Lycopersicon Esculentum Lectin Research Articles

Labeling and sorting of avian primordial germ cells utilizing Lycopersicon Esculentum lectin.

Avian species are essential resources for human society, with their preservation and utilization heavily dependent on primordial germ cells (PGCs). However, efficient methods for isolating live PGCs from embryos remain elusive in avian species beyond chickens, and even in chickens, existing techniques have shown limited efficiency. In this study, we present a rapid, simple, and cost-effective method for labeling and sorting circulating-stage PGCs across various avian species, including Carinatae and Ratitae, using Lycopersicon Esculentum (Tomato) lectin (LEL). Notably, this method demonstrates high sorting efficiency by identifying a wide range of PGC subtypes while preserving the proliferative and migratory potential of chicken PGCs. This approach is anticipated to significantly contribute to the conservation, research, and agricultural industries related to avian species globally.

Proteome Analysis of Serum Purified Using Solanum tuberosum and Lycopersicon esculentum Lectins.

Serum and plasma exhibit a broad dynamic range of protein concentrations, posing challenges for proteome analysis. Various technologies have been developed to reduce this complexity, including high-abundance depletion methods utilizing antibody columns, extracellular vesicle enrichment techniques, and trace protein enrichment using nanobead cocktails. Here, we employed lectins to address this, thereby extending the scope of biomarker discovery in serum or plasma using a novel approach. We enriched serum proteins using 37 different lectins and subjected them to LC-MS/MS analysis with data-independent acquisition. Solanum tuberosum lectin (STL) and Lycopersicon esculentum lectin (LEL) enabled the detection of more serum proteins than the other lectins. STL and LEL bind to N-acetylglucosamine oligomers, emphasizing the significance of capturing these oligomer-binding proteins when analyzing serum trace proteins. Combining STL and LEL proved more effective than using them separately, allowing us to identify over 3000 proteins from serum through single-shot proteome analysis. We applied the STL/LEL trace-protein enrichment method to the sera of systemic lupus erythematosus model mice. This revealed differences in >1300 proteins between the systemic lupus erythematosus model and control mouse sera, underscoring the utility of this method for biomarker discovery.

Low-temperature resin embedding of the whole brain for various precise structures dissection

Resin embedding combined with ultra-thin sectioning has been widely used in microscopic and electron imaging to acquire precise structural information of biological tissues. However, the existing embedding method was detrimental to quenchable fluorescent signals of precise structures and pH-insensitive fluorescent dyes. Here, we developed a low-temperature chemical polymerization method named HM20-T to maintain weak signals of various precise structures and to decrease background fluorescence. The fluorescence preservation ratio of green fluorescent protein (GFP) tagged presynaptic elements and tdTomato labeled axons doubled. The HM20-T method was suitable for a variety of fluorescent dyes, such as DyLight 488 conjugated Lycopersicon esculentum lectin. Moreover, the brains also retained immunoreactivity after embedding. In summary, the HM20-T method was suitable for the characterization of multi-color labeled precise structures, which would contribute to the acquisition of complete morphology of various biological tissues and to the investigation of composition and circuit connection in the whole brain.

MP05-09 IDENTIFICATION OF ABERRANT GLYCOSYLATION OF OSTEOPONTIN ON URINARY STONE FORMATION IN UROLITHIASIS PATIENTS AND RENAL STONE FORMATION RATS

