FITC-labeled Lectins Research Articles

Effect of a water-soluble form of dihydroquercetin on age-dependent LPS-induced gliovascular remodeling of the substantia nigra in rats

Background. The molecular and cellular mechanisms of action of the water-soluble form of dihydroquercetin (DHA-VF) on neurogliovascular units, age-related disturbances of intrasystem connections that may underlie neuroinflammatory and neurodegenerative diseases, remain unclear.The aim: to study structural changes of microcirculatory vessels and functional responses of micro- and astroglial cells in the substantia nigra of young and old rats in response to intranigral injection of lipopolysaccharide (LPS) and subsequent oral administration of DHA-VF.Methods. Young (250-320 g) and old (390-450 g) Wistar rats were injected with 2 μL LPS solution at a concentration of 0.01 μL/mL (experimental group; n=24) or 2 μL sterile physiological solution (control group; n=12) into the substantia nigra area of the brain using a stereotaxic device. Half of the animals in the experimental groups (6 animals of each age group) were given 2 ml of a solution containing DHA-VF at a concentration of 3 mg/mL daily by gavage with a probe. After 8 weeks, the animals were decapitated and cryostat sections were obtained for histochemical (FITC-labeled tomato lectin) and immunohistochemical (antibodies against GFAP and CD-11β) staining of vascular endothelium and glial cells, respectively. Results. 8 weeks after LPS administration to old rats in the CS, a significant excess of areas occupied by cell bodies and processes of microglial and astroglial cells as well as the number of vessels at standard sites was found compared to both young animals exposed to similar exposure and old control animals. Oral administration of DHA-VF to rats significantly reduced LPS-induced glial activation in young and old animals. In addition, administration of DHA-VF to old animals reduced the intensity of LPS-induced microvascular remodeling of the CS. Conclusions. LPS administration in the rat CS induces neuroinflammation and vascular angiogenesis, which are maximally expressed in old animals. Administration of DHA-VF for 8 weeks significantly reduced these LPS-induced changes.

Biofilm inhibitor Con A lectin feed additive protects shrimp against pathogenic Vibrio harveyi and V. parahaemolyticus

Extracellular matrix (ECM) is a major component of all bacterial biofilms that protect the bacterial cells living within. It consists mainly of carbohydrates, proteins, lipids, and nucleic acids. We characterized the ECM of an acute hepatopancreatic necrosis disease (AHPND) causing Vibrio parahaemolyticus isolate 3HP and two luminescent shrimp disease-causing V. harveyi isolates (1114GL or VH0 and its Siphoviridae phage-lysogenized isolate, VH1) using 11 FITC-labeled plant lectins with different terminal sugar specificities. Canavalia ensiformis lectin (Con A), wheat germ lectin (Triticum vulgaris, WGA), Griffonia simplicifolia lectin II (GSL-II), and Maclura pomifera Lectin (MPL) were found to react with the biofilms of all three isolates. Based on these results and availability, Con A and WGA and their respective specific sugars (D-mannose or glucose and N-acetyl-D-glucosamine) were tested for effects on V. harveyi and V. parahaemolyticus biofilm formations. Biofilms of these bacteria were evaluated by thickness after growth with and without the two lectins and with and without their respective sugar targets at different concentrations. Only Con A at 0.1 mg/ml and 0.5 mg/ml (with and without D-mannose or d-glucose) were found to inhibit biofilms of all three Vibrio isolates in a dose-dependent manner. Con A was then tested for efficacy in protecting shrimp against V. harveyi (VH1) and V. parahaemolyticus 3-HP by its addition to shrimp feed at 0.05 mg/g and 0.5 mg/g and fed for seven days before they were immersion challenged with either VH1 or 3HP on day 8. After the challenge, the shrimp continued to be fed with their respective feed, and their health and survival were monitored for another 11 and 12 days for the VH1 and 3HP experiments, respectively. From both experiments, the groups fed with Con A-supplemented feed gave significantly higher survival rates [51.9% and 53.6% (VH1) and 52.8% and 62.9% (3HP)] than those fed with un-supplemented feed [38.3% (VH1) and 21.2% (3HP)] (p < 0.05). These results suggested that D-mannose/d-glucose plays an essential role in V. harveyi and V. parahaemolyticus biofilm formations, and Con A lectin might be helpful in monitoring and controlling biofilms to prevent Vibrio diseases.

