LFchimera: a synthetic mimic of the two antimicrobial domains of bovine lactoferrin.

Saliva is essential for the maintenance of oral health. When salivary flow is impaired, the risk of various oral diseases such as caries and candidiasis increases drastically. Under healthy conditions, saliva provides effective protection against microbial colonization by the collaborative action of numerous host-defense molecules. This review describes how saliva has been the guideline for the design and characterization of a heterodimeric antimicrobial construct called LFchimera. This construct mimics the helical parts of two antimicrobial domains in the crystal structure of bovine lactoferrin. It shows high antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria, fungi, and parasites including biowarfare agents such as Bacillus anthracis, Burkholderia pseudomallei, and Yersinia pestis. Further, sublethal concentrations of LFchimera inhibited biofilm formation, the invasiveness of HeLa cells by Yersinia spp., and prevented haemolysis of enteropathogenic Escherichia coli, demonstrating the versatility of these peptides.

Identification of peptides derived from the human antimicrobial peptide LL-37 active against biofilms formed by Pseudomonas aeruginosa using a library of truncated fragments.

Persistent Pseudomonas aeruginosa infections are a major cause of morbidity and mortality in cystic fibrosis (CF) patients and are linked to the formation of a biofilm. The development of new biofilm inhibition strategies is thus a major challenge. LL-37 is the only human antimicrobial peptide derived from cathelicidin. The effects on the P. aeruginosa PAO1 strain of synthetic truncated fragments of this peptide were compared with the effects of the original peptide. Fragments of LL-37 composed of 19 residues (LL-19, LL13-31, and LL7-25) inhibited biofilm formation. The strongest antibiofilm activity was observed with the peptides LL7-37 and LL-31, which decreased the percentage of biomass formation at a very low concentration. Some peptides were also active on the bacteria within an established biofilm. LL7-31, LL-31, and LL7-37 increased the uptake of propidium iodide (PI) by sessile bacteria. The peptide LL7-37 decreased the height of the biofilm and partly disrupted it. The peptides active within the biofilm had an infrared spectrum compatible with an α-helix. LL-37, but not the peptides LL7-31 and LL7-37, showed cellular toxicity by permeabilizing the eukaryotic plasma membrane (uptake of ethidium bromide and release of lactate dehydrogenase [LDH]). None of the tested peptides affected mitochondrial activity in eukaryotic cells. In conclusion, a 25-amino-acid peptide (LL7-31) displayed both strong antimicrobial and antibiofilm activities. The peptide was even active on cells within a preformed biofilm and had reduced toxicity toward eukaryotic cells. Our results also suggest the contribution of secondary structures (α-helix) to the activity of the peptides on biofilms.

Involvement of P2X(7) purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: The antimicrobial peptide LL-37, derived from human neutrophils, can directly chemoattract leukocytes and up-regulate the expression of several immune-related genes in various cell types. In this study, we wanted to determine the immunoregulatory effect of LL-37 on interleukin-8 (IL-8) expression in human gingival fibroblasts (HGFs) and to characterize intracellular signaling pathway(s) and receptor(s) involved in IL-8 induction. MATERIAL AND METHODS: Cultured fibroblasts were treated with different concentrations of LL-37 or interleukin-1ß (IL-1ß), as a positive control, for specific periods of time in the presence or absence of various inhibitors. RT-PCR and real-time PCR were conducted to analyze the expression of IL-8 mRNA, and the IL-8 levels in cell-free culture media were measured using ELISAs. The MTT assay was performed to determine the cytotoxicity of LL-37. RESULTS: Nontoxic concentrations of LL-37 (up to 10 µm) and IL-1ß significantly up-regulated the expression of IL-8 mRNA in a dose-dependent manner (p < 0.05). The IL-8 protein levels were consistently significantly elevated in conditioned media of LL-37-treated HGFs (p < 0.05). IL-8 up-regulation by LL-37 was completely abrogated by 20 µm U0126, consistent with transient phosphorylation of p44/42 MAP kinases. Moreover, pretreatment with Brilliant Blue G (a selective antagonist of the P2X(7) receptor) and the neutralizing antibody against P2X(7) blocked IL-8 up-regulation in a dose-dependent manner, consistent with expression of the P2X(7) receptor in HGFs. CONCLUSION: These findings indicate that LL-37 induces IL-8 expression via the P2X(7) receptor and the MEK1/2-dependent p44/42 MAP kinases in HGFs, suggesting both direct and indirect involvement of LL-37 in neutrophil recruitment into an inflammatory site within diseased periodontal tissues.

