Pharmacological activation of SIRT6 triggers lethal autophagy in human cancer cells

Sirtuin 6 (SIRT6) is a member of the NAD+-dependent class III deacetylase sirtuin family, which plays a key role in cancer by controlling transcription, genome stability, telomere integrity, DNA repair, and autophagy. Here we analyzed the molecular and biological effects of UBCS039, the first synthetic SIRT6 activator. Our data demonstrated that UBCS039 induced a time-dependent activation of autophagy in several human tumor cell lines, as evaluated by increased content of the lipidated form of LC3B by western blot and of autophagosomal puncta by microscopy analysis of GFP-LC3. UBCS039-mediated activation of autophagy was strictly dependent on SIRT6 deacetylating activity since the catalytic mutant H133Y failed to activate autophagy. At the molecular level, SIRT6-mediated autophagy was triggered by an increase of ROS levels, which, in turn, resulted in the activation of the AMPK-ULK1-mTOR signaling pathway. Interestingly, antioxidants were able to completely counteract UBCS039-induced autophagy, suggesting that ROS burst had a key role in upstream events leading to autophagy commitment. Finally, sustained activation of SIRT6 resulted in autophagy-related cell death, a process that was markedly attenuated using either a pan caspases inhibitor (zVAD-fmk) or an autophagy inhibitor (CQ). Overall, our results identified UBCS039 as an efficient SIRT6 activator, thereby providing a proof of principle that modulation of the enzyme can influence therapeutic strategy by enhancing autophagy-dependent cell death

Trifunctionalized Naphthalene Diimides and Dimeric Analogues as G-Quadruplex-Targeting Anticancer Agents Selected by Affinity Chromatography

A focused library of newly designed monomeric and dimeric naphthalene diimides (NDIs) was analyzed in its ability to recognize specific G-quadruplex (G4) structures discriminating duplex DNA. The best G4 ligands—according to an affinity chromatography-based screening method named G4-CPG—were tested on human cancer and healthy cells, inducing DNA damage at telomeres, and in parallel, showing selective antiproliferative activity on HeLa cancer cells with IC50 values in the low nanomolar range. CD and fluorescence spectroscopy studies allowed detailed investigation of the interaction in solution with different G4 and duplex DNA models of the most promising NDI of the series, as determined by combining the biophysical and biological assays’ data

Glycohistochemical study of the toadfish <i>Halobatrachus didactylus</i> (Scheider, 1801) stomach

Toadfish Halobatrachus didactylus gastric mucosa was studied using conventional and lectin histochemistry. Conventional histochemistry revealed neutral glycoconjugates predominating over acidic ones in the apical zone of both surface epithelial cells and pit cells. The neck cells contained a few neutral glycoconjugates, whereas gastric glands were negative to PAS and AB staining. Lectin histochemistry showed different oligosaccharide expression along the columnar cells. The sub-nuclear cytoplasm was stained with RCA120, SBA, HPA, GSA I-B4, GSA II, UEA I, LTA, Con A, KOH-sialidase-WGA. The Golgi zone reacted with RCA120, DBA, SBA, HPA, KOH-sialidase-WGA, GSA I-B4, GSA II, UEA I, LTA, MAL II, SNA, and showed an increase in DBA staining after KOH-sialidase treatment. The granules of the apical zone stained with PNA, UEA I, LTA and showed increased PNA reactivity after KOH-sialidase treatment. The luminal cell coat reacted with PNA, HPA, Con A, KOH-sialidase-WGA, UEA I, LTA, MAL II, SNA and KOH-sialidase-PNA. Pit cells showed a minor expression of lectin-binding sites with respect to columnar cells. Neck cells linked UEA I and LTA and gastric glands reacted with PNA, DBA, SBA, HPA, Con A, GSA I-B4, KOH-sialidase-WGA and KOH-sialidase-DBA. The results suggest that the stomach of the toadfish H. didactylus is characterised by a species-specific glycoconjugate pattern

The epidemiology of mumps in Italy

In Italy, although vaccination has been recommended for a number of years, vaccination coverage for mumps is still sub-optimal. The objective of the present study was to evaluate the seroprevalence of mumps antibodies in the Italian population, stratified by age, gender and geographical area. The proportion of individuals positive for mumps antibodies remained stable in the age classes 0-11 months and 1 year (25.4% and 30.8%, respectively) and showed a continuous increase after the second year of life. The percentage of susceptible individuals was higher than 20% in persons 2-14 years of age and exceeded 10% in persons 15-39 years of age. No statistically significant differences were observed by gender or geographical area. Comparison between these results and the data obtained from a 1996 survey showed a statistically significant increase in seroprevalence in the age class 2-4 years. No changes were observed in the other age-groups. The results of this study confirm that the efforts made in recent years to improve vaccination coverage within the second year of life should be strengthened. \ua9 2008 Elsevier Ltd. All rights reserved

