Gretchen B. Caughman

Correlation of cytotoxicity, filler loading and curing time of dental composites.

Previous studies have shown that dental resin composites tested in cell culture produce cytotoxic effects on human gingival tissues. In this study, the cytotoxic potential of resin composites on primary human gingival fibroblast cultures was evaluated, based on inhibition of cellular protein synthesis measured by [35S] methionine incorporation. Both resin content and percentage of monomer conversion were considered as potential causes of cytotoxicity. Three resin composites were selected to provide a range of filler content from 45 to 86 wt%. Duplicate sample discs (1 mm thick x 10 mm diameter) of each composite were polymerized for 15, 30 and 60 s, followed by heat (110 degrees C, 10 min), and the degree of monomer conversion for each sample group was measured using Fourier transform infrared spectrophotometry. Identically fabricated discs were placed into 35 mm culture dishes with gingival fibroblasts and incubated for 24 h at 37 degrees C. The cell monolayers then were labelled at 24 h with [35S] methionine, washed and solubilized; then incorporated radioactivity was quantitated by liquid scintillation spectrometry. For each composite, as the percentage of monomer conversion increased, cellular toxicity decreased. In comparing different composites having similar monomer conversions, it was found that the filler/resin ratio was not the only factor determining the composites relative toxicity.

Regulation of equine herpesvirus type 1 gene expression: Characterization of immediate early, early, and late transcription

The regulation of equine herpesvirus type 1 (EHV-1) transcription was examined in infected rabbit kidney cells using metabolic inhibitors. In order to map EHV-1 immediate early, early, and late transcripts, viral RNA was 32P-labeled in vivo and hybridized to EHV-1 DNA restriction fragments immobilized on nitrocellulose filters. Immediate early viral RNA was mapped to one region of the viral genome within the inverted repeat DNA sequences (map units 0.78-0.83 and 0.95-1.0). Northern blot hybridization analysis using a 32P-labeled cloned DNA probe from this region identified a single immediate early viral transcript (approximately 6 kb). Transcription of early and late genes was not restricted to any specific region on the viral genome as indicated by the ability of 32P-labeled early and late RNA to hybridize to EHV-1 restriction endonuclease fragments from both the long and short components of EHV-1 DNA. Additional experiments performed without the use of metabolic inhibitors confirmed that EHV-1 transcription is temporally regulated. The characterization of EHV-1 transcription during productive infection will serve as a reference for the analysis of viral transcripts in oncogenically transformed and persistently infected cells.

Glass ionomer and composite resin cements: Effects on oral cells

Because the cement interfaces of restorations can approximate the periodontium, it is critical to determine the biocompatibility of cements. In this study, the cytotoxic potential of resin luting agents on cultures of gingival fibroblasts and oral epithelial cells was evaluated for direct microscopic cytotoxicity, cell morbidity, impaired adherence, and inhibition of macromolecular synthesis. Visible effects ranged from severe toxicity with inadequately polymerized composite resin to no detectable morphologic cell damage by a glass ionomer cement, but inhibition of protein and RNA synthesis varied with the material and cell type. The glass ionomer cement demonstrated no morphologic damage, but exhibited inhibition of macromolecular synthesis in gingival fibroblasts. These results confirmed that in vitro metabolic assays are appropriate for examining the biologic effects of materials.

Characterization of equine herpesvirus type 1 immediate early proteins.

EHV-1 immediate early (IE) gene expression in lytic infection results in the production of four high mol wt immediate early polypeptides (IEPs), designated IE1, IE2, IE2, and IE4; however, IE transcription is limited to the synthesis of a single 6-kb mRNA. Together, these findings raised questions as to whether the four IEPs were related products of the same gene. In the present study the IEPs were characterized with respect to their structural similarities, antigenic relatedness, and postsynthetic modifications. IE1 was the most abundant IEP, in that it accounted for approximately 80% of the IEP-incorporated radiolabel in infected rabbit kidney cells labeled under IE conditions with [35S]methionine or 14C-labeled amino acid mixtures. IE1 also was the major phosphorylated species. Limited proteolytic digestion of isolated radiolabeled IEP bands with Staph V8 protease yielded virtually identical fragment profiles in SDS-PAGE, as did digestions with chymotrypsin and N-chlorosuccinimide. Monospecific rabbit antisera raised against each of the four isolated IEPs reacted with all the IEP species in immunoblotting assays. Pulse-chase experiments indicated that all the IEPs were detectable immediately after a 15-min pulse and that several alterations in the IEP profile occurred during subsequent chase periods. Thus, the EHV-1 IEPs are closely related structurally and antigenically and appeared to be either produced simultaneously or processed to yield the individual forms immediately.

