Agglutinin Binding Research Articles

T-Antigen Expression Causes Macrothrombocytopenia and Extensive Hemophagocytosis in Mice Lacking the Sialyltransferase ST3Gal-I Specifically in Megakaryocytes

Changes in glycans expression have been associated with defects in blood platelet counts. However, the role of posttranslational modifications on platelet production is poorly understood. Six genes encoding sialyltransferases (ST)3Gal-I to -VI that form a2-3 sialic acid linkage have been identified in the mammalian genome, and deletion of St3gal1 and St3gal4 genes has been associated with macrothrombocytopenia in mice. We and others have shown previously that St3gal4-null platelets are cleared by the hepatic Ashwell-Morell receptor. Loss of ST3Gal-I activity has been associated with core 1 O-glycan Galβ1-3GalNAcα1-Ser/Thr expression, also known as tumor-associated or Thomsen-Friedenreich antigen (T antigen). We here investigated the detailed mechanisms of macrothrombocytopenia associated with St3gal1 deficiency by generating St3gal1loxP/PF4+ mice that lack ST3Gal-I specifically in the megakaryocyte (MK) lineage. Blood platelet counts were reduced by ~50% in St3gal1loxP/PF4+ mice, compared to control mice. Other blood cell counts were normal in St3gal1loxP/PF4+ mice. The clearance rate of St3gal1-null platelets was increased by ~15%, as determined by in vivo platelet biotinylation. Bone marrow MK numbers were normal in St3gal1loxP/PF4+ mice, compared to control mice, indicating that mechanisms other than clearance regulate circulating platelet counts in St3gal1loxP/PF4+ mice. Both St3gal1loxP/PF4+ platelets and bone marrow MKs had increased T antigen expression, as evidenced by flow cytometry using peanut agglutinin (PNA) binding. St3gal1loxP/PF4+ mice had increased bone marrow macrophage numbers, as evidenced by immunohistochemistry and flow cytometry using the macrophage marker F4/80. Macrophages in St3gal1loxP/PF4+ mice had increased expression of CD68 (macrosialin), as determined by immunohistochemistry and flow cytometry, indicative of an activated macrophage state. Consistently, St3gal1loxP/PF4+ bone marrow smears stained with May-Grunwald/Giemsa revealed increased hemophagocytosis. Macrophage ablation by in vivo injection of clodronate-encapsulated liposomes normalized blood platelet counts and size, and significantly reduced the numbers of activated macrophages in St3gal1loxP/PF4+ mice. Together, our data indicates that platelet production in the bone marrow is reliant on correct glycosylation on MK surface proteins and that the intimate interaction between MKs and macrophages play an important role in regulating platelet production and bone marrow homeostasis. DisclosuresNo relevant conflicts of interest to declare.

Altered glycosylation of complexed native IgG molecules is associated with disease activity of systemic lupus erythematosus.

In addition to the redundancy of the receptors for the Fc portion of immunoglobulins, glycans result in potential ligands for a plethora of lectin receptors found in immune effector cells. Here we analysed the exposure of glycans containing fucosyl residues and the fucosylated tri-mannose N-type core by complexed native IgG in longitudinal serum samples of well-characterized patients with systemic lupus erythematosus. Consecutive serum samples of a cohort of 15 patients with systemic lupus erythematosus during periods of increased disease activity and remission were analysed. All patients fulfilled the 1982 American College of Rheumatology classification criteria. Sera of 15 sex- and age-matched normal healthy blood donors served as controls. The levels and type of glycosylation of complexed random IgG was measured with lectin enzyme-immunosorbent assays. After specifically gathering IgG complexes from sera, biotinylated lectins Aleuria aurantia lectin and Lens culinaris agglutinin were employed to detect IgG-associated fucosyl residues and the fucosylated tri-mannose N-glycan core, respectively. In sandwich-ELISAs, IgG-associated IgM, IgA, C1q, C3c and C-reactive protein (CRP) were detected as candidates for IgG immune complex constituents. We studied associations of the glycan of complexed IgG and disease activity according to the physician's global assessment of disease activity and the systemic lupus erythematosus disease activity index 2000 documented at the moment of blood taking. Our results showed significantly higher levels of Aleuria aurantia lectin and Lens culinaris agglutinin binding sites exposed on IgG complexes of patients with systemic lupus erythematosus than on those of normal healthy blood donors. Disease activity in systemic lupus erythematosus correlated with higher exposure of Aleuria aurantia lectin-reactive fucosyl residues by immobilized IgG complexes. Top levels of Aleuria aurantia lectin-reactivity were found in samples taken during the highest activity of systemic lupus erythematosus. Our results show that native circulating IgG complexes from active systemic lupus erythematosus patients expose fucosyl residues and their glycan core is accessible to soluble lectins. Two putative mechanisms may contribute to the increased exposure of these glycans: (1) the canonical N-glycosylation site of the IgG-CH2 domain; (2) an IgG binding non-IgG molecule, like complement or C-reactive protein. In both cases the complexed IgG may be alternatively targeted to lectin receptors of effector cells, e.g. dendritic cells.