Glycoconjugates in the Senegalese sole testis

20 p., tablas, figuras y bibliografíaThe localization and characterization of oligosaccharide sequences in the testis of Senegalese sole Solea senegalensis was investigated using 12 lectins in combination with KOH saponification and sialidase digestion (K-s). The interstitial compartment contained all the sugar residues investigated, those bearing oligosaccharides terminating with sialic acid (Neu5Ac)alpha2,3Galbeta1,4GlcNAc, Neu5AcGalNAcalpha1,3(LFucbeta1,2)Galbeta1,3/4GlcNAcbeta1 and GalNAcalpha1,3(LFuc1,2) Galbeta1,3/4GlcNAcbeta1 being more abundant in the medullar region than in the cortex. The melano-macrophage centres found in the interstitial compartment displayed glycans terminating with Galbeta1,3GalNAc. The basal lamina separating the germinal and interstitial compartments exhibited glycans with terminal/internal mannose, internal betaGlcNAc, and terminal Neu5Acalpha2,6Gal/GalNAc, and Neu5AcGalbeta1,3GalNAc, Galbeta1,3GalNAc (PNA), Galbeta1,4GlcNAc, GalNAc, alphaGal, and alphaL-Fuc. In the germinal compartment, the Sertoli cells expressed only glycans terminating with Neu5Acalpha2,3Galbeta1,4GlcNAc in the apical and supra-nuclear lateral surface of the spermatonial cysts located in the distal part of the seminiferous lobules. Primary spermatocytes exhibited oligosaccharides terminating with Galbeta1,3GalNAc and alphaGalNAc in the cytoplasm and nucleus, respectively. The spermatids contained highly mannosylated glycans terminating with GalNac, alphaGal, and alphaL-Fuc. The head of spermatozoa expressed a more complex glycosylation pattern characterized by the additional presence of oligosaccharides terminating with Neu5Acalpha2,3Galbeta1,4GlcNAc, Neu5AcGalbeta1,3GalNAc, Neu5AcGalNAcalpha1,3(LFuca1,2)Galbeta1,3/4GlcNAcbeta1, GalNAcalpha1,3(LFucalpha1,2 Galbeta1,3/4GlcNAcbeta1. The comparison with previous lectin histochemical studies carried out in other fish species reveals a specific glycosylation pattern of Senegalese sole testicular structures and spermatozoa head.This research was funded by the Spanish Ministry of Education and Science-MEC-(Project AGL2006-13777

Expresión glucohistoquímica en el estómago del pez sapo, Halobatrachus didactylus (Schneider,1801)

11 pages, 6 figures, 2 tables.[EN] Toadfish Halobatrachus didactylus gastric mucosa was studied using conventional and lectin histochemistry. Conventional histochemistry revealed neutral glycoconjugates predominating over acidic ones in the apical zone of both surface epithelial cells and pit cells. The neck cells contained a few neutral glycoconjugates, whereas gastric glands were negative to PAS and AB staining. Lectin histochemistry showed different oligosaccharide expression along the columnar cells. The sub-nuclear cytoplasm was stained with RCA120, SBA, HPA, GSA I-B4, GSA II, UEA I, LTA, Con A, KOH-sialidase-WGA. The Golgi zone reacted with RCA120, DBA, SBA, HPA, KOH-sialidase-WGA, GSA I-B4, GSA II, UEA I, LTA, MAL II, SNA, and showed an increase in DBA staining after KOH-sialidase treatment. The granules of the apical zone stained with PNA, UEA I, LTA and showed increased PNA reactivity after KOH-sialidase treatment. The luminal cell coat reacted with PNA, HPA, Con A, KOH-sialidase-WGA, UEA I, LTA, MAL II, SNA and KOH-sialidase-PNA. Pit cells showed a minor expression of lectin-binding sites with respect to columnar cells. Neck cells linked UEA I and LTA and gastric glands reacted with PNA, DBA, SBA, HPA, Con A, GSA I-B4, KOH-sialidase-WGA and KOH-sialidase-DBA. The results suggest that the stomach of the toadfish H. didactylus is characterised by a species-specific glycoconjugate pattern.[ES] Se ha estudiado la expresión de residuos glucídicos de glucoconjugados en la mucosa gástrica del pez sapo, Halobatrachus didactylus usando histoquímica convencional y de lectinas. La histoquímica clásica reveló la presencia de glicoconjugados neutros predominando sobre los ácidos en la zona apical de la superficie de las células epiteliales y de la criptas gástricas. Las células del cuello contienen algunas glicoproteínas neutras, mientras las glándulas gástricas han sido negativas al PAS y al Azul Alcián (AB). La histoquímica de lectinas mostró diferente expresión de oligosacáridos a lo largo de las células columnares. El citoplasma sub-nuclear reaccionó con las lectinas RCA, SBA, HPA, GSA, I-B4, GSA II, UEA I, LTA, Con A, KOH-sialidasa-WGA. La expresión de RCA120, DBA, SBA, HPA, KOH-sialidasa-WGA, GSA I-B4, GSA II, UEA I, LTA, MAL II y SNA se localizó en la zona del Golgi, donde se observó un incremento de la reactividad de la lectina DBA después del tratamiento con KOH. Los gránulos de la zona apical reaccionaron con PNA, UEA I, LTA, mostrando un incremento de la reactividad hacia la lectina PNA después del tratamiento con KOH. El borde de las células del lumen reaccionó con PNA, HPA, ConA, KOH-sialidasa-WGA, UEA I, LTA, MAL II, SNA, KOH-sialidasa-PNA. Los sitios de unión de la expresión de las lectinas-glucoconjugados fue menor en las células del cuello de las criptas gastricas que en las células columnares epiteliales. Las células del cuello reaccionaron con la UEA I y LTA, y las glándulas gástricas con PNA, DBA, SBA, HPA, Con A, GSA I-B4, KOH-sialidasa-WGA y KOH-sialidasa-DBA. Los resultados sugieren que el estómago del pez sapo, Halobatrachus didactylus, se caracteriza por un patrón especie-específico de expresión de glicoconjugados.Peer reviewe