A Water-soluble Form of Dihydroquercetin Reduces LPS-induced Astrogliosis, Vascular Remodeling, and mRNA VEGF-A Levels in the Substantia Nigra of Aged Rats

Background: The age-dependent sporadic form of PD is characterized by the degeneration of dopaminergic (DA) neurons in the Substantia Nigra (SN), gliosis, and vascular changes. Vascular changes may contribute to the onset of the disease and exacerbate the neurodegenerative process, as some vascular changes occur before the onset of neuronal loss. To demonstrate the anti-neuroinflammatory efficacy of a new compound, a water-soluble form of dihydroquercetin (DHQ-WF), we studied the structural changes of microcirculatory vasculature, astroglial GFAP, and vascular endothelial growth factor –A (VEGF-A) mRNA expression in the SN of young and old rats after unilateral nigral treatment by lipopolysaccharide (LPS) and oral administration of DHQ-WF. Materials and methods: The experiments were performed on 18 young (8 weeks - 10 weeks old; 250 g - 320 g) and 18 old (18 months - 19 months old; 390 g - 450 g) male Vistar rats. Young and adult rats from the experimental groups were stereotactically injected with 2 μL LPS solution (LPS from Escherichia coli; 0,01 μL/mL) into one side of the SN. Control young and old rats were similarly injected with 2 μL sterile saline. Half of the animals in both the control and experimental groups (6 animals in each group) received a 2 ml solution containing DHQ-WF at a concentration of 3 mg/ml orally every day. After 8 weeks, brains were harvested and serial cryostat sections were prepared for histochemical (FITC-labeled tomato lectin), immunohistochemical (anti-GFAP Antibody, Cy3 Conjugate) staining, and real-time PCR (mRNA VEGF-A). Results: Eight weeks after LPS injection into the SN, a significant excess of areas occupied by cell bodies and processes of astroglial cells, the density of microcirculatory vessels, and mRNA VEGF-A expression was observed in old animals compared to control old animals and young LPS-treated rats. Oral administration of DHQ-WF to LPS-treated rats resulted in a significant reduction of these parameters in old animals. Conclusion: Injection of LPS into rat SN induces neuroinflammation and vascular angiogenesis, maximally expressed in old animals. Administration of DHQ-WF for 8 weeks significantly reduces these LPS-induced changes. DHQ-WF may be an effective treatment for reducing the effects of neuroinflammation in the aging brain.

MO261: Sparsentan, The Dual Endothelin Angiotensin Receptor Antagonist (DEARA), Attenuates Albuminuria and Protects from the Development of Renal Injury to a Greater Extent Than Losartan in the GDDY Mouse Model of IGA Nephropathy: A 16-Week Study

Abstract BACKGROUND AND AIMS gddY mice are an IgA nephropathy (IgAN)-prone mouse model that develops albuminuria by 8 weeks (wks) of age with glomerular IgA, IgG and C3 deposits and progressive mesangioproliferative glomerulonephritis. We have previously shown that sparsentan (SP), a novel, highly selective, first-in-class, single-molecule dual endothelin angiotensin receptor antagonist (DEARA) being developed for the treatment of focal segmental glomerulosclerosis and IgAN, protected gddY mice from the development of albuminuria and glomerulosclerosis in an 8-week study. Here we compare the impact of SP and losartan (LS), an angiotensin II subtype 1 receptor antagonist (AT1R), on albuminuria, % of sclerotic glomeruli (GS), podocyte number, and glycocalyx damage in gddY mice treated for 16 weeks. METHOD gddY mice at 4 weeks of age were treated for 8 or 16 weeks either with chow as a control (C) (n = 10) or containing SP at 900 ppm (SP900) (n = 12) or with control chow and LS in drinking water to deliver 30 mg/kg of LS (LS30) (n = 12). Systolic blood pressure (BP) was measured by tail-cuff plethysmography at 8, 12 and 18 weeks of age. Albuminuria/creatinine ratio (ACR) was assessed every 2 weeks until 20 weeks of age. Kidney biopsies (n = 4–5 per group) were taken from a subset of mice sacrificed at age 12 weeks and at the end of the study at 20 weeks of age (5–7 per group) and processed for determination of GS, the number of podocytes assessed by immunohistochemistry using an anti-WT-1 antibody (30 glomeruli per animal), and measurement of the glycocalyx area using FITC-labeled lectin (10 glomeruli per animal). RESULTS gddY mice treated with SP900 or LS30 had an equivalent and significant (P &amp;lt; .01) reduction in BP compared with C mice at 12 and 18 weeks of age (18 weeks of age mean BP ± SD mmHg: C, 147 ± 28; SP900, 100 ± 6; LS30, 101 ± 8). Despite similar systemic hemodynamic effects, SP900 treatment resulted in a greater and more rapid reduction in ACR from baseline compared with either mice treated with C or LS30 during the first 4 weeks of treatment (Fig. 1). Development of GS was significantly attenuated in mice at 20 weeks of age treated with SP900 (P &amp;lt; .001) compared with C mice, but not in those treated with LS30 (mean sclerosis score ± SD: C, 28 ± 17; SP900, 3 ± 1; LS30, 19 ± 21). Compared with C mice at 20 weeks of age, SP900 (P &amp;lt; .0001) and LS30 (P &amp;lt; .05) significantly prevented the reduction in WT-1 immunoreactivity (WT-1 positive cells/glomerulus, mean ± SD: C, 7.3 ± 0.5; SP900, 10.0 ± 0.5; LS30 8.1 ± 0.8). Notably, SP900-treated mice had significantly greater WT-1 staining (P &amp;lt; .0001) and glycocalyx area (Fig. 2) than LS30 mice. CONCLUSION gddY mice treated from 4–20 weeks of age with sparsentan demonstrated a more rapid attenuation of ACR and greater protection from GS than mice treated with losartan. Sparsentan protected the glomeruli from loss of podocytes and glycocalyx to a greater extent than losartan despite equivalent lowering of BP. These results suggest that the ability of sparsentan to protect gddY mice from progression of IgAN nephropathy may go beyond effects mediated through reduction in blood pressure and antagonism of AT1R alone and may be attributed to its dual mechanism of action. If translated to the clinic, these data may support sparsentan as a new approach to the treatment of IgAN.

