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Biomedical subjects

K M Pruitt

Publications and source records attributed to K M Pruitt.

At least 37 records · Page 2Linked to original sources
PubMed1983

Effect of growth phase and cell envelope structure on susceptibility of Salmonella typhimurium to the lactoperoxidase-thiocyanate-hydrogen peroxide system.

The lactoperoxidase-thiocyanate-hydrogen peroxide system was found to have both bacteriostatic and bactericidal activities against strains of Salmonella typhimurium. The bactericidal activity was clearly dependent on the permeability of the bacterial cell envelope. The deep rough mutant TA1535, with the most permeable cell envelope, was killed both at neutral and acid pH, whereas very little or no killing was observed with the intact cells of the parent strain hisG46. The delta gal mutant, TA1530, representing an intermediate in cell envelope permeability, was inhibited to a much lesser extent than TA1535. Bacteria in log phase of growth were more sensitive to the bactericidal effects than were those in stationary phase. Growth phase had little influence on the bacteriostatic effects. The hisG46 strain produced significant quantities of acid in the presence of glucose. This acid production was inhibited by the lactoperoxidase-thiocyanate-hydrogen peroxide system, and, in contrast to results obtained with several strains of streptococci, this inhibition was not reversed by addition of a reducing agent (2-mercaptoethanol).

Cell Membrane Permeability↗
PubMed1983

A mouthrinse which optimizes in vivo generation of hypothiocyanite.

We report here the properties of a mouthrinse which enhances one of the natural defense factors in human saliva, the salivary peroxidase system. Concentrations of the antimicrobial agent, the hypothiocyanite (OSCN-) ion, can be increased in vivo to bacteriostatic levels by use of a mouthrinse which is 4 mM (0.014%) in hydrogen peroxide and 1 mM (0.0097%) in potassium thiocyanate at pH 5.5. The volume of the rinse, the H2O2 concentrations, and the pH were shown to be determinants of the concentration of OSCN- generated by the rinse.

Anti-Infective Agents↗
PubMed1982

Human milk peroxidase is derived from milk leukocytes.

Peroxidase enzymes present in human colostrum, saliva, polymorphonuclear leukocytes, and bovine milk were compared with respect to their molecular exclusion chromatographic behavior and immunological cross-reactivity. Human milk peroxidase gave an elution profile similar to myeloperoxidase derived from blood polymorphonuclear leukocytes. Human salivary peroxidase reacted with an antibody directed against bovine lactoperoxidase, but with the same antibody preparation no reaction was detected either with human milk peroxidase or leukocyte myeloperoxidase. We conclude that the peroxidase enzyme in human milk is different from the human salivary and the bovine enzymes and is probably derived from milk leukocytes.

Colostrum↗
PubMed1982

Kinetics of hypothiocyanite production during peroxidase-catalyzed oxidation of thiocyanate.

We report here a kinetic study of the generation of hypothiocyanite (OSCN-), a product of lactoperoxidase-catalyzed oxidation of thiocyanate ion. Previous studies have measured OSCN- by reactions involving the oxidation of sulfhydryl compounds. Our results show that a more suitable kinetic analysis of OSCN- can be based on absorbance changes measured at 235 nm. About 90% of the oxidation products of SCN- observed at 235 nm were reactive with sulfhydryls and could be reduced with 2-mercaptoethanol. Both thiocyanate and peroxide were rate-limiting and the formation of OSCN- was proportional to the initial concentration of H2O2 until an equimolar concentration of H2O2 and SCN- was reached. This equimolar concentration gave the maximum generation of OSCN-. High concentrations (greater than 100 micrograms/ml) of lactoperoxidase decreased OSCN- generation, but only if the SCN- was added to the enzyme prior to addition of H2O2. With lactoperoxidase concentrations exceeding 1 microgram/ml, the reaction velocity was rapid, but the decay of OSCN- was slow. Free H2O2 in the reaction mixture always resulted in rapid decay of OSCN-. Addition of varying concentrations of peroxide to solutions containing 1 microgram/ml of enzyme and [SCN-] = 5 mM gave a family of hyperbolic A235 vs. time curves. Both the initial slopes and the plateaus of these curves increased linearly with increasing initial peroxide concentrations up to [H2O2] = 0.4 mM, remained relatively constant in the range [H2O2] = 0.4 to 0.8 mM, and decreased rapidly above [H2O2] = 0.8 mM. These results are consistent with the following kinetic model: Hydrogen peroxide reacts rapidly with lactoperoxidase to produce compound I. This compound I oxidizes SCN- to OSCN- and also oxidizes OSCN- to O2SCN-. The OSCN- also reacts with SCN-. The formation of OSCN- is associated with the appearance of an absorbance peak at 225-235 nm. The oxidation of OSCN- by excess peroxide or its decomposition at high concentrations is associated with a decrease in A235 and the appearance of a peak at 245-255 nm. The extinction coefficient for OSCN- was determined to be 1.29 x 10(3) M-1 . cm-1. The second-order rate constant for the oxidation of thiocyanate by compound I was estimated to be 2 x 10(5) M-1 . s-1.