Histatin 1 Enhances Cell Adhesion to Titanium in an Implant Integration Model.

Cellular adhesion is essential for successful integration of dental implants. Rapid soft tissue integration is important to create a seal around the implant and prevent infections, which commonly cause implant failure and can result in bone loss. In addition, soft tissue management is important to obtain good dental aesthetics. We previously demonstrated that the salivary peptide histatin 1 (Hst1) causes a more than 2-fold increase in the ability of human adherent cells to attach and spread on a glass surface. Cells treated with Hst1 attached more rapidly and firmly to the substrate and to each other. In the current study, we examine the potential application of Hst1 for promotion of dental implant integration. Our results show that Hst1 enhances the attachment and spreading of soft tissue cell types (oral epithelial cells and fibroblasts) to titanium (Ti) and hydroxyapatite (HAP), biomaterials that have found wide applications as implant material in dentistry and orthopedics. For improved visualization of cell adhesion to Ti, we developed a novel technique that uses sputtering to deposit a thin, transparent layer of Ti onto glass slides. This approach allows detailed, high-resolution analysis of cell adherence to Ti in real time. Furthermore, our results suggest that Hst1 has no negative effects on cell survival. Given its natural occurrence in the oral cavity, Hst1 could be an attractive agent for clinical application. Importantly, even though Hst1 is specific for saliva of humans and higher primates, it stimulated the attachment and spreading of canine cells, paving the way for preclinical studies in canine models.

Cell surface sialic acid and the invasive and metastatic potential of T-cell hybridomas.

T-cell hybridomas prepared by fusion of non-invasive non-metastatic BW5147 T-lymphoma cells and activated normal T-cells were found to be highly invasive in vitro and highly metastatic in vivo upon tail vein injection. By prolonged culturing and subcloning, non-invasive, non-metastatic hybrids were selected with modal DNA/cell contents close to the diploid value of both fusion partners. Since normal activated T-cells were invasive in vitro in hepatocyte cultures, these data suggest that invasiveness of the hybrids is derived from the parental normal T-cells and is one of the properties responsible for the metastatic potential of these cells. Analysis of a large panel of T-cell hybrids with fluorescein isothiocyanate conjugated lectins, specific for terminal galactose and/or N-acetylgalactosamine sugar residues, showed an inverse correlation between expression of lectin receptor sites and invasive and metastatic potential of the hybrids. Soybean agglutinin, as well as peanut agglutinin and Ricinus communis agglutinin, reacted strongly with non- or low-invasive hybrids but only weakly with invasive hybrids. The difference in lectin binding between both types of hybrids appeared to be due to masking of receptor sites by sialic acid. Removal of cell surface sialic acid by neuraminidase treatment unmasked the lectin receptor sites of invasive hybrids to the level of the corresponding sites of non- or low-invasive cells. This increase in active lectin binding sites was simultaneously accompanied by a striking decrease of invasiveness to the level of the low-invasive hybrids. Conversely, the blocking of R. communis agglutinin receptors by sialic acid allowed selection of invasive hybrids from segregating cell populations with the toxic lectin R. communis agglutinin. The results taken together indicate that sialylation of particular cell surface carbohydrate residues on the T-cell hybridomas is associated with the invasive and metastatic potential of these hybrids. The reduction of invasive potential after removal of cell surface sialic acid provides further evidence for a functional role of this sugar residue in invasiveness of the T-cell hybrids.

Modification of cell surface carbohydrates and invasive behavior by an alkyl lysophospholipid.

