Bovine Submaxillary Mucin Research Articles

Role of sialidase in glycoprotein utilization by Tannerella forsythia

The major bacterial pathogens associated with periodontitis include Tannerella forsythia. We previously discovered that sialic acid stimulates biofilm growth of T. forsythia, and that sialidase activity is key to utilization of sialoconjugate sugars and is involved in host–pathogen interactions in vitro. The aim of this work was to assess the influence of the NanH sialidase on initial biofilm adhesion and growth in experiments where the only source of sialic acid was sialoglycoproteins or human oral secretions. After showing that T. forsythia can utilize sialoglycoproteins for biofilm growth, we showed that growth and initial adhesion with sialylated mucin and fetuin were inhibited two- to threefold by the sialidase inhibitor oseltamivir. A similar reduction (three- to fourfold) was observed with a nanH mutant compared with the wild-type. Importantly, these data were replicated using clinically relevant serum and saliva samples as substrates. In addition, the ability of the nanH mutant to form biofilms on glycoprotein-coated surfaces could be restored by the addition of purified NanH, which we show is able to cleave sialic acid from the model glycoprotein fetuin and, much less efficiently, 9-O-acetylated bovine submaxillary mucin. These data show for the first time that glycoprotein-associated sialic acid is likely to be a key in vivo nutrient source for T. forsythia when growing in a biofilm, and suggest that sialidase inhibitors might be useful adjuncts in periodontal therapy.

Genome wide analysis of the bovine mucin genes and their gastrointestinal transcription profile.

BackgroundMucins are large glycoproteins implicated in protection of all mucosal surfaces. In humans and rodents, the mucin gene family has been well described and previous studies have investigated the distribution and function of mucins in the gastrointestinal (GI) tract. In contrast, little data is available on the mucin gene family in polygastric species, such as cattle. The aim of the current study was to identify all members of the bovine mucin family by genome mining and subsequently investigate the transcription pattern of these mucins in the GI tract.ResultsNine bovine membrane-associated mucins (MUC1, MUC3A, MUC4, MUC12, MUC13, MUC15, MUC16, MUC20 and MUC21) and six secreted mucins (MUC2, MUC5AC, MUC5B, MUC6, MUC7 and MUC19) were identified in the bovine genome. No homologues could be identified for MUC3B, MUC8 and MUC17. In general, domain architecture of the membrane-associated mucins was found to be similar between humans and cattle, while the protein architecture of the gel-forming mucins appeared to be less conserved. Further analysis of the genomic organization indicated that the previously reported bovine submaxillary mucin (BSM) may be part of a larger gene encoding for MUC19. Analysis of the transcription profile showed that the secreted mucins were transcribed from the abomasum onwards, whereas the membrane associated mucins MUC1 and MUC20 were transcribed throughout the whole GI tract. In contrast to humans, MUC5B transcript was found in both the small and large intestine, but was absent in oesophageal tissue.ConclusionsThis study provides the first characterization of the mucin gene family in cattle and their transcriptional regulation in the GI tract. The data presented in this paper will allow further studies of these proteins in the physiology of the GI tract in ruminants and their interactions with pathogens.

Enhanced Bioavailability of Polyaromatic Hydrocarbons in the Form of Mucin Complexes

Increasing exposure of biological systems to large amounts of polycyclic aromatic hydrocarbons is of great public concern. Organisms have an array of biological defense mechanisms, and it is believed that mucosal gel (which covers the respiratory system, the gastrointestinal tract, etc.) provides an effective chemical shield against a range of toxic materials. However, in this work, we demonstrate, for the first time, that, upon complexation of polyaromatic hydrocarbons with mucins, enhanced bioavailability and, therefore, toxicity are obtained. This work was aimed to demonstrate how complexation of various highly hydrophobic polycyclic aromatic hydrocarbons with representative mucin glycoprotein could lead to the formation of previously undescribed materials, which exhibit increased toxicity versus pristine polycyclic aromatic hydrocarbons. In the present work, we show that a representative mucin glycoprotein, bovine submaxillary mucin, has impressive and unprecedented capabilities of binding and solubilizing water-insoluble materials in physiological solution. The complexes formed between the mucin and a series of polycyclic aromatic hydrocarbons were comprehensively characterized, and their toxicity was evaluated by both in vivo and in vitro assays. In addition, the bioavailability and membrane-penetration capabilities were tested using an internalization assay. Our results provide, for the first time, evidence of an unknown route by which hydrophobic materials may achieve higher bioavailability, penetrating some of the biological defense systems, in the form of water-soluble complexes with mucosal proteins.

