Effects of subtoxic concentrations of benzoyl peroxide on cell lipid metabolism.

Benzoyl peroxide (BP), a tumor promoter, has been shown to cause free-radical-induced lipid peroxidation and membrane damage at toxic concentrations. However, its effects on lipid metabolism at concentrations that were not overtly toxic have not been investigated. The purpose of the current study was to examine the effects of BP and its final degradation product, benzoic acid (BA), on lipid metabolism. Two cell lines, hamster cheek pouch (HCP) and human monocytes (THP-1), were used to determine the effects of BP, BA, and BP combined with FeCl2 on cell lipid metabolism. Cells were exposed to BP and 14C acetate for 24 h, or cells with prelabeled lipids were harvested, and the lipids were extracted and separated with the use of thin-layer chromatography. Lipid metabolism of some neutral lipids such as triglycerides was altered for both cell types in response to BP. Also, cholesterol content was reduced in THP-1 cells and a phospholipid, phosphatidylethanolamine (PE), was reduced in HCP cells. The final degradation product of BP, BA, failed to elicit any response in lipid metabolism. Subtoxic concentrations of BP induced changes in neutral lipids such as triglycerides and cholesterol. The metabolism of major phospholipids except PE remained unchanged. The effects were related to BP and its degradation and varied with the cell type.

Transforming growth factor beta 1 dysregulation in a human oral carcinoma tumour progression model.

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-beta 1 on this model system, we analysed the responsiveness of those cells to transforming growth factor-beta 1 and explored the potential mechanism underlying the transforming growth factor-beta 1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor-beta 1, although to different degrees. Transforming growth factor-beta 1 induced the expression of cyclin-dependent kinase inhibitors p15(INK4B), p21WAF1/(CIP1) and p27(KIP1). In contrast, transforming growth factor-beta 1 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-beta 1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression.

The equine herpesvirus 1 immediate-early protein interacts with EAP, a nucleolar-ribosomal protein.

The equine herpesvirus 1 (EHV-1) immediate-early (IE) phosphoprotein is essential for the activation of transcription from viral early and late promoters and regulates transcription from its own promoter. The IE protein of 1487 amino acids contains a serine-rich tract (SRT) between residues 181 and 220. Deletion of the SRT decreased transactivation activity of the IE protein. Previous results from investigation of the ICP4 protein, the IE homolog of herpes simplex virus 1 (HSV-1), revealed that a domain containing a serine-rich tract interacts with EAP (Epstein-Barr virus-encoded small nuclear RNA-associated protein), a 15-kDa nucleolar-ribosomal protein (R. Leopardi, and B. Roizman, Proc. Natl. Acad. Sci. USA 93, 4572-4576, 1996). DNA binding assays revealed that (i) glutathione S-transferase (GST)-EAP disrupted the binding of HSV-1 ICP4 to its cognate DNA in a dose-dependent manner, (ii) GST-EAP interacted with the EHV-1 IE protein, but did not disrupt its binding to its cognate site in viral DNA. GST-pulldown assays indicated that the SRT of the IE protein is required for physical interaction with EAP. The IE protein and EAP colocalized in the cytoplasm of the infected equine ETCC cells at late times of the infection cycle. This latter finding may be important in EHV-1 gene regulation since late viral gene expression is greatly influenced by the EICP0 trans-activator protein whose function is antagonized by the IE protein.

Chemopreventive effects of green tea polyphenols correlate with reversible induction of p57 expression.

Green tea polyphenols are known to induce apoptosis in certain types of tumor cells. However, the mechanism(s) that enables normal cells to evade the apoptotic effect is still not understood. In this study, Western blot analysis combined with cycloheximide treatment was used to examine the effects of green tea polyphenols on the expression levels of p57, a cyclin-dependent kinase and apoptosis inhibitor, in normal human keratinocytes and in the oral carcinoma cell lines SCC25 and OSC2. The results showed that the most potent green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), induced p57 in normal keratinocytes in a dosage- and time-dependent manner, while the levels of p57 protein in oral carcinoma cells were unaltered. The differential response in p57 induction was consistent with the apoptosis status detected by annexin V assay. The data suggest that the chemopreventive effects of green tea polyphenols may involve p57-mediated cell cycle regulation in normal epithelial cells.

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.

The EICP22 protein of equine herpesvirus 1 physically interacts with the immediate-early protein and with itself to form dimers and higher-order complexes.

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 glutathione S-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.

Alterations in cell lipid metabolism by glycol methacrylate (HEMA).

