In vivo and in vitro characterization of novel microparticulates based on hyaluronan and chitosan hydroglutamate

This study examined the application of previously characterized microparticles composed of hyaluronan (HA) and chitosan hydroglutamate (CH) as well as novel microparticles consisting of both polymers (HA/CH) to improve the nasal delivery of a model drug. The rabbit bioavailabilities of gentamicin incorporated in HA, CH, and HA/CH microparticles were increased 23-, 31-, and 42-fold, respectively, compared with the control intranasal solution of gentamicin, indicating that all test microparticles were retained for longer periods on the nasal mucosa of the rabbits as supported by previous in vitro dissolution as well as frog palate mucoadhesion studies, thereby improving drug absorption. The higher bioavailabilities of CH-based formulations (CH and HA/CH) suggest the penetration-enhancing effects of CH may also be partially responsible for the improvement. A model was developed, based on a glass impinger device, to deliver dry powder formulations reproducibly onto the surface of cultured cell monolayers. In vitro permeability and fluorescence microscopy studies on the tight junctions of the 16HBE14o- cell lines further confirmed the ability of CH-based formulations to enhance penetration. Furthermore, the in vitro absorption profile from cell culture studies was consistent with those determined from in vivo studies. The complementary effect from the mucoadhesive nature of HA coupled with the penetration-enhancing effects of CH makes the novel HA/CH formulation a promising nasal delivery system.     

Characterization of porous PLGA/PLA microparticles as a scaffold for three dimensional growth of breast cancer cells

We have designed and evaluated biodegradable porous polymeric microparticles as a scaffold for cell growth. The hypothesis was that microparticles with optimized composition and properties would have better cell adhesion and hence cell growth into a tissue-like structure. Solvent-evaporation method was modified using sucrose as an additive to form large porous microparticles of poly(D,L-lactic-co-glycolic) (PLGA) and polylactide (PLA) polymers. Microparticles containing hydrophilic polymers (poly(vinyl alcohol) and chitosan) incorporated in their internal matrix structure were also formulated. Different formulations of microparticles were evaluated for physical properties, cell adhesion, and cell growth in culture. PLA microparticles containing poly(vinyl alcohol) (PVA) in the matrix structure (PLA-PVA) and treated with serum prior to cell seeding demonstrated better cell adhesion and cell growth than other formulations of microparticles. Cells were seen to grow into clumps, engulfing microparticles completely with time, and forming a 3-D tissue-like structure. Cell density of 1.5 x 10(6) cells per mg of microparticles was achieved in 9 days of culture, which was a 7-fold increase from the initial seeding cell density. The mechanism of better cell growth on PLA-PVA microparticles appears to be due to the PVA associated with the internal matrix structure of microparticles. These microparticles demonstrated better wetting in culture and also cell adhesion. In addition to tissue engineering applications, microparticles with cancer cells grown into a tissue-like structure in vitro can be potentially used as a model system for preclinical evaluation of the cytotoxic effect of anticancer agents.     

Mucoadhesive Properties of Eudragit®RS100, Eudragit®S100, and Poly(ε-caprolactone) Nanocapsules: Influence of the Vehicle and the Mucosal Surface

The use of polymers as mucoadhesive materials has been explored in several drug delivery systems. It is well known that the resulting mucoadhesiveness not only depends on the polymers by themselves, but also on the way they are delivered and on the application target. However, little attention has been given to the combined effect of such characteristics. Therefore, the objective of this study is to analyze the mucoadhesion resulting from combined effects of nanocapsules produced with polymers of different ionic properties, Eudragit®RS100, Eudragit®S100, or poly(ε-caprolactone), when they are incorporated into different vehicles (suspension, hydrogel, and powder) and applied on different mucosal surfaces (mucin, porcine vaginal, and buccal mucosa). Mucoadhesion was measured by a tensile stress tester. Our findings show that polymeric self-assembling as nanocapsules improved the mucoadhesion of the polymers. Eudragit®RS100 nanocapsules have the best performance, independently of the vehicle and surface used. Regarding the vehicle, hydrogels showed higher adhesion when compared to suspensions and powders. When considering different types of surfaces, mucin presented a similar pattern like the animal mucosa, but it overestimated the mucoadhesiveness of all formulations. In conclusion, this study demonstrated that the best strategy to achieve high mucoadhesive formulations is by incorporating Eudragit®RS100 nanocapsules in hydrogels. Moreover, mucin is a suitable substrate to compare and screen different formulations but not as a conclusive estimation of the mucoadhesion values that can be achieved. These results are summarized in a decision tree that can help to understand different strategies of combination of these factors and the expected outcomes.     

