Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwp1.

The pathogenesis of candidiasis involves invasion of host tissues by filamentous forms of the opportunistic yeast Candida albicans. Morphology-specific gene products may confer proinvasive properties. A hypha-specific surface protein, Hwp1, with similarities to mammalian small proline-rich proteins was shown to serve as a substrate for mammalian transglutaminases. Candida albicans strains lacking Hwp1 were unable to form stable attachments to human buccal epithelial cells and had a reduced capacity to cause systemic candidiasis in mice. This represents a paradigm for microbial adhesion that implicates essential host enzymes.

Do proline-rich proteins modulate a transglutaminase catalyzed mechanism of candidal adhesion?

Previously, we reported that a membrane-bound epithelial enzyme, transglutaminase (TGase), catalyzes the covalent cross-linking of acidic proline-rich proteins (APRPs) to surface proteins of buccal epithelial cells (BECs). The purpose of this study was twofold: (1) to provide evidence that TGase stabilizes C. albicans adhesion by covalently cross-linking C. albicans and BEC surface proteins and (2) to implicate PRPs in the modulation of this adhesive mechanism. The reactivity of candidal cell wall proteins with TGase was assessed in two separate experiments. Initially, following incubation with native BECs, the cross-linking of iodinated candidal cell wall proteins into high-molecular-weight complexes, as shown by SDS-PAGE/autoradiography, was inhibited by the TGase inhibitor iodoacetamide. Additionally, [14C]putrescine in the presence of purified TGase, but not [14C]putrescine alone, was shown by SDS-PAGE/fluorography to be cross-linked into surface proteins of both morphogenetic forms (blastospore > hyphal forms) of C. albicans. In adherence assays, a component of both blastospore and hyphal form Candida/BEC adherence was shown to be resistant to detachment by heating adherent cells in 1% SDS at 100 degrees C. However, pretreatment of BECs with iodoacetamide decreased SDS resistant adherence of both forms of C. albicans by approximately 75%. When incubated with [125I]APRPs and purified TGase, both morphogenetic forms of C. albicans bound dramatically more APRP than controls without TGase. [125I]APRP binding in experimental, but not control, samples was resistant to repeated extraction (48 h) with 4% SDS/10% beta-mercaptoethanol at 65 degrees C, suggesting that [125I]APRPs were cross-linked to the Candida surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of bone cell metabolism.

Bone formation and resorption are normal physiologic processes. In pathologic states such as in periodontal disease or osteoporosis a shift in the balance of these two processes occurs, resulting in a net loss of mineralized tissue. Osteoclasts have historically been considered to be the primary bone resorbing cells, but current research has lead to the hypothesis that osteoblastic cells play an integral role in bone resorption as well. It appears that osteoblasts respond to bone resorbing agents via a series of intracellular responses after interactions with specific surface receptors. Two basic pathways involving different "second messengers" have been identified. The first pathway involves cyclic 3',5' adenosine monophosphate (cAMP) production and the second involves membrane phospholipids, diacylglycerol and calcium. A cytosolic enzyme, protein kinase C (PKC), has been shown to affect both cAMP as well as calcium fluxes and may act to regulate both these pathways. It is the purpose of this paper to discuss current studies and hypotheses concerning the nature of mechanisms involved in regulation of bone metabolism with emphasis on second messenger systems. Information of this nature is critical to the development of rationale regarding diagnosis, treatment and management of systemic and local pathoses of bone.

Comparison of a lesion-inducing isolate and a non-lesional isolate of Candida albicans in an immunosuppressed rat model of oral candidiasis.

Two distinct strain-related patterns of organism-host interaction on dorsal tongue of immunocompetent rats have been identified for Candida albicans: some isolates induce mucosal lesions, while other isolates penetrate the keratin layer but do not produce a lesion. This study examined the behavior of each of the two types of isolates in a cyclosporin-immunosuppressed rat model. Groups B (normal) and D (cyclosporin) were orally inoculated with a lesion-inducing isolate of C. albicans, while a non-lesional isolate was given to Groups A (normal) and C (cyclosporin). A typical dorsal tongue lesion developed in 4/18 rats in Group B and in 13/16 in Group D (P = 0.00267). No significant difference in infection rate between the normal and cyclosporin-treated animals was seen for the non-lesional isolate. The lack of a host inflammatory response associated with the non-lesional isolate may represent an ecologic advantage for the organism.