Components of dental resins such as dimethylaminoethyl methacrylate (DMAEMA) can alter cell lipid composition, presumably by esterase-mediated hydrolysis. The resulting dimethylethanolamine is incorporated into cell phospholipids, while the methacrylic acid may alter several metabolic pathways. We hypothesize that HEMA is cleaved in a similar manner and the released ethylene glycol is incorporated into cell lipids, yielding phosphatidylethylene glycol (PtEG), and the methacrylic acid alters other lipid pathways in a manner similar to that of methacrylic acid released from hydrolysis of DMAEMA. Cultures of hamster buccal pouch (HCP) and rabbit kidney (RK13) epithelial cells were exposed to subtoxic concentrations of HEMA in the presence of [14C]-acetate or [3H]-oleic acid. Other cultures were prelabeled with [14C]-acetate followed by exposure to various concentrations of HEMA. Cell lipids were extracted by the method of Bligh and Dyer and separated by thin layer chromatography on silica gel K-6 plates or SG-81 silica gel loaded chromatography paper. The fate of the ethylene glycol was traced using [14C]-ethylene glycol. Radioactive lipids were located using autoradiography and known standard lipids and quantitated by liquid scintillation spectrometry. In the presence of HEMA several classes of lipids were altered. Among the neutral lipids, the most notable changes involved sterol precursors, triglycerides, fatty acids, and cholesterol esters, while phosphatidylcholine was affected among the phospholipids. The results differed quantitatively between the two cell types. Results also suggest that EG, including that released by hydrolysis of HEMA, is incorporated into cell phospholipids, producing PtEG. The changes in neutral lipid labeling may occur by alteration of lipid synthetic pathways utilizing acetyl Co-A as well as inhibition of enzymes involved in synthesis of cholesterol from sterol precursors and hydrolysis of cholesterol esters. Synthesis of PtEG may take place via phospholipase D-mediated headgroup exchange. Alterations in the cellular lipids may affect cell membrane properties and associated cell functions.

Cell lipid alterations resulting from prolonged exposure to dimethylaminoethylmethacrylate.

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.

Structural and antigenic identification of the ORF12 protein (alpha TIF) of equine herpesvirus 1.

The equine herpesvirus 1 (EHV-1) homolog of the herpes simplex virus type 1 (HSV-1) tegument phosphoprotein, alpha TIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12) and shown to transactivate immediate early (IE) gene promoters. However, a specific virion protein corresponding to the ORF12 product has not been identified definitively. In the present study the ORF12 protein, designated ETIF, was identified as a 60-kDa virion component on the basis of protein fingerprint analyses in which the limited proteolysis profiles of the major 60-kDa in vitro transcription/ translation product of an ORF12 expression vector (pT7-12) were compared to those of purified virion proteins of similar size. ETIF was localized to the viral tegument in Western blot assays of EHV-1 virions and subvirion fractions using polyclonal antiserum and monoclonal antibodies generated against a glutathione-S-transferase-ETIF fusion protein. Northern and Western blot analyses of EHV-1-infected cell lysates prepared under various metabolic blocks indicated that ORF12 is expressed as a late gene, and cross reaction of polyclonal anti-GST-ETIF with a 63.5-kDa HSV-1 protein species suggested that ETIF and HSV-1 alpha TIF are antigenically related. Last, DNA band shift assays used to assess ETIF-specific complex formation indicated that ETIF participates in an infected cell protein complex with the EHV-1 IE promoter TAATGARAT motif.

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.

Expression and function of the equine herpesvirus 1 virion-associated host shutoff homolog.

The ability of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) to repress host cell protein synthesis early in infection has been studied extensively and found to involve the activities of the UL41 gene product, the virion-associated host shutoff (vhs) protein. To date, UL41 homologs have been identified in the genomes of three other alphaherpesviruses: equine herpesvirus 1 (EHV-1), varicella-zoster virus, and pseudorabies virus, but very little is known about the putative products of these homologous genes. Our earlier observations that no rapid early host protein shutoff occurred in EHV-1-infected cells led us to test EHV-1 vhs activity more thoroughly and to examine the expression and function of the EHV-1 UL41 homolog, ORF19. In the present study, the effects of EHV-1 and HSV-1 infections on cellular protein synthesis and mRNA degradation were compared at various multiplicities of infection in several cell types under an actinomycin D block. No virion-associated inhibition of cellular protein synthesis or vhs-induced cellular mRNA degradation was detected in cells infected with any of three EHV-1 strains (Ab4, KyA, and KyD) at multiplicities of infection at which HSV-1 strain F exhibited maximal vhs activity. However, further analyses revealed that (i) the EHV-1 vhs homolog gene, ORF19, was transcribed and translated into a 58-kDa protein in infected cells; (ii) the ORF19 protein was packaged into viral particles in amounts detectable in Western blots (immunoblots) with monoclonal antibodies; (iii) in cotransfection vhs activity assays, transiently-expressed ORF19 protein had intrinsic vhs activity comparable to that of wild-type HSV-1 vhs; and (iv) this intrinsic vhs activity was ablated by in vitro site-directed mutations in which either the functionally inactive HSV-1 vhs1 UL41 mutation (Thr at position 214 replaced by Ile [Thr-214-->Ile]) was recreated within ORF19 or two conserved residues within the putative poly(A) binding region of the ORF19 sequence were altered (Tyr-190, 192-->Phe). From these results we conclude that EHV-1's low vhs activity in infected cells is not a reflection of the ORF19 protein's intrinsic vhs activity but may be due instead to the amount of ORF19 protein associated with viral particles or to modulation of ORF19 protein's intrinsic activity by another viral component(s).

