Intracranial graft of bioresorbable polymer scaffolds loaded with human Dental Pulp Stem Cells in stab wound murine injury model.

The prevalence of central nervous system (CNS) dysfunction as a result of disease or trauma remains a clinically unsolved problem which is raising increased awareness in our aging society. Human Dental Pulp Stem Cells (hDPSCs) are excellent candidates to be used in tissue engineering and regenerative therapies of the CNS due to their neural differentiation ability and lack of tumorigenicity. Accordingly, they have been successfully used in animal models of spinal cord injury, stroke and peripheral neuropathies. The ideal therapy in brain injury should combine strategies aiming to protect the damaged lesion and, at the same time, accelerate brain tissue regeneration, thus promoting fast recovery while minimizing side or long-term effects. The use of bioresorbable nanopatterned poly(lactide-co-ɛ-caprolactone) (PLCL) polymeric scaffolds as hDPCSs carriers can represent an advantage for tissue regeneration. In this chapter, we describe the surgical procedures to implant functionalized bioresorbable scaffolds loaded with hDPSCs to improve the brain lesion microenvironment in an intracranial stab wound injury model severing the rostral migratory stream (RMS) that connects the brain subventricular zone (SVZ) and the olfactory bulb in nude mice. Additionally, we also describe the technical steps after animal sacrifice for histological tissue observation and characterization.

Testicular Germ Cell Tumours and Proprotein Convertases.

Testicular Germ Cell Tumours (TGCT) are widely considered a "curable cancer" due to their exceptionally high survival rate, even if it is reduced by many years after the diagnosis due to metastases and relapses. The most common therapeutic approach to TGCTs has not changed in the last 50 years despite its multiple long-term side effects, and because it is the most common malignancy in young Caucasian men, much research is needed to better the quality of life of the many survivors. Proprotein Convertases (PC) are nine serine proteases responsible for the maturation of inactive proproteins with many diverse functions. Alterations in their expression have been associated with various diseases, including cancer and inflammation. Many of their substrates are adhesion molecules, metalloproteases and proinflammatory molecules, all of which are involved in tumour development. Inhibition of certain convertases has also been shown to slow tumour formation, demonstrating their involvement in this process. Considering the very established link between PCs and inflammation-related malignancies and the recent studies carried out into the immune microenvironment of TGCTs, the study of the involvement of PCs in testicular cancer may open up avenues for being both a biomarker for diagnosis and a therapeutic target.

Zona Pellucida sperm-binding protein 3 receptor distribution during Gopc-/- globozoospermic spermatogenesis.

Globozoospermia is a type of teratozoospermia characterized by round morphology of the sperm head. Gopc-/- infertile globozoospermic murine model has failures during spermiogenesis, such as the incorrect biogenesis of the acrosome, disorganized acroplaxome and manchette, round nuclei and spiral flagella. In this study, Western blot, RT-PCR, immunohistochemistry and immunogold were done for the localization of the acrosome protein Zona Pellucida sperm-binding protein 3 receptor (ZP3R), also called sp56, in wild type and Gopc-/- mice testis. The ZP3R protein was located in the acrosome and pseudo-acrosome vesicles of wild type and Gopc-/- mice, respectively. Also, it is distributed through the cytoplasm of the haploid spermatids only. The incorrect spermiogenesis of Gopc-/- mice causes a deregulation in the expression of ZP3R in the globozoospermic spermatids. Our results suggest that although the lack of GOPC causes a failure during the transport of the pre-acrosomal vesicles, the acrosome protein ZP3R is localized in the acrosome and is distributed through the cytoplasm only during spermiogenesis. Furthermore, the failure in spermiogenesis does not impair the synthesis of ZP3R and its localization in the pre-acrosomal vesicles.

Identification of sugar moieties in chief cells of the rat fundic gastric glands.

