Hypothiocyanite and host-microbe interactions.

The pseudohypohalous acid hypothiocyanite/hypothiocyanous acid (OSCN- /HOSCN) has been known to play an antimicrobial role in mammalian immunity for decades. It is a potent oxidant that kills bacteria but is non-toxic to human cells. Produced from thiocyanate (SCN- ) and hydrogen peroxide (H2 O2 ) in a variety of body sites by peroxidase enzymes, HOSCN has been explored as an agent of food preservation, pathogen killing, and even improved toothpaste. However, despite the well-recognized antibacterial role HOSCN plays in host-pathogen interactions, little is known about how bacteria sense and respond to this oxidant. In this work, we will summarize what is known and unknown about HOSCN in innate immunity and recent advances in understanding the responses that both pathogenic and non-pathogenic bacteria mount against this antimicrobial agent, highlighting studies done with three model organisms, Escherichia coli, Streptococcus spp., and Pseudomonas aeruginosa.

Safety Assessment of the Modified Lactoperoxidase System-In Vitro Studies on Human Gingival Fibroblasts.

One strategy in caries prevention is to inhibit the formation of cariogenic biofilms. Attempts are being made to develop oral hygiene products enriched with various antimicrobial agents. One of them is lactoperoxidase-an enzyme that can oxidise (pseudo)halide ions to reactive products with antimicrobial activity. Currently, commercially available products utilise thiocyanate as a substrate; however, several alternatives that are oxidised to products with greater antimicrobial potential have been found. In this study, toxicity against human gingival fibroblasts of the lactoperoxidase system was evaluated using four different (pseudo)halide substrate systems-thiocyanate, iodide, selenocyanate, and a mixture of thiocyanate and iodide. For this purpose, cells were treated with the systems and then apoptosis, cell cycle, intracellular glutathione concentration, and mitochondrial superoxide production were assessed. The results showed that each system, after generating 250 µM of the product, inhibited cell divisions, increased apoptosis, and increased the percentage of dead cells. It was concluded that the mechanism of the observed phenomena was not related to increased superoxide production or the depletion of glutathione concentration. These findings emphasised the need for the further in vitro and in vivo toxicity investigation of the modified lactoperoxidase system to assess its safety and the possibility of use in oral hygiene products.

Modified Lactoperoxidase System as a Promising Anticaries Agent: In Vitro Studies on Streptococcus mutans Biofilms.

The lactoperoxidase (LPO) system shows promise in the prevention of dental caries, a common chronic disease. This system has antimicrobial properties and is part of the non-specific antimicrobial immune system. Understanding the efficacy of the LPO system in the fight against biofilms could provide information on alternative strategies for the prevention and treatment of caries. In this study, the enzymatic system was modified using four different (pseudo)halide substrates (thiocyanate, thiocyanate-iodide mixture, selenocyanate, and iodide). The study evaluated the metabolic effects of applying such modifications to Streptococcus mutans; in particular: (1) biofilm formation, (2) synthesis of insoluble polysaccharides, (3) lactate synthesis, (4) glucose and sucrose consumption, (5) intracellular NAD+ and NADH concentrations, and (6) transmembrane glucose transport efficiency (PTS activity). The results showed that the LPO-iodide system had the strongest inhibitory effect on biofilm growth and lactate synthesis (complete inhibition). This was associated with an increase in the NAD+/NADH ratio and an inhibition of glucose PTS activity. The LPO-selenocyanate system showed a moderate inhibitory effect on biofilm biomass growth and lactate synthesis. The other systems showed relatively small inhibition of lactate synthesis and glucose PTS but no effect on the growth of biofilm biomass. This study provides a basis for further research on the use of alternative substrates with the LPO system, particularly the LPO-iodide system, in the prevention and control of biofilm-related diseases.

Deactivation efficiency of stabilized bactericidal emulsions.