The effect of the alkyl lysophospholipid racemic-1-O-octadecyl-2-O-methyl glycero-3-phosphocholine on the expression of cell surface carbohydrates of four matched pairs of normal and malignant cells was studied using chromatographic techniques. After treatment with alkyl lysophospholipid, glycopeptides proteolytically derived from normal and malignant cells displayed a shift in the size distribution profiles obtained by gel filtration. These drug-induced changes in molecular weight distribution were expressed most strongly in untransformed cells and resembled the carbohydrate alterations found after their malignant transformation. Desialylation abolished the effect of alkyl lysophospholipid, thus suggesting an increased amount of sialic acid in the surface carbohydrates of drug-treated cells. Chromatography of glycopeptides on concanavalin A-Sepharose, Ricinus communis agglutinin I-agarose, and Bio-Gel P-4 columns excluded a higher degree of branching but suggested addition of extra terminal sialic acid residues as the major cause of the observed alterations. Alkyl lysophospholipid stimulated glycoprotein sialylation of normal cells to the level observed in malignant cells, thus inducing a "malignant-like" surface phenotype. The drug-induced carbohydrate changes in normal chick heart tissue prevented its being invaded by tumor cells when tested in an organotypic assay. The alkyl lysophospholipid thus appears to modulate in a nontoxic fashion the expression of surface molecules implicated in various cellular interactions including invasiveness.

Effect of cancer-related and drug-induced alterations in surface carbohydrates on the invasive capacity of mouse and rat cells.

The effect of alterations in cell surface carbohydrates on invasion of mouse and rat cells into embryonic chick heart fragments in organ culture was studied. Matching pairs of malignant and nonmalignant cells, including all categories of carcinogenic induction (i.e., viral, chemical, or oncogenic), were compared for their alterations in cell surface carbohydrates and invasive behavior. Glycopeptides derived from the surface of malignant cells expressed cancer-related changes in carbohydrate composition, demonstrated by gel filtration chromatography as a shift in size distribution in comparison with those from nonmalignant counterparts. This phenotypic property strictly correlated with the acquisition of the invasive capacity. Morphological transformation of cells without simultaneous alteration in surface carbohydrates was, however, insufficient for invasion. To test the possible mechanistic role of altered surface carbohydrates in the invasive capacity of cells, the surface molecules of noninvasive cells were modified by incubation with an alkyl-lysophospholipid (racemic 1-O-octadecyl-2-O-methyl glycero-3-phosphocholine). Alkyl-lysophospholipid induced an increase in surface sialylation resembling the changes found in malignant and invasive cells. After pretreatment with alkyl-lysophospholipid, morphologically transformed but nonmalignant and noninvasive cells became able to invade chick heart tissue. These findings indicate that alterations in cell surface carbohydrates, induced by entirely different mechanisms, endowed cells with invasive capacity.

Genetic polymorphism of MUC7: allele frequencies and association with asthma.

MUC7 encodes a small salivary mucin, previously called MG2, a glycoprotein with a putative role in facilitating the clearance of oral bacteria. The central domain of this glycoprotein was previously shown to comprise five or six tandemly repeated units of 23 amino-acids which carry most of the O-linked glycans. The polymorphism of these two allelic forms (MUC7*5 or MUC7*6) has been confirmed in this study in which we have analysed a large cohort of subjects (n = 375) of various ethnic origins. We have also identified a novel rare allele with eight tandem repeats (MUC7*8). MUC7*6 was the most common allele (0.78-0.95) in all the populations tested. The tandem repeat arrays of 22 MUC7*5 alleles and 34 MUC7*6 alleles were sequenced. No sequence differences were detected in any of the MUC7*6 alleles. Twenty-one MUC7*5 alleles sequenced lacked the 4th tandem repeat (structure TR12356), while one showed the structure TR12127. The structure of the MUC7*8 allele was TR12343456. Because of the known role of MUC7 in bacterial binding, and thus its potential involvement in susceptibility to chest disease we also tested MUC7 in our previously described series of Northern European atopic individuals with and without associated asthma. The MUC7*5 allele was rarer in the atopic asthmatics than in the atopic non-asthmatics (P = 0.014, OR for no asthma in atopic individuals 3.13, CI 1.01-6.10), and the difference in frequency between all asthmatics and all non-asthmatics was statistically significant (P = 0.009) while there was no difference between atopy and non-atopy (P = 0.199). In this study we also report the electrophoretic analysis of the MUC7 glycoprotein in saliva from individuals of different MUC7 genotype.