Polyelectrolyte nanogels decorated with monoclonal antibody for targeted drug delivery

Novel surface-functionalized cross-linked nanogels were developed as a platform to allow conjugation of monoclonal antibodies (mAb) for targeted drug delivery. Well-defined diblock copolymers of poly(ethylene glycol)- b-poly(methacrylic acid) (PEG- b-PMA) with PEG terminal aldehyde functionality were synthesized by atom transfer radical polymerization (ATRP) and characterized by GPC and 1H NMR. These copolymers were used to prepare nanogels via condensation of PEG- b-PMA with Ca 2+ ions into micelle-like aggregates, cross-linking of the PMA/Ca 2+ cores and removal of Ca 2+ ions. The resulting nanogels represent highly swollen spherical polyelectrolyte particles with free terminal aldehyde functionalities at the nonionic PEG chains. A reductive amination reaction between aldehyde groups and amino groups of mAb resulted in effective conjugation to the nanogels of mAb CC49 against tumor-associated glycoprotein 72 (TAG-72). The mAb retained the binding affinity to bovine submaxillary mucin after conjugation as shown by surface plasmon resonance (SPR). Therefore, aldehyde-functionalized nanogels can be linked to mAb using a simple, one-step approach. They may have potential for targeted delivery of diagnostic and therapeutic agents to tumors.

Effect and Limitation of Excess Ammonium on the Release of O-Glycans in Reducing Forms from Glycoproteins under Mild Alkaline Conditions for Glycomic and Functional Analysis

Ammonium-based alkali-catalyzed β-elimination under nonreducing conditions was investigated in detail for the stability of the released mucin-type O-glycan chains with β1,3-linked cores. In contrast to the previously studied β1,4-linkage of the N-glycan-type, which was shown to be stable under the ammonium-based alkaline conditions, the β1,3-linkage is labile toward alkaline treatment and considerable peeling was observed with both model heptasaccharides and standard glycoproteins. The former include eight reducing glucoheptasaccharides with different and commonly occurring linkages (α1,2-, β1,2-, α1,3-, β1,3-, α1,4-, β1,4-, α1,6-, and β1,6-linkages), and the latter include mucin-type bovine submaxillary mucin and bovine fetuin, which contains both O- and N-glycans. The results indicated that complete prevention of peeling under nonreducing alkali-catalyzed hydrolysis conditions remains difficult. The yields of released O- and N-glycans were also assessed by use of the two glycoproteins as models. Compared with conventional procedures, Carlson degradation for O-glycan release and PNGase F digestion for N-glycan release, the nonreducing ammonium-based alkaline hydrolysis gave lower yields. Great care has to be taken when employing such nonreducing alkaline conditions in glycomic analysis and in obtaining glycoprotein glycans for functional studies.