Biocide emulsions stabilized with various stabilizing agents were prepared and characterized, and their efficiency in bacteria deactivation was evaluated. A number of stabilizing agents were tested for their stabilizing effect on emulsions of thiocyanomethylthiobenzothiazole (TCMTB) biocide. Two agents, the most successful in stabilizing the biocide, were chosen for further studies: high molecular weight polyethyleneimine (PEI) and an amphiphilic block copolymer of poly(caprolactone)-b-poly(acrylic acid) (PCL(33)-b-PAA(33)). The emulsion droplet sizes varied between 325 and 500 nm. Deactivation of bacteria was studied by exposing E. coli ATCC 11229 bacteria dispersions to emulsions stabilized by positively charged PEI or negatively charged PCL-b-PAA micelles and by measuring their absorbance; E. coli do not grow with time in the presence of biocide emulsions. PEI molecules alone act as biocide and deactivate the bacteria. PCL-b-PAA micelles as stabilizing agent do not affect the growth of the E. coli ; bacteria are deactivated by TCMTB released from the emulsion droplets. The kinetics of emulsion dissolution studies revealed for both stabilizing agents a decrease in droplet size with time while the emulsions were subjected to dialysis. The biocide was released from the emulsions within ∼250 min; the droplet shells consist mostly of PEI or PCL-b-PAA insoluble complexes with the biocide, which do not dissolve during dialysis. SEM images confirm the presence of residual crumbled shells with holes after 24 h of dialysis.

Effect of lactoperoxidase system containing toothpaste on cariogenic bacteria in children with early childhood caries.

BACKGROUND AND OBJECTIVES: Lactoperoxidase system contains Lactoperoxidase, Hydrogen peroxide and Thiocyanate ions, which have inhibitory action against cariogenic oral microflora. The present study was undertaken to assess the effect of lactoperoxidase system containing toothpaste on cariogenic microflora in children with early childhood caries. METHODS: Study group included 30 children with Early Childhood Caries. 15 were considered as test group who used the test product Biotene toothpaste and other 15 as control group who used Colgate Active as control product. Salivary samples were analyzed for mutans streptococci (MS) and Lactobacilli, and for the levels of Thiocyanate ions. RESULTS: Showed significant increase in the levels of Thiocyanate ion in saliva during experimental period. Compared to the control group test group showed significant increase in the levels of thiocyanate ions during experimental and washout period, whereas the number of colonies of MS and Lactobacilli were significantly reduced in test group during experimental period. CONCLUSION: The levels of thiocyanate ions can be increased in vivo by supplementing the saliva with natural enzymes like lactoperoxidase. This increased concentration of thiocyanate will reduce the number of cariogenic microflora in children with Early Childhood Caries.

Reactions of thiocyanate in the mixture of nitrite and hydrogen peroxide under acidic conditions: investigation of the reactions simulating the mixture of saliva and gastric juice.

Nitrite and SCN(-) in saliva can mixes with H(2)O(2) in the stomach. The mixing can result in the formation of ONOOH. It is not yet known how salivary SCN(-) reacts with ONOOH. An objective of the present study was to elucidate the reaction between ONOOH and SCN(-). In nitrite/H(2)O(2) systems at pH 2, SCN(-) inhibited the consumption of nitrite and the formation of O(3)(-). SCN(-) enhanced the decomposition of ONOOH and H(2)O(2) in HNO(2)/H(2)O(2) systems. Accompanying the reactions, sulfate was formed, suggesting that ONOOH oxidized SCN(-). SCN(-) inhibited the nitration of phenolics induced by HNO(2)/H(2)O(2). The inhibition is discussed taking SCN(-)-dependent reduction of ONOOH to HNO(2) into consideration. SCN(-) also inhibited H(2)O(2)-induced consumption of nitrite and nitration of phenolics in acidified saliva. The result obtained in this study suggests that salivary SCN(-) can reduce ONOOH to O(2)(-)/HNO(2) inhibiting nitrating reactions in the stomach.

Dual-function of thiocyanate on nitrite-induced formation of reactive nitrogen oxide species in human oral cavity: inhibition under neutral and enhancement under acidic conditions.