Morphometric and ultrastructural features of the mare oviduct epithelium during oestrus

Morphometric, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) investigations have displayed regional differences in the mare oviductal epithelium. The entire mucosa of the oviduct was lined with a pseudostratified epithelium, which consisted of two distinct cell types, ciliated and non-ciliated. Ciliated cells were predominant in the three different segments of the oviduct and their percentage increased from fimbriae to ampulla and significantly decreased in the isthmus. SEM revealed in the infundibulum finger-like mucosal folds, some of them interconnected, in the ampulla numerous and elaborated branched folds of the mucosa, whereas the isthmus displayed a narrow lumen, short and non-branched mucosal folds. In the ampulla and isthmus the majority of non-ciliated cells showed apical blebs provided or not of short microvilli. TEM displayed different ultrastructural features of ciliated and non-ciliated cells along the oviduct. Isthmus ciliated cells presented a more electron-dense cytoplasm than in infundibulum and ampulla cells and its cilia were enclosed in an amorphous matrix. The non-ciliated cells of infundibulum did not contain secretory granules but some apical endocytic vesicles and microvilli coated by a well developed glycocalyx. Non-ciliated cells of ampulla and isthmus contained secretory granules. Apical protrusions of ampulla displayed two types of secretory granules as well as occasional electron-lucent vesicles. Isthmus non-ciliated cells showed either electron-lucent or electron-dense cytoplasm and not all contained apical protrusions. The electron-dense non-ciliated cells displayed microvilli coated with a well developed glycocalyx. Three types of granules were observed in the isthmus non-ciliated cells. The regional differences observed along the epithelium lining the mare oviduct suggest that the epithelium of the each segment is involved in the production of a distinctive microenvironment with a unique biochemical milieu related to its functional role

Lectin-binding pattern of the senegalese sole Solea senegalensis oogenesis

The glycoconjugate pattern of developing ovarian follicles in wild and cultured Senegalese sole Solea senegalensis was investigated by means of lectin histochemistry. Ovaries from cultured fish contained oocytes up to the late vitellogenic stage, whereas they reached the hydration stage in wild specimens. The follicular cells bound MAL II, SBA, HPA, DBA, Con A, KOH-sialidase (K-s)-WGA, GSA I-B4 in the late vitellogenic stage, and in wild fish also SNA and K-s-PNA, whereas in the hydration stage SBA, HPA, DBA, and GSA I-B4 only. The zona radiata reacted with SBA, HPA, DBA, Con A, and GSA I B4 in the late vitellogenic stage and in cultured fish also with UEA I, whereas in the hydration stage it stained with SBA only. The cortical alveoli bound SBA, HPA, RCA120 during the late vitellogenic stage, also SNA, PNA, K-s-PNA, GSA I-B4 in cultured fish, DBA, and K-s-WGA in wild ones which stained with SBA, HPA, and GSA I-B4 in the hydrated stage. The yolk reacted with Con A in the late vitellogenic oocytes, and also with MAL II, SNA, K-s-PNA, SBA, HPA, K-s-WGA, GSA I-B4, UEA I in the hydrated ones. From perinucleolus to late vitellogenic stages, the oocyte nucleoplasm bound Con A, GSA I-B4, GSA II, UEA I, and in wild fish also MAL II, SNA, LTA but only GSA I-B4 reactivity in the early maturation stage. These findings demonstrate that the glycan pattern of fish ovarian follicles changes during the maturative stages and that it is affected by culture-rearing conditions.Peer reviewe