A d-galactose-binding lectin purified from coronate moon turban, Turbo (Lunella) coreensis, with a unique amino acid sequence and the ability to recognize lacto-series glycosphingolipids

A divalent, cation-independent d-galactose-binding lectin was purified from coronate moon turban Turbo (Lunella) coreensis. This lectin recognizes d-galactose and is a 38-kDa dimeric protein consisting disulphide-bonded 22-kDa polypeptides under non-reducing and reducing conditions of sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. Haemagglutination activity was inhibited by D-galactose, N-acetyl D-galactosamine, melibiose, lactose, porcine stomach mucin, asialofetuin and bovine submaxillary mucin. The lectin has tolerance for pH 5-11 and temperature until 50°C for 1h. The lectin strongly aggregated Gram-negative bacteria, such as Vibrio parahaemolyticus and Salmonella O7, but weakly Gram-positive strain as Staphylococcus aureus and Bacillus subtilis. The glycan-binding profile of this lectin was evaluated using frontal affinity chromatography technology and the lectin appeared to recognize oligosaccharides such as lacto-series glycosphingolipids contained in blood type A and H substances in addition to complex-type N-linked glycoproteins. Partial primary structures of 139 amino acid residues of this lectin were determined from N-terminus polypeptides and 8 peptides derived by cleavage with lysyl-endopeptidase. The primary structure was slightly similar to other known sequences of lectin; however, a repeating motif has been included.

Investigation of lectinized liposomes as M-cell targeted carrier-adjuvant for mucosal immunization

In the present investigation hepatitis B surface antigen (HBsAg) encapsulated liposomes were developed and coupled with Ulex europaeus agglutinin 1 (UEA-1) to increase transmucosal uptake by M-cells of the Peyer's patches. The liposomes were characterized for shape, size, polydispersity and encapsulation efficiency. Bovine submaxillary mucin (BSM) was used as a biological model for the in vitro determination of lectin activity and specificity. Dual staining technique was used to investigate targeting of lectinized liposomes to the M-cells. Anti-HBsAg IgG response in serum and anti-HBsAg sIgA level in various mucosal fluids was estimated by using ELISA, following oral immunization with lectinized and non-lectinized liposomes in Balb/c mice. Additionally, interleukin-2 (IL-2) and interferon-γ (IFN-γ) level in the spleen homogenates was determined. The results suggest that lectinized liposomes were successfully developed, exhibited increased activity with BSM as compared to non-lectinized liposomes and α- l-fucose specificity of the lectinized liposomes was also maintained. The lectinized liposomes were predominantly targeted to the M-cells. The serum anti-HBsAg IgG titre obtained after 3 consecutive days oral immunizations with HBsAg encapsulated lectinized liposomes and boosting after third week was comparable with the titre recorded after single intramuscular prime and third week boosting with alum-HBsAg. Moreover, lectinized liposomes induced higher sIgA level in mucosal secretions and cytokines level in the spleen homogenates. The results showed that the developed surface modified liposomes could be a potential module for the development of effective mucosal vaccines.

Lectin anchored PLGA nanoparticles for oral mucosal immunization against hepatitis B

Present study aimed at exploring the potential of α-l-fucose specific, LTA (Lotus tetragonolobus from Winged or Asparagus pea) as a homing device for nanocarriers to target the M cell for elicitation of strong immune response. LTA grafted poly(lactic-co-glycolic acids) (PLGA) nanoparticles encapsulating hepatitis B surface antigen (HBsAg) was developed and characterized for shape, size, polydispersity index, zeta potential, and antigen loading efficiency. The peyer’s patch uptake was studied by using confocal laser scanning microscopy technique using dual staining technique. The immune stimulating potential was assessed by measuring anti-HBsAg titer in serum of balb/c mice. Induction of the mucosal immunity was assessed by estimating secretory immunoglobulin A level in the salivary, intestinal, and vaginal secretion and cytokine (interleukin-2 and interferon-γ) levels in the spleen homogenates. Furthermore, IgG1 and IgG2a isotype were determined to confirm the TH1/TH2 mixed immune response. The LTA anchored PLGA nanoparticles have demonstrated approximately four-fold increase in the degree of interaction with the bovine submaxillary mucin (BSM). The results demonstrated that LTA anchored PLGA nanoparticles elicited strong mucosal and systemic response and hence could be a promising carrier adjuvant for the M cell targeted oral mucosal immunization against Hepatitis B.