Salivary nitrate is reduced to nitric oxide (NO) via nitrite in the human oral cavity. The nitrite and NO formed can be transformed to reactive nitrogen oxide species (RNOS). In this investigation, RNOS formed in mixed whole saliva and its fractions were detected by the oxidation of aminophenyl fluorescein (APF) and the transformation of 3-amino-4-monomethylamino-2',7'-difluorofluorecein (DAF-FM) to its triazol form (DAF-FMT). Nitrite-induced oxidation of APF and formation of DAF-FMT increased as pH was decreased from 7 to 5 and SCN- inhibited the oxidation of APF and the formation of DAF-FMT around neutral pH and enhanced at pH about 5. The SCN- -dependent inhibition was due to the suppression of salivary peroxidase and the enhancement was due to the formation of NOSCN from HNO2 and SCN-. It is deduced that the increase in the concentrations of nitrite and H+ in the oral cavity may result in the enhanced formation of

Formation of the thiocyanate conjugate of chlorogenic acid in coffee under acidic conditions in the presence of thiocyanate and nitrite: possible occurrence in the stomach.

The objective of the present study was to elucidate how chlorogenic acid in coffee was transformed under acidic conditions simulating the mixture of saliva and gastric juice. When coffee was incubated in acidified saliva that contained nitrite and SCN-, in addition to nitric oxide (NO), four major components were detected. Two of the four components (components 3 and 4) were generated when chlorogenic acid was incubated in acidified saliva and when incubated in an acidic buffer solution in the presence of both nitrite and SCN-. By the incubation of chlorogenic acid in acidic nitrite in the absence of SCN-, components 3 and 4 were not formed but the quinone of chlorogenic acid and nitrated chlorogenic acid were formed. The result indicates that SCN- was indispensable for nitrous acid induced formation of components 3 and 4. Component 4 was isolated and its structure was determined to be (E)-5'-(3-(7-hydroxy-2-oxobenzo[d] [1,3]oxathiol-4-yl)acryloyloxy)quinic acid. Component 3, which was suggested to be 2-thiocyanatochlorogenic acid, seemed to be formed by the reaction between SCN- and the quinone of chlorogenic acid. As it has been reported that the quinone of chlorogenic acid can react with thiols and can decompose producing H2O2, the formation of component 4 can reduce the toxic effects of the quinone of chlorogenic acid.

HCO-3-activated adenosine triphosphatase in intestinal mucosa of the eel.

An HCO-3-activated and SCN--inhibited ATPase (ATP phosphohydrolase, EC 3.6.1.3) found in homogenates of intestinal mucosa of the eel was solubilized by Triton X-100. Optimal HCO-3-concentration and pH for the enzyme were 25 mM and 8.7, respectively. HCO-3-ATPase activity in both homogenate and solubilized preparations increased after seawater adaptation. This adaptive increase in enzyme activity was also observed in the gills and the kidney. The HCO-3-ATPase seems to be related to transport mechanisms, especially for Cl-, in osmoregulatory surfaces of the eel.

Hydrolysis of neutral glycerides by lipases of rat brain microsomes.

The hydrolysis of monoacylglycerol and diacylglycerol by rat brain microsomes was followed by measuring the release of glycerol and monooleylglycerol from dispersions of water insoluble glyceryl esters of oleic acid. The microsomes showed three lipolytic activities. One activity, optimal at pH 4.8, catalyzed the hydrolysis of diacylglycerol but not monoacylglycerol. Two other lipolytic activities, optimal at pH 8.0-8.6, catalyzed the hydrolysis of both diacylglycerol and monoacylglycerol. The pH 8.0-8.6 activities were sensitive to heat and SH-reagents. Detergents were inhibitory in all cases. Extraction of the microsomes with KCl, KSCN, urea or Triton X-100 did not change the ratio of diacylglycerol hydrolysis at pH 4.8 and 8.0. The results of subcellular fractionation studies showed that there was no significant enrichment of the acid lipase in any fraction.

Location and ion-binding of membrane-associated valinomycin, a proton nuclear magnetic resonance study.

Valinomycin, incorporated in small unilamellar vesicles of perdeuterated dimyristoylphosphatidylcholine, reveals several well-resolved 1H-NMR resonances. These resonances were used to examine the location, orientation and ion-binding of membrane-bound valinomycin. The order of affinity of membrane-bound valinomycin for cations is Rb+ greater than K+ greater than Cs+ greater than Ba2+, and binding is sensitive to surface change. The exchange between bound and free forms is fast on the NMR time scale. The intrinsic binding constants, extrapolated to zero anion concentration, are similar to those determined in aqueous solution. Rb+ and K+ show 1:1 binding to valinomycin, whereas the stoichiometry of Cs+ and Ba2+ is not certain. Paramagnetic chemical shift reagents and nitroxide spin label relaxation probes were used to study the location and orientation of valinomycin in the membrane. Despite relatively fast exchange of bound cations, the time average location of the cation-free form of valinomycin is deep within the bilayer under the conditions of these experiments. Upon complexation to K+, valinomycin moves closer to the interfacial region.