You have accessJournal of UrologyCME1 Apr 2023MP05-09 IDENTIFICATION OF ABERRANT GLYCOSYLATION OF OSTEOPONTIN ON URINARY STONE FORMATION IN UROLITHIASIS PATIENTS AND RENAL STONE FORMATION RATS Go Anan, Tohru Yoneyama, Takuo Hirose, Makoto Sato, Takefumi Mori, and Chikara Ohyama Go AnanGo Anan More articles by this author , Tohru YoneyamaTohru Yoneyama More articles by this author , Takuo HiroseTakuo Hirose More articles by this author , Makoto SatoMakoto Sato More articles by this author , Takefumi MoriTakefumi Mori More articles by this author , and Chikara OhyamaChikara Ohyama More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003216.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Osteopontin (OPN) is one of the most important components in the calcium stone matrix. However, the relationship between glycosylation of OPN and urinary stone formation remains unknown. This study aims to identify the aberrant glycosylation profile of OPN related to urolithiasis in urolithiasis patients and renal calcium oxalate stone formation rats. METHODS: We retrospectively measured urinary glycosylated OPN normalized by urinary full-length-OPN (uFL-OPN) levels in 110 urolithiasis patients and 157 healthy volunteers. Also, we used rats with ethylene glycol-induced renal stones to evaluate the aberrant glycosylation of OPN in renal tissue. The urinary full-length-OPN levels were measured using enzyme-linked immunosorbent assay and glycosylated OPN was measured using a lectin array and lectin blotting. The assays were evaluated using the area under the receiver operating characteristics curve to discriminate stone forming urolithiasis patients. RESULTS: In the retrospective cohort, urinary Gal3C-S lectin reactive- (Gal3C-S-) OPN/uFL-OPN, was significantly higher in the stone forming urolithiasis patients than in the healthy volunteers (p<0.0001), with good discrimination (AUC, 0.953), 90% sensitivity, and 92% specificity. The Lycopersicon esculentum lectin (LEL) analysis of uFL-OPN showed that uFL-OPN in stone forming urolithiasis patients had a polyLacNAc structure that was not observed in healthy volunteers. The rats study showed that the levels of LEL-reactive OPN that had a polyLacNAc structure were increased in the renal cortex, even though total OPN levels did not increase. CONCLUSIONS: The aberrant glycosylation of OPN occurs in urine of urolithiasis patients and in renal cortex of renal stone formation rats. The aberrant glycosylation of OPN might be a marker of accelerated renal stone formation. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e47 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Go Anan More articles by this author Tohru Yoneyama More articles by this author Takuo Hirose More articles by this author Makoto Sato More articles by this author Takefumi Mori More articles by this author Chikara Ohyama More articles by this author Expand All Advertisement PDF downloadLoading ...

Morphometric study of the vestibuloauditory organ of the African clawed frog, Xenopus laevis.

Independent auditory end-organs appear first in amphibians in vertebrate phylogeny. In amphibians, sound detection is carried out by the amphibian papilla, basilar papilla and macula saccule. Amphibians inhabit distinct habitats and exhibit specific behaviours and sound frequency responses, so the amphibian vestibuloauditory system is an excellent model for considering the relationships between behaviour and physiological/anatomical vestibuloauditory properties. The African clawed frog, Xenopus laevis, lives in shallow water throughout its life and is thought to use sound in a higher frequency range compared with terrestrial anurans. In this study, the size of each vestibuloauditory end-organ and the distribution of ganglion cells in the vestibuloauditory ganglion were examined using haematoxylin and eosin staining and lectin histochemistry in Xenopus laevis. This study revealed that the size ratios among end-organs in Xenopus are similar to those in terrestrial anurans. Large and small cells were observed in the ganglion, but their distribution patterns are different from those in general terrestrial anurans. Lycopersicon esculentum lectin stained a large number of ganglion cells. Lectin-stained cells were found throughout the whole ganglion, but were especially abundant in the caudal part. These results suggested a unique distribution pattern of the vestibuloauditory ganglion cells in Xenopus.

Salivary Glycopatterns as Potential Non-Invasive Biomarkers for Diagnosing and Reflecting Severity and Prognosis of Diabetic Nephropathy.