Regional specific adaptation of the endothelial glycocalyx dimension in tail-suspended rats.

Previous animal studies by using tail-suspended hindlimb-unloaded rat model have shown that simulated microgravity-induced vessel structural and functional remodeling may be anatomic region dependent. However, little care has been taken to assess the structural adaptation of the endothelial glycocalyx, the apical surface of the endothelium, the key mechanosensor mediating nitric oxide (NO) production, and the natural protective barrier of the vasculature. Therefore, the present study extended simulated microgravity-induced vessel remodeling to the endothelial glycocalyx level. The percents of bone mineral density (BMD) change from both control and tail-suspended (TS) rats were measured by micro-computed tomography (Micro-CT). Structural parameters such as the luminal diameter (D), the thickness of each layer, the ratio of intima to media (IMR), the cross-sectional areas of the intima (CSAI) and media (CSAM) of vessels from three different regions (the common carotid artery, abdominal aorta, and femoral artery) were assessed by hematoxylin and eosin staining. Dimensions of the glycocalyx above, below, and away from the endothelial cell nucleus were examined by fluorescein isothiocyanate-labeled wheat germ agglutinin (WGA-FITC) binding to the cryosection of vessels. Our results show that 3-week tail suspension of rats increases the thickness and CSA of the abdominal aortic endothelium by 23.7 and 21.1%, respectively, thickens the media layer of the common carotid artery by 34.0%, and increases the luminal diameter, the CSA of the intima and media of the femoral artery by 75.7, 93, and 61.2%, respectively. Correspondingly, the dimension of the glycocalyx away from the common carotid arterial and the abdominal aortic endothelial cell nucleus from tail-suspended rats shows a 1.66- and 1.64-fold increase respectively, while it shows a 0.79-fold reduction on the top of the femoral endothelial cells. These results suggest that simulated microgravity induces vascular endothelial glycocalyx remodeling in a regional-dependent manner. The perturbation of the endothelial glycocalyx at the lower body artery may be the first event of vascular remodeling initiating endothelial dysfunction, contributing to postspaceflight orthostatic intolerance.

Trophoblast specialisations during pregnancy in the tammar wallaby, Macropus eugenii: A morphological and lectin histochemical study