Salivary thiocyanate/nitrite inhibits hydroxylation of 2-hydroxybenzoic acid induced by hydrogen peroxide/Fe(II) systems under acidic conditions: possibility of thiocyanate/nitrite-dependent scavenging of hydroxyl radical in the stomach.

Formation of OH radicals in the stomach is possible by Fenton-type reactions, as gastric juice contains ascorbic acid (AA), iron ions and H2O2. An objective of the present study is to elucidate the effects of salivary SCN- and NO2- on the hydroxylation of salicylic acid which was induced by H2O2/Fe(II) and AA/H2O2/Fe(II) systems. Thiocyanate ion inhibited the hydroxylation of salicylic acid by the above systems in acidic buffer solutions and in acidified saliva. The inhibition by SCN- was deduced to be due to SCN- -dependent scavenging of OH radicals. Nitrite ion could enhance the SCN- -dependent inhibition of the hydroxylation induced by AA/H2O2/Fe(II) systems. The enhancement was suggested to be due to scavenging of OH radicals by NO which was formed by the reactions among AA, HNO2 and SCN- contained in the reaction mixture. The concentrations of SCN- and NO2-, which were effective for the inhibition, were in ranges of their normal salivary concentrations. These results suggest that salivary SCN- can cooperate with NO2- to protect stomach from OH radicals formed by AA/H2O2/Fe(II) systems under acidic conditions.

Is there a plasma membrane-located anion-sensitive ATPase? III. Identity of the erythrocyte enzyme with (Ca2+ + Mg2+)-ATPase.

The characteristics of the anion-sensitive Mg2+-ATPase activity of the rabbit erythrocyte have been studied in a lyophilized ghost preparation. The enzyme appears to be different from the anion-sensitive Mg2+-ATPase activity of other tissues in many parameters, such as optimal pH, effects of various anions, oligomycin sensitivity and effects of Triton X-100. The enzyme is insensitive towards inhibition by irreversibly bound 4,4'-diisothiocyano-dihydrostilbene-2,2'-disulfonic acid (H2DIDS). This excludes a relationship between the enzyme and the "band 3" protein, which is thought to be involved in the anion exchange over the erythrocyte membrane. From the effects of ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), CaCl2, chlorpromazine and ruthenium red it is concluded that the enzyme activity does not represent a separate entity but is part of the (Ca2+ + Mg2+)-ATPase system of the erythrocyte membrane. A reported stimulatory effect of carbonic anhydrase is attributed to a contamination of the carbonic anhydrase preparation by calcium and/or (Ca2+ + Mg2+)-ATPase activator protein.

Purification and properties of the intact P-700 and Fx-containing Photosystem I core protein.

The intact Photosystem I core protein, containing the psaA and psaB polypeptides, and electron transfer components P-700 through FX, was isolated from cyanobacterial and higher plant Photosystem I complexes with chaotropic agents followed by sucrose density ultracentrifugation. The concentrations of NaClO4, NaSCN, NaI, NaBr or urea required for the functional removal of the 8.9 kDa, FA/FB polypeptide was shown to be inversely related to the strength of the chaotrope. The Photosystem I core protein, which was purified to homogeniety, contains 4 mol of acid-labile sulfide and has the following properties: (i) the FX-containing core consists of the 82 and 83 kDa reaction center polypeptides but is totally devoid of the low-molecular-mass polypeptides; (ii) methyl viologen and other bipyridilium dyes have the ability to accept electrons directly from FX; (iii) the difference spectrum of FX from 400 to 900 nm is characteristic of an iron-sulfur cluster; (iv) the midpoint potential of FX, determined optically at room temperature, is 60 mV more positive than in the control; (v) there is indication by ESR spectroscopy of low-temperature heterogeneity within FX; and (vi) the heterogeneity is seen by optical spectroscopy as inefficiency in low-temperature electron flow to FX. The constraints imposed by the amount of non-heme iron and labile sulfide in the Photosystem I core protein, the cysteine content of the psaA and psaB polypeptides, and the stoichiometry of high-molecular-mass polypeptides, cause us to re-examine the possibility that FX is a [4Fe-4S] rather than a [2Fe-2S] cluster ligated by homologous cysteine residues on the psaA and psaB heterodimer.