The EICP22 Protein of Equine Herpesvirus 1 Physically Interacts with the Immediate-Early Protein and with Itself To Form Dimers and Higher-Order Complexes

ABSTRACT The EICP22 protein (EICP22P) of Equine herpesvirus 1(EHV-1) is an early protein that functions synergistically with other EHV-1 regulatory proteins to transactivate the expression of early and late viral genes. We have previously identified EICP22P as an accessory regulatory protein that has the ability to enhance the transactivating properties and the sequence-specific DNA-binding activity of the EHV-1 immediate-early protein (IEP). In the present study, we identify EICP22P as a self-associating protein able to form dimers and higher-order complexes during infection. Studies with the yeast two-hybrid system also indicate that physical interactions occur between EICP22P and IEP and that EICP22P self-aggregates. Results from in vitro and in vivo coimmunoprecipitation experiments and glutathioneS-transferase (GST) pull-down studies confirmed a direct protein-protein interaction between EICP22P and IEP as well as self-interactions of EICP22P. Analyses of infected cells by laser-scanning confocal microscopy with antibodies specific for IEP and EICP22P revealed that these viral regulatory proteins colocalize in the nucleus at early times postinfection and form aggregates of dense nuclear structures within the nucleoplasm. Mutational analyses with a battery of EICP22P deletion mutants in both yeast two-hybrid and GST pull-down experiments implicated amino acids between positions 124 and 143 as the critical domain mediating the EICP22P self-interactions. Additional in vitro protein-binding assays with a library of GST-EICP22P deletion mutants identified amino acids mapping within region 2 (amino acids [aa] 65 to 196) and region 3 (aa 197 to 268) of EICP22P as residues that mediate its interaction with IEP.

Effects of an aminomethacrylate on epithelial cell lipid metabolism

Methacrylates can affect cell functions by surfactant-like effects or by altering cell lipid composition. Dimethylaminoethyl methacrylate (DMAEMA), an activator widely used in visible-light polymerized dental resins has been shown to elute readily into aqueous environments. The current study examined the metabolism of this material by oral epithelial cells (HCP) and its subsequent effects on cell lipids. Cells were plated in culture medium, then exposed to DMAEMA in the presence of 14C-acetate, a precursor which labeled the cell lipids. Other cultures were prelabeled with radioisotope, then exposed to DMAEMA. After incubation, the cell lipids were extracted and separated by TLC. Radioactive lipids were located and quantitated. Exposure of the cells to DMAEMA resulted in decreased synthesis of cholesterol with a concomitant increase in sterol precursors. Cholesterol esters and triacylglycerides also increased. Among the polar lipids, phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) decreased in response to DMAEMA. However, dimethylphosphatidyl ethanolamine (DMPE), a precursor of PC not detectable in control cultures, accumulated to a significant extent in cells exposed to DMAEMA. Furthermore, changes in PC and DMPE levels persisted in the cells for at least 48 h after removal of the DMAEMA. The results indicate that DMAEMA produces alterations in the relative amounts of several cellular neutral and polar lipids. Such alterations, especially of the normal phospholipid composition, along with an alteration in cellular cholesterol, could result in altered membrane-associated cell functions.

Changes in cell phospholipid metabolism in vitro in the presence of HEMA and its degradation products.