Many studies have been conducted to determine the composition of the glycoconjugates of the mucus-secreting cells of the fundic glands of the stomach. However, the chief cells of these glands have been largely ignored because they secrete mainly zymogens with a lower glycosylation. The aim of this work was to analyze the glycoconjugates of the gastric chief cells by a battery of 17 different lectins, recognizing Fucose, N-acetylgalactosamine, Galactose, N-acetylneuraminic acid, N-acetylglucosamine and Mannose containing oligosaccharides. Histochemical techniques were performed with several lectins and also combined with two pre-treatments; ß-elimination, which removes O-linked oligosaccharides, and incubation with Peptide-N-Gycosidase F, which removes N-linked oligosaccharides. In addition, acid hydrolysis was performed before WGA histochemistry, and incubation with glucose oxidase before Con A labeling. Many lectins did not stain the chief cells. In addition, the presence of O-glycans in the apical cell membrane was demonstrated with the lectins AAL, HPA, MPA/MPL, PNA, RCA-I, and WGA. Some of these O-glycans were resistant to short-term ß-elimination pre-treatments. Mannose-binding lectins stained the basal cytoplasm of the chief cells. The level of glycosylation of the chief cells was lower than that of the mucous cells. The presence of O-glycans in the apical cell membrane is consistent with the presence of mucins such as MUC1 in the apical membrane of chief cells. Moreover, Mannose-binding lectins revealed N-glycosylation in the basal cytoplasm. The knowledge of gastric chief cell glycoconjugates is relevant because of their potential involvement not only in in physiological but also in pathological processes, such as cancer.

Different Glycoconjugate Content in Mucus Secreting Cells of the Rat Fundic Gastric Glands.

The fundic glands of the stomach contain two types of mucous cells: surface mucous cells (SMCs) located at the surface of the stomach and the pits, and mucous neck cells (MNCs) situated in the neck of the glands. They produce mucins, highly glycosylated proteins. Very little is known about the glycan composition of these mucins and of gastric secretion in general. We used several lectins combined with deglycosylation pretreatments to analyze the glycan composition of SMCs and MNCs. The results showed the presence of terminal sialic acid and subterminal Gal and GalNAc, which is consistent with previous knowledge about glycosylation in mucins. Our results also support previous reports that showed a different expression of mucins in the SMCs, depending on their superficial or deep location in the pit. Some lectins labeled only the perinuclear region of the SMCs, but not the apical region, where the secretory granules are stored. This suggests that the lectins are labeling sugar residues that are accessible to lectins during the first steps of glycan synthesis, which occurs in the endoplasmic reticulum and Golgi apparatus. Our results indicate that SMCs and MNCs produce a mucus secretion with a different glycoconjugate composition. The secretion is more varied in SMCs. As our results coincide with what we know about glycosylation of mucins, we can conclude that most of the glycans detected belong to mucins, and the differences in glycosylation observed in each cell type may be due, mainly, to the different secreted mucins. Anat Rec, 301:2128-2144, 2018. © 2018 Wiley Periodicals, Inc.

Transdifferentiation of mucous neck cells into chief cells in fundic gastric glands shown by GNA lectin histochemistry.

The epithelium of the gastric mucosa and its glands in the corpus of rat stomach contains mucous surface cells (MSCs), parietal cells, mucous neck cells (MNCs), zymogenic or chief cells (ZCs), several types of enteroendocrine cells, and intermediate cells with characteristics between MNCs and ZCs also called transitional or prezymogenic cells (pre-ZCs). The aim of our work was to analyze the expression of Mannose (Man) in the rat gastric glands by means of Galanthus nivalis lectin (GNA) histochemistry to identify the differences between MNC, pre-ZCs and ZCs and to establish the relationships between these cells. Most of the cytoplasm of MNCs was negative for GNA histochemistry. Intensity of GNA labeling in the gastric gland showed a graduation from pre-ZCs (weak labeling) to ZCs (moderate labeling). Labeling of ZCs was stronger at the perinuclear and apical cytoplasm. In the last years, strong evidence has been reported supporting that ZCs differentiate from MNCs. Our work also supports the origin of ZCs from MNCs, because the GNA labeling graduation might be due to oligosaccharides which are not expressed in MNCs, start to express in pre-ZCs and are more abundant in ZCs, indicating that differentiation from MNCs to ZCs is a process in which glycans with Man moieties are synthesized.

Characterization by Lectin Histochemistry of Two Subpopulations of Parietal Cells in the Rat Gastric Glands.

Parietal cells undergo a differentiation process while they move from the isthmus toward the pits and the base region of the gastric gland. The aim of this work was to analyze the rat gastric glands by lectin histochemistry to show the glycans expressed by upper (young) and lower (old) parietal cells. We used lectins recognizing the most frequent sugar moieties in mammals. Each lectin was assayed alone and in combination with several deglycosylation pretreatments: (1) ß-elimination, which removes O-linked oligosaccharides; (2) incubation with Peptide-N-glycosidase F, to remove N-linked glycans; (3) acid hydrolysis, which removes terminal sialic acid moieties; (4) methylation-saponification, to remove sulfate groups from sugar residues; and (5) glucose oxidase, a technique carried out with the lectin concanavalin A to convert glucose into gluconic acid. The lectins from Helix pomatia, Dolichos biflorus (DBA), Glycine max (soybean), Maclura pomifera, Arachis hypogaea (peanut), Bandeiraea simplicifolia (lectin I-B4), and Datura stramonium showed a different glycan expression in the parietal cells throughout the gastric gland. This difference supports that parietal cells undergo a maturation/degeneration process while the cells descend along the gland. The role of DBA as a marker of parietal cells previously reported should be taken with caution because these cells showed different reactivity for the lectin, ranging from negative to strong labeling.