A Cl-/HCO-3-ATPase in the gills of Carassius auratus. Its inhibition by thiocyanate.

An ATPase is demonstrated in plasma membrane fractions of goldfish gills. This enzyme is stimulated by Cl- and HCO-3, inhibited by SCN-. Biochemical characterization shows that HCO-3 stimulation (Km = 2.5 mequiv./l) is specifically inhibited in a competitive fashion by SCN- (Ki = 0.25 mequiv./l). This residual Mg2+-dependent activity is weakly affected by SCN-. In the microsomal fraction chloride stimulation of the enzyme occurs in the presence of HCO-3 (Km for chloride = 1 mequiv/l); no stimulation is observed in the absence of HCO-3. Thiocyanate exhibits a mixed type of inhibition (Ki = 0.06 mequiv./l) towards the Cl- stimulation of the enzyme. Bicarbonate-dependent ATPase from the mitochondrial fraction is stimulated by Cl-, but this enzyme has a relatively weak affinity for this substrate (Km = 14 mequiv./l).

Transport of sodium into apical membrane vesicles prepared from fetal sheep alveolar type II cells.

A method is described for isolating apical plasma membrane vesicles from fetal alveolar type II cells. The procedure yields purified apical membranes which are enriched 24-fold with the brush-border enzyme marker, alkaline phosphatase. Contamination of this fraction by basolateral membranes and organelles is minimal. Evidence for transport of Na+ into an intravesicular space is demonstrated by: (1) time-dependent uptake of Na+ with release of accumulated Na+ by treatment with detergent; (2) a linear inverse correlation between Na+ uptake and medium osmolarity. In addition, Na+ uptake is shown to be anion dependent (SCN- greater than Cl- greater than gluconate-) and sensitive to amiloride inhibition at a concentration of 1 mM.

Effect of chemical modification on the crystallization of Ca2+-ATPase in sarcoplasmic reticulum.

The influence of chemical modification on the morphology of crystalline ATPase aggregates was analyzed in sarcoplasmic reticulum (SR) vesicles. The Ca2+-ATPase forms monomer-type (P1) type crystals in the E1 and dimer-type (P2) crystals in the E2 conformation. The P1 type crystals are induced by Ca2+ or lanthanides; P2 type crystals are observed in Ca2+-free media in the presence of vanadate or inorganic phosphate. P1- and P2-type Ca2+-ATPase crystals do not coexist in significant amounts in native sarcoplasmic reticulum membrane. The crystallization of Ca2+-ATPase in the E2 conformation is inhibited by guanidino-group reagents (2,3-butanedione and phenylglyoxal), SH-group reagents, phospholipases C or A2, and detergents, together with inhibition of ATPase activity. Amino-group reagents (fluorescein 5'-isothiocyanate, pyridoxal phosphate and fluorescamine) inhibit ATPase activity but do not interfere with the crystallization of Ca2+-ATPase induced by vanadate. In fluorescamine-treated sarcoplasmic reticulum the vanadate-induced crystals contain significant P1-type regions in addition to the dominant P2 form.

Initial characterizations of nontransformed and transformed [3H ]aldosterone-type I receptor complexes in brain cytosol.