Oral mucosal pellicle. Adsorption and transpeptidation of salivary components to buccal epithelial cells.

The present investigation was carried out to examine the mechanism(s) whereby salivary molecules interact with human buccal epithelial cells. By utilizing antiserum against human parotid saliva, selected salivary components were detected by electrophoretic-transfer analysis of 1.5% SDS extracts of epithelial cells. Incubation of the cells and their aqueous cell-free extracts with 125I-labelled parotid saliva resulted in the formation of an iodinated high-molecular-mass complex which was not present in 125I-labelled saline alone. Formation of this complex was time-dependent and was inhibited by treating the buccal epithelial cells or their cell-free extracts with EGTA, iodoacetamide, N-ethylmaleimide or by heating at 100 degrees C for 15 min. The epithelial cells also promoted incorporation of [14C]putrescine into high-molecular-mass complexes whose formation was inhibited by iodoacetamide, unlabelled putrescine and EGTA. Cell extracts mediated cross-linking of monodansylcadaverine into alpha-casein, and this interaction was inhibited by iodoacetamide. Significant amounts of radioactivity were recovered with the epithelial-cell envelopes after exhaustive extraction of 125I-saliva- or [14C]putrescine-treated epithelial cells with 4% (w/v) SDS/10% (v/v) beta-mercaptoethanol. The incorporation of radioactivity into epithelial-cell envelopes was inhibited by pretreatment of the cells with putrescine, EGTA, iodoacetamide, or heating at 100 degrees C for 15 min. These data suggest that: (1) oral mucosal pellicle is formed by the selective adsorption of saliva to the epithelial-cell plasma membrane and its associated cytoskeleton; and (2) the adsorbed salivary components may be cross-linked to each other or the epithelial cytoskeleton by epithelial transglutaminases.

Modulation of herpes simplex virus type 1 replication by human salivary secretions.

Saliva functions to protect the oral cavity from pathogenic invasion by modulating the ability of microbes to colonize the oral surfaces or limiting their growth and/or viability. Although the role of salivary secretions in the modulation of the oral bacteria flora has received considerable attention, little is known concerning its role in viral pathogenesis. Accordingly, the purpose of this study was to assess the effect of salivary secretions on herpes simplex virus type 1 (HSV-1) replication. Initially, HSV-1 plaque and titer reduction assays were performed to determine the ability of human submandibular/sublingual (HSMSL) and parotid (HPS) salivas to inhibit the early stages of HSV-1 infection (adsorption and penetration). Our results suggested that both HSMSL and HPS possess cell-protective and virus neutralization activities, with HSMSL being more active than HPS. Additional experiments were performed to determine the effect of saliva on the yield of virus progeny. Again, HSMSL caused a greater reduction of HSV-1 replication than did HPS. A similar effect could not be obtained using vaccinia, suggesting that this inhibitory activity of human saliva is selective. Collectively, these results suggest that human salivary secretions can modulate the replication of HSV-1 in vitro.

Prostaglandin-induced changes in calcium uptake and cAMP production in osteoblast-like cells: role of protein kinase C.

Phorbol esters were used to evaluate the putative effect of protein kinase C (PKC) activation on prostaglandin E2 (PGE2)-induced increases in calcium uptake and cAMP production in the human osteoblastic osteosarcoma cell line, Saos-2. The cells were pretreated for 15 min with phorbol myristate acetate (PMA) followed by a 5 min incubation with PGE2. Calcium uptake was measured with 45Ca and cAMP by radioimmunoassay. A significant increase in calcium uptake was noted in the PGE-treated cells compared with controls and preincubation with the PMA caused a significant decrease in this response. Preincubation with PMA also inhibited the PGE2-induced increase in cAMP under identical conditions. The effect of PMA on the cAMP response was not influenced by the addition of a phosphodiesterase inhibitor. PMA had no effect on the basal levels of either calcium uptake or cAMP production. Likewise, the inactive phorbol esters, phorbol 12,13-didecanoate (PDD) and 4 alpha-phorbol 12-myristate, 13-acetate (4 alpha), had no effect on either basal levels of these parameters or on the PGE2-induced increases. These results suggest that PKC is involved in the down-regulation of PGE2-induced increases in calcium uptake and cAMP production in the Saos-2 osteoblastic cell line.