IntroductionThe tammar wallaby has a short gestation (26.5 days) and vascular modifications to expedite transport during that brief pregnancy. Here we examine trophoblast structural attributes that would facilitate materno-fetal exchange. Materials and methodsFour specimens of Macropus eugenii between days 23 and 26 gestation were examined using electron microscopy and 24 lectins to characterise glycosylated secretions and their internalisation. ResultsTwo trophoblast phenotypes were found, flattened cells generally in contact with the underlying uterine epithelium and giant cells associated with histiotrophe. The latter appeared to penetrate uterine clefts, occasionally detach and become necrotic. Lectin histochemistry and ultrastructure indicated the presence of many lysosomes and residual bodies especially in trophoblast giant cells; these contained glycans, mainly apically, which were also detected in secretions and cell debris. Trophoblast basal membranes bore extensive filopodia. Giant cells were less common in vascular trilaminar areas and here the trophoblast barrier became thinner near term. DiscussionLoss of Maackia amurensis agglutinin binding suggested cleavage of terminal sialic acid residues as an early post-internalisation event in the trophoblast. Lectin staining indicated degradation occurred in an apical–basal direction, and the heavily glycosylated basal membrane appeared specialised for transport out of the cell. ConclusionGranules seen ultrastructurally and histochemically, particularly in giant trophoblast cells of the bilaminar area, suggest that internalised histiotrophe is broken down here and nutrients transferred to the embryo via the specialised basal plasma membrane. The trilaminar vascular area contained mostly flattened trophoblast cells, supporting the suggestion that gaseous exchange is its primary function.

Actin depolymerizing factor but not cofilin‐1 is necessary to have a normal intestinal brush border (1109.16)

Two highly conserved isoforms of the cofilin gene family (actin severing proteins expressed in intestinal epithelial cells) are cofilin‐1 (COF) and actin depolymerizing factor (ADF). COF and ADF take part in Arp2/3 remodeling and seem to play overlapping roles since both rescue the knockdown (KD) phenotypes of the other. Their precise role in the intestine is unknown. We hypothesized that COF and/or ADF change local actin polymerization at the base of epithelial cell microvilli by terminal web remodeling. COF and ADF were KD using lentivirus/puromycin in Caco‐2 cells, a polarized intestinal Na‐absorptive cell line. Both were normally subapical, as well as diffusely cytosolic in the cells. Structural studies were performed using confocal and electron microscopy with brush border (BB) characterization by phalloidin and wheat germ agglutinin binding. ADF KD, but not COF, significantly changed epithelial cell structure with a) shorter cell heights; b) greater cell diameter; c) shorter/sparser BB. In addition, Western blot showed increased B‐actin and villin amount in ADF KD cells, normalized to GAPDH. In contrast, COF KD had less dramatic changes in intestinal cell structure: a) slight increase in cell height; b) taller/denser microvilli; c) minimally increased B‐actin and villin. We conclude that in spite of similar sub‐apical localization, ADF but not COF is necessary to have a normal intestinal BB, including the microvilli.Grant Funding Source: Supported by APS UGSRF, NIH NIDDK R01DK26523, R01DK61765, P01DK72084, P30DK89502

Carbohydrate-binding agents act as potent trypanocidals that elicit modifications in VSG glycosylation and reduced virulence inTrypanosoma brucei

The surface of Trypanosoma brucei is covered by a dense coat of glycosylphosphatidylinositol-anchored glycoproteins. The major component is the variant surface glycoprotein (VSG) which is glycosylated by both paucimannose and oligomannose N-glycans. Surface glycans are poorly accessible and killing mediated by peptide lectin-VSG complexes is hindered by active endocytosis. However, contrary to previous observations, here we show that high-affinity carbohydrate binding agents bind to surface glycoproteins and abrogate growth of T. brucei bloodstream forms. Specifically, binding of the mannose-specific Hippeastrum hybrid agglutinin (HHA) resulted in profound perturbations in endocytosis and parasite lysis. Prolonged exposure to HHA led to the loss of triantennary oligomannose structures in surface glycoproteins as a result of genetic rearrangements that abolished expression of the oligosaccharyltransferase TbSTT3B gene and yielded novel chimeric enzymes. Mutant parasites exhibited markedly reduced infectivity thus demonstrating the importance of specific glycosylation patterns in parasite virulence.