N-Glycans in Xenopus laevis testis characterised by lectin histochemistry.

Analysis of glycan chains of glycoconjugates is difficult because of their considerable variety. Despite this, several functional roles for these glycans have been reported. N-Glycans are oligosaccharides linked to asparagine residues of proteins. They are synthesised in the endoplasmic reticulum (ER) in a unique way, and later modified in both the ER and Golgi apparatus, developing different oligosaccharide chains. An essential role for complex N-glycans in mammalian spermatogenesis has been reported. The aim of the present study was to analyse the N-glycans of the Xenopus laevis testis by means of lectin histochemistry. Five lectins were used that specifically recognise mannose-containing and complex glycans, namely Galanthus nivalis agglutinin (GNA) from snowdrops, concanavalin A (Con A) from the Jack bean, Lens culinaris agglutinin (LCA) from lentils and Phaseolus vulgaris erythroagglutinin (PHA-E) and P. vulgaris leukoagglutinin (PHA-L) from the common bean. GNA and Con A labelled the interstitium and most of the germ cell types, whereas LCA and PHA-E showed affinity only for the interstitium. A granular cytoplasmic region was labelled in spermatogonia and spermatocytes by GNA and PHA-L, whereas GNA and LCA labelled a spermatid region that is probably associated with the centriolar basal body of the nascent flagellum. There was no specific labelling in the acrosome. Some unexpected results were found when deglycosylative pretreatments were used: pre-incubation of tissue sections with peptide N glycosidase F, which removes N-linked glycans, reduced or removed labelling with most lectins, as expected. However, after this pretreatment, the intensity of labelling remained or increased for Con A in the follicle (Sertoli) and post-meiotic germ cells. The ß-elimination procedure, which removes O-linked glycans, revealed new labelling patterns with GNA, LCA and PHA-L, suggesting that some N-glycans were masked by O-glycans, and thus they became accessible to these lectins only after removal of the O-linked oligosaccharides. The functional role of the glycan chains identified could be related to the role of N-glycans involved in mammalian spermatogenesis reported previously.

miR-148a plays a pivotal role in the liver by promoting the hepatospecific phenotype and suppressing the invasiveness of transformed cells.

UNLABELLED: MicroRNAs (miRNAs) are evolutionary conserved small RNAs that post-transcriptionally regulate the expression of target genes. To date, the role of miRNAs in liver development is not fully understood. By using an experimental model that allows the induced and controlled differentiation of mouse fetal hepatoblasts (MFHs) into mature hepatocytes, we identified miR-148a as a hepatospecific miRNA highly expressed in adult liver. The main finding of this study revealed that miR-148a was critical for hepatic differentiation through the direct targeting of DNA methyltransferase (DNMT) 1, a major enzyme responsible for epigenetic silencing, thereby allowing the promotion of the "adult liver" phenotype. It was also confirmed that the reduction of DNMT1 by RNA interference significantly promoted the expression of the major hepatic biomarkers. In addition to the essential role of miR-148a in hepatocyte maturation, we identified its beneficial effect through the repression of hepatocellular carcinoma (HCC) cell malignancy. miR-148a expression was frequently down-regulated in biopsies of HCC patients as well as in mouse and human HCC cell lines. Overexpressing miR-148a led to an enhancement of albumin production and a drastic inhibition of the invasive properties of HCC cells, whereas miR-148a silencing had the opposite consequences. Finally, we showed that miR-148a exerted its tumor-suppressive effect by regulating the c-Met oncogene, regardless of the DNMT1 expression level. CONCLUSION: miR-148a is essential for the physiology of the liver because it promotes the hepatospecific phenotype and acts as a tumor suppressor. Most important, this report is the first to demonstrate a functional role for a specific miRNA in liver development through regulation of the DNMT1 enzyme.

Potential applications of miRNAs as diagnostic and prognostic markers in liver cancer.

