The immunostaining patterns of the zebrafish olfactory system are distinct from those of other fish to some extent, suggesting interspecific differences. We also showed that the olfactory pathway of zebrafish expresses markers of mammalian olfactory ensheathing cells. The olfactory systems of vertebrates have similarities but there are also marked variations between them. The issue of whether regional and interspecific differences in immunostaining patterns of olfactory pathway markers have functional significance requires further investigation.

Acoustic organ of European anchovy from Adriatic Sea was described through histological and immunohistochemical analyses, in order to define a new approach for future studies of quali-quantitative variations in specimens according to sex, age and distribution.