Adhesion of microorganisms to bovine submaxillary mucin coatings: effect of coating deposition conditions

The adhesion of Staphylococcus epidermidis, Escherichia coli, and Candida albicans on mucin coatings was evaluated to explore the feasibility of using the coating to increase the infection resistance of biomaterials. Coatings of bovine submaxillary mucin (BSM) were deposited on a base layer consisting of a poly(acrylic acid-b-methyl methacrylate) (PAA-b-PMMA) diblock copolymer. This bi-layer system exploits the mucoadhesive interactions of the PAA block to aid the adhesion of mucin to the substratum, whereas the PMMA block prevents dissolution of the coating in aqueous environments. The thickness of the mucin coating was adjusted by varying the pH of the solution from which it was deposited. Thin mucin coatings decreased the numbers of bacteria but increased the numbers of C. albicans adhering to the copolymer and control surfaces. Increasing the mucin film thickness resulted in a further lowering of the density of adhering S. epidermidis cells, but it did not affect the density of E. coli. In contrast, the density of C. albicans increased with an increase in mucin thickness.

Purification and Characterization of a Novel Thermostable Mycelial Lectin from Aspergillus terricola

Lectin has been isolated from mycelia of Aspergillus terricola by single step purification on porcine stomach mucin-Sepharose 4B affinity column. Lectin could be effectively purified with 75% recovery and 4.47-fold increase in specific activity. Lectin migrated as a single band on SDS-PAGE with an apparent molecular mass of 32.5 kDa. Sugar inhibition assay revealed that the lectin did not strongly interact with most carbohydrates and their derivatives tested while strong binding affinity to D-glucose, D-sucrose, N-acetyl-D-galactosamine, asialofetuin, porcine stomach mucin, and bovine submaxillary mucin was indicated. Neuraminidase and protease treatment to erythrocytes enhanced lectin titre. Lectin activity was stable within the pH range of 7.0-10.5. A. terricola lectin displayed remarkable thermostability and remained unaffected upon incubation at 70 degrees C for 2.5 h. Lectin did not require metal ions for its activity. Incubation with denaturants (urea, thiourea, and guanidine-HCl) substantially reduced lectin activity. Carbohydrate analysis revealed that it is a glycoprotein with 9.76% total sugars.

Purification, characterization and cDNA cloning of a novel lectin from the jellyfish Nemopilema nomurai

A lectin (designated NnL) from a jellyfish Nemopilema nomurai was purified by ion-exchange chromatography followed by affinity chromatography. The apparent molecular mass of NnL was 28kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. NnL agglutinated horse erythrocytes, the gram-positive bacterium Bacillus subtilis and gram-negative bacterium Escherichia coli K12. The hemagglutinating activity of NnL was inhibited by N-acetyl-D-galactosamine N-acetylneuraminic acid and N-glycolylneuraminic acid. Bovine submaxillary mucin was the potent inhibitor of the hemagglutinating activity of NnL among the glycoproteins tested. cDNA cloning of NnL revealed that its primary structure contained part of a fibrinogen-like domain. The deduced amino acid sequence of NnL showed no significant sequence identities to other known lectins. On the other hand NnL showed high sequence identity to a predicted protein of the sea anemone Nematostella vectensis suggesting that NnL may belong to a novel lectin family in metazoans. This is the first report to describe the purification characterization and cDNA cloning of a jellyfish lectin.

