O-linkage of N-acetylglucosamine to Sp1 activation domain inhibits its transcriptional capability.

The posttranslational modification of eukaryotic intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc) monosaccharides is essential for cell viability, yet its precise functional roles are largely unknown. O-GlcNAc transferase utilizes UDP-GlcNAc, the end product of hexosamine biosynthesis, to catalyze this modification. The availability of UDP-GlcNAc correlates with glycosylation levels of intracellular proteins as well as with transcriptional levels of some genes. Meanwhile, transcription factors and RNA polymerase II can be modified by O-GlcNAc. A linkage between transcription factor O-GlcNAcylation and transcriptional regulation therefore has been postulated. Here, we show that O-GlcNAcylation of a chimeric transcriptional activator containing the second activation domain of Sp1 decreases its transcriptional activity both in an in vitro transcription system and in living cells, which is in concert with our observation that O-GlcNAcylation of Sp1 activation domain blocks its in vitro and in vivo interactions with other Sp1 molecules and TATA-binding protein-associated factor II 110. Furthermore, overexpression of O-GlcNAc transferase specifically inhibits transcriptional activation by native Sp1 in cells. Thus, our studies provide direct evidence that O-GlcNAcylation of transcription factors is involved in transcriptional regulation.

Stage-sensitive blockade of pituitary somatomammotrope development by targeted expression of a dominant negative epidermal growth factor receptor in transgenic mice.

The epidermal growth factor receptor (EGFR) and its ligands EGF and transforming growth factor-alpha (TGF alpha) are expressed in the anterior pituitary, and overexpression of TGF alpha in the lactotrope cells of the pituitary gland in transgenic mice results in lactotrope hyperplasia and adenomata, suggesting a role for EGFR signaling in pituitary cell proliferation. To address the role of EGFR signaling in pituitary development in vivo, we blocked EGFR signaling in transgenic mice using the dominant negative properties of a mutant EGFR lacking an intracellular protein kinase domain (EGFR-tr). We directed EGFR-tr expression to GH- and PRL- producing cells using GH and PRL promoters, and a tetracycline-inducible gene expression system, to allow temporal control of gene expression. EGFR-tr overexpression in GH-producing cells during embryogenesis resulted in dwarf mice with pituitary hypoplasia. Both somatotrope and lactotrope development were blocked. However, when EGFR-tr overexpression was delayed to the postnatal period either by directing its expression with the PRL promoter or by delaying the onset of induction with tetracycline in the GH cells, no specific phenotype was observed. Lactotrope hyperplasia during pregnancy also occurred normally in the PRL-EGFR-tr mice. These data suggest that EGFR signaling is required for the differentiation and/or maintenance of somatomammotropes early in pituitary organogenesis but not later in life. (Molecular Endocrinology 15: 600-613, 2001)

Human Sug1/p45 is involved in the proteasome-dependent degradation of Sp1.

The transcription factor Sp1 was previously shown to undergo proteasome-dependent degradation when cells were glucose-starved and stimulated with the adenylate cyclase inducer, forskolin. However, the control of the Sp1 degradation process is largely unknown. Using in vitro and in vivo interaction studies, we show in the present study that Sp1 interacts with human Sug1 [hSug1, also known as p45 or thyroid-hormone-receptor interacting protein ('TRIP1')], an ATPase subunit of the 26 S proteasome and a putative transcriptional modulator. This interaction with Sp1 occurs through the C-terminus of hSug1, the region that contains the conserved ATPase domain in this protein. Both in vitro studies, in reconstituted degradation assays, and in vivo experiments, in which hSug1 is overexpressed in normal rat kidney cells, show that full-length hSug1 is able to stimulate the proteasome-dependent degradation of Sp1. However, hSug1 truncations that lack either the N- or C-terminal domain of hSug1 act as dominant negatives, inhibiting Sp1 degradation in vitro. Also, an ATPase mutant of hSug1, while still able to bind Sp1, acts as a dominant negative, blocking Sp1 degradation both in vitro and in vivo. These results demonstrate that hSug1 is involved in the degradation of Sp1 and that ATP hydrolysis by hSug1 is necessary for this process. Our findings indicate that hSug1 is an exchangeable proteasomal component that plays a critical regulatory role in the proteasome-dependent degradation of Sp1. However, hSug1 is not the factor limiting Sp1 degradation in the cells treated with glucosamine. This and other considerations suggest that hSug1 co-operation with other molecules is necessary to target Sp1 for proteasome degradation.