Alarm Pheromone Is Detected by the Vomeronasal Organ in Male Rats

It is widely known that a stressed animal releases specific pheromones, possibly for alarming nearby conspecifics. We previously investigated an alarm pheromone in male rats and found that this alarm pheromone evokes several responses, including increases in the defensive and risk assessment behaviors in a modified open-field test, and enhancement of the acoustic startle reflex. However, the role of the vomeronasal organ in these pheromone effects remains unclear. To clarify this point, vomeronasal organ-excising or sham surgeries were performed in male rats for use in 2 experimental models, after which they were exposed to alarm pheromone. We found that the vomeronasal organ-excising surgery blocked the effects of this alarm pheromone in both the modified open-field test and acoustic startle reflex test. In addition, the results of habituation/dishabituation test and soybean agglutinin binding to the accessory olfactory bulb suggested that the vomeronasal organ-excising surgery completely ablated the vomeronasal organ while preserving the functioning of the main olfactory system. From the above results, we showed that the vomeronasal organ plays an important role in alarm pheromone effects in the modified open-field test and acoustic startle reflex test.

Total IgA and IgA reactivity to antigen I/II epitopes in HLA-DRB1*04 positive subjects.

Bacterial adherence to the acquired dental pellicle, important in dental caries (caries), is mediated by receptor-adhesins such as salivary agglutinin binding to Streptococcus mutans antigen I/II (I/II). Ten selected I/II epitopes were chosen to determine their reactivity to human salivary IgA. Previous studies suggested that a specific HLA biomarker group (HLA-DRB1*04) may have differential influence of immune responses to I/II. However, it was not known whether secretory IgA (SIgA) responses to the selected epitopes from HLA-DRB1*04 positive subjects were different compared to controls, or across other caries-related factors such as total IgA (TIgA). Thirty-two total subjects were matched according to HLA type, gender, ethnicity and age. HLA genotyping, oral bacterial, immunoglobulin and antibody analyses were performed. A large observed difference emerged with regard to the natural immune reservoir of TIgA in HLA-DRB1*04 positive subjects, specifically, a 27.6% reduction compared to controls. In contrast to all other epitopes studied, HLA-DRB1*04 positive subjects also exhibited reduced reactivity to I/II epitope 834–853. HLA-DRB1*04 positive subjects exhibited lower specific SIgA activity/TIgA to 834–853 and also a lower specific reactivity to 834–853/whole cell S. mutans UA159. Furthermore, HLA-DRB1*04 positive subjects exhibited lower responses to I/II in its entirety. The large observed difference in TIgA and the 834–853 reactivity pattern across multiple measures suggest potentially important connections pertaining to the link between HLA-DRB1*04 and caries.

Expression of GALNT2 in human extravillous trophoblasts and its suppressive role in trophoblast invasion

Extravillus trophoblast (EVT) invasion plays a critical role in placental development. Integrins bind to extracellular matrix (ECM) proteins to mediate EVT cell adhesion, migration, and invasion. Changes in O-glycans on β1-integrin have been found to regulate cancer cell behavior. We hypothesize that O-glycosyltransferases can regulate EVT invasion through modulating the glycosylation and function of β1-integrin. Here, we found that the GALNT1 and GALNT2 mRNA were highly expressed in HTR8/SVneo and first trimester EVT cells. Immunohistochemstry and immunofluorescence staining showed that GALNT2 was expressed in subpopulations of EVT cells in deciduas, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. The percentage of GALNT2-positive EVT cells increased with gestational ages. Overexpression of GALNT2 in HTR8/SVneo cells significantly enhanced cell-collagen IV adhesion, but suppressed cell migration and invasion. Notably, we found that GALNT2 increased the expression of Tn antigen (GalNAc-Ser/Thr) on β1-integrin as revealed by Vicia Villosa agglutinin (VVA) binding. Furthermore, GALNT2 suppressed the phosphorylation of focal adhesion kinase (FAK), a crucial downstream signaling molecule of β1-integrin. Our findings suggest that GALNT2 is a critical initiating enzyme of O-glycosylation for regulating EVT invasion.