Kinetics↗
PubMed1982

Lactoperoxidase-catalyzed oxidation of thiocyanate: polarographic study of the oxidation products.

The lactoperoxidase-catalyzed oxidation of thiocyanate (SCN-) was studied by two different polarographic techniques: direct current polarography and linear sweep voltammetry. The main oxidation product at pH 6.5, with a half-wave potential (E1/2) of -0.39 to -0.44 V, was identified as hypothiocyanite (OSCN-) ion. The E1/2 for OSCN- was not available in the literature. The identification of OSCN- was based on a close correlation between the current of the OSCN- peak and the concentration of chemically assayed OSCN-. Also the specific rates of decay of the current and that of chemically detectable OSCN- were similar, and both curves followed apparent first-order kinetics. Subsequently, the addition of a reducing agent (2-mercaptoethanol) resulted in immediate disappearance of both chemically detectable OSCN- and the OSCN- wave in the polarograms. All three components of the lactoperoxidase (LPO) system (SCN-, H2O2, and LPO) were needed to produce the OSCN- peak. Addition of excess H2O2 or H2O2-LPO to an OSCN--SCN- mixture resulted in a formation of a new peak with a characteristic peak potential (Ep) of -0.20 to -0.25 V. The generation of this new peak was associated with a simultaneous, markedly enhanced decrease of OSCN- concentration, indicating a possible reaction between H2O2 (or H2O2-LPO) and OSCN-. No equivalent reaction was obtained by the addition of buffer alone. This new peak may represent higher oxy acids of SCN- (O2SCN-, O3SCN-), formed in the oxidation of OSCN- by H2O2 or by H2O2-LPO. This type of reaction can explain why, in solutions which already contain OSCN- (e.g., in saliva), the addition of H2O2 results in the formation of highly reactive, short-lived antimicrobial products in addition to OSCN-.

Animals↗
PubMed1982

Peroxidase antimicrobial system of human saliva: hypothiocyanite levels in resting and stimulated saliva.

The antimicrobial oxidizing agent hypothiocyanite ion (OSCN-) was measured in resting (drooling) and stimulated (expectorated) whole saliva. Stimulation of the saliva flow rate resulted in a rapid decrease in OSCN- concentration, whereas the thiocyanate ion (SCN-) concentration and peroxidase activity were increased. The decrease in OSCN- levels was greater than could be accounted for by dilution of the whole saliva volume. Assuming that the antimicrobial activity of the salivary peroxidase system is proportional to OSCN- concentration, this system may be more effective in resting saliva than in stimulated saliva.

Adult↗
PubMed1982

Ellipsometric studies of film formation on tooth enamel and hydrophilic silicon surfaces.

The uptake of salivary substances onto hydrophilic negatively charged surfaces of silicon or onto enamel surfaces was studied in vitro with ellipsometry. There was a rapid initial uptake of substances followed by a slow but continuous uptake probably of other substances, which could be removed from saliva by absorption with a S. mutans serotype c strain. the kinetics for the uptake onto enamel and silicon surfaces and the thicknesses of the layers were similar. The combination of ellipsometry and silicon surfaces is suggested as a model for obtaining increased information on dental pellicle formation.

Chemical Phenomena↗
PubMed1982

Interaction of specific and innate factors of immunity: IgA enhances the antimicrobial effect of the lactoperoxidase system against Streptococcus mutans.