Restored invasion of mouse MO4 cells into chick heart in vitro through mutual conditioning at reduced temperature.

Invasion of malignant mouse MO4 cells into embryonic chick heart fragments in confronting organ cultures was arrested for 7 days when the temperature of incubation was lowered to 28 degrees C. Afterwards invasion resumed and progression between days 10 and 17 at 28 degrees C was comparable to that between days 0 and 7 at 37 degrees C. This pattern of progression of MO4 cell invasion at 28 degrees C was unaltered when either MO4 cells or heart fragments or both were preincubated separately at 28 degrees C for 14 days before confrontation with each other. Invasion at 28 degrees C resumed only when MO4 cells and heart tissue had been in immediate contact for at least 7 days. Metabolic labelling with [3H]fucose showed a correlation in time between transient suppression of invasion and transient inhibition of incorporation of fucosylation-precursor molecules into glycoproteins by MO4 cells. The latter activity was far less temperature-sensitive in heart cells. Our observations suggest that metabolic cooperation between invading MO4 cells and heart tissue is essential for progression of invasion in vitro.

Decreased fucose incorporation in cell surface carbohydrates is associated with inhibition of invasion.

Invasion of malignant MO4 cells into embryonic chick heart fragments in an organ culture assay was arrested for at least 7 days when the temperature was lowered to 28 degrees C. Prolonged culturing of MO4 cells at 28 degrees C on tissue culture substrates showed no recuperation of fucose incorporation into cell surface glycopeptides. However, invasion was restored after 10 days of organ culture in confrontation with chick heart tissue at 28 degrees C. A histoautoradiographic study showed that the regained capability to invade was accompanied by an increase in fucose labeling of the MO4 cells in the invading areas. At 28 degrees C the incorporation of [3H]fucose into total cell protein was drastically reduced, whereas [3H]leucine incorporation as a measure for protein synthesis was less affected. Cell surface glycopeptides, metabolically labeled with either fucose or glucosamine at 28 degrees C, showed a time-dependent decrease in the incorporation of fucose but not of glucosamine and no changes in overall size distribution. Low temperature did not reduce fucosyltransferase activity but the relative accumulation of fucose-1-P suggested inhibited conversion towards GDP-fucose. Moreover, mouse L cells which were incapable of invading chick heart tissue appeared also deficient in fucose incorporation, owing to low levels of fucosyltransferase activity. According to the results, fucosylation of surface carbohydrates may be required for invasive capacity and restored in MO4 cells invading at 28 degrees C by metabolic cooperation with the host tissue.

Detection and quantification of MUC7 in submandibular, sublingual, palatine, and labial saliva by anti-peptide antiserum.

The large carbohydrate moiety of low-Mr salivary mucin MUC7 (originally referred to as MG2) is subject to variations. Biochemical analysis and quantification of MUC7 in saliva samples require recognition tools that are independent of the carbohydrate moiety. Therefore, we have evoked three antisera to synthetic peptides of MUC7. One of these (CpMG2), raised against the C-terminal peptide, recognized native MUC7 in saliva and was characterized further. Recognition of MUC7 by CpMG2 turned out to be specific, resistant to dissociating and reductive treatments, and independent of glycosylation differences, as indicated by Western analysis and ELISA. The antiserum could be used to monitor MUC7 during purification procedures. MUC7 was demonstrated in small volumes of saliva from all (sero)mucous glands, including the palate and lip. Analysis with antibodies and lectins indicated large variations in amount as well as in glycosylation of MUC7. An ELISA was developed to determine the relative quantity of MUC7 in the glandular salivas: mean values of approximately 220, 980, and 100 microg mucin per mL were found in submandibular, sublingual, and palatine saliva, respectively.