Phosphorylation of human glutamine:fructose-6-phosphate amidotransferase by cAMP-dependent protein kinase at serine 205 blocks the enzyme activity.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the rate-limiting enzyme in glucosamine synthesis. Prior studies from our laboratory indicated that activation of adenylate cyclase was associated with depletion of O-GlcNAc modification. This finding and evidence that human GFAT (hGFAT) might be regulated by cAMP-dependent protein kinase (PKA) led us to investigate the role of PKA in hGFAT function. We confirmed that adenylate cyclase activation by forskolin results in diminished O-GlcNAc modification of several cellular proteins which can be overcome by exposure of the cells to glucosamine but not glucose, suggesting the PKA activation results in depletion of UDP-GlcNAc for O-glycosylation. To determine if GFAT is indeed regulated by PKA, we expressed the active form of the enzyme using a vaccinia virus expression system and showed that the activity of the enzyme was to decrease to undetectable levels by PKA phosphorylation. We mapped the PKA phosphorylation sites with the aid of matrix-assisted laser desorption ionization mass spectroscopy and showed that the protein was stoichiometrically phosphorylated at serine 205 and also phosphorylated, to a lesser extent at serine 235. Mutagenesis studies indicated that the phosphorylation of serine 205 by PKA was necessary for the observed inhibition of enzyme activity while serine 235 phosphorylation played no observable role. The activity of GFAT is down-regulated by cAMP, thus placing regulation on the hexosamine pathway that is in concert with the energy requirements of the organism. During starvation, hormones acting through adenylate cyclase could direct the flux of glucose metabolism into energy production rather than into synthetic pathways that require hexosamines.

Glucose stimulates protein modification by O-linked GlcNAc in pancreatic beta cells: linkage of O-linked GlcNAc to beta cell death.

The pancreatic beta cell can respond in the long term to hyperglycemia both with an increased capacity for insulin production and, in susceptible individuals, with apoptosis. When glucose-induced apoptosis offsets the increasing beta cell capacity, type 2 diabetes results. Here, we tested the idea that the pathway of glucose metabolism that leads to the modification of intracellular proteins with the O-linked monosaccharide N-acetylglucosamine (O-GlcNAc) is involved in the glucose-induced apoptosis. This idea is based on two recent observations. First, the beta cell expresses much more O-GlcNAc transferase than any other known cell, and second, that the beta cell-specific toxin, streptozotocin (STZ), itself a GlcNAc analog, specifically blocks the enzyme that cleaves O-GlcNAc from intracellular proteins. As a consequence, we now show that hyperglycemia leads to the rapid and reversible accumulation of O-GlcNAc specifically in beta cells in vivo. Animals pretreated with STZ also accumulate O-GlcNAc in their beta cells when hyperglycemic, but this change is sustained upon re-establishment of euglycemia. In concert with the idea that STZ toxicity results from the sustained accumulation of O-GlcNAc after a hyperglycemic episode, we established a low-dose STZ protocol in which the beta cells' toxicity of STZ was manifest only after glucose or glucosamine administration. Transgenic mice with impaired beta cell glucosamine synthesis treated with this protocol are resistant to the diabetogenic effect of STZ plus glucose yet succumb to STZ plus glucosamine. This study provides a causal link between apoptosis in beta cells and glucose metabolism through glucosamine to O-GlcNAc, implicating this pathway of glucose metabolism with beta cell glucose toxicity.