Identification of a Surface Protein from <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> JCM1081 That Adheres to Porcine Gastric Mucin and Human Enterocyte-Like HT-29 Cells

Adhesion of lactobacilli to the host gastrointestinal (GI) tract is considered an important factor in health-promoting effects. However, studies addressing the molecular mechanisms of the adhesion of lactobacilli to the host GI tract have not yet been performed. The aim of this work was to identify Lactobacillus reuteri surface molecules mediating adhesion to intestinal epithelial cells and mucins. Nine strains of lactobacilli were tested for their ability to adhere to human enterocyte-like HT-29 cells. The cell surface proteins involved in the adhesion of Lactobacillus to HT-29 cells and gastric mucin were extracted. The active fractions were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with horseradish peroxidase-labeled mucin and NHS-Biotin-labeled HT-29 cells. Furthermore, tandem mass spectrometry analysis was performed to identify the surface protein that participates in adhesion. It was shown that the ability of lactobacilli to adhere to HT-29 cells in vitro varied considerably among different strains. The most adhesive strain was the chicken intestinal tract isolate Lactobacillus reuteri JCM1081 (495.07 +/- 80.03 bacterial cells/100 HT-29 cells). The adhesion of L. reuteri JCM1081 to HT-29 cells appeared to be mediated by a cell surface protein, with an approximate molecular mass of 29 kDa. The peptides generated from the 29-kDa protein significantly matched the Lr0793 protein sequence of L. reuteri strain ATCC55730 (~71.1% identity) and displayed significant sequence similarity to the putative ATP-binding cassette transporter protein CnBP.     

Amine-reactive biodegradable diblock copolymers

A new class of diblock copolymers was synthesized from biodegradable poly(lactic acid) and poly(ethylene glycol)minus signmonoamine. These polymers were activated by covalently attaching linkers such as disuccinimidyl tartrate or disuccinimidyl succinate to the hydrophilic polymer chain. The polymers were characterized by (1)H NMR spectroscopy, (13)C NMR spectroscopy and gel permeation chromatography (GPC). These investigations indicated that the polymers were obtained with the correct composition, in high purities, and the expected molecular weight. By using dyes containing primary amine groups such as 5-aminoeosin as model substrates, it was possible to show that the polymers are able to bind such compounds covalently. The diblock copolymers were developed to suppress unspecific protein adsorption and allow the binding of bioactive molecules by instant surface modification. The polymers are intended to be used for tissue engineering applications where surface immobilized cell adhesion peptides or growth factors are needed to control cell behavior.     

Selective adhesion of functional microtubules to patterned silane surfaces.

We show that microtubule polymers can be immobilized selectively on lithographically patterned silane surfaces while retaining their native properties. Silane films were chemisorbed on polished silicon wafers or glass coverslips and patterned using a deep UV lithographic process developed at the Naval Research Laboratory. Hydrocarbon and fluorocarbon alkyl silanes, as well as amino and thiol terminal alkyl silanes, were investigated as substrates for microtubule adhesion with retention of biological activity. Microtubules were found to adhere strongly to amine terminal silanes while retaining the ability to act as substrates for the molecular motor protein kinesin. Aminosilane patterns with linewidths varying from 1 to 50 microns were produced lithographically and used to produce patterns of selectively adhered microtubules. Microtubules were partially aligned on the patterned lines by performing the immobilization in a fluid flow field. Patterns were imaged with atomic force microscopy and differential interference contrast microscopy. Motility assays were carried out using kinesin-coated beads and observed with differential interference contrast microscopy. Kinesin bead movement on the patterned microtubules was comparable to movement on microtubule control surfaces.     