Primary liver tumors are mainly represented by hepatocellular hepatocarcinoma (HCC), one of the most aggressive and resistant forms of cancer. Numerous studies have reported the key role of microRNAs (miRNAs) in development, cell proliferation, apoptosis, and tumor biology. The alteration of cancer-related miRNA expression can be associated with tumorigenesis. In HCC, deregulated miRNAs frequently act as oncogenes or altered tumor suppressors. Distinct subtypes of hepatic cancer can also be related to an aberrant expression of particular miRNAs, arguing for the significance of using miRNAs as tumor biomarkers in order to refine the HCC grading assessment. In this article, we review the latest reports regarding miRNA profiling and the potential of small RNAs in HCC diagnosis. The relevance of cancer-related miRNA signatures for the prognosis and better understanding of liver cancer outcome is then considered.

Inhibition of natural killer cells protects the liver against acute injury in the absence of glycine N-methyltransferase.

UNLABELLED: Glycine N-methyltransferase (GNMT) catabolizes S-adenosylmethionine (SAMe), the main methyl donor of the body. Patients with cirrhosis show attenuated GNMT expression, which is absent in hepatocellular carcinoma (HCC) samples. GNMT(-/-) mice develop spontaneous steatosis that progresses to steatohepatitis, cirrhosis, and HCC. The liver is highly enriched with innate immune cells and plays a key role in the body's host defense and in the regulation of inflammation. Chronic inflammation is the major hallmark of nonalcoholic steatohepatitis (NASH) progression. The aim of our study was to uncover the molecular mechanisms leading to liver chronic inflammation in the absence of GNMT, focusing on the implication of natural killer (NK) / natural killer T (NKT) cells. We found increased expression of T helper (Th)1- over Th2-related cytokines, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-R2/DR5, and several ligands of NK cells in GNMT(-/-) livers. Interestingly, NK cells from GNMT(-/-) mice were spontaneously activated, expressed more TRAIL, and had strong cytotoxic activity, suggesting their contribution to the proinflammatory environment in the liver. Accordingly, NK cells mediated hypersensitivity to concanavalin A (ConA)-mediated hepatitis in GNMT(-/-) mice. Moreover, GNMT(-/-) mice were hypersensitive to endotoxin-mediated liver injury. NK cell depletion and adoptive transfer of TRAIL(-/-) liver-NK cells protected the liver against lipopolysaccharide (LPS) liver damage. CONCLUSION: Our data allow us to conclude that TRAIL-producing NK cells actively contribute to promote a proinflammatory environment at early stages of fatty liver disease, suggesting that this cell compartment may contribute to the progression of NASH.

SAMe and HuR in liver physiology: usefulness of stem cells in hepatic differentiation research.

S-Adenosylmethionine, abbreviated as SAM, SAMe or AdoMet, is the principal methyl group donor in the mammalian cell and the first step metabolite of the methionine cycle, being synthesized by MAT (methionine adenosyltransferase) from methionine and ATP. About 60 years after its identification, SAMe is admitted as a key hepatic regulator whose level needs to be maintained within a specific range in order to avoid liver damage. Recently, in vitro and in vivo studies have demonstrated the regulatory role of SAMe in HGF (hepatocyte growth factor)-mediated hepatocyte proliferation through a mechanism that implicates the activation of the non-canonical LKB1/AMPK/eNOS cascade and HuR function. Regarding hepatic differentiation, cellular SAMe content varies depending on the status of the cell, being lower in immature than in adult hepatocytes. This finding suggests a SAMe regulatory effect also in this cellular process, which very recently was reported and related to HuR activity. Although in the last years this and other discoveries contributed to throw light into the tangle of regulatory mechanisms that govern this complex process, an overall understanding is still a challenge. For this purpose, the in vitro hepatic differentiation culture systems by using stem cells or fetal hepatoblasts are considered as valuable tools which, in combination with the methods used in current days to elucidate cell signaling pathways, surely will help to clear up this question.

Potentiation of proliferation of some but not all human colon carcinoma cell lines by immobilized hepatic asialoglycoprotein receptor 1.