Electrochemical characterization of globotriose-containing self-assembled monolayers on nanoporous gold and their binding of soybean agglutinin

Self-assembled monolayers (SAMs) of α-d-Gal-(1→4)-β-d-Gal-(1→4)-β-d-Glc-mercaptooctane (globotriose, Gb3-C8-SH) were prepared both as single-component SAMs and as mixed SAMs with either octanethiol (OCT) or 8-mercapto-3,6-dioxaoctanol (HO-PEG2-SH), on flat gold and on nanoporous gold (NPG) electrodes. The binding of soybean agglutinin (SBA) to the globotriose (Gb3) unit in the SAMs was then studied using electrochemical impedance spectroscopy (EIS), which is a label free method found to be quite sensitive to SAM composition and to the differences in SAM structure on NPG versus on flat Au. The affinity of SBA to the mixed SAM of HO-PEG2-SH and Gb3-C8-SH on NPG is found to be greater on NPG than on flat gold, and indicates a potential advantage for NPG as a substrate. The SAMs of HO-PEG2-SH were found to resist protein adsorption on either NPG or flat gold. The non-specific adsorption of SBA to OCT SAMs on flat Au was observed in EIS by the increase in charge transfer resistance; whereas, the increase seen on the NPG surface was smaller, and suggests that EIS measurements on NPG are less affected by non-specific protein adsorption. Atomic force microscopy (AFM) images of the SBA binding to mixed SAM of HO-PEG2-SH and Gb3-C8-SH on NPG showed a greater number of proteins on top of the OCT containing SAMs.

Population-Based Study Reveals New Risk-Stratification Biomarker Panel for Barrett's Esophagus

The risk of progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) is low and difficult to calculate. Accurate tools to determine risk are needed to optimize surveillance and intervention. We assessed the ability of candidate biomarkers to predict which cases of BE will progress to EAC or high-grade dysplasia and identified those that can be measured in formalin-fixed tissues. We analyzed data from a nested case-control study performed using the population-based Northern Ireland BE Register (1993-2005). Cases who progressed to EAC (n = 89) or high-grade dysplasia ≥ 6 months after diagnosis with BE were matched to controls (nonprogressors, n = 291), for age, sex, and year of BE diagnosis. Established biomarkers (abnormal DNA content, p53, and cyclin A expression) and new biomarkers (levels of sialyl Lewis(a), Lewis(x), and Aspergillus oryzae lectin [AOL] and binding of wheat germ agglutinin) were assessed in paraffin-embedded tissue samples from patients with a first diagnosis of BE. Conditional logistic regression analysis was applied to assess odds of progression for patients with dysplastic and nondysplastic BE, based on biomarker status. Low-grade dysplasia and all biomarkers tested, other than Lewis(x), were associated with risk of EAC or high-grade dysplasia. In backward selection, a panel comprising low-grade dysplasia, abnormal DNA ploidy, and AOL most accurately identified progressors and nonprogressors. The adjusted odds ratio for progression of patients with BE with low-grade dysplasia was 3.74 (95% confidence interval, 2.43-5.79) for each additional biomarker and the risk increased by 2.99 for each additional factor (95% confidence interval, 1.72-5.20) in patients without dysplasia. Low-grade dysplasia, abnormal DNA ploidy, and AOL can be used to identify patients with BE most likely to develop EAC or high-grade dysplasia.

Association of Immunostaining of Galectin-3 and Sambucus nigra Agglutinin with Invasion, Metastasis and Poor Progression of Gallbladder Adenocarcinoma

To study the expression of galectin-3 (gal-3) and Sambucus nigra agglutinin (SNA) binding site and to detect their clinicopathological significances in the benign and malignant lesions of gallbladder. We used immunohistochemistry to detect gal-3 expression and ABC affinity-cytochemistry to detect SNA binding site in specimens of adenocarcinoma, peritumoral tissues, polyp and chronic cholecystitis. The positive expression rates of gal-3 and SNA binding site were significantly higher in adenocarcinoma (62.0%, 66.7%) than those in peritumoral tissues (39.1%, 45.6%), polyp (26.7%, 33.3%) and chronic cholecystitis (11.4%, 11.4%) (p<0.05). A high consistency was found between the levels of expression of gal-3 expression and SNA binding site in adenocarcinoma (χ²=9.51, p<0.01). Univariate Kaplan-Meier analysis showed that increased expression of gal-3 (p=0.028) or SNA binding site (p=0.030) was associated with decreased overall survival. Multivariate Cox regression analysis showed that increased expression of gal-3 (p=0.012) or SNA binding site (p=0.030) was an independent prognostic predictor in gallbladder adenocarcinoma. These results suggest that expression of gal-3 and SNA binding site might have important effects on the carcinogenesis, progression and biological behaviors of gallbladder cancer.

