Immunogenic and protective potential of mutans streptococcal glucosyltransferase peptide constructs selected by major histocompatibility complex class II allele binding.

Mutans streptococcal glucosyltransferases (GTF) have been demonstrated to be effective components of dental caries vaccines. We had previously selected peptide subunits of GTF for vaccine development based on putative functional significance and conservation of GTF primary structure among enzyme isoforms. In this study, 20 20-mer linear GTF peptides were synthesized, 17 identified on the basis of the highest potential major histocompatibility complex (MHC) class II-binding activity using computer-generated algorithms (Epimatrix and ProPred) and 3 with previously demonstrated functional significance. The immunoreactivities of these peptides were explored with rodent systems. Sera from GTF-immunized rats, assessed for binding to linear peptides by enzyme-linked immunosorbent assay, demonstrated immunoglobulin G antibody reactivity with peptides 6 and 11 and a T-cell proliferation response to peptides 6, 9, 11, and 16. Multiple antigenic peptide (MAP) constructs were synthesized from promising linear sequences. Rats that were immunized with MAP 7, 11, or 16, respectively, responded well to the immunizing MAP. Most importantly, a robust immune response (antibody and T-cell proliferation) was observed to native GTF following MAP 11 (amino acids 847 to 866; VVINNDKFVSWGITDFEM) immunization. This response inhibited GTF enzyme function. Two dental caries pathogenesis experiments were performed wherein rats were immunized with MAP constructs 11, 16, and/or 11 plus 16, followed by infection with cariogenic Streptococcus sobrinus. In both experiments cariogenic bacterial recoveries were reduced relative to total streptococci in the MAP 11- and MAP 11 plus 16-immunized groups, and the extent of dental caries was also significantly reduced in these groups. Thus, we have identified a peptide with projected avid MHC-binding activity that elicited immunoreactivity with native GTF and demonstrated protection against dental caries infection after immunization, implying that this peptide may be important in a subunit dental caries vaccine.

Polarized Caco-2 cells. Effect of reactive oxygen metabolites on enterocyte barrier function.

Reactive oxygen metabolites are implicated in gastrointestinal disease and enterocyte injury associated with ischemia-reperfusion, bacterial translocation, inflammatory bowel disease, and necrotizing enterocolitis. The ileal-like, human colon carcinoma cell line, Caco-2, was used to investigate oxidative damage. After challenging Caco-2 cells with reactive oxygen metabolites, the permeability, viability, and energy charge of Caco-2 cells were assessed. Permeability was determined by transepithelial electrical potential and flux of small molecules. Viability was determined by release of 51Cr. Cell energy was evaluated by determining adenylate energy charge. The source of reactive oxygen metabolites, with the exception of menadione, did not affect viability of Caco-2 cells; cell permeability was increased. The increased varied with the source and location of the reactive oxygen metabolite. There was no change in energy charge. This study suggests that reactive oxygen metabolites could cause enterocyte damage and that the source of the reactive oxygen metabolite is an important variable in determining the extent of damage. Antioxidants might prevent injury.

Platelet adenylate cyclase and monoamine oxidase in women with alcoholism or a family history of alcoholism.

PURPOSE: Characteristic changes of platelet membrane monoamine oxidase and adenylate cyclase activities have been described in men with alcoholism. We studied the occurrence of these changes in abstinent alcoholic women and in nonalcoholic female control subjects with and without family histories of alcoholism. METHODS: Blood samples were collected from 23 female alcoholics and 39 nonalcoholic female social drinkers. Platelet membrane assays were performed for monoamine oxidase and adenylate cyclase activities. RESULTS: Alcoholic women had lower basal adenylate cyclase (p < 0.01) and adenylate cyclase activities stimulated by cesium fluoride (p < 0.001), by the guanine nucleotide analog 5'-guanylylimidodiphosphate (p < 0.02), and by prostaglandin E1 (p < 0.01). Female control subjects with family histories of alcoholism also had lower basal adenylate cyclase (p < 0.01) and adenylate cyclase activities enhanced by incubation with cesium fluoride (p < 0.005) and 5'-guanylylimidodiphosphate (p < 0.001). Monoamine oxidase activity levels measured with (p < 0.001) and without ethanol (p < 0.01) were higher for alcoholic women. No significant differences were found between female control subjects with and without family histories of alcoholism for monoamine oxidase in the absence or presence of ethanol. DISCUSSION: In vitro platelet adenylate cyclase activity may facilitate a diagnosis of alcoholism in women and may be a biologic indicator of vulnerability in the offspring of alcoholics.

Selenium regulation of glutathione peroxidase in human hepatoma cell line Hep3B.