A rapid solid-phase fluorimetric assay for measuring bacterial adherence, using DNA-binding stains.

In this report, we describe the validation of a rapid, single-step, microtiter plate method for quantifying bacterial adherence, based on fluorescent labeling of microorganisms with cell-permeable fluorescent DNA-binding probes. We have tested the binding to saliva-coated microtiter plates of bacteria, including Helicobacter pylori and viridans streptococci (S. mitis, S. gordonii, S. sanguis), known to interact with salivary components. Furthermore, we tested the short-term and longer-term temporal stability of a saliva-mediated adherence of these bacteria in a healthy population (N=30). The assay exhibited excellent reliability statistics, yielding within-assay variability coefficients ranging from 4.9% to 11%. A range of approximately 5 x 10(4)-1 x 10(7) cells could be detected. This method may be generally applicable to study surface binding of virtually any microbial species, while obviating the need of radioactive materials or specific antibodies for quantification, thus providing a procedure that is useful to both basic and clinical research.

Effect of lipid derivatives on invasion in vitro and on surface glycoproteins of three rodent cell types.

The antiinvasive activity on MO4 mouse cells of the following lipid derivatives was tested in vitro: an alkyl-lysophospholipid derivative (ET-18-OCH3), a thioether-phospholipid derivative (BM 41.440), an alkyl-linked lipoidal amine (CP-46,665) and a naturally occurring ester-linked phospholipid (2-LPC). In this test, BM 41.440 had the same antiinvasive potency as ET-18-OCH3, whereas CP-46,665 and 2-LPC had no effect on invasion. Comparison of the antiinvasive effect of ET-18-OCH3 on three types of cells showed the following ranking: 12R1C-RK rat kidney adenovirus type 12 transfected cells greater than MO4 mouse cells greater than LLC-H61 Lewis lung carcinoma cells. This ranking was not reflected in ET-18-OCH3-induced changes of cell surface exposed glycopeptides derived from the three types of cells metabolically labeled with radioactive fucose. The present and previous experiments suggested that changes in invasion caused by lipid derivatives depended upon relative cell surface fucosyl-glycopeptide alterations in both the invasive cells and the normal tissue.

CNS myelin induces regulatory functions of DC-SIGN-expressing, antigen-presenting cells via cognate interaction with MOG.

Myelin oligodendrocyte glycoprotein (MOG), a constituent of central nervous system myelin, is an important autoantigen in the neuroinflammatory disease multiple sclerosis (MS). However, its function remains unknown. Here, we show that, in healthy human myelin, MOG is decorated with fucosylated N-glycans that support recognition by the C-type lectin receptor (CLR) DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) on microglia and DCs. The interaction of MOG with DC-SIGN in the context of simultaneous TLR4 activation resulted in enhanced IL-10 secretion and decreased T cell proliferation in a DC-SIGN-, glycosylation-, and Raf1-dependent manner. Exposure of oligodendrocytes to proinflammatory factors resulted in the down-regulation of fucosyltransferase expression, reflected by altered glycosylation at the MS lesion site. Indeed, removal of fucose on myelin reduced DC-SIGN-dependent homeostatic control, and resulted in inflammasome activation, increased T cell proliferation, and differentiation toward a Th17-prone phenotype. These data demonstrate a new role for myelin glycosylation in the control of immune homeostasis in the healthy human brain through the MOG-DC-SIGN homeostatic regulatory axis, which is comprised by inflammatory insults that affect glycosylation. This phenomenon should be considered as a basis to restore immune tolerance in MS.

The antimicrobial peptide, LL-37, inhibits in vitro osteoclastogenesis.