The Fast Track for Intestinal Tumor Cell Differentiation and In Vitro Intestinal Models by Inorganic Topographic Surfaces.

Three-dimensional (3D) complex in vitro cell systems are well suited to providing meaningful and translatable results in drug screening, toxicity measurements, and biological studies. Reliable complex gastrointestinal in vitro models as a testbed for oral drug administration and toxicity are very valuable in achieving predictive results for clinical trials and reducing animal testing. However, producing these models is time-consuming due to the lengthy differentiation of HT29 or other cells into mucus-producing goblet cells or other intestinal cell lineages. In the present work, HT29 cells were grown on an inorganic topographic surface decorated with a periodic pattern of micrometre-sized amorphous SiO2 structures for up to 35 days. HT29 cells on topographic surfaces were compared to undifferentiated HT29 in glucose-containing medium on glass or culture dish and with HT29 cells differentiated for 30 days in the presence of methotrexate (HT29-MTX). The cells were stained with Alcian blue for mucus, antibodies for mucus 2 (goblet cells), villin (enterocytes), lysozyme (Paneth cells), and FITC-labeled lectins to identify different cells, glycomic profiles, and cell features. We observed that HT29 cells on topographic surfaces showed more similarities with the differentiated HT29-MTX than with undifferentiated HT29. They formed islands of cell clusters, as observed for HT29-MTX. Already after 2 days, the first mucus secretion was shown by Alcian blue stain and FITC-wheat germ agglutinin. After 4–6 days, mucus was observed on the cell surface and in the intercellular space. The cell layer was undulated, and in 3D reconstruction, the cells showed a clear polarisation with a strong actin signal to one membrane. The lectins and the antibody-staining confirmed the heterogeneous composition of differentiated HT29 cells on topographic surfaces after 6–8 days, or after 6–8 days following MTX differentiation (30 days).

Buffalo sperm surface proteome profiling reveals an intricate relationship between innate immunity and reproduction

BackgroundLow conception rate (CR) despite insemination with morphologically normal spermatozoa is a common reproductive restraint that limits buffalo productivity. This accounts for a significant loss to the farmers and the dairy industry, especially in agriculture-based economies. The immune-related proteins on the sperm surface are known to regulate fertility by assisting the spermatozoa in their survival and performance in the female reproductive tract (FRT). Regardless of their importance, very few studies have specifically catalogued the buffalo sperm surface proteome. The study was designed to determine the identity of sperm surface proteins and to ascertain if the epididymal expressed beta-defensins (BDs), implicated in male fertility, are translated and applied onto buffalo sperm surface along with other immune-related proteins.ResultsThe raw mass spectra data searched against an in-house generated proteome database from UniProt using Comet search engine identified more than 300 proteins on the ejaculated buffalo sperm surface which were bound either by non-covalent (ionic) interactions or by a glycosylphosphatidylinositol (GPI) anchor. The singular enrichment analysis (SEA) revealed that most of these proteins were extracellular with varied binding activities and were involved in either immune or reproductive processes. Flow cytometry using six FITC-labelled lectins confirmed the prediction of glycosylation of these proteins. Several beta-defensins (BDs), the anti-microbial peptides including the BuBD-129 and 126 were also identified amongst other buffalo sperm surface proteins. The presence of these proteins was subsequently confirmed by RT-qPCR, immunofluorescence and in vitro fertilization (IVF) experiments.ConclusionsThe surface of the buffalo spermatozoa is heavily glycosylated because of the epididymal secreted (glyco) proteins like BDs and the GPI-anchored proteins (GPI-APs). The glycosylation pattern of buffalo sperm-surface, however, could be perturbed in the presence of elevated salt concentration or incubation with PI-PLC. The identification of numerous BDs on the sperm surface strengthens our hypothesis that the buffalo BDs (BuBDs) assist the spermatozoa either in their survival or in performance in the FRT. Our results suggest that BuBD-129 is a sperm-surface BD that could have a role in buffalo sperm function. Further studies elucidating its exact physiological function are required to better understand its role in the regulation of male fertility.