Is BMP‐2 a component of the particulate matrix that is responsible for epithelial/mesenchymal transformation during heart development

Early cardiac development involves an epithelial/mesenchymal transformation (EMT) of endothelial cells in the atrioventricular (AV) canal and outflow tract (OT) regions of the heart. It has been shown that the myocardium secrets factors, collectively called particulate matrix that induce this process. Previous reports from our laboratory showed that, only myocardial conditioned media (MyoCM) or EDTA extract can replace the myocardium and drive mesenchyme formation. Reports from other laboratories suggest that bone morphogenetic protein 2 (BMP‐2) has a role in EMT. So, we asked whether BMP‐2 is a part of the particulate matrix. Initial experiments using immunostaining of stage 15–16 chick embryo sections with anti BMP‐2 antibody revealed that BMP‐2 signals were detected within the myocardium of the cushion forming regions and in a group of cells in the endocardial layer of these regions in the heart, but not in the extracellular matrix. To confirm these results, whole heart lysate, EDTA extract from stage 15–16 embryos and soybean agglutinin (SBA) binding proteins from MyoCM will be examined for the presence of BMP‐2 by western blotting analysis.Grant Funding Source : none

Serum from patients with erectile dysfunction inhibits circulating angiogenic cells from healthy men: relationship with cardiovascular risk, endothelial damage and circulating angiogenic modulators

Erectile dysfunction (ED) is an early manifestation of arteriosclerosis associated with endothelial damage/dysfunction and to a blunted ability of cultured mononuclear circulating cells (MNCs) to differentiate circulating angiogenic cells (CACs), putatively involved in endothelial damage repair. Here we explored effects of human serum (HS) from patients with ED and cardiovascular risk factors (VRFs) but no clinical atherosclerosis, on cultured MNCs of healthy men to differentiate CACs and to form colonies. Effect of HS on number of CACS and of colony forming units (CFUs) was correlated with circulating markers of endothelial damage and with angiogenic modulators. MNCs from healthy men were cultured in standard conditions or with 20% HS from 35 patients with ED and from 10 healthy men. CACs were identified after 7 days of culture by uptake of acetylated low-density lipoprotein with concomitant binding of Ulex europaeus agglutinin I. CFUs were counted after 5 days of culture. Enzyme-linked immunosorbent assays assessed plasmatic soluble (s) form of E-selectin, Endothelin (ET)-1, tissue type plasminogen activator (tPA), vascular endothelial growth factor (VEGF)(165) and sVEGF receptor (R)-1. The number of CACs and of CFUs from healthy men was reduced after culturing MNCs with HS compared to standard medium. The inhibitory effect was significantly higher with HS from ED patients with higher or lower VRF exposure compared to healthy men. Inhibition was positively correlated with VRFs exposure, with ED severity, with common carotid artery intima media thickness measured using B-mode ultrasound, and to a lesser extent with plasmatic sE-Selectin, sET-1 and sVEGFR-1. Dysfunction of cells involved in vascular homoeostasis is induced by soluble factors still unknown and already present in a very initial systemic vascular disease in men with ED and VRFs.

Molecular imaging using fluorescent lectins permits rapid endoscopic identification of dysplasia in Barrett's esophagus

Barrett's esophagus is an example of a pre-invasive state, for which current endoscopic surveillance methods to detect dysplasia are time consuming and inadequate. The prognosis of cancer arising in Barrett's esophagus is improved by early detection at the stage of mucosal carcinoma or high-grade dysplasia. Molecular imaging methods could revolutionize the detection of dysplasia, provided they permit a wide field of view and highlight abnormalities in real time. We show here that cell-surface glycans are altered in the progression from Barrett's esophagus to adenocarcinoma and lead to specific changes in lectin binding patterns. We chose wheat germ agglutinin as a candidate lectin with clinical potential. The binding of wheat germ agglutinin to human tissue was determined to be specific, and we validated this specific binding by successful endoscopic visualization of high-grade dysplastic lesions, which were not detectable by conventional endoscopy, with a high signal-to-background ratio of over 5.