Conditional expression of the ErbB2 oncogene elicits reversible hyperplasia in stratified epithelia and up-regulation of TGFalpha expression in transgenic mice.

The ErbB2 receptor tyrosine kinase (RTK) is expressed in basal cells of squamous epithelia and the outer root sheath of hair follicles. We previously showed that constitutive expression of activated ErbB2 directed to these sites in the skin by the keratin 14 (K14) promoter produces prominent hair follicle abnormalities and striking skin hyperplasia in transgenic mice. However, perinatal lethality precluded the establishment of a transgenic line for analysis of ErbB2 function in adult animals. To investigate the significance of ErbB2 signaling in epithelial tissues during and post development, we developed a K14-rtTA/TetRE-ErbB2 'Tet-On' bitransgenic mouse system. These mice were normal until the ErbB2 transgene was induced by exposure to doxycycline (Dox). Prenatal induction resulted in perinatal death. Postnatally, ErbB2 transgene expression was observed at 4 h after the initiation of Dox, and reached a plateau at 24 h. Skin hyperplasia followed after 2 days and these changes reverted to normal upon Dox withdrawal. In adults, as in the neonates, prolonged ErbB2 induction caused prominent skin and hair follicle hyperplasias. Severe hyperplasias in the cornea, eye lids, tongue and esophagus were also observed. ErbB2 transgene induction was accompanied by increased expression of TGFalpha, a ligand of epidermal growth factor receptor (EGFR), and to a lesser extent, EGFR, further enhancing RTK signal transduction. We conclude that ErbB2 plays important roles in both development and maintenance of hair follicles and diverse squamous epithelia and that this ligand-inducible and tissue-specific 'Tet-On' transgenic mouse system provides a means to study transgenes with perinatal toxicity.

An N-terminal region of Sp1 targets its proteasome-dependent degradation in vitro.

The transcription factor Sp1 is important for the expression of many cellular genes. Previously, it was shown that reduced O-glycosylation of Sp1 is associated with increased proteasome susceptibility. Sp1 undergoes proteasome-dependent degradation in cells stressed with glucose deprivation and adenylate cyclase activation, and this process is blocked in cells treated with glucosamine. In this study, using a reconstituted in vitro system, we identified the principal structural determinant in Sp1 that targets Sp1 for proteasome-dependent degradation. We found by using deletion analysis that the N-terminal 54 amino acids of Sp1 is required for Sp1 degradation. This element can act as an independent processing signal by directing degradation of an unrelated protein. Recognition of this Sp1 element by the proteasome-dependent system is saturable, and ubiquitination of this element is not required for recognition. Time course experiments revealed that Sp1 degradation is a two-step process. First, a discrete endoproteolytic cleavage occurs downstream of the target region immediately C-terminal to Leu56. The Sp1 sequence C-terminal to the cleavage site is subsequently degraded, whereas the N-terminal peptide remains intact. The identification of this Sp1 degradation-targeting signal will facilitate the identification of the critical proteins involved in the control of Sp1 proteasome-dependent degradation and the role of OGlcNAc in this process.

Streptozotocin, an analog of N-acetylglucosamine, blocks the removal of O-GlcNAc from intracellular proteins.