Intra-Amniotic Sildenafil Treatment Promotes Lung Growth and Attenuates Vascular Remodeling in an Experimental Model of Congenital Diaphragmatic Hernia

Background: Defective lung development resulting in lung hypoplasia and an attenuated and hypermuscularized pulmonary vasculature contributes to significant postnatal mortality in congenital diaphragmatic hernia (CDH). We hypothesize that deficient embryonic pulmonary blood flow contributes to defective lung development in CDH, which may therefore be ameliorated via enhancement of embryonic pulmonary blood flow. Methods: The mouse nitrofen model of CDH was utilized to measure embryonic pulmonary blood flow by in utero intracardiac injection of FITC-labeled tomato lectin and color-flow Doppler ultrasound. The effect of prenatal intra-amniotic treatment with sildenafil on survival, lung growth, and vascular morphology in the nitrofen model was determined. Results: Nitrofen-treated embryos exhibited decreased blood flow in the lung periphery compared to controls, and intra-amniotic sildenafil significantly improved embryonic pulmonary blood flow. Similar to nitrofen alone, pups delivered after nitrofen treatment and intra-amniotic injection of dextrose control exhibited respiratory distress and never survived beyond 6 h. Intra-amniotic sildenafil ameliorated respiratory distress in nitrofen-treated pups and improved postnatal survival to 82%. Following intra-amniotic sildenafil treatment at embryonic day (E)10.5, nitrofen-treated P0 lungs were larger with increased left lobe weight, reduced small pulmonary arterial wall muscularization, and increased airway branching complexity compared to controls. Intra-amniotic sildenafil treatment later at E15.5 also resulted in improved survival, lung growth, and attenuation of vascular remodeling in nitrofen-treated embryos. Conclusions: Defective embryonic pulmonary blood flow may contribute to lung maldevelopment in CDH. Enhancement of embryonic pulmonary blood flow via intra-amniotic sildenafil results in lung growth and attenuation of pulmonary vascular remodeling and may have therapeutic potential for CDH.

Effects of Sphingosine‐1‐Phosphate on Endothelial Barrier Function Following Hypoxic Injury and Hemorrhagic Shock

Our lab has previously demonstrated that S1P reduces hemorrhagic shock and resuscitation (HSR)‐induced microvascular hyperpermeability. HSR can cause poor perfusion and hypoxia in the gut, leading to endothelial apoptosis and injury to the endothelial surface layer (glycocalyx). In the current study our objective was to investigate the extent to which S1P can prevent apoptotic signaling, shedding of the (glycocalyx), and elevated microvascular permeability in response to HSR in vivo or hypoxia in a cultured endothelial cell model. For our in vivo studies, we used a rat model of HSR, in which S1P was administered in the intravenous resuscitation fluid. The mesenteric microvasculature was dissected and protein was extracted for the assessment of pro‐apoptotic proteins by western blot. We also performed immunofluorescence of the mesenteric microvasculature to evaluate paracellular junction protein interaction. As an indicator of glycocalyx health, we used an intravenous injection of FITC‐labeled lectin and measured the amount of lectin bound to the microvascular endothelium. For the in vitro studies, we placed cultured primary endothelial cells in a low‐oxygen environment and applied S1P during the hypoxic challenge. We tested whether S1P has the potential to protect barrier function of endothelial cells grown on Transwell membranes (0.4 μm pores) from hypoxia‐induced hyperpermeability by determining permeability to FITC‐albumin. We have found that HSR causes an increase in pro‐apoptotic protein BAK in vivo, but S1P fails to blunt this increase. However, S1P seems to decrease levels of cytosolic cytochrome c compared to HSR‐treated rats. Furthermore, we found that the amount of lectin bound to the mesenteric microvasculature is higher in HSR animals treated with S1P compared to HSR only. In our in vitro studies, we found that S1P prevents hypoxia‐induced permeability in different types of endothelial cells, characterized by a decrease in endothelial permeability. Taken together, our results suggest that S1P has the potential to protect microvascular barrier dysfunction caused by HSR by preventing glycocalyx shedding. However, how S1P reduces the activation of apoptotic cascade following HSR remains elusive.Support or Funding InformationSupported by NIH grants R01HL098215 and R01GM120774.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glycopattern analysis of acidic secretion in the intestine of the red-eared slender turtle; Trachemys scripta elegans (Testudines: Emydidae).