Incubation of [3H]aldosterone-type I receptor complexes in mouse brain cytosol with the chaotropic anion thiocyanate increased the fraction of receptors retained by DNA-cellulose from less than 10% to over 40%, whereas it decreased the fraction retained by protamine sulfate from more than 90% to less than 10%. Thiocyanate-induced transformation to the DNA-binding species was also accompanied by a 2.1-fold decrease in the rate of [3H]aldosterone dissociation from type I receptors as well as by an increase in the apparent positive charge and hydrophobicity of the surface of these receptors, as revealed by DEAE Bio-Gel ion exchange and pentyl agarose hydrophobic interaction chromatography. Sucrose density gradient sedimentation and Sephacryl S-300 gel exclusion chromatography revealed a reduction in the sedimentation coefficient and Stokes radius of the steroid-receptor complex from 9.6S and 8.0 nm before to 4.7S and 6.1 nm after transformation, respectively. These changes in hydrodynamic parameters were found to correspond to a 2.8-fold reduction in the apparent molecular mass from 331,000 before to 120,000 after transformation. In view of these various findings as well as the known differential affinity of protamine sulfate for the 90K heat shock protein, we suggest that thiocyanate-induced transformation is initiated by the dissociation of two molecules of heat shock protein from each steroid/DNA-binding type I receptor subunit.

Effect of cigarette smoke on oral peroxidase activity in human saliva: role of hydrogen cyanide.

Peroxidase activity in human saliva is composed of salivary peroxidase (80%), of salivary glandular origin, and myeloperoxidase (20%), of leukocyte origin. The term oral peroxidase (OPO) is used here to denote the total activity of both peroxidase species. Using the 2-nitrobenzoic acid-thiocyanate assay, OPO activity was measured in the saliva of nonsmokers after exposure to gas-phase cigarette smoke (CS) in an in vitro system using three puffs of CS in 1 h. A marked decrease of 76% of activity was observed following three puffs of CS. In order to elucidate the mechanism by which CS caused loss of OPO activity, several oxidants and antioxidants were applied to saliva in vitro in the presence and absence of CS. No protection for CS-induced loss of OPO activity occurred in the presence of glutathione, N-acetylcysteine, ascorbic acid, or Desferal. Exposure of saliva to purified aldehydes present in CS did not significantly affect OPO loss of activity. Similarly, ascorbic acid in the presence of FeCl(3) and nicotine also had no effect on OPO activity. Exposure of OPO to cyanate at levels present in CS caused a 65-70% loss of OPO activity, which was reversible after 24 h of dialysis. Moreover, hydroxocobalamin, a known cyanate chelator, could prevent CS- and potassium cyanide-induced inactivation of OPO by 70-90%. The results show that hydrogen cyanide, known to be present in microgram amounts per cigarette, is likely to be the species in CS responsible for loss of salivary OPO activity. The finding of reduced salivary OPO levels after CS exposure may represent a contributory mechanism for CS-related compromises in antimicrobial defenses in the aerodigestive tract.

Inhibition of herpes simplex virus type 1, respiratory syncytial virus and echovirus type 11 by peroxidase-generated hypothiocyanite.

The human mouth is an important route of viral transmission and evidence exists that human saliva can neutralize some viruses, e.g. herpes simplex type 1 (HSV-1) and human immunodeficiency virus (HIV) in vitro. However, little is known of the actual antiviral agents in saliva. We have analyzed how hypothiocyanite (HOSCN/-OSCN) ions, present in human saliva and generated by salivary peroxidase systems, affect the viability of three different types of viruses; HSV-1 (capable of inducing oral lesions), respiratory syncytial virus (RSV, respiratory infections), and echovirus 11 (EV 11, enteric diseases). Viral suspensions were pretreated (30 min) with HOSCN/-OSCN concentrations up to 180 microM both at pH 6.0 and 7.1 and inoculated into human gingival fibroblasts. The cultures were incubated at 37 degrees C for 18-48 h, fixed and the infected cells were counted after immunoperoxidase staining. HSV-1 was most sensitive to HOSCN/-OSCN with an IC50 of 8.5 microM at pH 6.0 and an IC50 of 20 microM at pH 7.1, respectively. RSV was inhibited by HOSCN/-OSCN only at pH 6.0 with an IC50 of 8.0 microM. EV 11 was also resistant at neutral pH, but sensitive at pH 6.0 with an IC50 of 68 microM. In contrast to HSV-1 and RSV, the inhibition of EV 11 was not dependent on the concentration of HOSCN/-OSCN. The inhibition was in all cases stronger at pH 6.0 than at neutral pH. Our results suggest that hypothiocyanite, a normal component of human whole saliva, in physiological concentrations effectively inhibits HSV-1 and RSV at acidic pH, whereas EV 11 is more resistant in vitro.