Exogenous incorporation of neugc-rich mucin augments n-glycolyl sialic acid content and promotes malignant phenotype in mouse tumor cell lines

BackgroundCarbohydrates embedded in the plasma membrane are one of the main actors involved in the communication of cells with the microenvironment. Neuraminic sialic acids are glycocalyx sugars that play important roles in the modulation of malignant cell behaviour. N-glycolylneuraminic acid (NeuGc) is synthesized by the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH), an enzyme expressed in all mammals except humans. In mice, this sugar is synthesized in several somatic tissues.MethodsWe used the B16 melanoma and F3II mammary carcinoma mouse tumor cell lines. By CMAH directed RT-PCR and NeuGc detection with the specific anti-NeuGc-GM3 antibody 14F7 we evaluated enzyme and ganglioside expression in tumor cells, respectively. Expression of NeuGc-GM3 ganglioside was reached by in vitro incubation with NeuGc-rich bovine submaxillary mucin and evaluated by slot-blot and immunohistochemistry assays using the 14F7 antibody. Tumor cells treated with mucin or purified NeuGc were injected s.c. and i.v. in syngeneic mice to evaluate tumor and metastatic growth.ResultsIn the present work we demonstrated the absence of expression of CMAH enzyme in B16 melanoma and F3II mammary carcinoma cells. In vitro incubation of these NeuGc-negative cells with NeuGc-rich mucin increased the presence of NeuGc in cell membranes for at least 48-72 h, as a component of the GM3 ganglioside. Preincubation with NeuGc-rich mucin reduced tumor latency and increased the metastatic potential of tumor cells in syngeneic animals. Similar results were obtained when cells were incubated with purified NeuGc alone.ConclusionOur results indicate that B16 and F3II mouse tumor cell lines do not express NeuGc in cell membranes but they are able to incorporate NeuGc from an exogenous source, contributing to the malignant phenotype of melanoma and mammary carcinoma cells.

Expression of active human sialyltransferase ST6GalNAcI in Escherichia coli

BackgroundThe presence of terminal, surface-exposed sialic acid moieties can greatly enhance the in vivo half-life of glycosylated biopharmaceuticals and improve their therapeutic efficacy. Complete and homogeneous sialylation of glycoproteins can be efficiently performed enzymically in vitro but this process requires large amounts of catalytically active sialyltransferases. Furthermore, standard microbial hosts used for large-scale production of recombinant enzymes can only produce small quantities of glycosyltransferases of animal origin, which lack catalytic activity.Results and conclusionIn this work, we have expressed the human sialyltransferase ST6GalNAc I (ST6), an enzyme that sialylates O-linked glycoproteins, in Escherichia coli cells. We observed that wild-type bacterial cells are able to produce only very small amounts of soluble ST6 enzyme. We have found, however, that engineered bacterial strains which possess certain types of oxidative cytoplasm or which co-express the molecular chaperones/co-chaperones trigger factor, DnaK/DnaJ, GroEL/GroES, and Skp, can produce greatly enhanced amounts of soluble ST6. Furthermore, we have developed a novel high-throughput assay for the detection of sialyltransferase activity and used it to demonstrate that the bacterially expressed ST6 enzyme is active and able to transfer sialic acid onto a desialylated O-glycoprotein, bovine submaxillary mucin. To the best of our knowledge, this is the first example of expression of active human sialyltransferase in bacteria. This system may be used as a starting point for the evolution of sialyltransferases with better expression characteristics or altered donor/acceptor specificities.

Characterization of a new lectin involved in the protoplast regeneration of Bryopsis hypnoides

A group of coenocytic marine algae differs from higher plants, whose totipotency depends on an intact cell (or protoplast). Instead, this alga is able to aggregate its extruded protoplasm in sea water and generate new mature individuals. It is thought that lectins play a key role in the aggregation process. We purified a lectin associated with the aggregation of cell organelles in Bryopsis hypnoides. The lectin was ca. 27 kDa with a pI between pH 5 and pH 6. The absence of carbohydrate suggested that the lectin was not a glycoprotein. The hemagglutinating activity (HA) of the lectin was not dependent on the presence of divalent cations and was inhibited by N-Acetylgalactosamine, N-Acetylglucosamine, and the glycoprotein bovine submaxillary mucin. The lectin preferentially agglutinated Gram-negative bacterium. The HA of this lectin was stable between pH 4 to pH 10. Cell organelles outside the cytoplasm were agglutinated by the addition of lectin solution (0.5 mg ml−1). Our results suggest that the regeneration of B. hypnoides is mediated by this lectin. We also demonstrated that the formation of cell organelle aggregates was inhibited by nigericin in natural seawater (pH 8.0). Given that nigericin dissipates proton gradients across the membrane, we hypothesize that the aggregation of cell organelles was proton-gradient dependent.