Streptozotocin (STZ), an analog of N-acetylglucosamine (GlcNAc), is a specific toxin for the pancreatic beta cell. We found that treatment of rats with STZ results in an early beta-cell-specific increase in the level of intracellular protein modification by O-linked GlcNAc (O-GlcNAc). Using a model O-GlcNAc peptide based on the transcription factor Sp1, we show that treatment of cultured cells with STZ during peptide biosynthesis results in hyperglycosylation of the peptide as a result of the ability of STZ to specifically inhibit the activity of O-GlcNAc-selective N-acetyl-beta-D-glucosaminidase. Although this inhibitory activity of STZ probably can occur in all cells, we found, using in situ hybridization, that beta cells express very high levels of the mRNA encoding the enzyme responsible for cytoplasmic protein O-glycosylation, O-GlcNAc transferase (OGT). These findings suggest that the pancreatic beta cell is particularly sensitive to the toxicity of STZ because it expresses such high levels of OGT. When STZ blocks O-GlcNAc removal from intracellular proteins, the cell with the most rapid on-rate for O-GlcNAc, the beta cell, will experience the most rapid accumulation of this protein modification. Because we also show that the on-rate of O-GlcNAc is substrate driven in several cell types, we speculate that the beta cell, with its high level of OGT, may also respond to elevations of blood sugar with increased protein modification by O-GlcNAc. Thus, this proposed mechanism of STZ toxicity on the beta cell may result from an exaggeration of a heretofore unrecognized physiological response to glucose mediated through the high level of OGT in these cells.

Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles.

The erbB-2 proto-oncogene belongs to a receptor tyrosine kinase family that includes the epidermal growth factor receptor, erbB-2, erbB-3, and erbB-4. erbB-2 is expressed in basal cells of the squamous epithelia and the outer root sheath of the hair follicles, but its function in epidermal development has not been well studied. To investigate its role in the skin, we created transgenic mice harboring an activated erbB-2 oncogene under the control of the human keratin 14 promoter. The keratin 14 promoter directed its expression to cells in which erbB-2 is normally expressed, whereas the activated receptor gene ensured increased signaling. All transgenic founder mice exhibited extensive and striking skin phenotype, including epidermal hyperplasia, preneoplasia, papilloma, hyperkeratosis, and dyskeratosis. The majority of the hair follicles were replaced by bizarre hyperproliferative intradermal squamous invaginations, whereas the rest of the follicles exhibited severe hyperplasia and disorganization. All but one of the transgenic mice died before or shortly after birth, probably as a consequence of defects in the skin and esophagus. These observations demonstrate that the skin is sensitive to erbB-2 signaling, suggesting an important role for this receptor tyrosine kinase in epidermal growth, differentiation, and hair follicle morphogenesis.

Targeted expression of a dominant negative epidermal growth factor receptor in the mammary gland of transgenic mice inhibits pubertal mammary duct development.

The epidermal growth factor (EGF) system has been thought to play an important role in normal mammary development and carcinogenesis. To study the role of the EGF receptor (EGFR) in mammary development, we developed a transgenic mouse model in which a C-terminal truncated mouse EGFR (EGFR-TR) was expressed in the mouse mammary epithelium under the control of the mouse mammary tumor virus long terminal repeat. The EGFR-TR lacks most of the cytoplasmic domain of the receptor, including the entire protein tyrosine kinase domain. In cultured cells, we show that it acts in a dominant negative manner in EGF-signaled EGFR autophosphorylation. Several lines of mice were characterized and shown to express the transgene at the mRNA and protein levels not only in the mammary gland but also in the salivary glands, epididymis, and prostate. In postpubertal virgin female mice, the expression of the EGFR-TR in the mammary glands was greater than the expression of the endogenous wild type EGFR. In these virgin mice, inhibition in mammary ductal development and a decrease of mammary epithelial DNA synthesis were observed beginning at 5-6 weeks. The inhibition of duct development was most apparent by 15-16 weeks, resulting in a significant defect in ductal branching and outgrowth and an apparent overall decrease in the size of the mammary glands. However, during pregnancy, expression of the endogenous wild type EGFR was markedly increased relative to the EGFR-TR and, at this stage, normal presecretory alveoli developed from the hypoplastic duct tree. Postpartum, normal lactation occurred. Despite EGFR-TR expression in other tissues, no morphological abnormalities were observed. This model demonstrates that the EGFR-TR behaves as a dominant negative regulator of the EGFR system in vivo and that the EGFR system plays an important role in mammary ductal development.