Use of the photoaffinity cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid to characterize salivary-glycoprotein-bacterial interactions.

The present study has utilized the iodinatable cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid (ASA) to examine the specific interaction between the proline-rich glycoprotein (PRG) of human parotid saliva and Streptococcus sanguis G9B. The binding of 125I-ASA-PRG to Streptococcus sanguis G9B displayed saturation kinetics, reversibility and was inhibited by unlabelled PRG. Inhibition studies with other glycoproteins and saccharides indicated that binding was mediated by a bacterial adhesin with specificity towards N-acetylneuraminic acid, galactose, and N-acetylgalactosamine. After cross-linking, the 125I-ASA-PRG-adhesin complex could be extracted with SDS and separated from uncoupled 125I-ASA-PRG by gel filtration on Sepharose CL-6B. Approx. 1% of the 125I-ASA-PRG was cross-linked to the bacterial surface. Examination of the 125I-ASA-PRG-adhesin complex by SDS/polyacrylamide-gel electrophoresis/fluorography on 5% -(w/v)-polyacrylamide gels revealed that PRG was bound to two bacterial components. These findings support our previous suggestion that human salivary glycoproteins can specifically interact with oral streptococci and that these interactions occur between the glycoprotein's carbohydrate units and lectin(s) on the bacterial cell surface.

Formation of salivary-mucosal pellicle: the role of transglutaminase.

The present investigation was carried out to identify salivary components of mucosal pellicles in vivo and explore further the mechanism of interaction between salivary molecules and buccal epithelial cells. By using specific antisera and immunoprotein blotting, high-(MG1) and low-(MG2) molecular-mass salivary mucins, amylase, salivary cystatins and proline-rich proteins were detected within mucosal pellicle in vivo. In addition, the data indicated that the mucins and proline-rich proteins could be cleaved into lower-molecular-mass products, whereas the proline-rich proteins could also be cross-linked into higher-molecular-mass complexes. The role of buccal epithelial cell transglutaminase in these interactions was further studied by utilizing purified iodinated amylase, neutral cystatin SN and acidic proline-rich proteins 1 and 3 (APRP1 and 3). After incubation with buccal epithelial cells in vitro 125I-labelled APRPs appeared to undergo a greater degree of cross-linking than 125I-labelled cystatin SN, as determined by SDS/PAGE/autoradiography. Amylase did not appear to be cross-linked at all. Recovery of 125I-labelled APRPs and 125I-labelled cystatin SN with epithelial cell envelopes after repeated extraction suggested that both molecules were cross-linked to envelope proteins, but that 125I-labelled APRPs were cross-linked to a greater degree than 125I-labelled cystatin SN. Cross-linking in buccal epithelial cell preparations was inhibited by an excess of methylamine hydrochloride, a transglutaminase substrate. In a further assessment of amylase, cystatin and APRPs as transglutaminase substrates, only APRP3 and a partially purified preparation of APRPs acted as an amine acceptor for the cross-linking of [14C]methylamine by purified transglutaminase, as determined by SDS/PAGE/fluorography. This reaction was completely inhibited by excess EDTA. The combined data from this study suggest that during mucosal pellicle formation multiple components of saliva adsorb to buccal epithelial cell surfaces, and that, within this group, selected components are enzymically cross-linked by an epithelial transglutaminase and/or proteolytically cleaved into smaller fragments.

Effects of caffeine and nicotine administration on growth and ossification of the ICR mouse fetus.