Glutathione peroxidase is an important enzyme in cellular antioxidant defense systems, detoxifying peroxides and hydroperoxides. As a component of the glutathione cycle, it protects the liver from reactive oxygen metabolites. Selenocysteine is present at the catalytic site of glutathione peroxidase, and selenium availability regulates glutathione peroxidase enzyme activity. Hep3B cells, a well-differentiated human hepatoma-derived cell line, exhibited time-dependent decrease in glutathione peroxidase activity (nmol NADPH oxidized/min/mg protein, mean +/- SE) when incubated in selenium-free medium for 10 days (Day 0, 21.8 +/- 7.3; Day 2, 10.9 +/- 1.2; Day 4, 7.9 +/- 0.8; Day 6, 4.0 +/- 0.7; Day 8, 4.5 +/- 0.6; Day 10, 1.6 +/- 0.4). With the reintroduction of selenium, glutathione peroxidase activity returned. A second human hepatoma cell line, HepG2, demonstrated a similar pattern when depleted of and then repleted with selenium. To assess protein synthesis, glutathione peroxidase activity was measured in deficient and replete Hep3B cells incubated with and without selenium and with and without cycloheximide. Deficient cells (mean +/- SE) (4.9 +/- 0.2) showed an increase in glutathione peroxidase activity after 24 h in selenium-containing medium (11.6 +/- 0.2), but not when cycloheximide was included in the medium (6.9 +/- 0.5) or when cycloheximide and no selenium was included (5.3 +/- 0.8). Replete Hep3B cells (40.1 +/- 1.1) demonstrated decreased glutathione peroxidase after 24 h in medium without selenium (34.0 +/- 1.4), medium with both cycloheximide and selenium (34.0 +/- 2.6), and medium without selenium and containing cycloheximide (37.6 +/- 1.3). These data suggest that protein synthesis is needed for selenium repletion to exert control on glutathione peroxidase activity. Using a cDNA for human glutathione peroxidase (GPx1), selenium-deficient and replete Hep3B cell RNA was analyzed by Northern blot. mRNA for GPx was quantified by densitometry. The steady-state mRNA level for glutathione peroxidase in deficient cells was 40% of that in replete cells. Nuclear run-on studies to determine the rate of GPx-specific mRNA synthesis showed no difference between nuclei from selenium-replete and selenium-deficient cells. This finding eliminated the possibility of differential transcription rates as an explanation for the observed reduction in mRNA brought about by selenium deficiency and suggested instead a stabilization of mRNA in selenium-replete cells. While selenium deficiency decreased mRNA levels by 60%, glutathione peroxidase enzyme activity decreased by 93%, suggesting a co- and/or post-translational control mechanism in addition to the effect on mRNA stability.

Synthesis of only two heat shock proteins is required for thermoadaptation in cultured cowpea cells.

Cell cultures of a heat sensitive genotype of cowpea (Vigna unguiculata) were adapted to tolerate moderate levels of heat by maintaining cells at 32, 36, and 38 degrees C over many cell generations. Cells adapted to 32 and to 36 degrees C did not produce the typical heat shock proteins (HSP). Cells adapted to 38 degrees C synthesized two new proteins, which appear to be a subset of the HSP. In many temperature sensitive organisms it is thought that HSP confer thermotolerance. However, we hypothesize that specific proteins are associated with heat tolerance in cowpea, other heat tolerant plants (species such as sorghum and millet), and adapted cells which provide them with enhanced heat tolerance. From present data we suggest two proteins (70 and 80 kilodaltons) are strongly associated with heat tolerance and heat adaptation.

Fatty acid transfer between multilamellar liposomes and fatty acid-binding proteins.

A simple experimental system was developed for studying the movement of long-chain fatty acids between multilamellar liposomes and soluble proteins capable of binding fatty acids. Oleic acid was incorporated into multilamellar liposomes containing cholesterol and egg yolk lecithin and incubated with albumin or hepatic fatty acid-binding protein. It was found that the fatty acid transferred from the liposomes to either protein rapidly and selectively under conditions where phospholipid and cholesterol transfer did not occur. More than 50% of the fatty acid contained within liposomes could become protein bound, suggesting that the fatty acid moved readily between and across phospholipid bilayers. Transfer was reduced at low pH, and this reduction appeared to result from decreased dissociation of the protonated fatty acid from the bilayer. Liposomes made with dimyristoyl or dipalmitoyl lecithin and containing 1 mol per cent palmitic acid were used to show the effect of temperature on fatty acid transfer. Transfer to either protein did not occur at temperatures where the liposomes were in a gel state but occurred rapidly at temperatures at or above the transition temperatures of the phospholipid used.