The secretion of the goblet cells in the intestine of Trachemys scripta elegans was studied in situ by histochemical methods to analyze the diversity of sugar chains, with particular regard to the acidic glycans. Conventional histochemical stains (Periodic acid-Schiff, Alcian Blue pH 2.5, High Iron Diamine) and binding with ten FITC-labelled lectins combined with chemical and enzymatic pre-treatments were used to characterize the oligosaccharidic chains. The intestine can be divided into three regions, i.e. a duodenum, a small intestine and a large intestine. Goblet cells were observed in all the three tracts and presented an acidic secretion. WGA, LFA, PNA and SBA binding was observed only after desulfation. Glycans secreted by the three tracts consist mainly of sulfosialomucins with 1,2-linked fucose, mannosylated, glucosaminylated and subterminal galactosyl/galactosaminylated residuals. Differences among tracts are quantitative rather than qualitative, with sulfated, galactosaminylated and glycosaminylated residuals increasing from duodenum to large intestine, and galactosylated and fucosylated residuals showing an opposite trend. Variation is observed also between apices and bases of villi in both duodenum and small intestine, where sulphation decreases from the base to the apex and glycosylation shows an opposite trend. Functional implication of these findings is discussed in a comparative context.

Retained fertilizing capability in cryopreserved feline spermatozoa.

Sperm cryopreservation offers a long-term preservation of male genetic materials for future assisted reproductive technologies. However, dramatic changes in temperature during freezing and thawing injure sperm cells. While motility is essential for AI and membrane integrity is crucial for in vitro fertilization (IVF), sperm DNA integrity is a common index of fertilizing capability required for AI, IVF and intracytoplasmic sperm injection (ICSI). In endangered felids died unexpectedly, attempts have been made to recover as many DNA intact spermatozoa as possible from epididymis and testis to increase the opportunity to produce offspring in future. Although sperm from caudal epididymis has shown retained fertilizing capability after freezing and thawing (27.3% conception rate after unilateral intrauterine insemination), sperm recovery from the corpus epididymis has been suggested as an alternative to increase the amount of preserved genetic materials. To improve epididymal sperm quality, pre-treatment with single-layer centrifugation resulted in selection of sperm cells with intact DNA while post-thaw treatment with extracellular ATP incubation promoted the blastocyst rate. Cold storage of domestic cat testis for 7days at 4°C demonstrated <1% of sperm cells with fragmented DNA. Moreover, isolated testicular sperm cells, stored for 7days at 4°C, produced after ICSI poorer percentages of cleavage, morula and blastocyst than the fresh control. In wild felids, a death-to-necropsy time of 2hr after a jungle cat (Felis chaus) aged 10years died during anaesthesia plus another necropsy-to-sperm recovery time of 25hr has been reported to yield the post-thawed testicular sperm with 22.2% intact DNA. In summary, the chromatin structure of feline ejaculated and epididymal sperm seems to be tolerated to cold storage and cryopreservation; thus, fertilizing capability is well protected. In contrast, the cat testicular sperm DNA is generally damaged through the cryopreservation.

Fluorescent imaging of endothelial glycocalyx layer with wheat germ agglutinin using intravital microscopy.

Endothelial glycocalyx (GCX) is located on the apical surface of vascular endothelial cells and is composed of a negatively-charged network of proteoglycans and glycoproteins. The GCX plays an important role in maintaining the integrity of vascular walls and preventing leakage of plasma. Therefore, degradation of the GCX is believed to lead to pathological leakage of plasma. Because the GCX is a very thin layer, its ultrastructural image has been demonstrated on electron microscope. To explore the function of the GCX, it should be visualized by a microscope in vivo. Thus, we developed in vivo visualization technique of the GCX under fluorescence microscopy using a mouse dorsal skinfold chamber (DSC) model. To label and visualize the GCX, we used fluorescein isothiocyanate (FITC)-labeled lectin, which has a high specificity for sugar moieties. We examined the affinity of the different lectins to epivascular regions under an intravital fluorescent microscope. Among seven different lectins we examined, FITC labeled Triticum vulgaris (wheat germ) agglutinin (WGA) delineated the GCX most clearly. Binding of WGA to the GCX was inhibited by chitin hydrolysate, which contained WGA-binding polysaccharide chains. Furthermore, the septic condition attenuated this structure, suggesting structural degradation of endothelial GCX layer. In conclusion, FITC-labeled WGA lectin enabled visualization of endothelial GCX under in vivo fluorescence microscopy.