The association of biologically active lipids with the development of transfusion-related acute lung injury: a retrospective study.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is clinically similar to the adult respiratory distress syndrome (ARDS) and has been linked to the transfusion of leukocyte antibodies in blood components. Animal model have implicated neutrophil (PMN)-priming agents in ARDS; however, two agents were required. Previous studies showed the generation of PMN-priming agents during blood storage. Thus the association of PMN-priming agents with TRALI was examined. STUDY DESIGN AND METHODS: Ten patients with TRALI and 10 with febrile or urticarial reactions (control group) were evaluated. The presence of PMN-priming activity was tested in the patients' pretransfusion and posttransfusion blood samples by incubating PMNs with these samples followed by activation of the respiratory burst. Plasma lipids were separated by normal-phase high-performance liquid chromatography (HPLC), and the priming activity was evaluated. The presence of leukocyte antibodies was determined in the blood donors and patients with TRALI. RESULTS: Significantly more PMN-priming activity was present in the posttransfusion sera (11.4 +/- 1.8 nmol superoxide anion/min, mean +/- SEM; n = 10) and plasma of patients with TRALI than in their pretransfusion sera (6.5 +/- 1.5: n = 10) or in the pretransfusion and posttransfusion sera (5.1 +/- 1.3, n = 10; and 4.5 +/- 1.4, n = 10, respectively) and from the controls (p < 0.05). HPLC separation of lipids demonstrated that three active species were present in the posttransfusion plasma samples of TRALI patients. All the patients with TRALI had underlying clinical factors, such as infection, cytokine administration, recent surgery, or massive transfusion, while only 2 of 10 control patients had these clinical conditions. None of the donors had significant titers of HLA or HLA-DR antibodies; however, 50 percent had weak positivity for granulocyte antibodies. CONCLUSION: TRALI is the result of two clinical events, the first being a predisposing clinical condition and the second being the transfusion of biologically active lipids in stored blood.

MDA468 growth inhibition by EGF is associated with the induction of the cyclin-dependent kinase inhibitor p21WAF1.

Epidermal growth factor (EGF) usually stimulates the proliferation of a variety of normal and malignant cells. In contrast, MDA468, a human breast cancer cell line with a very high number of EGF receptors, is growth inhibited in response to concentrations of EGF that stimulate most other cells. The purpose of this study was to elucidate the cellular mechanisms involved in EGF-induced growth inhibition. EGF treatment stimulated the sustained expression of the cyclin-dependent kinase (CDK) inhibitor p21WAF1. The p21WAF1 induction in EGF-treated MDA468 cells is probably p53-independent since these cells contain no active p53. The promoter for p21WAF1 gene contains binding sites for signal transducer and activator of transcription (STAT) and EGF is known to activate members of this family of transcription factors. Using electrophoretic mobility shift assays (EMSA), we found that EGF activates STAT1 and STAT3 in the MDA468 cells. These activated STATs specifically recognized the three conserved STAT-responsive elements in the p21WAF1 gene promoter, suggesting that STATs may be responsible for the p21WAF1 induction by EGF in MDA468 cells. The sustained rise in p21WAF1 in response to EGF is proposed to be a means of growth inhibition in these cells.

Role of glucosamine synthesis in the stimulation of TGF-alpha gene transcription by glucose and EGF.

Transforming growth factor-alpha (TGF-alpha) gene transcription is regulated by both epidermal growth factor (EGF) and glucose. Previous studies have suggested that the metabolism of glucose to glucosamine through the enzyme L-glutamine: D-fructose-6-phosphate amidotransferase (GFAT) plays a critical role in the glucose signaling. In this paper, we compared the role of GFAT in the glucose and EGF signals. We found that, although EGF stimulates GFAT mRNA accumulation in MDA-MB-468 cells, this effect of EGF occurred several hours after TGF-alpha transcription increased. MDA-MB-468 cells also exhibited a TGF-alpha transcriptional response to low concentrations of glucose. The TGF-alpha response to glucose but not EGF could be inhibited by a blocker of GFAT activity. Blockade of GFAT was confirmed by using Western blotting with the RL2 antibody, which recognizes an epitope on proteins containing N-acetylglucosamine. Exposure of cells to glucose increased the RL2 signal on several polypeptides, but this change could be blocked by inhibition of GFAT. These results support the notion that glucose stimulation of TGF-alpha expression requires GFAT, but EGF stimulation does not.