Discriminating between diabetic nephropathy (DN) and non-diabetic renal disease (NDRD) can help provide more specific treatments. However, there are no ideal biomarkers for their differentiation. Thus, the aim of this study was to identify biomarkers for diagnosing and predicting the progression of DN by investigating different salivary glycopatterns. Lectin microarrays were used to screen different glycopatterns in patients with DN or NDRD. The results were validated by lectin blotting. Logistic regression and artificial neural network analyses were used to construct diagnostic models and were validated in in another cohort. Pearson’s correlation analysis, Cox regression, and Kaplan–Meier survival curves were used to analyse the correlation between lectins, and disease severity and progression. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) and bioinformatics analyses were used to identify corresponding glycoproteins and predict their function. Both the logistic regression model and the artificial neural network model achieved high diagnostic accuracy. The levels of Aleuria aurantia lectin (AAL), Lycopersicon esculentum lectin (LEL), Lens culinaris lectin (LCA), Vicia villosa lectin (VVA), and Narcissus pseudonarcissus lectin (NPA) were significantly correlated with the clinical and pathological parameters related to DN severity. A high level of LCA and a low level of LEL were associated with a higher risk of progression to end-stage renal disease. Glycopatterns in the saliva could be a non-invasive tool for distinguishing between DN and NDRD. The AAL, LEL, LCA, VVA, and NPA levels could reflect the severity of DN, and the LEL and LCA levels could indicate the prognosis of DN.

Changes in Gastric Mucosal Glycosylation Before and After Helicobacter pylori Eradication Using Lectin Microarray Analysis.

Glycosylation is a common post-translational modification, and it has been reported that alterations in the glycosylation patterns on cells are related to cell proliferation, differentiation, tissue adhesion, and carcinogenesis. This study aimed to investigate the relationship between Helicobacter pylori infection and gastric mucosal glycosylation using a lectin microarray system. Gastric mucosal samples were obtained from 10 Helicobacter pylori-non-infected patients, 10 H. pylori-infected patients, and 10 after H. pylori-eradicated patients who underwent gastric mucosal biopsy by endoscopy in our institute. The gastric gland cells which were isolated from formalin-fixed, paraffin-embedded gastric mucosal biopsy samples using laser capture microdissection were used for lectin microarray to obtain lectin-glycan interaction values. Comparison of the lectin-glycan interaction values before and after eradication in the same patients showed significant increases for Ricinus communis agglutinin 120, Trichosanthes japonica agglutinin II, Euonymus europaeus lectin, jacalin, Amaranthus caudatus agglutinin, and Maclura pomifera agglutinin and significant decreases for Urtica dioica agglutinin, Lycopersicon esculentum lectin, Ulex europaeus agglutinin, Sambucus nigra agglutinin, Sambucus sieboldiana agglutinin, and Trichosanthes japonica agglutinin I. Furthermore, jacalin and MPA in the gastric antrum were significantly decreased with H. pylori infection compared with the without infection group and improved to the levels seen without infection as a result of eradication. Lycopersicon esculentum lectin, Sambucus nigra agglutinin, Sambucus sieboldiana agglutinin, and Trichosanthes japonica agglutinin I in the gastric body were significantly increased with H. pylori infection and improved to the level seen without infection as a result of eradication. H. pylori infection changes the lectin binding state which is related to various cancers on the gastric mucosal cell. Furthermore, those changes are reversible by H. pylori eradication.

A simple fluorescent strategy for liver capillary labeling with carbon quantum dot-lectin nanoprobe.

Taking the hepatic sinusoid (HS) as the main delivery area of liver nutrients and metabolic waste, recognizing its structure is important for a deep understanding of liver function. In this paper, based on lycopersicon esculentum lectin (LEL), with targeting ability for endothelial cells, and carbon quantum dots (CQDs), with high biosafety, an LEL-coupled CQD immunofluorescence probe (CQD@LEL) that can label microvessels is designed and used for the fluorescence labeling and imaging of HS in liver tissue sections. The CQD size is approximately 2 nm. Blue fluorescence is emitted under excitation; its optimal excitation wavelength is 400 nm while the emission is at about 450 nm. Gel electrophoresis and capillary electrophoresis confirm that glutaraldehyde can couple LEL to CQD, and the obtained CQD@LEL retains the fluorescence property and has good stability. Optimization experiments show that its labeling effect is positively correlated with time and probe concentration for dyeing the blood vessels of mouse liver slices. In order to improve the effect further, a probe concentration of 0.17 mg mL-1 and incubation time of 3 h were chosen to label the liver tissue sections. The results show that the liver microvessels are formed by interstitial structures among the hepatic cords, and the HS presents a granular or patchy appearance. H&E and ultrathin section TEM show that the microvascular wall of the liver is composed of discontinuous endothelial cells, and there are Kupffer cells and other cells in the tubes, proving that our probe can clearly label the structure and morphology of liver microvessels. This work is of great significance for the visualization of HS.