Polymer coated liposomes for use in the oral cavity – a study of the in vitro toxicity,effect on cell permeability and interaction with mucin

In this study we investigated the in vitro toxicity, impact on cell permeability and mucoadhesive potential of polymer-coated liposomes intended for use in the oral cavity. A TR146 cell line was used as a model. The overall aim was to end up with a selection of safe polymer coated liposomes with promising mucoadhesive properties for drug delivery to the oral cavity. The following polymers were tested: chitosan, low-methoxylated pectin (LM-pectin), high-methoxylated pectin (HM-pectin), amidated pectin (AM-pectin), Eudragit, poly(N-isopropylacrylamide-co-methacrylic acid) (p(NIPAAM-co-MAA)), hydrophobically modified hydroxyethyl cellulose (HM-HEC), and hydrophobically modified ethyl hydroxyethyl cellulose (HM-EHEC). With chitosan as an exception, all the systems exhibited no significant effect on cell viability and permeability at the considered concentrations. Additionally, all the formulations showed to a varying degree an interaction with mucin (BSM type I-S); the positively charged formulations exhibited the strongest interaction, while the negatively and neutrally charged formulations displayed a moderate or low interaction. The ability to interact with mucin makes all the liposomal formulations promising for oromucosal administration. Although the chitosan-coated liposomes affected the cell viability, this formulation also influenced the cell permeability, which makes it an interesting candidate for systemic drug delivery from the oral cavity.     

Bacterial adhesion at synthetic surfaces.

A systematic investigation into the effect of surface chemistry on bacterial adhesion was carried out. In particular, a number of physicochemical factors important in defining the surface at the molecular level were assessed for their effect on the adhesion of Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli. The primary experiments involved the grafting of groups varying in hydrophilicity, hydrophobicity, chain length, and chemical functionality onto glass substrates such that the surfaces were homogeneous and densely packed with functional groups. All of the surfaces were found to be chemically well defined, and their measured surface energies varied from 15 to 41 mJ. m(-2). Protein adsorption experiments were performed with (3)H-labelled bovine serum albumin and cytochrome c prior to bacterial attachment studies. Hydrophilic uncharged surfaces showed the greatest resistance to protein adsorption; however, our studies also showed that the effectiveness of poly(ethyleneoxide) (PEO) polymers was not simply a result of its hydrophilicity and molecular weight alone. The adsorption of the two proteins approximately correlated with short-term cell adhesion, and bacterial attachment for L. monocytogenes and E. coli also correlated with the chemistry of the underlying substrate. However, for S. aureus and S. typhimurium a different pattern of attachment occurred, suggesting a dissimilar mechanism of cell attachment, although high-molecular-weight PEO was still the least-cell-adsorbing surface. The implications of this for in vivo attachment of cells suggest that hydrophilic passivating groups may be the best method for preventing cell adsorption to synthetic substrates provided they can be grafted uniformly and in sufficient density at the surface.     

Enhancement of Bioavailability of Cefpodoxime Proxetil Using Different Polymeric Microparticles

Poorly water-soluble drugs such as cefpodoxime proxetil (400 μg/ml) offer a challenging problem in drug formulation as poor solubility is generally associated with poor dissolution characteristics and thus poor oral bioavailability. According to these characteristics, preparation of cefpodoxime proxetil microparticle has been achieved using high-speed homogenization. Polymers (methylcellulose, sodium alginate, and chitosan) were precipitated on the surface of cefpodoxime proxetil using sodium citrate and calcium chloride as salting-out agents. The pure drug and the prepared microparticles with different concentrations of polymer (0.05–1.0%) were characterized in terms of solubility, drug content, particle size, thermal behavior (differential scanning calorimeter), surface morphology (scanning electron microscopy), in vitro drug release, and stability studies. The in vivo performance was assessed by pharmacokinetic study. The dissolution studies demonstrate a marked increase in the dissolution rate in comparison with pure drug. The considerable improvement in the dissolution rate of cefpodoxime proxetil from optimized microparticle was attributed to the wetting effect of polymers, altered surface morphology, and micronization of drug particles. The optimized microparticles exhibited excellent stability on storage at accelerated condition. The in vivo studies revealed that the optimized formulations provided improved pharmacokinetic parameter in rats as compared with pure drug. The particle size of drug was drastically reduced during formulation process of microparticles.     