Uncoupled bone resorption leads to net alveolar bone loss in periodontitis. The deficiency of LL-37, the only human antimicrobial peptide in the cathelicidin family, in patients with aggressive periodontitis suggests that LL-37 may play a pivotal role in the inhibition of alveolar bone destruction in periodontitis. We aimed to investigate a novel function of LL-37 in osteoimmunity by blocking osteoclastogenesis in vitro. Human osteoclast progenitor cells were isolated from a buffy coat of blood samples. The cells were cultured in the presence of various concentrations of LL-37 during an in vitro induction of osteoclastogenesis. Non-toxic doses of LL-37 could block multinuclear formation of the progenitor cells and significantly diminish the number of tartrate-resistant acid-phosphatase-positive cells and the formation of resorption pits (p < 0.05), whereas these concentrations induced cellular proliferation, as demonstrated by increased expression of proliferating cell nuclear antigen. Expression of several osteoclast genes was down-regulated by LL-37 treatment. It was demonstrated that nuclear translocation of nuclear-factor-activated T-cells 2 (NFAT2) was blocked by LL-37 treatment, consistent with a significant reduction in the calcineurin activity (p < 0.005). Collectively, our findings demonstrate that LL-37 inhibits the in vitro osteoclastogenesis by inhibiting the calcineurin activity, thus preventing nuclear translocation of NFAT2.

Histatins enhance wound closure with oral and non-oral cells.

The role of human saliva in oral wound-healing has never been fully elucidated. We previously demonstrated that parotid-salivary histatins enhance in vitro wound closure. The question remains whether other salivary-gland secretions enhance wound closure, and also the effects of histatins on primary and non-oral cells. Since the presence of histatins is not limited to parotid saliva, we expected to observe wound-closure activity of other salivary-gland secretions. However, here we show that non-parotid saliva does not stimulate wound closure, most probably due to the presence of mucins, since the addition of MUC5B to parotid saliva abolished its effect. Furthermore, we found that histatins stimulated wound closure of (primary) cells of both oral and non-oral origin. This suggests that the cellular receptor of histatins is widely expressed and not confined to cells derived from the oral cavity. These findings encourage the future therapeutic application of histatins in the treatment of all kinds of wounds.

The electrochemical proton gradient generated by the fumarate-reductase system in Escherichia coli and its bioenergetic implications.

Proton translocation, coupled to electron transfer in the fumarate reductase system, generates and electrochemical potential gradient for protons (delta approximately mu H+). The magnitude of this free energy gradient has been determined in the Escherichia coli strains ML 208-225 and AN 283. The measurements were performed in (inverted) membrane particles, right-side out membrane vesicles and EDTA-treated intact cells in external media of various ionic compositions and pH. The maximal values of delta approximately mu H+ in these three systems were +103, -101 and -105 mV, respectively. This implicates that in E. coli, upon transition from oxygen to fumarate as electron acceptor, the magnitude of the delta approximately mu H+ decreases considerably. This change of delta approximately mu H+ has substantial consequences for the cellular metabolism.

Sulfomucins in the human body.

Mucins are widely distributed in mucous secretion fluids or are associated with plasma membranes. Up to now 9 genes of epithelial mucins have been identified, distributed over five chromosomes. Superposed on the genetic diversity, each type of mucin displays heterogeneity in oligosaccharide composition, including the terminal sugar residues. On top of that there is variation between individuals brought about by blood group antigens. Heterogeneity is further incited by the degree of sulfation. This tremendous structural heterogeneity endows mucin molecules with properties suggestive for a multifunctional role. The major biological function assigned to mucins is still the protection of tissues covered by the mucous gel. Current knowledge on the specific biological functions of the sulfate residues is fragmentary and periphrastic. Glycosylation including sulfation appears to be subject to modification under pathological conditions. There is evidence that sulfation rate-limits bacterial degradation of mucins. Moreover, accumulating data focus towards their involvement in recognition phenomena. Sulfate residues on blood group related structures provoke specific epitopes for selective interaction with microorganisms e.g. Helicobacter pylori. A distinct class of mucins acts as ligands for selectins, crucial in cellular recognition processes like cellular homing of lymphocytes. Whereas in earlier days mucins were only seen as water-binding molecules, protecting the underlying mucosa against harmful agents, the current picture of these molecules is characterized by the selective interaction with their environment, including epithelial-, and endothelial cells and microorganisms, thereby regulating a great number of biological processes. However, the specific role of sulfate remains to be further elucidated.