OBJECTIVES Diacylglycerol-kinase (DAG-kinase) is an enzyme that phosphorylates diacylglycerol (DAG) to phosphatidic acid (PA), which serves as a precursor to phosphoglycerides involved in cell signaling or as cell membrane structural components. DAG-kinase can be inhibited by diacylethylene glycols (DAEG). We hypothesize that 2-hydroxyethyl methacrylate (HEMA) may alter phosphorylation of DAG to PA following intracellular formation of DAEG. METHODS Cultured rabbit kidney (RK13) epithelial cells were treated with HEMA, EG, or known inhibitors of DAG-kinase for 24 h, then exposed to [32P]O4- in the presence of a synthetic diacylglycerol for 2 h. Other cultures were radiolabeled with [3H]-oleic acid for 24 h, then exposed to HEMA for an additional 24 h. The cells were harvested and the lipids extracted. Radioactive lipids were separated by thin layer chromatography, located by autoradiography, and quantitated as cpm/ug protein. Cell cultures treated with HEMA were homogenized and the DAG-kinase activity was assayed and expressed as cpm/ug protein. Data were analyzed by one-way ANOVA and Newman-Keuls Multiple Comparison Test. RESULTS Cultures exposed to HEMA or known DAG-kinase inhibitors exhibited reduced incorporation of radioactivity in the PA fraction compared to control cultures. Direct assays of DAG-kinase activity from cells exposed to HEMA demonstrated decreased enzyme activity. Evaluation of cell phospholipid synthesis showed altered formation of phosphatidylethanolamine and phosphatidylcholine. SIGNIFICANCE Results suggest that HEMA impairs formation of PA, possibly by acylation of EG released by hydrolysis of the HEMA and resultant production of the inhibitor DAEG. The decreased availability of PA may alter PA-dependent cell structural lipid pathways and lipid-dependent signaling pathways, altering cell growth.

Cell lipid alterations resulting from prolonged exposure to dimethylaminoethylmethacrylate.

Abstract Dimethylaminoethylmethacrylate (DMAEMA), a commonly-used component of visible-light polymerized dental resins, has the potential to elute and interact with tissue cells to cause cytotoxicity or sublethal metabolic changes. Short-term exposure of cultured oral epithelial cells to sublethal DMAEMA concentrations has been shown previously to affect cell neutral lipid and phospholipid metabolism, resulting in accumulation of significant quantities of dimethylphosphatidylethanolamine (DMPE). In non-treated cells, DMPE is a transient intermediate in phospholipid metabolism and is not detectable by standard methods. In the current study, the effects of prolonged exposure of cells to DMAEMA, and the mechanisms for formation of DMPE in the presence of DMAEMA were examined. Exposure of a keratinizing hamster buccal cheek pouch cell line (HCP cells) to 0.8 mM DMAEMA for 2, 3, 7, and 14 days resulted in reduced incorporation of [14C]acetate into several classes of phospholipids. DMPE was detectable at all time points in DMAEMA-exposed cultures and comprised between 12.48% and 18.33% of the total radiolabeled phospholipids. The results of short-term exchange experiments indicated that headgroup exchange was not the major reaction responsible for formation of DMPE in DMAEMA-treated cells; rather the formation appeared to occur through typical phospholipid metabolic pathways. The cells appeared able to re-establish and maintain homeostasis in the presence of this altered cell lipid composition.

Transforming growth factor β1 dysregulation in a human oral carcinoma tumour progression model

Abstract. A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor‐β1 on this model system, we analysed the responsiveness of those cells to transforming growth factor‐β1 and explored the potential mechanism underlying the transforming growth factor‐β1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor‐β1, although to different degrees. Transforming growth factor‐β1 induced the expression of cyclin‐dependent kinase inhibitors p15INK4B, p21WAF1/CIP1 and p27KIP1. In contrast, transforming growth factor‐β1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor‐β1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression.

An apparent unusual relationship between rampant caries and the oral mucosal manifestations of chronic graft-versus-host disease

Oral complications of chronic graft-versus-host disease are well known and may be present in up to 80% of persons with the condition. A case involving a 29-year-old woman with chronic graft-versus-host disease after allogeneic bone marrow transplantation is presented to illustrate how oral mucosal lesions may be adversely affected by severe caries. A variety of extensive mucosal lesions were continually irritated by the sharp edges of the carious teeth. Full-mouth extractions facilitated the complete resolution of the oral lesions. The patient has had no recurrence of oral lesions 4 years after the extractions and has shown no adverse mucosal changes 3 1/2 years after rehabilitation with complete dentures.