Relationships between denture base resin cytotoxicity and cell lipid metabolism.

Substances that elute from denture base resins may inhibit cell growth and disrupt various metabolic processes. This study investigated the effects on cell lipid metabolism of eluates from several denture base resins. Cultured oral epithelial cells were exposed in vitro to eluates of discs made from several denture base resins. Lipid metabolism of the cells was measured using isotopic labeling with 14C-acetate. Results demonstrated that the metabolism of several lipid classes found mainly in the cell membrane was altered by the resin eluates. Eluate from one resin caused the appearance of two previously unrecognized classes of lipids. The alterations of the cell lipids and the presence of the previously unrecognized lipids may be the basis for some clinically evident cytotoxic and allergic reactions.

Characterization of the regulatory function of the ICP22 protein of equine herpesvirus type 1.

The IR4 gene (inverted repeat gene 4) of equine herpesvirus type 1 (EHV-), the homolog of the herpes simplex virus type 1 ICP22 gene, is differentially expressed as a 1.4-kb early transcript and a 1.7-kb late transcript that encode a series of proteins that migrate between 42 to 47 kDa, localize to the nucleus of EHV-1-infected cells, and become packaged within EHV-1 virions (V. R. Holden, G. B. Caughman, Y. Zhao, R. N. Harty, and D. J. O'Callaghan, J. Virol. 68, 4329-4340, 1994). To assess the role of the IR4 protein in EHV-1 gene regulation, an IR4 expression vector was cotransfected with EHV-1 chimeric promoter-CAT reporter constructs and EHV-1 effector plasmids to determine the effects of the IR4 protein on the expression of immediate-early (IE), early, and late promoters. These studies revealed that the IR4 protein: (i) minimally trans-activates EHV-1 promoters, (ii) acts synergistically with the UL3 (ICP27) gene product to trans-activate the IE promoter, (iii) does not interfere with the trans-repression of the IE promoter by the IE protein, (iv) enhances transactivation of early promoters by the IE protein, (v) enhances the transactivation of both early and late promoters by the IE and UL3 proteins, and (vi) interacts synergistically with the IE protein to trans-activate the heterologous HSV-1 ICP4 promoter. These data suggest that the IR4 gene product plays a significant role in EHV-1 gene regulation.

Detection and intracellular localization of equine herpesvirus 1 IR1 and IR2 gene products by using monoclonal antibodies.

During lytic infection, two transcripts arise from the equine herpesvirus 1 (EHV-1) immediate-early (IE) gene (IR1): a single, spliced 6.0-kb IE mRNA and a 3'-coterminal 4.4-kb early mRNA (IR2). Previous studies demonstrated that transiently expressed IR1 and IR2 gene products are potent transcriptional regulators: IR1 proteins are capable of trans activating representative EHV-1 early and late promoters, while both IR1 proteins and the IR2 product, which lacks IR1 amino acid residues 1 to 322, trans repress the IR1 promoter. In the present study, monoclonal antibodies (MAbs) against the major IE protein, IE1, were developed, characterized as to their ability to detect IR1 and IR2 products, and used to examine extracellular virions for the presence of IE1-related proteins and to define the IR1 and IR2 protein synthesis and intracellular distribution in EHV-1-infected cells. The results demonstrated that (i) anti-IE1 MAbs representing three noncompetitive epitope-binding groups reacted with multiple IE protein species, as well as with a 146-kDa early protein identified as the putative IR2 gene product; (ii) the three reactive epitopes mapped to a region spanning amino acids 323 to 552 of IR1; (iii) anti-IE1 MAbs reacted with the 144-kDa in vitro-translated IR2 product and with a transiently expressed IR2 product similar in size; (iv) small amounts of IE1 and the 146-kDa protein were associated with the nucleocapsid-tegument fraction of mature virions; (v) in immunofluorescence assays of lytically infected cells, IR1-IR2 gene products were first detectable between 1 and 2 h postinfection as discrete, punctate, intranuclear foci; (vi) as the infection progressed, the intranuclear reactivity increased and redistributed into large, intensely stained nuclear compartments which corresponded to the sites of active viral DNA synthesis; (vii) fibrillar, as well as more generalized cytoplasmic staining, first observed at about 5 h postinfection, increased throughout infection; and (viii) while viral DNA synthesis was required for the progressive intranuclear redistribution, the cytoplasmic accumulation of IR1-IR2 proteins occurred subsequent to early infection events.