Sun Ginseng Protects Endothelial Progenitor Cells From Senescence Associated Apoptosis

Endothelial progenitor cells (EPC) are a population of cells that circulate in the blood stream. They play a role in angiogenesis and, therefore, can be prognostic markers of vascular repair. Ginsenoside Rg3 prevents endothelial cell apoptosis through the inhibition of the mitochondrial caspase pathway. It also affects estrogen activity, which reduces EPC senescence. Sun ginseng (SG), which is heat-processed ginseng, has a high content of ginsenosides. The purpose of this study was to investigate the protective effects of SG on senescence-associated apoptosis in EPCs. In order to isolate EPCs, mononuclear cells of human blood buffy coats were cultured and characterized by their uptake of acetylated low-density lipoprotein (acLDL) and their binding of Ulex europaeus agglutinin I (ulex-lectin). Flow cytometry with annexin-V staining was performed in order to assess early and late apoptosis. Senescence was determined by β-galactosidase (β-gal) staining. Staining with 4′-6-Diamidino-2-phenylindole verified that most adherent cells (93±2.7%) were acLDL-positive and ulex-lectin-positive. The percentage of β-gal-positive EPCs was decreased from 93.8±2.0% to 62.5±3.6% by SG treatment. A fluorescence-activated cell sorter (FACS) analysis showed that 4.9% of EPCs were late apoptotic in controls. Sun ginseng decreased the apoptotic cell population by 39% in the late stage of apoptosis from control baseline levels. In conclusion, these results show antisenescent and antiapoptotic effects of SG in human-derived EPCs, indicating that SG can enhance EPC-mediated repair mechanisms.

Cone outer segment extracellular matrix as binding domain for interphotoreceptor retinoid‐binding protein

Cones are critically dependent on interphotoreceptor retinoid-binding protein (IRBP) for retinoid delivery in the visual cycle. Cone-dominant vertebrates offer an opportunity to uncover the molecular basis of IRBP's role in this process. Here, we explore the association of IRBP with the interphotoreceptor matrix (IPM) of cones vs. rods in cone dominant retinas from chicken (Gallus domesticus), turkey (Meleagris gallopavo), and pig (Sus scrofa). Retinas were detached and fixed directly or washed in saline prior to fixation. Disassociated photoreceptors with adherent matrix were also prepared. Under 2 mM CaCl(2) , insoluble matrix was delaminated from saline washed retinas. The distribution of IRBP, as well as glycans binding peanut agglutinin (cone matrix) and wheat germ agglutinin (rod/cone matrix), was defined by confocal microscopy. Retina flat mounts showed IRBP diffusely distributed in an interconnecting, lattice-like pattern throughout the entire matrix. Saline wash replaced this pattern with fluorescent annuli surrounding individual cone outer segments. In isolated cones and matrix sheets, IRBP colocalized with the peanut agglutinin binding matrix glycans. Our results reveal a wash-resistant association of IRBP with a matrix domain immediately surrounding cone outer segments. The cone matrix sheath may be responsible for IRBP-mediated cone targeting of 11-cis retinoids.