Interaction of N-acylated and N-alkylated chitosans included in liposomes with lipopolysaccharide of gram-negative bacteria

The interactions of lipopolysaccharide (LPS) with the polycation chitosan and its derivatives — high molecular weight chitosans (300 kDa) with different degree of N-alkylation, its quaternized derivatives, N-monoacylated low molecular weight chitosans (5.5 kDa) — entrapped in anionic liposomes were studied. It was found that the addition of chitosans changes the surface potential and size of negatively charged liposomes, the magnitudes of which depend on the chitosan concentration. Acylated low molecular weight chitosan interacts with liposomes most effectively. The binding of alkylated high molecular weight chitosan with liposomes increases with the degree of its alkylation. The analysis of interaction of LPS with chitoliposomes has shown that LPS-binding activity decreased in the following order: liposomes coated with a hydrophobic chitosan derivatives > coated with chitosan > free liposomes. Liposomes with N-acylated low molecular weight chitosan bind LPS more effectively than liposomes coated with N-alkylated high molecular weight chitosans. The increase in positive charge on the molecules of N-alkylated high molecular weight chitosans at the cost of quaternization does not lead to useful increase in efficiency of binding chitosan with LPS. It was found that increase in LPS concentration leads to a change in surface ζ-potential of liposomes, an increase in average hydrodynamic diameter, and polydispersity of liposomes coated with N-acylated low molecular weight chitosan. The affinity of the interaction of LPS with a liposomal form of N-acylated chitosan increases in comparison with free liposomes. Computer simulation showed that the modification of the lipid bilayer of liposomes with N-acylated low molecular weight chitosan increases the binding of lipopolysaccharide without an O-specific polysaccharide with liposomes due to the formation of additional hydrogen and ionic bonds between the molecules of chitosan and LPS.     

In vivo degradation and tissue response to poly(5-ethylene ketal ε-caprolactone-co-D,L-lactide)

Low molecular weight poly(5-ethylene ketal ε-caprolactone-co-D,L-lactide) (PEKCDLLA) is being considered as a viscous liquid, injectable depot for localized drug delivery. This polymer degrades in vitro via surface erosion, which is potentially advantageous for the proposed application. However, the in vivo degradation rate and mechanism, and tissue response, to polymers based on 5-ethylene ketal ε-caprolactone have not yet been reported. The purpose of this study was to measure the in vivo weight loss and change in polymer properties and assess the tissue response to PEKCDLLA after subcutaneous injection in rats. The tissue response was assessed histologically using Masson's trichrome staining and immunohistochemically by staining for CD68 positive cells. The polymer lost weight with time in a nearly linear fashion but did not exhibit significant changes in number average molecular weight, polydispersity index, and glass transition temperature or monomer ratio, consistent with a surface erosion process. The tissue response to the polymer was moderate and comparable to that reported in the literature for other degradable polymers used in clinical applications. These findings indicate that PEKCDLLA is a promising candidate for injectable drug delivery.     