The objective of this study was to determine how fetal effects are altered when nicotine (N) and caffeine (CA) are administered concurrently at dosages that individually produce minimal effects to the fetus. Female ICR mice were bred overnight and were assigned to four groups: CA (125 mg/kg), N (12mg/kg), CA plus N (125 mg/kg plus 12 mg/kg, respectively) treated, and control (distilled water) groups. Dams were intubated with these dosages three times daily during gestational days (GD) 6-18 and were euthanized on GD 18. Live fetuses were sexed, weighed, and examined for external malformations. One-half of the fetuses were fixed in 10% formalin and examined for internal malformations using Wilson's method. The remaining half was fixed in 95% ethanol (ETOH), stained, and cleared (Inouye's method) for skeletal examinations. Ossification was assessed by staging and measuring craniofacial bones, and counting ossification centra in sternbrae and in cervical and sacrococcygeal vertebrae. Data were analyzed by analysis of variance (ANOVA) followed by Student-Newman-Keuls post-hoc tests set at p < .05 significance level. The litter was used as the unit of measure and the ANOVA main effects were CA, N, and an interaction term (CA+N). In comparison to controls, CA treatment resulted in reduced bone measurements or reduced ossification scores in 5 of the 19 parameters examined, whereas for N only five parameters were significant. The main effects for interaction of CA+N were significant for seven parameters measured. Although it is difficult to assign the specific type of drug interaction that occurred because results were not completely consistent for all parameters measured, it may be concluded that in most parameters measured both CA and CA+N were different from controls, but CA was not different from CA+N. Under the experimental conditions of this study, we found that of the two drugs, caffeine had a significantly greater effect on fetal growth and ossification than nicotine.

Second messenger systems stimulated by bradykinin in osteoblastic cells: evidence for B2 receptors.

The effects of bradykinin, analogs and inhibitors on the human osteoblastic osteosarcoma cell lines Saos-2 and G292 and on normal rat calvarial osteoblastic cells were investigated. In all cell types, bradykinin (1 nM-100 microM) caused significant time- and dose-dependent changes in the levels of inositol phosphates. Neomycin inhibited the inositol phosphate response to bradykinin, while indomethacin had no effect. Bradykinin also elicited a dose-dependent increase in free cytosolic calcium concentration. Bradykinin and T-kinin did not affect cyclic AMP levels in these cells. Doses of des-Arg9-bradykinin, a B1 receptor agonist, up to 100 nM did not stimulate the osteoblastic inositol phosphate response. In addition, the bradykinin-stimulated inositol phosphate response was unaffected by des-Arg9-[Leu8]-bradykinin, a B1 receptor antagonist, while it was inhibited by D-Arg-[Hyp3-[beta-(2-thienyl)-Ala]5,8-D-Phe7]-bradykinin, a B2 receptor antagonist. These results suggest that in osteoblastic cells the mechanism of action of bradykinin involves stimulation of the phosphoinositide metabolism and increases in cytosolic calcium levels through activation of B2 receptors.

Effects of inositol trisphosphate on calcium mobilization in bone cells.

The effect of inositol 1,4,5 trisphosphate (IP3) on calcium mobilization was studied in human osteosarcoma lines, Saos-2 and G292, as well as isolated rat osteoblastic and osteoclastic cells. Cells were permeabilized with saponin and calcium mobilization was studied with the fluorescent dye, fura-2 in a recording spectrofluorometer. IP3 (10 microM) increased calcium release in all cell types studied. The effect was dependent on ATP and occurred in the presence of mitochondrial inhibitors. The effect was not seen with inositol 1-phosphate (IP) or inositol 1,4-diphosphate (IP2). Inositol 1,3,4,5 tetrakisphosphate (IP4) appeared to elicit a decrease in the calcium released. Depletion of the intracellular pool with the calcium ionophore, ionomycin, as well as incubation with the inhibitor of intracellular calcium mobilization, TMB-8, obliterated the IP3 effect. The results are consistent with the hypothesis that increases in IP3 can cause a rapid elevation of bone cell cytosolic calcium.