Organic Reactions Promoted by Mucin Glycoproteins

Mucins are heavily glycosylated proteins that exhibit a broad range of adhesive interactions with various hydrophobic organic materials. However, there has been no clear evidence that mucins are capable of promoting chemical reactions. Here we provide the first demonstration that bovine submaxillary mucin and porcine gastric mucin accelerate the rate of fatty acid p-nitrophenol ester hydrolysis up to 337 times and a carbon-carbon bond-forming Diels-Alder reaction between N-propylmaleimide and anthracene up to 200 times relative to the rates of the reference processes, which were performed in water and chloroform, respectively. The discovered property of mucins to accelerate organic reactions provides a new and unique example of natural nonenzymatic proteins capable of promoting reactions of hydrophobic materials in aqueous solution. A better understanding of the interactions of mucins with hydrophobic molecules is needed for the development of superior drug-delivery systems and implantable devices.

Spectroscopic Ellipsometry of Mucin Layers on an Amphiphilic Diblock Copolymer Surface

Both visible and infrared (IR) spectroscopic ellipsometry have been employed to study the structure of thin layers of bovine submaxillary mucin (BSM) adsorbed on poly(acrylic acid-block-methyl methacrylate) (PAA-b-PMMA) copolymer and poly(methyl methacrylate) (PMMA) surfaces at three pH values (3, 7, and 10). The adsorbed mucin layer on the copolymer surface had the greatest thickness (17 nm) when adsorbed from a mucin solution at a pH of 3. For the first time, IR ellipsometry was used to identify adhesive interactions and conformational changes in mucin/polymer double layers. After applying the regularized method of deconvolution in the analysis, the formation of hydrogen bonds between the carboxyl groups of the BSM and PAA-b-PMMA copolymer in double layers has been found. The IR ellipsometry data, in agreement with the visible ellipsometry analysis, indicate the pH dependence of adhesion of mucin to the copolymer surface. There is an increase in the amount of hydrogen-bonded carboxyl groups in mucin deposited at a pH of 3. There is no evidence that the amide groups of the mucin participate in this bonding. At the lower pH, the IR ellipsometry spectra after deconvolution reveal an increase in the proportion of beta-sheets in the BSM upon adsorption on the copolymer surface, indicating a more unfolded, aggregated structure. The IR ellipsometry data also indicated some changes in the conformational states of the side groups in the copolymer induced by entanglements and bonding interactions with the mucin macromolecules. Deconvolution provides an unprecedented level of information from the IR ellipsometry spectra and yields important insights.

Ion mobility-mass spectrometry analysis of isomeric carbohydrate precursor ions

The rapid separation of isomeric precursor ions of oligosaccharides prior to their analysis by mass spectrometry to the nth power (MS(n)) was demonstrated using an ambient pressure ion mobility spectrometer (IMS) interfaced with a quadrupole ion trap. Separations were not limited to specific types of isomers; representative isomers differing solely in the stereochemistry of sugars, in their anomeric configurations, and in their overall branching patterns and linkage positions could be resolved in the millisecond time frame. Physical separation of precursor ions permitted independent mass spectra of individual oligosaccharide isomers to be acquired to at least MS(3), the number of stages of dissociation limited only practically by the abundance of specific product ions. IMS-MS(n) analysis was particularly valuable in the evaluation of isomeric oligosaccharides that yielded identical sets of product ions in tandem mass spectrometry experiments, revealing pairs of isomers that would otherwise not be known to be present in a mixture if evaluated solely by MS dissociation methods alone. A practical example of IMS-MS(n) analysis of a set of isomers included within a single high-performance liquid chromatography fraction of oligosaccharides released from bovine submaxillary mucin is described.