Targeting cell surface glycans with lectin-coated fluorescent nanodiamonds.

Glycosylation is arguably the most important functional post-translational modification in brain cells and abnormal cell surface glycan expression has been associated with neurological diseases and brain cancers. In this study we developed a novel method for uptake of fluorescent nanodiamonds (FND), carbon-based nanoparticles with low toxicity and easily modifiable surfaces, into brain cell subtypes by targeting their glycan receptors with carbohydrate-binding lectins. Lectins facilitated uptake of 120 nm FND with nitrogen-vacancy centers in three types of brain cells – U87-MG astrocytes, PC12 neurons and BV-2 microglia cells. The nanodiamond/lectin complexes used in this study target glycans that have been described to be altered in brain diseases including sialic acid glycans via wheat (Triticum aestivum) germ agglutinin (WGA), high mannose glycans via tomato (Lycopersicon esculentum) lectin (TL) and core fucosylated glycans via Aleuria aurantia lectin (AAL). The lectin conjugated nanodiamonds were taken up differently by the various brain cell types with fucose binding AAL/FNDs taken up preferentially by glioblastoma phenotype astrocyte cells (U87-MG), sialic acid binding WGA/FNDs by neuronal phenotype cells (PC12) and high mannose binding TL/FNDs by microglial cells (BV-2). With increasing recognition of glycans having a role in many diseases, the lectin bioconjugated nanodiamonds developed here are well suited for further investigation into theranostic applications.

Effectiveness of Selenium on Chondrocyte Glycoprotein Glycosylation Which Play Important Roles in the Pathogenesis of an Endemic Osteoarthritis, Kashin-Beck Disease.

In this study, we aimed to explore the effectiveness of selenium on the chondrocyte glycoprotein glycosylation which plays important roles in the pathogenesis of Kashin-Beck disease (KBD). Cartilage samples were collected from KBD patients after total knee replacement surgery. Chondrocytes were cultured with sodium selenium. The group of chondrocytes which were cultured without adding sodium selenium was considered as control group. Lectin microarray was used to screen the differences in lectin levels between KBD and KBD with selenium groups. Stronger signals for Bandeiraea simplicifolia (BS-I), Hippeastrum hybrid lectin (HHL), Pisum sativum agglutinin (PSA), Psophocarpus tetragonolobus lectin I (PTL-I), Psophocarpus tetragonolobus lectin II (PTL-II), Sophora japonica agglutinin (SJA), Lotus tetragonolobus lectin (LTL), and Triticum vulgaris (WGA) were observed in the KBD group. Meanwhile, Aleuria aurantia lectin (AAL), Lens culinaris agglutinin (LCA), Lycopersicon esculentum (tomato) lectin (LEL), Peanut agglutinin (PNA), and Sambucus nigra lectin (SNA) signals were lower in the KBD group. Selenium may have the function of influence the expression levels of carbohydrate chains Galα1,3-Gal, high mannose, and GlcNAc.

Identification of Microglial Cell Molecular Markers During Chick Embryo Brain Development and Human Xenograft Tumor Formation