Modulation and adherence of intestinal mucus by commensal bacteria and the pathogen C. difficile

Gut pathogens initially encounter the host at the gastrointestinal (GI) mucus layer making its regulation a crucial aspect of gut health. Few studies have addressed bacteria‐mucus adherence or mucus oligosaccharide utilization by commensal or pathogenic bacteria. To address this gap in knowledge, the interaction between mucus (human stool, HT‐29‐MTX cells and human ileal enteroids) and commensal bacteria (B. thetaiotaomicron, C. coccoides, C. butryicum, B. producta, A. muciniphila, F. prausnitzii, R. torques, L. reuteri) or the pathogen C. difficile was examined. In vitro cultures in tryptone‐yeast extract media revealed that C. difficile and B. producta were capable of using multiple types of oligosaccharides (fucose, mannose, galactose, GlcNAc, GalNAC, sialic acid) as a sole carbon source, while other commensal bacteria exhibited specific oligosaccharide preferences. Commensal bacteria had enhanced adherence to mucus from human stool compared to HT‐29‐MTX mucus. Additionally, FITC‐labeled oligosaccharide specific lectins demonstrated that all tested commensal bacteria were capable of altering mucus oligosaccharide composition. Select commensal species were also able to stimulate mucus secretion. In contrast, the pathogen C. difficile adhered to multiple oligosaccharides in human stool mucus, but had limited adherence to HT‐29‐MTX mucus. C. difficile did not stimulate mucus secretion in HT‐29‐MTX cells and decreased secretion in enteroids. Together this data demonstrates that commensal and pathogenic bacteria adhere directly to GI mucus, but this interaction is mucin type and composition dependent. Manipulation or adherence to intestinal mucus may represent a method of bacterial‐host modulation. T32DK07644

In utero intra-cardiac tomato-lectin injections on mouse embryos to gauge renal blood flow.

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis (which is the branching off of major vessels) and vasculogenesis (de novo vessel formation), perfusion mapping techniques such as resin casts, in vivo ultrasound imaging, and micro-dissection have been limited in demonstrating the intimate relationships between these two processes and developing renal structures within the embryo. Here, we describe the procedure of in utero intra-cardiac ultrasound-guided FITC-labeled tomato lectin microinjections on mouse embryos to gauge the ontogeny of renal perfusion. Tomato lectin (TL) was perfused throughout the embryo and kidneys harvested. Tissues were co-stained for various kidney structures including: nephron progenitors, nephron structures, ureteric epithelium, and vasculature. Starting at E13.5 large caliber vessels were perfused, however peripheral vessels remained unperfused. By E15.5 and E17.5, small peripheral vessels as well as glomeruli started to become perfused. This experimental technique is critical for studying the role of vasculature and blood flow during embryonic development.

The effect of AMA0428, a novel and potent ROCK inhibitor, in a model of neovascular age-related macular degeneration.

Rho kinase (ROCK) is associated with VEGF-driven angiogenesis, as well as with inflammation and fibrosis. Therefore, the effect of AMA0428, a novel ROCK inhibitor, was studied in these processes, which highly contribute to the pathogenesis of neovascular AMD. The effect of AMA0428 (0.5-5.0 μM) on human umbilical vein endothelial cells (HUVECs), human brain microvascular endothelial cells (HBMECs), and human brain microvascular pericytes (HBVPs) was determined using cell viability (WST-1), apoptosis (caspase 3/7), and migration (scratch and under-agarose) assays. The in vivo response was investigated using a laser-induced choroidal neovascularization (CNV) mouse model, in which intravitreal injections of AMA0428, murine anti-VEGF-R2 mAb (DC101), or placebo was given. Outcome was assessed by analysis of inflammation (CD45), angiogenesis (FITC-dextran), vessel leakage (Texas Red-conjugated Dextran and FITC-labeled lectin) and fibrosis (Sirius Red/Collagen I). The AMA0428 dose-dependently reduced proliferation and VEGF-induced migration of HUVEC and HBMEC (P < 0.05). No significant effect was seen on HBVP proliferation; however, migration and pericyte recruitment were enhanced (P < 0.05) by AMA0428 administration. There was no apoptosis induction. The AMA0428 significantly reduced CNV and vessel leakage 2 weeks after laser treatment, comparable to DC101. In addition, AMA0428 inhibited inflammation on day 5 by 42% (P < 0.05) and collagen deposition on day 30 by 43% (P < 0. 05), whereas DC101 had no effect on inflammation nor on fibrosis. The results suggest that targeting ROCK with AMA0428 not only reduces neoangiogenesis, but also blocks inflammation and fibrosis (contrary to VEGF suppression). These results point to a potential therapeutic benefit of ROCK inhibition in neovascular AMD.