Analysis of the priming activity of lipids generated during routine storage of platelet concentrates.

BACKGROUND: Compounds generated during the routine storage of platelet concentrates may have deleterious effects on the transfusion recipient. STUDY DESIGN AND METHODS: Daily plasma samples from platelet concentrates, both apheresis platelets and those separated from whole blood, were obtained serially during routine storage. These plasma samples were assayed for their ability to prime the NADPH oxidase in isolated human neutrophils. Quantitative and qualitative analysis of the priming agents was completed by lipid extraction, high-pressure liquid chromatography separation, and gas chromatography/mass spectroscopy. RESULTS: Compounds were generated in both apheresis and whole-blood platelets that significantly primed the NADPH oxidase after 24 and 48 hours of storage, respectively. The priming activity was maximal by component outdate: 2.6-fold that of the buffer-treated control neutrophils (apheresis) and 3.9-fold that of the buffer-treated control neutrophils (whole blood). These agents were generated by cellular constituents, as stored plasma did not demonstrate such priming activity. Inhibition of this priming activity by WEB 2170, a specific platelet-activating factor receptor antagonist, suggested that the observed priming involved the platelet-activating factor receptor. A portion of the priming activity from platelet concentrates was organically extractable: 69 percent of that from apheresis platelets and 46 percent of that from whole-blood platelets. Further purification of the lipid's priming activity by normal-phase high-pressure liquid chromatography demonstrated a single peak of priming activity at the retention time of lysophosphatidylcholines. Because 46 percent of the priming activity from whole-blood platelets was chloroform insoluble and because it has been reported that interleukin 8 is generated during routine storage of whole-blood platelets, the effects of interleukin 8 on the NADPH oxidase were examined. Recombinant monocyte interleukin 8 rapidly primed the oxidase but was not inhibited by WEB 2170. CONCLUSION: Lipids were generated during the routine storage of platelet concentrates that prime the NADPH oxidase, and they may play a role in the severe complications of transfusion therapy. Other non-lipid compounds, such as interleukin 8, that are generated in whole-blood platelets may also contribute to the observed priming activity of plasma.

Targeting of transforming growth factor-alpha expression to pituitary lactotrophs in transgenic mice results in selective lactotroph proliferation and adenomas.

The PRL-secreting cells of the pituitary gland normally express transforming growth factor-alpha (TGF alpha). To determine the effect of increasing TGF alpha expression in the pituitary, a transgenic mouse model was created in which overexpression of human TGF alpha was directed to the pituitary lactotrophs using the rat PRL promoter. Of the four gene-positive mouse lines, two expressed the messenger RNA corresponding to the transgenic in the pituitary glands. However, in both these lines, expression could only be detected in the female animals. Expression of the transgenic could be detected as early as 1 month of age, but no pathology or developmental abnormalities were detected until the animals reached 6 months, at which time, hyperplasia of the lactotrophs. By the age of 12 months, all of the homozygous transgenic females had developed pituitary adenomas that were immunopositive for PRL. The other hormone-producing cells of the pituitary showed no obvious pathology. The male transgenics developed neither hyperplasia nor adenomas, nor did the gene-positive transgenic lines that did not express the transgene. In no case was an aggressive pituitary tumor seen. This transgenic mouse model indicates that TGF alpha overexpression by lactotrophs stimulates the growth of these pituitary cells. Furthermore, TGF alpha has a highly localized action in the pituitary gland, resulting only in lactotroph hyperplasia and prolactinomas. These observations suggest that TGF alpha might play a role in the development of prolactinomas.

p53 stimulates transcription from the human transforming growth factor alpha promoter: a potential growth-stimulatory role for p53.