Twenty-three human colorectal carcinoma cell lines were examined for the binding of recombinant hepatic asialoglycoprotein receptor 1 (ASGR1), which is known to be exclusively expressed on hepatic parenchymal cells. The effects of the binding were assessed by adhesion to and proliferation on immobilized recombinant ASGR1. Recombinant ASGR1 bound strongly to six cell lines and moderately to 15 cell lines out of 23 lines tested, as shown by flow cytometric analysis. The first six cell lines (group A) also exhibited strong adherence to immobilized ASGR1, whereas 11 of the 15 cell lines of the second group (group B) showed significant adhesion with smaller enhancement by ASGR1 than the cell lines in group A. With a representative cell line (DLD-1 cells categorized in group B), a significant portion of the adhesion was inhibited by preincubation of ASGR1 with asialofetuin, a competitive inhibitor of the carbohydrate recognition by ASGR1. The growth rates of 13 cell lines (two of group A and 11 of group B) were significantly accelerated when they were cultured on immobilized recombinant ASGR1. The results indicate that ASGR is a potential organ-specific microenvironmental factor for colorectal carcinoma growth and metastasis formation in livers.

The expression of a novel cxcr4 gene in Xenopus embryo.

The aim of the present work was to identify a homologue of zebrafish cxcr4b in Xenopus, which could be involved in primordial germ cell (PGC) guidance migration. Following a BLAST analysis, the clone gi 27519681, homologous to the zebrafish gene z-cxcr4b, was identified, inserted into pCMV-SPORT6 plasmid and cloned in Escherichia coli. Embryonic expression of x-cxcr4b was analyzed by RT-PCR. X-cxcr4b was weakly expressed maternally but sharply increased after the mid-blastula transition (MBT), declining significantly at stage 45 when PGCs migration is complete. In contrast, RT-PCR of isolated presumptive PGCs showed strong maternal expression at stage 8, which decreased by stage 10 post-MBT and was not detected at stage 14. Whole mount in situ hybridization of x-cxcr4b mRNA showed that this gene is expressed in neural and haematopoietic tissues, and should be linked to important processes during embryonic development of these organs. Although weak staining could be seen in some samples within the anterior endoderm, expression of x-cxcr4b was never coincident with that of Xpat mRNA, which labels PGCs restricted to the posterior endoderm. Therefore, maternal x-cxcr4b is specifically downregulated within PGCs at pre-migratory stages while it is expressed in other tissues.

Impaired liver regeneration in mice lacking glycine N-methyltransferase.

UNLABELLED: Hepatic S-adenosylmethionine (SAMe) is maintained constant by the action of methionine adenosyltransferase I/III (MATI/III), which converts methionine into SAMe and glycine N-methyltransferase (GNMT), which eliminates excess SAMe to avoid aberrant methylation reactions. During liver regeneration after partial hepatectomy (PH) MATI/III activity is inhibited leading to a decrease in SAMe. This injury-related reduction in SAMe promotes hepatocyte proliferation because SAMe inhibits hepatocyte DNA synthesis. In MATI/III-deficient mice, hepatic SAMe is reduced, resulting in uncontrolled hepatocyte growth and impaired liver regeneration. These observations suggest that a reduction in SAMe is crucial for successful liver regeneration. In support of this hypothesis we report that liver regeneration is impaired in GNMT knockout (GNMT-KO) mice. Liver SAMe is 50-fold higher in GNMT-KO mice than in control animals and is maintained constant following PH. Mortality after PH was higher in GNMT-KO mice than in control animals. In GNMT-KO mice, nuclear factor kappaB (NFkappaB), signal transducer and activator of transcription-3 (STAT3), inducible nitric oxide synthase (iNOS), cyclin D1, cyclin A, and poly (ADP-ribose) polymerase were activated at baseline. PH in GNMT-KO mice was followed by the inactivation of STAT3 phosphorylation and iNOS expression. NFkappaB, cyclin D1 and cyclin A were not further activated after PH. The LKB1/AMP-activated protein kinase/endothelial nitric oxide synthase cascade was inhibited, and cytoplasmic HuR translocation was blocked despite preserved induction of DNA synthesis in GNMT-KO after PH. Furthermore, a previously unexpected relationship between AMPK phosphorylation and NFkappaB activation was uncovered. CONCLUSION: These results indicate that multiple signaling pathways are impaired during the liver regenerative response in GNMT-KO mice, suggesting that GNMT plays a critical role during liver regeneration, promoting hepatocyte viability and normal proliferation.

Evidence for LKB1/AMP-activated protein kinase/ endothelial nitric oxide synthase cascade regulated by hepatocyte growth factor, S-adenosylmethionine, and nitric oxide in hepatocyte proliferation.