Proteomics Characterization of Haptoglobin Alpha-2 Subunit in Non-Small Cell Lung Cancer Serum and Various Human Materials

Non-small cell lung cancer (NSCLC) is detected in all lung cancer patients and the serum samples as potential source are often used as a potential source for early diagnosis. In this study, we used the proteomic approaches combining 2-DE, 2-D DIGE and mass spectrometry to identify the potential serum biomarkers in NSCLC serum samples, and compare the result to normal serum samples. The haptoglobin alpha 2 subunit (HAP2) is one of the proteins we are interested in and showed the overexpression level in NSCLC serum amongst the NSCLC subtypes. The identity of HAP2 was confirmed by LC-MS/MS and NH2-terminal amino acid sequencing analyses, while the high expression level of HAP2 in NSCLC serum samples was validated using co-immunoprecipitation and western blotting. Interestingly, the high expression of HAP2 has specificity in human serum samples, but nonspecific expression in lung cancer tissues and is not observed in lung cancer cell lines except HepG2 hepatocellular carcinoma cell line. This indicates that HAP2 is not produced from lung tissues and/or cells, but may be secreted from the liver. In addition, HAP2 isoforms and its post-translational modifications (PTMs) could be detected by various staining methods such as phosphoprotein, glycoprotein stains and FITC-labeled lectins stain. The different carbohydrate specificities of HAP2 isoforms in NSCLC serum samples, especially sialyl Lewis x, was observed and may correlate to the development or metastasis of lung cancer. We suggest that HAP2 may become a potentially useful serum biomarker for early diagnostic and therapeutic applications.

The influence of carbohydrates in the interaction of Paracoccidioides brasiliensis with CCL-6 cells in vitro

Little is known about the early events in the interaction between Paracoccidioides brasiliensis and its host. To understand the effect of carbohydrates in the interaction between the fungus and epithelial cell in culture, we analyzed the influence of different carbohydrate solutions on the adhesion of P. brasiliensis yeast cells to CCL-6 cells in culture. Fungal cells were cultivated with the epithelial cell line, and different concentrations of D-fucose, N-acetyl-glucosamine, D-mannose, D-glucosamine, D-galactosamine, sorbitol and fructose were added at the beginning of the experiment. Six hours after the treatment, the cells were fixed and observed by light microscopy. The number of P. brasiliensis cells that were adhered to the CCL-6 monolayer was estimated. The number of adhesion events was diminished following treatments with D-fucose, N-acetyl-glucosamine, D-mannose, D-glucosamine and D-galactosamine as compared to the untreated controls. Sorbitol and fructose-treated cells had the same adhesion behavior as the observed in the control. P. brasiliensis propagules were treated with fluorescent lectins. The FITC-labeled lectins WGA and Con-A bound to P. brasiliensis yeast cells, while SBA and PNA did not. The perceptual of adhesion between P. brasiliensis and CCL-6 cells decreased with the use of D-mannose, N-acetyl-glucosamine and D-glucosamine. The assay using FITC-labeled lectins suggests the presence of N-acetyl-glucosamine, α-mannose and α-glucose on the P. brasiliensis cell surface. An enhanced knowledge of the mediators of adhesion on P. brasiliensis could be useful in the future for the development of more efficient and less harmful methods for disease treatment and control.

The mechanism of adhesion and germination in the carpospores of Porphyra spiralis var. amplifolia (Rhodophyta, Bangiales)

Spore release is the primary means of dispersion employed by red algae, and it provides insight into the elements linking the stages of their life history. In most red algae, spores are released within a sheath-like envelope of mucilage, which is responsible for their primary attachment. However, few studies have characterized the polysaccharides involved in the adhesion of seaweed spores. Therefore, in this paper, the process of spore germination and adhesion in Porphyra spiralis var. amplifolia is described, as representative of the germination pattern of the Naccaria type. Using FITC-labeled lectins, we discovered high concentrations of α- d-mannose, α- d-glucose and β- d-galactose in the mucilage. The germ tube reacted with RCA-FITC, indicating the presence β- d-galactose, and the rhizoidal cells showed the presence of α- d-mannose, α- d-glucose and β- d-galactose, indicating their importance to substrate adhesion. Using light and transmission electron microscopy, we also conducted an analysis of spore ultrastructure. We found that the differentiation of a vacuole in the spore is one of the most important processes marking the initial stage of germination. Thus, as the degree of vacuolation increases, whole cell contents move towards the germ tube, which undergoes several successive divisions forming the sporophytic phase. Therefore, we can conclude that germination in Porphyra spiralis var. amplifolia is characterized by (1) the fixation of carpospores in the substrate by sugars present in the mucilage and (2) the polarization of cell contents by the processes of vacuolization and germ tube formation.