Glioblastoma (GBM) is the most aggressive and invasive human brain tumor, which despite advancement in surgery and immunology, only 10% of patients survive 18 months after diagnosis. The Galileo lab studies GBM, focusing on the role of the protein L1CAM on increasing the motility, proliferation, and invasiveness of GBM cells using in vitro cell tracking and a novel xenograft chick embryo brain tumor model. The work here focused on microglial cells, which are resident brain immune system cells that are not well understood. Studies in rodents and GBM patients showed that microglia play a critical role in promoting GBM growth via an activation of complex network of cytokines, chemokines and other metabolic factors that occur in the extracellular matrix. In the context of brain tumors, microglia promote neuroinflammation, which induces the BBB breakdown and inhibits antitumor immune responses. The first aim for my project was to find biomarkers that specifically identify microglial cells in normal chick embryo brain during development. The second aim was to use those markers in xenograft tumors in the chick embryo brain to look for any proinflammatory changes in microglial cells. Chick embryo brains were fixed, frozen, and cut into serial cryostat sections or fixed and vibratome sectioned, followed by immunofluorescent staining using antibodies or lectins that specifically identified microglial cells and endothelial cells in other model organisms. The optimal staining protocol required overnight primary antibody/lectin with 0.1% Triton X‐100 detergent and 5% normal goat serum in phosphate buffered saline. The antibodies and lectins investigated included Isolectin B4, Tomato (Lycopersicon esculentum) lectin, anti‐ferritin, anti‐laminin and CD45. Immunofluorescence analysis revealed CD45 to stain microglial cells with the most specificity. CD45 staining reveal microglial cell morphology to progressively change from a compact ameboid shape (E5) into ramified branched shape (E12 and E15). In addition to the increased quantity of ramified microglial cells in chick brain with xenograft human tumors, the microglia also appeared to be localized near endothelia. Further investigations are needed to verify the increased presence of reactive microglial cells and their interaction with tumor cells and angiogenesis.Support or Funding InformationThis work was supported by the Delaware INBRE program, with a grant from the National Institute of General Medical Sciences‐NIGMS (8 P20 GM103446‐16) from the National Institutes of Health, a grant to DSG from the National Cancer Institute, and a generous gift from Pamela S. Simpson ‘97 Translational Cancer Research Fund. I would like to thank Tyler Hellmig and Emily Kollenbroich for their guidance.

The Impact of Glycosylation of Osteopontin on Urinary Stone Formation.

Osteopontin (OPN) is a matrix glycoprotein of urinary calculi. This study aims to identify the role of aberrant glycosylation of OPN in urolithiasis. We retrospectively measured urinary glycosylated OPN normalized by urinary full-length-OPN levels in 110 urolithiasis patients and 157 healthy volunteers and 21 patients were prospectively longitudinal follow-up during stone treatment. The urinary full-length-OPN levels were measured using enzyme-linked immunosorbent assay and glycosylated OPN was measured using a lectin array and lectin blotting. The assays were evaluated using the area under the receiver operating characteristics curve to discriminate stone forming urolithiasis patients. In the retrospective cohort, urinary Gal3C-S lectin reactive- (Gal3C-S-) OPN/full-length-OPN, was significantly higher in the stone forming urolithiasis patients than in the healthy volunteers (p < 0.0001), with good discrimination (AUC, 0.953), 90% sensitivity, and 92% specificity. The Lycopersicon esculentum lectin analysis of urinary full-length-OPN showed that urinary full-length-OPN in stone forming urolithiasis patients had a polyLacNAc structure that was not observed in healthy volunteers. In the prospective longitudinal follow-up study, 92.8% of the stone-free urolithiasis group had Gal3C-S-OPN/full-length-OPN levels below the cutoff value after ureteroscopic lithotripsy (URS), whereas 71.4% of the residual-stone urolithiasis group did not show decreased levels after URS. Therefore, Gal3C-S-OPN/full-length-OPN levels could be used as a urolithiasis biomarker.

314P - An EGF motif of Del1 inhibits efficient angiogenesis and suppresses tumour growth in vivo