The antimicrobial effect of the lactoperoxidase (LPO) system (enzyme with the thiocyanate ion and hydrogen peroxide) on Streptococcus mutans NCTC 10449 (serotype c) was significantly enhanced when the system was combined with secretory IgA. Similar enhancement was observed with LPO-myeloma IgA1 or IgA2 combinations. This enhancement of the antimicrobial efficiency was not dependent on the presence of specific antibodies to S. mutans in the IgA preparation, but seemed to require binding between LPO and immunoglobulin. However, neither human polyclonal nor myeloma IgG or IgM nor rabbit IgG enhanced the antibacterial activity of the LPO system. None of the immunoglobulins, when added alone, produced antimicrobial effects. LPO was shown to bind to colostral secretory IgA, myeloma IgA1, IgA2, and to a lesser degree to monoclonal and polyclonal IgG and monoclonal IgM. This binding had a stabilizing effect on the enzyme activity. Our results suggest that IgA significantly enhances the antibacterial efficiency of one of the innate immune factors--the LPO system.

Animals↗
PubMed1981

Lactoperoxidase-catalyzed inactivation of hexokinase.

The enzymatic activity of hexokinase (ATP : D-hexose 6-phosphotransferase, EC 2.7.1.1) decreased rapidly when the enzyme was exposed to the lactoperoxidase antimicrobial system (consisting of lactoperoxidase, H2O2 and SCN-). Inactivation did not begin until the reaction of one sulfhydryl group per hexokinase monomer was completed. Loss of enzyme activity accompanied the reaction of at least one additional sulfhydryl group per monomer. Covalent incorporation of 14C-labeled SCN- into hexokinase increased as the inactivation reaction progressed. The rate of the hexokinase activity loss dependent on temperature, pH and the presence of glucose and phosphate ion. When H2O2 and SCN- were applied to a Sepharose column bearing covalently attached lactoperoxidase, the column eluate inactivated hexokinase. This demonstrated that the lactoperoxidase molecule itself need not be in contact with hexokinase in order to catalyze hexokinase inactivation. The sulfhydryl-reactive oxidation product of SCN- which is generated by the column is sufficient. The results are consistent with a two-stage reaction in which the exposed, non-essential sulfhydryl groups on the hexokinase molecule react first to produce an enzymatically active but unstable form of hexokinase. This modified form of hexokinase then undergoes a spontaneous, temperature-dependent structural change, which allows reaction of previously shielded, essential sulfhydryl groups. The phenomenon described here suggests a possible mechanism for the antimicrobial effects of the lactoperoxidase system.

Glucose↗
PubMed1981

Inhibition of dental plaque acid production by the salivary lactoperoxidase antimicrobial system.

Resting human whole saliva inhibited acid production by glucose-stimulated, homologous plaque. The degree of inhibition of plaque acid production correlated positively with the concentration of hypothiocyanite (OSCN-) ions in saliva. Supplementation of saliva with an appropriate combination of thiocyanate and hydrogen peroxide resulted in a significant increase in the concentration of OSCN- ions and in more effective inhibition of plaque acid production. In most cases, the inhibition was complete when the supplements were added directly to saliva-plaque mixtures. Acid production resumed when the inhibitory effect of OSCN- was reversed by addition of thiols. Among the oral defense factors, the salivary lactoperoxidase system seems to play an important role by producing highly reactive antibacterial products (including OSCN-) which can regulate bacterial metabolism in the human mouth. The concentration of OSCN- in normal human whole saliva seems to be just below the threshold level required for plaque inhibition. Therefore, enhancement of this system in vivo may be effective in the regulation of plaque acid production.

Animals↗
PubMed1979

Lactoperoxidase binding to streptococci.

There have been conflicting reports regarding the binding of lactoperoxidase to bacterial cell surfaces. We describe here the effects of cell-bound lactoperoxidase on acid production by suspensions of Streptococcus mutans (NCTC 10449) in the presence of hydrogen peroxide and thiocyanate. Saline suspensions of log-phase bacteria were treated with 0.1 mg of lactoperoxidase per ml and were then washed thoroughly. The addition of hydrogen peroxide and thiocyanate markedly reduced the acid production of these lactoperoxidase-treated bacteria but had no effect on the acid production of untreated controls. After a 3-h incubation in saline, the lactoperoxidase-treated bacteria produced acid in the presence of hydrogen peroxide and thiocyanate at the same rate as untreated bacteria. These observations suggest that lactoperoxidase is initially bound to the cell surface in an enzymatically active form at a concentration sufficient to inhibit acid production. The lactoperoxidase is slowly degraded or desorbed as the bacteria stand in saline suspension.