Alteration of epithelial cell lipid synthesis by N-nitrosonornicotine.

Changes in the lipid composition of a cell membrane due to the binding of one cell modulator may affect binding of a second modulator, whether that binding is receptor-mediated (specific) or non-receptor-mediated (nonspecific). Such altered binding interactions have been demonstrated in oral epithelial cells, wherein N-nitrosonornicotine (NNN), a nonspecific ligand, enhances phorbol ester binding. To characterize membrane changes that may be responsible for such an effect, the current study examined lipid changes in hamster oral epithelial (HCP) cells associated with NNN binding. HCP cultures at two cell densities, 5 x 10(6) cells/100 mm plate (subconfluent cultures) or 10 x 10(6) cells/100 mm plate (confluent cultures) were incubated in Keratinocyte-Serum-Free Medium and exposed to 10 microM NNN or DMSO (solvent control) for 48 h. Lipids were labeled with 14C-acetate, then extracted, separated by thin layer chromatography, and the 14C-lipids located by autoradiography and counted. Exposure of subconfluent cultures to NNN for 48 h, with 14C-acetate present during the final 24 h, resulted in altered phospholipid and fatty acid labeling. Phospholipid labeling increased slightly in the presence of NNN compared to controls, while fatty acid labeling showed a modest but significant decrease in the presence of NNN. Similar changes occurred in the confluent cultures. Prelabeling of lipids in subconfluent cultures, followed by exposure to NNN in the absence of radiolabel, resulted in significantly (P < 0.05) greater phospholipid labeling in the presence of NNN compared to control cultures. At the same time, fatty acid labeling decreased significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and localization of the equine herpesvirus 1 major DNA binding protein.

In previous studies of equine herpesvirus 1 (EHV-1) gene regulation, we observed an abundant early infected cell polypeptide (ICP), designated ICP130, which appeared in reduced amounts in cells infected with defective interfering particle-rich EHV-1 stocks compared to standard EHV-1-infected cells. To characterize this ICP further, a monoclonal antibody (MAb) was developed to EHV-1 ICP130 and used to (1) affinity purify ICP130, (2) examine ICP130's ability to bind DNA, and (3) define the synthesis and intracellular localization of ICP130 during productive EHV-1 infections. Although anti-ICP130 MAbs did not crossreact with any HSV-1 protein in immunoblots, a polyclonal antiserum against HSV-2 major DNA binding protein (ICSP11,12) did react with purified EHV-1 ICP130. DNA band shift assays indicated that (1) the mobility of shifted bands representing DNA/EHV-1-infected cell protein complexes was further decreased by the addition of either anti-ICP130 MAbs or anti-ICSP11,12, but not by the addition of irrelevant MAbs, (2) the ability of ICP130 to complex with DNA was not sequence dependent, (3) ICP130 associated with both single- and double-stranded oligomers, and (4) similar supershifted patterns were produced using affinity-purified ICP130 and anti-ICP130 MAbs. During productive infection, ICP130 initially localized rapidly and exclusively to the infected cell's nucleus in a generalized, fine granular pattern. Over the course of infection, this pattern typically progressed to include several large, intensely reactive intranuclear granules, and by 6 hr p.i. some cytoplasmic reactivity also was visible. In < 5% of the cells, a dense, fibrillar network surrounding the nucleus was observed instead. The progressive changes in nuclear localization depended upon the onset of viral DNA replication, and once the late pattern was established, ongoing DNA synthesis was required to maintain it. The results indicate that ICP130 is the previously reported EHV-1 counterpart of the HSV major DNA-binding protein and is similar, but not identical, in many aspects.

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.

Identification and characterization of the ICP22 protein of equine herpesvirus 1.