Expression of β1,3-N-acetylglucosaminyltransferases during differentiation of human acute myeloid leukemia cells

The expressions of β1,3-N-acetylglucosamonyltransferase-2 and -8 (β3GnT-2, β3GnT-8),-the two main glycosyltransferases responsible for the synthesis of poly-N-acetyllactosamine (polyLacNAc) in glycans, and β3GnT-5 participating in the syntheses of sphingoglycolipids were studied in leukemia cell lines during differentiation using RT-PCR method. β3GnT-2 and β3GnT-8 distribute widely in six myeloid and monocytoid leukemia cell lines with different abundances, while β3GnT-4 was only present in NB4 cells. ATRA (all-trans retinoic acid) and dimethylsulfoxide (DMSO), which induce the differentiation of HL-60 and NB4 (two human acute myeloid leukemia cell lines) to myelocytic lineage, up-regulated these two enzymes with various degrees at 2 and 72 h of treatment. In HL-60 cells treated with ATRA, the increase of β3GnT-8 was more than β3GnT-2, while in NB4 cells treated with DMSO, the increase of β3GnT-2 was more than β3GnT-8. However, when HL-60 and NB4 were differentiated to monocytic lineage induced by phorbol 12-myristate 13-acetate the expressions of β3GnT-2 and β3GnT-8 showed no alterations or the increase of expressions was far less than those in myelocytic differentiation. By means of FITC-labeled tomato lectin affinity staining and flow-cytometry, it was found that the product of β3GnT-2 and -8, polyLacNAc was also increased on the cell surface of HL-60 and NB4 treated with ATRA or DMSO, but unchanged when treated with PMA. These results were in accordance with the up-regulation of the mRNAs of β3GnT-2 and -8. The expression of β3GnT-5, however, was not changed both in myelocytic and monocytic differentiations. The difference in the up-regulation of β3GnT-2 and -8, especially their products may become a useful index to discriminate the myelocytic and monocytic differentiation of leukemia cells.

Distribution of Glycoproteins on Feline Testicular Sperm, Epididymal Sperm and Ejaculated Sperm

Glycoproteins (GPs) are known to be involved in the phenomenon of sperm maturation and capacitation. In the present study, we investigated the attachment of GPs on sperm cell membrane during the process of feline sperm maturation from testicular sperm to ejaculated sperm by using 8 FITC-labeled lectins. The results showed that 3 types of GPs were presented on testicular sperm and 7 on caput epididymal sperm. Corpus and cauda epididymal sperm and ejaculated sperm had GPs detected by 8 FITC-labeled lectins used in the present study. This study demonstrates the part of the characteristic of GPs that are present on the feline sperm cell membrane during the process of sperm maturation.

Microalgal Cell Surface Carbohydrates as Recognition Sites for Particle Sorting in Suspension-Feeding Bivalves

Cell surface carbohydrates play important roles in cell recognition mechanisms. Recently, we provided evidence that particle selection by suspension-feeding bivalves can be mediated by interactions between carbohydrates associated with the particle surface and lectins present in mucus covering bivalve feeding organs. In this study, we used lectins tagged with fluorescein isothiocyanate (FITC) to characterize carbohydrate moieties on the surface of microalgal species and evaluate the effect of oyster mucus on lectin binding. These analyses revealed that concanavalin A (Con A), one of six lectins tested, bound to Isochrysis sp., while Nitzschia closterium reacted with Pisum sativum agglutinin (PNA) and peanut agglutinin (PEA). The cell surface of Rhodomonas salina bound with PNA and Con A, and Tetraselmis maculata cell surface was characterized by binding with PNA, PEA, and Con A. Pre-incubation of microalgae with oyster pallial mucus significantly decreased the binding of FITC-labeled lectins, revealing that lectins present in mucus competitively blocked binding sites. This decrease was reversed by washing mucus-coated microalgae with specific carbohydrates. These results were used to design a feeding experiment to evaluate the effect of lectins on sorting of microalgae by oysters. Crassostrea virginica fed with an equal ratio of Con A-labeled Isochrysis sp. and unlabeled Isochrysis sp. produced pseudofeces that were significantly enriched in Con A-labeled Isochrysis sp. and depleted in unlabeled microalgae. Selection occurred even though two physical-chemical surface characteristics of the cells in each treatment did not differ significantly. This work confirms the involvement of carbohydrate-lectin interaction in the particle sorting mechanism in oysters, and provides insights into the carbohydrate specificity of lectins implicated in the selection of microalgal species.