BackgroundRecently, the gene therapy is attacked attention for a new cancer therapy. We invented for cancer gene therapy by a Del1 fragment. Del1 protein is composed of five domains, including three epidermal growth factor (EGF) repeats (E1–E3) and two discoidin domains (C1, C2). It consists of E2 has been reported to contain an RGD sequence that binds to integrin receptors and supports endothelial cell survival. The C1 domain is essential for the deposition of Del1 in the ECM. Additionally, the E3 domain can increase transfection efficiency. The Del1 protein was deposited in ECM, increased apoptosis, and enhanced the efficiency of a following transfection.At high concentrations, E3 induces apoptosis. And more, Dell is known to show pro-angiogenic or anti-angiogenic activities depending on the experimental conditions. In the present study, mouse ex-planted tumors were treated with the Del1 protein by a non-viral vector. MethodsCells of the human oral squamous cell carcinoma cell line, SCCKN, were injected into nude mice to generate explanted tumors. cDNAs encoding, the E3 and C1 domains of Del1 (E3C1), were inserted into pcDNA3D (pE3C1)and injected into the tumors every 7 days with a transfection reagent, jet-PEI. Tumor angiogenesis was evaluated by immunohistochemistry with antibodies for PECAM, von Willebrand factor and PDGF-beta and intravenous injection of lycopersicon esculentum lectin. The signal transduction of Notch was analyzed by western blotting. ResultsTreatment with pE3C1 suppressed the growth of explanted tumors and improved life prognosis of mice. In localization with immunostaining of PECAM or von Willebrand factor, and angioglaphy with lycopersicon esculentum lectin, vasculature without lumen were increased by gene therapy of Del1 fragment. Among the treatment with pE3C1 and control, the PDGF beta staining cells, which is marker of tip cells in angiogenesis, were increased. Western blotting of human umbilical endothelial cells cultured with an E3C1 recombinant protein showed the decreased expression of active Notch and hey1. ConclusionsIn the present study, we provide evidence that an E3C1 fragment suppresses Notch signaling and angiogenesis in explanted tumor model. Legal entity responsible for the studyHisataka Kitano. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Receptor-destroying enzyme (RDE) from Vibrio cholerae modulates IgE activity and reduces the initiation of anaphylaxis

IgE plays a key role in allergies by binding to allergens and then sensitizing mast cells through the Fc receptor, resulting in the secretion of proinflammatory mediators. Therefore, IgE is a major target for managing allergies. Previous studies have reported that oligomannose on IgE can be a potential target to inhibit allergic responses. However, enzymes that can modulate IgE activity are not yet known. Here, we found that the commercial receptor-destroying enzyme (RDE) (II) from Vibrio cholerae culture fluid specifically modulates IgE, but not IgG, and prevents the initiation of anaphylaxis. RDE (II)-treated IgE cannot access its binding site on bone marrow-derived mast cells, resulting in reduced release of histamine and cytokines. We also noted that RDE (II)-treated IgE could not induce passive cutaneous anaphylaxis in mouse ears. Taken together, we concluded that RDE (II) modulates the IgE structure and renders it unable to mediate allergic responses. To reveal the mechanism by which RDE (II) interferes with IgE activity, we performed lectin microarray analysis to unravel the relationship between IgE modulation and glycosylation. We observed that RDE (II) treatment significantly reduced the binding of IgE to Lycopersicon esculentum lectin, which recognizes poly-N-acetylglucosamine and poly-N-acetyllactosamine. These results suggest that RDE (II) specifically modulates branched glycans on IgE, thereby interfering with its ability to induce allergic responses. Our findings may provide a basis for the development of drugs to inhibit IgE activity in allergies.

Isolation and molecular characterization of hemocyte sub-populations in kuruma shrimp Marsupenaeus japonicus

Crustacean hemocytes, which have usually been classified morphologically based on methods using Giemsa or May-Giemsa stains, have recently been categorized using monoclonal antibodies or marker genes. However, these latter techniques are not yet widely used, and different classification methods are used for hemocytes among laboratories. Therefore, we aimed to develop a molecular classification method that can be widely used by researchers. The method we have developed uses lectins and magnetic-activated cell sorting (MACS) to isolate sub-populations of hemocytes. Two lectins, wheat germ agglutinin (WGA) and tomato lectin (Lycopersicon esculentum lectin; LEL), characteristically bind to hemocytes, which allows them to be classified into three sub-populations. Furthermore, different sub-populations of hemocyte can be isolated by using LEL and MACS. These sub-populations were characterized as non-granular and granular hemocytes, and the accumulation patterns of the gene transcripts were consistent with the results of a functional analysis reported previously. The lectin-based hemocyte isolation method developed in this study has good reproducibility.