Enzymatic amplification involving glycosyltransferases forms the basis for the increased size of asparagine-linked glycans at the surface of NIH 3T3 cells expressing the N-ras proto-oncogene.

Expression of ras oncogenes in NIH 3T3 fibroblasts results in the acquisition by these cells of an invasive potential concomitant with the appearance of cell surface asparagine-linked complex-type glycan structures of a higher average molecular weight (Bolscher, J.G. M., van der Bijl, M. M. W., Neefjes, J. J., Hall, A., Smets, L.A., and Ploegh, H.L. (1988) EMBO J. 7, 3361-3368). We have investigated the enzymatic basis for the altered glycosylation by assessing the activities of all major Golgi glycosyltransferases involved in the synthesis of these structures. Use was made of a stable transfectant cell line (T15) containing the N-ras-protooncogene under the control of a glucocorticoid-inducible mouse mammary tumor virus promoter. Upon induction of the ras gene with dexamethasone: 1) the levels of N-acetylglucosaminyltransferase I and II were essentially unaltered, indicating an unaffected potential to synthesize complex-type glycans; 2) the activities of the branching N-acetylglucosaminyltransferase III and V were elevated 2- to 2.5-fold suggesting the formation of increased amounts of bisected glycans and of structures carrying a Gal beta 1----GlcNAc beta 1----6Man-branch; 3) the levels of the elongating beta 4-galactosyltransferase and beta 3-N-acetylglucosaminyl-transferase were increased 5- to 7-fold indicating a strongly enhanced capacity to synthesize polylactosaminoglycan chains; 4) the level of the major chain-terminating enzyme, alpha 3-galactosyltransferase, was slightly decreased (0.7-fold), whereas those of the alpha 3- and alpha 6-sialyltransferases were slightly elevated (1.3- and 2-fold, respectively), suggesting a shift from termination by alpha-galactosyl residues to termination by sialic acid moieties. Studies on the acceptor specificities of the different glycosyltransferases indicate that these changes occur in a coordinated manner in which the effects of altered glycosyltransferase expression levels amplify each other. Analysis of the size of cell surface complex-type glycopeptides before and after digestion with neuraminidase and endo-beta-galactosidase suggested an increased sialic acid density, an increase in the number and/or length of polylactosaminoglycan chains, and an increased branching of the glycans upon N-ras induction. The enzymatic results explain these structural changes and allow us to define the alterations in glycosylation pathways associated with ras expression.

Salivary proteins: protective and diagnostic value in cariology?

Saliva is essential for a lifelong conservation of the dentition. Various functions of saliva are implicated in the maintenance of oral health and the protection of our teeth: (i) The tooth surface is continuously protected against wear by a film of salivary mucins and proline-rich glycoprotein. (ii) The early pellicle proteins, proline-rich proteins and statherin, promote remineralization of the enamel by attracting calcium ions. (iii) Demineralization is retarded by the pellicle proteins, in concert with calcium and phosphate ions in saliva and in the plaque fluid. (iv) Several salivary (glyco)proteins prevent the adherence of oral microorganisms to the enamel pellicle and inhibit their growth. (v) The salivary bicarbonate/carbonate buffer system is responsible for rapid neutralization of acids. An overview is presented on the major antimicrobial systems in human saliva. Not only the well-known major salivary glycoproteins, including mucins, proline-rich glycoprotein and immunoglobulins, but also a number of minor salivary (glyco)proteins, including agglutinin, lactoferrin, cystatins and lysozyme, are involved in the first line of defense in the oral cavity. Besides, small cationic antimicrobial peptides, e.g. defensins, cathelicidin and the histatins, have come into focus. These are potentially suited as templates for the design of a new generation of antibiotics, since they kill a broad spectrum of microorganisms, while hardly evoking resistance, in contrast to the classical antibiotics.