Drying Using Supercritical Fluid Technology as a Potential Method for Preparation of Chitosan Aerogel Microparticles

Supercritical fluid technology offers several advantages in preparation of microparticles. These include uniformity in particle size, morphology, and drug distribution without degradation of the product. One of the recent advantages is preparation of porous aerogel carrier with proper aerodynamic properties. In this study, we aimed to prepare chitosan aerogel microparticles using supercritical fluid (SCF) technology and compare that with microparticles produced by freeze drying (FD). Loading the prepared carriers with a model drug (salbutamol) was also performed. Comparisons of the particle properties and physicochemical characterizations were undertaken by evaluating particle size, density, specific surface area, and porosity. In vitro drug release studies were also investigated. The effect of many variables, such as molecular weight of chitosan oligomers, concentrations of chitosan, and concentrations of tripolyphosphate on the release, were also investigated. Chitosan aerogels were efficiently produced by SCF technology with an average particle size of 10 μm with a tapped density values around 0.12 g/mL, specific surface area (73–103) m²/g, and porosity (0.20–0.29) cc/g. Whereas, microparticles produced by FD method were characterized as cryogels with larger particle size (64 microns) with clear cracking at the surface. Sustained release profile was achieved for all prepared microparticles of salbutamol produced by the aforementioned methods as compared with pure drug. The results also demonstrates that chitosan molecular weight, polymer concentration, and tripolyphosphate concentration affected the release profile of salbutamol from the prepared microparticles. In conclusion, SCF technology was able to produce chitosan aerogel microparticles loaded with salbutamol that could be suitable for pulmonary drug delivery system.KEY WORDS: aerodynamic, aerogels, chitosan, salbutamol, supercritical fluid technology     

Bacterial Adhesion at Synthetic Surfaces

A systematic investigation into the effect of surface chemistry on bacterial adhesion was carried out. In particular, a number of physicochemical factors important in defining the surface at the molecular level were assessed for their effect on the adhesion of Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli. The primary experiments involved the grafting of groups varying in hydrophilicity, hydrophobicity, chain length, and chemical functionality onto glass substrates such that the surfaces were homogeneous and densely packed with functional groups. All of the surfaces were found to be chemically well defined, and their measured surface energies varied from 15 to 41 mJ · m⁻². Protein adsorption experiments were performed with ³H-labelled bovine serum albumin and cytochrome c prior to bacterial attachment studies. Hydrophilic uncharged surfaces showed the greatest resistance to protein adsorption; however, our studies also showed that the effectiveness of poly(ethyleneoxide) (PEO) polymers was not simply a result of its hydrophilicity and molecular weight alone. The adsorption of the two proteins approximately correlated with short-term cell adhesion, and bacterial attachment for L. monocytogenes and E. coli also correlated with the chemistry of the underlying substrate. However, for S. aureus and S. typhimurium a different pattern of attachment occurred, suggesting a dissimilar mechanism of cell attachment, although high-molecular-weight PEO was still the least-cell-adsorbing surface. The implications of this for in vivo attachment of cells suggest that hydrophilic passivating groups may be the best method for preventing cell adsorption to synthetic substrates provided they can be grafted uniformly and in sufficient density at the surface.     

Polyelectrolyte Complexes: Interactions between Lignosulfonate and Chitosan

The interactions between high molecular weight chitosans (fraction of acetylated units (F(A)) = 0.10 or 0.50) and lignosulfonates of varying molecular weights (5000-400000 g/mol) and degrees of sulfonation (0.39-0.64) were studied. Lignosulfonates and chitosans form primarily insoluble polyelectrolyte complexes when mixed at pH 4.5, where the polymers are oppositely charged. In contrast, no complex formation occurred at pH 8, as shown by using a chitosan with F(A) = 0.50, which is soluble at this pH. Thus, a positively charged chitosan is a prerequisite for interactions leading to insoluble complexes with lignosulfonates. It is therefore unlikely that complex formation involves the formation of covalent sulfonylamide linkages as proposed in the literature. The composition of the complexes varied to some degree with the mixing ratio and molecular weight of lignosulfonate, but in most cases compact complexes with a sulfonate/amino ratio close to 1.0 were formed, suggesting that all sulfonate groups are accessible for interactions with chitosan. The influence of the ionic strength and temperature on the complex formation and the behavior of the precipitated complexes were in agreement with that expected for classical polyelectrolyte complexes where the associative phase separation is primarily governed by the increase in entropy due to the release of counterions.     