UNLABELLED: S-adenosylmethionine (SAMe) is involved in numerous complex hepatic processes such as hepatocyte proliferation, death, inflammatory responses, and antioxidant defense. One of the most relevant actions of SAMe is the inhibition of hepatocyte proliferation during liver regeneration. In hepatocytes, SAMe regulates the levels of cytoplasmic HuR, an RNA-binding protein that increases the half-life of target messenger RNAs such as cyclin D1 and A2 via inhibition of hepatocyte growth factor (HGF)-mediated adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. Because AMPK is activated by the tumor suppressor kinase LKB1, and AMPK activates endothelial nitric oxide (NO) synthase (eNOS), and NO synthesis is of great importance for hepatocyte proliferation, we hypothesized that in hepatocytes HGF may induce the phosphorylation of LKB1, AMPK, and eNOS through a process regulated by SAMe, and that this cascade might be crucial for hepatocyte growth. We demonstrate that the proliferative response of hepatocytes involves eNOS phosphorylation via HGF-mediated LKB1 and AMPK phosphorylation, and that this process is regulated by SAMe and NO. We also show that knockdown of LKB1, AMPK, or eNOS with specific interference RNA (iRNA) inhibits HGF-mediated hepatocyte proliferation. Finally, we found that the LKB1/AMPK/eNOS cascade is activated during liver regeneration after partial hepatectomy and that this process is impaired in mice treated with SAMe before hepatectomy, in knockout mice deficient in hepatic SAMe, and in eNOS knockout mice. CONCLUSION: We have identified an LKB1/AMPK/eNOS cascade regulated by HGF, SAMe, and NO that functions as a critical determinant of hepatocyte proliferation during liver regeneration after partial hepatectomy.

S-adenosylmethionine and proliferation: new pathways, new targets.

SAMe (S-adenosylmethionine) is the main methyl donor group in the cell. MAT (methionine adenosyltransferase) is the unique enzyme responsible for the synthesis of SAMe from methionine and ATP, and SAMe is the common point between the three principal metabolic pathways: polyamines, transmethylation and transsulfuration that converge into the methionine cycle. SAMe is now also considered a key regulator of metabolism, proliferation, differentiation, apoptosis and cell death. Recent results show a new signalling pathway implicated in the proliferation of the hepatocyte, where AMPK (AMP-activated protein kinase) and HuR, modulated by SAMe, take place in HGF (hepatocyte growth factor)-mediated cell growth. Abnormalities in methionine metabolism occur in several animal models of alcoholic liver injury, and it is also altered in patients with liver disease. Both high and low levels of SAMe predispose to liver injury. In this regard, knockout mouse models have been developed for the enzymes responsible for SAMe synthesis and catabolism, MAT1A and GNMT (glycine N-methyltransferase) respectively. These knockout mice develop steatosis and HCC (hepatocellular carcinoma), and both models closely replicate the pathologies of human disease, which makes them extremely useful to elucidate the mechanism underlying liver disease. These new findings open a wide range of possibilities to discover novel targets for clinical applications.

Histochemical demonstration of two subtypes of O-linked oligosaccharides in the rat gastric glands.

The gastric glands synthesize glycoproteins whose oligosaccharides are linked to the peptide core mainly by the O-glycosidic bond, specifically removed by beta-elimination procedure. Our aim was to research the possibility of the existence of two subtypes of O-linked oligosaccharides with a different behavior to the removal procedure. The lectins from peanut (PNA) and Maackia amurensis (MAA-I) were histochemically used as markers of the O-linked oligosaccharides. Sections were also pretreated with beta-elimination and/or peptide N-Glycosidase F (PNGase-F) for the specific removal of O- and N-linked oligosaccharides, respectively. The lectin GNA, which mainly labels to N-linked oligosaccharides, was used to test the correct working of PNGase-F. To test the possibility that the beta-elimination treatment could remove the terminal sialic acid residues, the lectin LFA was used. The surface epithelium was negative to PNA, while it became strongly positive when beta-elimination was performed for 1 day. This staining was resistant to PNGase-F, suggesting that PNA was labeling to O-linked oligosaccharides. However, after beta-elimination for 5 days this staining is not observed. A similar pattern appeared with MAA-I. We propose the existence of two subtypes of O-linked oligosaccharides: labile and resistant. The labile O-linked oligosaccharides are removed with beta-elimination for 1 day, unmasking the PNA-positive oligosaccharides. These oligosaccharides are resistant O-linked oligosaccharides because staining is abolished with longer treatment of beta-elimination. The results with MAA-I also support this suggestion. In summary, the labile O-linked oligosaccharides are removed with short treatment, while the resistant O-linked oligosaccharides need a stronger procedure (for 5 days).