Cell Membrane↗
PubMed1979

Practical application of generic growth theory and the significance of the growth curve parameters.

The generic growth curve is developed by plausibility arguments based on simple growth models. Parameters of the generic curve include the maximum size, maximum specific growth rate, and a dimensionless velocity constant which can be related to metabolic efficiency in the case of nutrient-limited growth. Parameter estimates are obtained from estimates of the size and time at the point of inflection, the size and time at any other arbitrarily selected point, and the maximum size. Parameter estimates thus obtained and used as initial estimates in nonlinear least squares analysis often give rapid convergence to a minimum error mean square. [For the growth of Escherichia coli K-12 in liquid medium, the generic curve could be simplified to a form containing only three parameters: the maximum specific growth rate, the maximum size, and the time required to reach maximum size. When the bacteria were inhibited by the addition of increasing amounts of the lactoperoxidase antimicrobial factor, there was no significant change in viable counts or maximum specific growth rate, but the time required to reach maximum growth increased linearly with increasing amounts of added antimicrobial factor.] Analysis of growth in terms of the generic growth curve can be a powerful technique for finding relationships which may not be apparent from qualitative consideration of the data.

Biometry↗
PubMed1978

The effects of human saliva on the hemolytic activity of complement.

Human saliva was tested for the presence of factors that affect the complement system. Parotid saliva and salivary fractions were incubated at 37 C with human serum as a source of complement. Samples removed from the mixtures within the first 15 minutes had higher levels of whole hemolytic complement activity than did appropriate controls. The final ionic strength of the saliva-serum mixtures was critical to the hemolytic activity of complement. After 60 minutes all serum-saliva mixtures had lower levels of hemolytic activity than did serum-buffer controls. With regard to whole saliva, the salivary sediment was found to be strongly complement-reactive.

Animals↗
PubMed1978

Kinetic assessment of alternative complement pathway activity in a hemolytic system. II. Influence of antibody on alternative pathway activation.

By using a kinetic assay, we have examined the role of antibody in the lysis of rabbit erythrocytes (RaRBC) through the alternative complement (C) pathway. Sera from some hypogammaglobulinemic (Hgamma) humans and all agammaglobulinemic chickens tested had subnormal activity in the assay. Heated normal human or chicken sera, but not heated Hgamma sera, restored activity to deficient Hgamma serum and initiated hemolysis in the presence of rabbit serum as C source. Absorption of heated normal human serum with RaRBC, but not with sheep erythrocytes or zymosan, removed its ability to reconstitute deficient Hgamma serum. Normal hemolytic activity could be resotred to Hgamma serum with human IgM, IgG, or colostral IgA, with goat anti-RaRBC IgG, or with an eluate from serum-sensitized RaRBC, but not with myeloma IgA. Restoration of hemolytic activity to Hgamma serum could be achieved in a dose-dependent fashion with the F(ab')2 fragment of IgG. These results suggest that antibody exerts a significant rate-limiting effect on alternative pathway activity in the RaRBC lytic system. This raises the possibility that antibody may be required for efficient alternative pathway activity in vivo and that the pyogenic infections that occur in Hgamma individuals are due to inefficient activation and fixation of C3 through either the classical or alternative pathway.

Agammaglobulinemia↗
PubMed1977

Enzyme activity of salivary lactoperoxidase adsorbed to human enamel.

Human saliva was incubated with human enamel powder, and the lactoperoxidase activity of the saliva was measured before and after incubation. Liquidphase lactoperoxidase activity was reduced in direct proportion to the weight of enamel powder added. Lactoperoxidase molecules were adsorbed to the enamel surface in an enzymatically active conformation, and this enamel-bound lactoperoxidase activity was also measured. The adsorption of lactoperoxidase was irreversible and produced at least a 40% increase in the concentration of lactoperoxidase in the enamel surface phase as compared with its concentration in the liquid phase. Enamel-bound lactoperoxidase, as well as the free enzyme, was capable of inactivating the key glycolytic enzyme hexokinase. The implications of the adsorption phenomenon for bacterial colonization are discussed.

Adsorption↗