Apolipoprotein A-I mimetic peptide D-4F promotes human endothelial progenitor cell proliferation, migration, adhesion though eNOS/NO pathway

Circulating endothelial progenitor cells (EPCs) have a critical role in endothelial maintenance and repair. Apolipoprotein A-I mimetic peptide D-4F has been shown to posses anti-atherogenic properties via sequestration of oxidized phospholipids, induction of remodeling of high density lipoprotein and promotion of cholesterol efflux from macrophage-derived foam cells. In this study, we test the effects of D-4F on EPC biology. EPCs were isolated from the peripheral venous blood of healthy male volunteers and characterized by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated LDL uptake and ulex europaeus agglutinin binding and flow cytometry. Cell proliferation, migration, adhesion, nitric oxide production and endothelial nitric oxide synthase (eNOS) expression in the absence and presence of D-4F or simvastatin (as a positive control), were assayed. We demonstrated that D-4F significantly enhanced EPC proliferation, migration and adhesion in a dose-dependent manner compared with vehicle. However, all of the favorable effects of D-4F on EPCs were dramatically attenuated by preincubation with NOS inhibitor L-NAME. Further, D-4F also increased nitric oxide production in culture supernatant and the levels of eNOS expression and phosphorylation. The stimulatory effects of D-4F (10μg/ml) on EPC biology were comparable to 0.5μM simvastatin. These results suggest that eNOS/NO pathway mediates the functional modulation of EPC biology in response to D-4F treatment and support the notion that the beneficial role of D-4F on EPCs may be one of the important components of its anti-atherogenic potential.

Helix pomatia Agglutinin Binding Glycoproteins in Thyroid Tumors

Binding of the lectin Helix pomatia agglutinin (HPA) has been shown to be associated with poor prognosis in many human cancers, but not in thyroid cancer. The aims of the present study were to assess alteration in cellular glycosylation, detected by HPA binding, in thyroid tumors; to determine if such altered glycosylation carries any prognostic significance; and to analyze qualitative and quantitative differences of HPA-binding glycoproteins in various thyroid tumors. Lectin histochemistry was performed on 110 archival paraffin wax embedded specimens of various thyroid tumors excised between the years 1983-1993. Demographic data, histological data, and time to death were recorded, and multivariate Cox regression analysis was performed to determine a prognostic model for patient survival based on the data. Helix pomatia agglutinin binding glycoproteins were isolated from 128 fresh specimens of various thyroid tissues by affinity chromatography, analyzed by SDS-PAGE and western blotting. There was a marked qualitative difference in the profile of HPA-binding glycoproteins, with the malignant thyroid tumors showing a heterogeneous profile of numerous HPA binding glycoprotein bands. Lectin histochemistry showed significant positive HPA binding in malignant thyroid tumors (p = 0.0009). Kaplan-Meir survival analysis revealed that among patients who died of their disease those with HPA-positive tumors had a significantly shorter survival (p = 0.009). This is the first study showing that HPA binding glycoproteins are synthesized by thyroid tumors. Thyroid cancers show a broader profile of HPA binding glycoproteins compared to benign thyroid tumors, and positive HPA binding is significantly associated with shorter survival and poorer prognosis in thyroid cancers.

Characterization and In Vitro Culture of Male Germ Cells from Developing Bovine Testis

The transition from male primitive germ cells (gonocytes) to type A spermatogonia in the neonatal testis is the initial process and a crucial process in spermatogenesis. However, in large domestic animals, the physiological and biochemical characteristics of germ cells during the developmental processes remain largely unknown. In this study, we characterized bovine germ cells in the developing testis from the neonatal stage to the adult stage. The binding of the lectin Dolichos biflorus agglutinin (DBA) and the expression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) were restricted to gonocytes in the neonatal testis and spermatogonia in the adult testis. Gonocytes also expressed a germ cell marker (VASA) and stem cell markers (NANOG and OCT3/4), while the expressions of these markers in the adult testis were restricted to differentiated spermatic cells and were rarely expressed in spermatogonia. We subsequently utilized these markers to characterize gonocytes and spermatogonia after culture in vitro. Spermatogonia that were collected from the adult testis formed colonies in vitro only for one week. On the other hand, gonocytes from the neonatal testis could proliferate and form colonies after every passage for 1.5 months in culture. These colonies retained undifferentiated states of gonocytes as confirmed by the expression of both germ cell and stem cell markers. Moreover, a transplantation assay using immunodeficient mice testes showed that long-term cultured cells derived from gonocytes were able to colonize in the recipient testis. These results indicated that bovine gonocytes could maintain germ cell and stem cell potential in vitro.