Comparison of the adsorption kinetics and surface arrangement of “as received” and purified bovine submaxillary gland mucin (BSM) on hydrophilic surfaces

The effect of bovine serum albumin (BSA) as impurity in a commercial bovine submaxillary gland mucin preparation (BSM; Sigma M3895) on the adsorption of BSM to hydrophilic surfaces (mica and silica) has been studied in terms of adsorption kinetics, amount and structure of the formed adlayer. The Surface Force Apparatus (SFA) was used to gain information about the extended and compressed structure of adsorbed “as received” BSM, purified BSM, BSA extracted from the “as received” BSM and mixtures of the latter purified proteins. The adsorbed amount was estimated using a combination of X-ray Photoelectron Spectroscopy (XPS), Enzyme-Linked Immuno Sorbent Assay (ELISA), Enzyme-Linked Lectin Assay (ELLA), Dual Polarization Interferometry (DPI) and Quartz Crystal Microbalance (QCM-D) measurements. Under the used conditions, purified BSM showed very low affinity for silica and only small amounts were found to adsorb on mica. Initially, the BSM molecules adopted an extended conformation on the mica surface with tails extending into the bulk phase. These tails were irreversibly compressed into a very thin (10 Å) layer upon applying a high load. “As received” BSM formed considerably thicker compressed layers (35 Å); however, the extended layer structure was qualitatively the same. When mixtures of purified BSM and BSA were coadsorbed on mica, a 9 wt-% albumin content gave a comparable layer thickness as the “as received” BSM and from XPS data we draw the conclusion that the albumin content in the layer adsorbed from “as received” BSM was approximately 5 wt-%. Adsorption from an equal amount of BSM and BSA revealed that even though the amount of BSM is scarce in the mixed layer, the few BSM molecules have a drastic effect on the adsorbed thickness and structure. Clearly, this study shows the importance of characterizing the mucin used since differences in purity give rise to different adsorption behaviours in terms of both adsorbed amount and layer structure.

Size-exclusion chromatography combined with small-angle X-ray scattering optics

Size-exclusion chromatography with on-line synchrotron radiation solution small-angle X-ray scattering optics, absorbance and/or refractive index detectors was evaluated by protein characterizations. The radius of gyration value and zero-angle scattering intensity of protein molecules eluted from the chromatography column were estimated using this measurement system. In addition, the characterization of the conformation of the eluted proteins was demonstrated for hen egg lysozyme and bovine submaxillary mucin. The present technique will be useful for not only the determination of the radius of gyration value and molecular weight of proteins with dimensions of 1–10 nm, but also for the structural characterization of the macromolecules during the chromatography.

Development and characterization of lectin-functionalized vesicular constructs bearing amphotericin B for bio-film targeting

The long-term goal of this work will be to develop a topical formulation for oropharyngeal candidosis. Liposomes were prepared by the vesicle extrusion technique from mixtures of dipalmitoylphosphatidylcholine, cholesterol, and stearylamine incorporating a reactive phospholipid, the m-maleimidobenzoyl-N-hydroxysuccinimide ester derivative of dipalmitoylphosphatidylethanolamine, which was conjugated with the N-succinimidyl-S-acetylthioacetate derivatives of succinyl concanavalin A. Morphology of liposomes was studied by transmission electron microscopy and bio-film architecture using fluorescence microscopy. Lectinized vesicles were put into contact with bovine submaxillary mucin, to determine the in vitro activity and specificity. Targeting study was performed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The liposomes were found to have a diameter in the range of 360–450 and 390–510 nm for uncoated and coated formulations, respectively. The MTT assay showed a strong association of lectin-bearing liposomes with candidal bio-film. The results suggest that surface-modified liposomes can effectively target candidal bio-film in vitro.