The equine herpesvirus 1 (EHV-1) homolog of herpes simplex virus type 1 ICP22 is differently expressed from the fourth open reading frame of the inverted repeat (IR4) as a 1.4-kb early mRNA and a 1.7-kb late mRNA which are 3' coterminal (V. R. Holden, R. R. Yalamanchili, R. N. Harty, and D. J. O'Callaghan, J. Virol. 66:664-673, 1992). To extend the characterization of IR4 at the protein level, the synthesis and intracellular localization of the IR4 protein were investigated. Antiserum raised against either a synthetic peptide corresponding to amino acids 270 to 286 or against a TrpE-IR4 fusion protein (IR4 residues 13 to 150) was used to identify the IR4 protein. Western immunoblot analysis revealed that IR4 is expressed abundantly from an open reading frame composed of 293 codons as a family of proteins that migrate between 42 to 47 kDa. The intracellular localization of IR4 was examined by cell fractionation, indirect immunofluorescence, and laser-scanning confocal microscopy. These studies revealed that IR4 is localized predominantly in the nucleus and is dispersed uniformly throughout the nucleus. Interestingly, when IR4 is expressed transiently in COS-1 or LTK- cells, a punctate staining pattern within the nucleus is observed by indirect immunofluorescence. Cells transfected with an IR4 mutant construct that encodes a C-terminal truncated (19 amino acids) IR4 protein exhibited greatly reduced intranuclear accumulation of the IR4 protein, indicating that this domain possesses an important intranuclear localization signal. Western blot analysis of EHV-1 virion proteins revealed that IR4 proteins are structural components of the virions. Surprisingly, the 42-kDa species, which is the least abundant and the least modified form of the IR4 protein family in infected cell extracts, was the most abundant IR4 protein present in purified virions.

Transcriptional control of the equine herpesvirus 1 immediate early gene.

Transient expression assays measuring induction of an equine herpesvirus 1 (EHV-1) immediate early (IE) promoter construct, pIE beta gal, were used to examine trans-induction of the IE gene (IR1) promoter by superinfection with intact EHV-1 (Kentucky A strain), uv-inactivated EHV-1, or EHV-1 stocks highly enriched for defective interfering particles (DIPs), and by cotransfection with plasmids containing EHV-1 DNA fragments. Our results confirm reports by others in that the IE promoter can be induced by both intact and uv-inactivated EHV-1 and provide evidence that DIP-rich virus stocks also possess transinducing activity. Furthermore, IE promoter activity was induced by cotransfection of the promoter construct with either of two plasmids containing restriction enzyme DNA fragments that span ORF12, the putative EHV-1 homolog of the herpes simplex virus 1 (HSV-1) alpha-trans-inducing factor (alpha-TIF) gene, UL48, or by cotransfection with an isolated DNA fragment containing only ORF12, while no transactivation was associated with plasmids linearized by restriction enzyme digestion at a single site within ORF12. These data indicate that, despite its predicted lack of an acidic carboxy terminus (a region essential in HSV-1 alpha-TIF for trans-inducing activity), the EHV-1 ORF12 product is a functional alpha-TIF.

Sterol metabolism and oral epithelial cell growth.

Previous studies have demonstrated that as the density of cultured oral epithelial cells increases, there is a concomitant increase in phospholipids and cholesterol ester synthesis and a decrease in that of cholesterol and sterol precursors. Other studies have suggested that the effects of exogenous cholesterol sulfate may be similar to growth responses and influence metabolic steps related to cell density. To further examine this possibility, in the present study lipid synthesis was monitored in hamster cheek pouch epithelial cells in cultures established at different cells densities and in the presence of varying amounts of exogenous cholesterol sulfate. Cell [14C]acetate incorporation into lipids was measured in cultures established at four densities ranging from very subconfluent to very dense (postconfluent) in two media, Dulbecco's modified Eagle's medium (DMEM) with 5% fetal bovine serum and KSFM, a non-serum containing keratinocyte medium. Results indicated that the relative proportion of radiolabel incorporated into different lipid classes changed with cell density. In DMEM, the percentage of radiolabel incorporated into total phospholipids and fatty acids increased significantly with increasing cell density whereas percent incorporation into cholesterol, sterol precursors, and cholesterol esters significantly decreased. In KSFM cultures, proportionate phospholipids labeling was significantly increased in more dense cultures whereas cholesterol and cholesterol esters labeling was significantly decreased. In subconfluent and confluent cultures exposed to 10 or 25 microM cholesterol sulfate, the relative proportions of phospholipid labeling also increased significantly compared to dimethyl sulfoxide (solvent) controls, whereas sterol precursors, fatty acids, and cholesterol esters labeling was significantly decreased. These results indicate that cholesterol sulfate can affect cellular lipid synthesis in a manner similar to that which occurs with increasing cell density, and strengthen the hypothesis that cholesterol sulfate may regulate lipid metabolic pathways related to growth and differentiation.