Lectins identify distinct populations of coelomocytes in Strongylocentrotus purpuratus.

Coelomocytes represent the immune cells of echinoderms, but detailed knowledge about their roles during immune responses is very limited. One major challenge for studying coelomocyte biology is the lack of reagents to identify and purify distinct populations defined by objective molecular markers rather than by morphology-based classifications that are subjective at times. Glycosylation patterns are known to differ significantly between cell types in vertebrates, and furthermore they can vary depending on the developmental stage and activation states within a given lineage. Thus fluorescently labeled lectins that recognize distinct glycan structures on cell surface proteins are routinely used to identify discrete cell populations in the vertebrate immune system. Here we now employed a panel of fifteen fluorescently-labeled lectins to determine differences in the glycosylation features on the surface of Strongylocentrotus purpuratus coelomocytes by fluorescence microscopy and flow cytometry. Eight of the lectins (succinylated wheat germ agglutinin, Len culinaris lectin, Pisum sativum agglutinin, Saphora japonica agglutinin, Solanum tuberosum lectin, Lycopersicon esculentum lectin, Datura stramonium lectin, Vicia villosa lectin) showed distinct binding patterns to fixed and live cells of three major coelomocyte classes: phagocytic cells, red spherule cells, and vibratile cells. Importantly, almost all lectins bound only to a subgroup of cells within each cell type. Lastly, we established fluorescently-labeled lectin-based fluorescence activated cell sorting as a strategy to purify distinct S. purpuratus coelomocyte (sub-)populations based on molecular markers. We anticipate that this will become a routine approach in future studies focused on dissecting the roles of different coelomocytes in echinoderm immunity.

Sorafenib induced alteration of protein glycosylation in hepatocellular carcinoma cells.

Sorafenib is a multikinase inhibitor and is effective in treating hepatocellular carcinoma (HCC). However, it remains unknown whether sorafenib induces the alteration of protein glycosylation. The present study treated HCC MHCC97L and MHCC97H cells with a 50% inhibitory concentration of sorafenib. Following this treatment, alteration of protein glycosylation was detected using a lectin microarray. Compared with the controls, the binding capacity of glycoproteins extracted from sorafenib-treated HCC cells to the lectins Bauhinia purpurea lectin, Dolichos biflorus agglutinin, Euonymus europaeus lectin, Helix aspersa lectin, Helix pomatia lectin, Jacalin, Maclura pomifera lectin and Vicia villosa lectin were enhanced; while, the binding capacities to the lectins Caragana arborescens lectin, Lycopersicon esculentum lectin, Limulus polyphemus lectin, Maackia amurensis lecin I, Phaseolus vulgaris leucoagglutinin, Ricinus communis agglutinin 60, Sambucus nigra lectin and Solanum tuberosum lectin were reduced (spot intensity median/background intensity median ≥2, P<0.05). This difference in glycoprotein binding capacity indicates that cells treated with sorafenib could increase α-1,3GalNAc/Gal, β-1,3 Gal, GalNAcα-Ser/Thr(Tn) and α-GalNAc structures and decrease GlcNAc, sialic acid, tetra-antennary complex-type N-glycan and β-1,4Gal structures. These results were additionally confirmed by lectin blotting. Expression levels of signaling molecules including erythroblastosis 26–1 (Ets-1), extracellular signal-related kinases (ERK) and phosphorylated-ERK were measured by western blotting. There was a reduction in the expression of Ets-1 and ERK phosphorylation in sorafenib or 1,4-Diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene treated cells suggesting that sorafenib may reduce the expression levels of Ets-1 by blocking the Ras/Raf/mitogen activated protein kinase signaling pathway. In the present study, it was clear that sorafenib could inhibit the proliferation of HCC cells and alter protein glycosylation. The findings of this study may lead to providing a novel way of designing new anti-HCC drugs.

Functional characterisation of phagocytes in the Pacific oyster Crassostrea gigas.