Differential expression of glycans in the urothelial layers of horse urinary bladder

Background: Urothelium is a multilayer epithelium covering the inner surface of the urinary bladder that acts as a blood-urine barrier and is involved in maintaining the wellbeing of the whole organism. Glycans serve in the maturation and differentiation of cells and thus play a key role in the morphology and function of the multilayered epithelium. The aim of the present study was to examine the glycoprotein pattern of the horse urinary bladder urothelium by lectin histochemistry.Methods: The study involved urinary bladders from four horse stallions. Tissue sections were stained with a panel of eleven lectins, in combination with saponification and sialidase digestion (Ks).Results: Basal cells displayed high-mannose N-glycans (Con A), alpha 2,6-linked sialic acid (SNA), and O-linked sialoglycans with sialic acids linked to Gal beta l,3GalNAc (T antigen) (KsPNA) and terminal N-acet-ylgalactosamine (Tn antigen) (KsSBA). The young intermediate cells expressed terminal N-acet-ylglucosamine (GlcNAc) (GSA II), galactose (GSA I-B4), T-and Tn antigens (PNA, SBA). The mature intermediate cells showed additional high-mannose N-glycans, O-linked sialoglycans (sialyl-T antigen, sialyl-Tn antigen), alpha 2,6-and alpha 2,3-linked sialic acid (MAL II), alpha 1,2-linked fucose (UEA I), and GlcNAc (KsWGA). The latter residue marked the boundary with the overlying surface layer. Few Con A positive intermediate cells were seen to cross the entire urothelium thickness. The surface cells showed additional glycans such as T antigen and sialic acids linked to GalNAc binding DBA (KsDBA). Few surface cells contained alpha 1,3-linked fucose (LTA), whereas some other cells displayed intraluminal secretion of mucin-type glycans terminating with GalNAc alpha 1,3(LFuc alpha 1,2)Gal beta 1,3/4GlcNAc beta 1 (DBA). The luminal surface expressed the most complex glycan pattern in the urothelium because only alpha 1,3-linked fucose lacked among the demonstrated glycans. Conclusions: This study showed that the glycan pattern becomes more complex from the basal to surface layer of the urothelium and that surface cells could modify the composition of urine via the secretion of glycoproteins.(c) 2022 Elsevier GmbH. All rights reserved

ULTRASTRUCTURAL CHARACTERISTICS OF OVINE BONE MARROW-DERIVED MESENCHYMAL STROMAL CELLS CULTURED WITH A SILICON STABILIZED TRICALCIUM PHOSPHATE BIOCERAMIC

Bioceramics are being used in experimental bone engineering application in association with bone marrow derived mesenchymal stem cells (BM-MSCs) as a new therapeutic tool, but their effects on the ultrastructure of BM-MSCs are yet unknown. In this study we report the morphological features of ovine (o)BM-MSCs cultured with Skelite, a resorbable bioceramic based on silicon stabilized tricalcium phosphate (SiTCP), able to promote the repair of induced bone defect in sheep model. oBM-MSCs were isolated from the iliac crest, cultured until they reached near-confluence and incubated with SiTCP. After 48 hours the monolayers were highly damaged and only few cells adhered to the plastic. Thus, SiTCP was removed, and after washing the cells were cultured until they became confluent. Then, they were trypsinizated and processed for transmission electron microscopy (TEM) and RT-PCR analysis. RT-PCR displayed that oBM-MSCs express typical surface marker for mesenchymal stem cells. TEM revealed the presence of electron-lucent cells and electron-dense cells, both expressing the CD90 surface antigen. The prominent feature of electron-lucent cells was the concentration of cytoplasmic organelles around the nucleus as well as large surface blebs containing glycogen or profiles of endoplasmic reticulum. The dark cells had a multilocular appearance by the presence of peripheral vacuoles. Some dark cells contained endocytic vesicles, lysosomes, and glycogen aggregates. oBM-MSCs showed different types of specialized interconnections. The comparison with ultrastructural features of untreated oBM-MSCs suggests the light and dark cells are two distinct cell types which were differently affected by SiTCP bioceramic