Wettability of polymers by mucin aqueous solutions

The wettability of poly(methyl methacrylate) and polyethylene by water and aqueous mucin solutions have been studied by sessile drop and under-water captive air bubble contact angles, respectively. From the sessile drop and octane under-water contact angles the polymer-water interfaces have been characterized in terms of works of adhesion and acid-base (polar) interactions. A large water-air contact angle hysteresis observed with poly(methyl methacrylate) surfaces has been attributed to side-chain beta relaxations of polymer ester methyl groups. The wettabilities of the polymers by mucin aqueous solutions have been studied as a function of protein concentration and related to the surface tensions. A positive slope of adhesion tension vs surface tension line, characteristic of polar surfaces, was found with poly(methyl methacrylate). By contrast, a change in the slope, explained as a change in mucin relative adsorption densities at solid/liquid and solid/vapour interfaces, was observed with polyethylene. This adhesion tension behavior appeared to be in agreement with previous data we have published concerning the quantity and state of mucin which are adsorbed to polymers characterized by different surface properties.     

Glycated polyelectrolyte multilayer films: differential adhesion of primary versus tumor cells

Glycated polymers have already been widely employed for cell transfection studies, as cells possess specific lectins. However, up to now, these glycated polymers have barely been investigated for their cell adhesive properties, save macrophages. In this work, we use polyelectrolyte multilayer films made of poly(L-lysine) and poly(L-glutamic) acid as polymeric substrates to investigate the role of sugar molecules (e.g., mannose and lactose) on the adhesion of primary cells as compared to that of a tumor cell line. The glycated polymeric films were compared to ungrafted and chemically cross-linked films, which are known to present opposite adhesive properties. A differential adhesion could be evidenced on mannose grafted films: primary chondrocytes adhere and proliferate well on these films, whereas chondrosarcoma cells do not grow well. Although present, the effect of lactose on cell adhesion was much less important. This adhesion, mediated by glycated polymers, appears to be specific. These results show that it is possible to use glycated polyelectrolytes not only as nonviral vectors but also as cell adhesive substrates.     

An ellipsometry study on the effect of aluminium chloride and ferric chloride formulations on mucin layers adsorbed at hydrophobic surfaces

Ellipsometry was used to investigate the effect of polyaluminium chloride (PAC) formulations of different degrees of hydrolysation on an adsorbed mucin film. The results were compared to the effect of aluminium chloride (AlCl₃) and ferric chloride. A compaction of the mucin film took place upon addition of the formulations and this occurred to different extents and at different concentrations for the different formulations. The compaction of PAC of a low degree of hydrolysis behaved similarly to AlCl₃. PAC of a high degree of hydrolysis showed a greater compaction effect than the other aluminium formulations. The initial compaction concentration was found to be 0.001 mM which is less than previously found for aluminium–mucin complex formation in bulk. The reversibility of the compaction was also investigated. The compaction of the mucin film was found to be partly reversible for AlCl₃ and PAC of low degree of hydrolysis. No reversibility was observed for the formulations of PAC of high hydrolysis grade or for ferric chloride. The results are consistent with previously observed effects of PAC of a low degree of hydrolysis on bacterial surfaces where a compaction of surface polymers was indicated by the reduced range of repulsive steric interactions.     

Poly (DL-lactide-co-glycolide) microparticles as carriers for antimycobacterial drug rifampicin

Poly (DL-lactide-co-glycolide) polymers were investigated as carriers for the first line antitubercular drug rifampicin. Different formulations of PLG microparticles viz. porous, non porous and hardened exhibited sustained release of rifampicin up to 7 weeks in vitro. However, hardened PLG microparticles exhibited the most sustained release in vivo in different organs up to 6 weeks. In case of free rifampicin, release was detected in vivo only up to 48 hr. In addition, no hepatotoxicity was observed on a biochemical basis (levels of SGPT, ALP and total bilirubin) in comparison to control animals. Taken together, these results suggest that polymer encapsulated antitubercular drug rifampicin may serve as an ideal therapeutic approach for treatment of tuberculous infections.     