Invertebrates lack canonical adaptive immunity and mainly rely on innate immune system to fight against pathogens. The phagocytes, which could engulf and kill microbial pathogens, are likely to be of great importance and have to undertake significant roles in invertebrate immune defense. In the present study, flow cytometry combined with histological and lectin staining was employed to characterise functional features of phagocytes in the Pacific oyster Crassostrea gigas. Based on the cell size and cellular contents, haemocytes were categorised into three cell types, i.e., granulocytes, semigranulocytes and agranulocytes. Agranulocytes with smaller cell volume and lower cytoplasmic-to-nuclear ratio did not show phagocytic activity, while semigranulocytes and agranulocytes exhibited larger cell volume, higher cytoplasmic-to-nuclear ratio and phagocytic activity. In addition, granulocytes with higher side scatter (SSC) exhibited higher phagocytic activity than that of semigranulocytes. When β-integrin and lectin-like receptors were blocked by RGD tripeptide and carbohydrates, respectively, the phagocytic activity of both granulocytes and semigranulocytes was significantly inhibited, indicating that β-integrin and certain lectin-like receptors were involved in phagocytosis towards microbes. Moreover, lipopolysaccharide but not peptidylglycan could enhance phagocytic activity of granulocytes and semigranulocytes towards Vibrio splendidus and Staphylococcus aureus. Lectin staining analysis revealed that Lycopersicon esculentum lectin (LEL), binding the epitope polylactosamine, was highly distributed on the extracellular cell surface of phagocytes, and could be utilized as a potential molecular marker to differentiate phagocytes from non-phagocytic haemocytes. The results, collectively, provide knowledge on the functional characters of oyster phagocytes, which would contribute to deep investigation of cell typing and cellular immunity in bivalves.

Differentiation of Glycan Diversity with Serial Affinity Column Set (SACS)

Targeted glycoproteomics is an effective way to discover disease-associated glycoproteins in proteomics and serial affinity chromatography (SAC) using lectin and glycan-targeting antibodies shows glycan diversity on the captured glycoproteins. This study suggests a way to determine glycan heterogeneity and structural analysis on the post-translationally modified proteins through serial affinity column set (SACS) using four Lycopersicon esculentum lectin (LEL) columns. The great advantage of this method is that it differentiates between glycoproteins on the basis of their binding affinity. Through this study, some proteins were identified to have glycoforms with different affinity on a single glycoprotein. It will be particularly useful in determining biomarkers in which the disease-specific feature is a unique glycan, or a group of glycans.

Photoperiodic Regulation of Cerebral Blood Flow in White-Footed Mice (Peromyscus leucopus).

Individuals living outside the tropics need to adjust their behavioral and physiological repertoires throughout the year to adapt to the changing seasons. White-footed mice (Peromyscus leucopus) reduce hippocampal volumes, hippocampal-dependent memory function, long-term potentiation, and alter neurogenesis in response to short (winter-like) day lengths (photoperiods). During winter, these mice putatively shunt energy away from the brain to maximize peripheral thermogenesis, immune function, and survival. We hypothesized that these changes in brain function are accompanied by alterations in brain vasculature. We maintained white-footed mice in short (8 h light/16 h dark) or long (16 h light/8 h dark) photoperiods for 8–9 weeks. Mice were then perfused with fluorescein isothiocyanate (FITC)-conjugated tomato (Lycopersicon esculentum) lectin to visualize the perfused cerebrovasculature. Short-day mice reduced hippocampal and cortical capillary density (FITC+ area); vessels isolated from short day-exposed mice expressed higher mRNA levels of the gelatinase matrix metalloproteinase 2 (MMP2). Additionally, short-day mice reduced cerebral blood flow ∼15% compared with their long-day counterparts, as assessed by laser speckle flowmetry. Immunohistochemistry revealed higher levels of MMP2 in the hippocampus of mice maintained in short days compared with long days, potentially contributing to the observed vascular remodeling. These data demonstrate that a discrete environmental signal (i.e., day length) can substantially alter cerebral blood flow in adult mammals.