Embryonal carcinoma cell adhesion: the role of surface galactosyltransferase and its 90K lactosaminoglycan substrate

Embryonal carcinoma (EC) cells possess a complex cell surface glycoconjugate called lactosaminoglycan, whose core structure is composed of repeating N-acetyllactosamine (Gal leads to GlcNAc) disaccharides. Recent studies suggest that the cell surface receptor for lactosaminoglycan is galactosyltransferase, which binds terminal GlcNAc residues on various side chains, thus anchoring the glycoconjugate to the cell surface (Shur, B. D. (1982). J. Biol. Chem. 257, 6871-6878.). The results described in this paper suggest that multivalent lactosaminoglycans mediate EC cell adhesions by binding to their surface galactosyltransferase receptors. In the presence of UDPgalactose, but not other sugar nucleotides, EC cell adhesion is reduced and preformed cell adhesions are dissociated. UDPgalactose interferes with EC cell adhesion by forcing the galactosyltransferase reaction to completion, thus dissociating the enzyme from its galactosylated substrate (i.e., lactosaminoglycan), and thereby dissociating EC cells from one another. Lactosaminoglycans purified from EC cell cultures rapidly agglutinate EC cells, and EC cells preferentially adhere to substrates irreversibly derivatized with protein- and lipid-free lactosaminoglycan side chains. Under identical conditions, EC cells do not adhere to either hyaluronate- or chondroitin sulfate-derivatized substrates, relative to underivatized control surfaces. EC cell adhesion to other cells and to lactosaminoglycan-derivatized surfaces can be inhibited by reagents that selectively interfere with surface galactosyltransferase activity. First, alpha-lactalbumin specifically reduces the galactosyltransferase's affinity for its lactosaminoglycan substrate and simultaneously inhibits adhesion. Similar levels of bovine serum albumin have no effect. Second, selective inhibition of surface galactosyltransferase with UDP-dialdehyde also inhibits adhesion, while similar levels of AMP-dialdehyde do not. Results show that 1 mM Ca2+ protects the surface galactosyltransferase activity from proteolysis, which suggests the galactosyltransferase is one of the Ca2+-dependent EC cell adhesion molecules. SDS-PAGE fluorography and gel chromatography analyses have determined that the principal lactosaminoglycan substrate for EC surface galactosyltransferase has an apparent molecular weight of 90K. Taken together, these results suggest that lactosaminoglycans participate in EC cell adhesion by binding to their surface galactosyltransferase receptors.     

Purification and characterization of a surface protein from Lactobacillus fermentum 104R that binds to porcine small intestinal mucus and gastric mucin

An adhesion-promoting protein involved in the binding of Lactobacillus fermentum strain 104R to small intestinal mucus from piglets and to partially purified gastric mucin was isolated and characterized. Spent culture supernatant fluid and bacterial cell wall extracts were fractionated by ammonium sulfate precipitation and gel filtration. The active fraction was purified by affinity chromatography. The adhesion-promoting protein was detected in the fractions by adhesion inhibition and dot blot assays and visualized by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, and Western blotting with horseradish peroxidase-labeled mucus and mucin. The active fraction was characterized by estimating the relative molecular weight and by assessing the presence of carbohydrates in, and heat sensitivity of, the active region of the adhesion-promoting protein. The purified protein was digested with porcine trypsin, and the peptides were purified in a SMART system. The peptides were tested for adhesion to horseradish peroxidase-labeled mucin by using the dot blot adhesion assay. Peptides which bound mucin were sequenced. It was shown that the purified adhesion-promoting protein on the cell surface of L. fermentum 104R is extractable with 1 M LiCl and low concentrations of lysozyme but not with 0.2 M glycine. The protein could be released to the culture supernatant fluid after 24 h of growth and had affinity for both small intestinal mucus and gastric mucin. In the native state this protein was variable in size, and it had a molecular mass of 29 kDa when denatured. The denatured protein did not contain carbohydrate moieties and was not heat sensitive. Alignment of amino acids of the adhering peptides with sequences deposited in the EMBL data library showed poor homology with previously published sequences. The protein represents an important molecule for development of probiotics.