Physical and chemical agents can damage the genome. Part of the protective response to this damage is the increased expression of p53. p53, a transcription factor, controls the expression of genes, leading to cell cycle arrest and apoptosis. Another protective mechanism is the proliferative response required to replace the damaged cells. This proliferation is likely to be signaled by growth factors. In this communication, we show that the transforming growth factor alpha (TGF-alpha) gene is a direct target for p53-mediated transcriptional activation. In a stable cell line containing an inducible p53 construct, p53 induction leads to a threefold accumulation of the native TGF-alpha mRNA. IN cotransfection assays using a TGF-alpha promoter reporter construct, we show that expression of wild-type but not mutant p53 increases transcriptional activity of the TGF-alpha promoter by approximately 2.5-fold. In vitro, wild-type p53 binds to a consensus binding site found in the proximal portion of the promoter, and this sequence is necessary for the p53 transcriptional response. Furthermore, this element confers p53 induction to the otherwise nonresponsive adenovirus major late promoter. In addition to these results, we found that the TGF-alpha promoter contains a nonconsensus but functional TATA box-binding protein-binding site approximately 30 bp upstream of the transcription start site. Although p53 can repress transcription from promoters containing a TATA box, the nonconsensus TGF-alpha TATA motif is resistant to this effect. On the basis of these results, we propose that p53 may play a dual role, which includes both the elimination of irreparably genetically damage cells and the proliferative response necessary for their replacement, in the response to physical-chemical damage.

Burning mouth syndrome: the relationship between the HAD scale and parafunctional habits.

This study investigated 84 patients with burning mouth syndrome (BMS), who were asked to complete a hospital anxiety and depression (HAD) scale questionnaire. A control group of 69 patients was also included. All patients were interviewed regarding parafunctional habits and were subjectively examined for signs of occlusal wear of the natural teeth or dentures. The results demonstrated that parafunctional habits were present in 61% of patients with BMS. There was a statistically significantly relationship between parafunctional habits and anxiety as indicated by the HAD scale, but not with depression.

Regulation of glutamine:fructose-6-phosphate amidotransferase gene transcription by epidermal growth factor and glucose.

In preparation for the cellular proliferation stimulated by growth factors, the rate of macromolecular synthesis must be increased to allow for the enlargement of the cell that proceeds mitosis. The increased glycoprotein synthesis that follows growth factor stimulation would consume the hexosamines required for protein modification. Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the rate-limiting enzyme controlling the synthesis of the hexosamines used in these biosynthetic pathways. We tested the idea that growth factors might activate the transcription of the GFAT gene to increase the cellular content of this rate-limiting enzyme in hexosamine synthesis. We employed a human breast cancer cell line, MDA468 cells, which express high numbers of epidermal growth factor (EGF) receptors, to determine whether EGF could stimulate transcription of the GFAT gene. Our experiments showed that EGF stimulated the accumulation of GFAT messenger RNA (mRNA) to a level 4-fold higher than that in unstimulated cells. This accumulation could be largely accounted for by an increase in transcription, as assessed by nuclear run-on experiments. Furthermore, the GFAT mRNA was highly stable and not further stabilized by EGF. This effect of EGF on GFAT gene transcription required stimulation for 12-16 h with EGF. Interestingly, when cells were exposed to 25 mM glucose instead of 5 mM glucose, this effect of EGF was blocked. Glucose had no effect on the stability of the GFAT mRNA, implying that the effect of glucose was to antagonize the transcriptional effect of EGF on the GFAT gene. Glucosamine had an effect opposite that of glucose, in that it stimulated GFAT mRNA accumulation and had an additive effect with EGF on the accumulation of this mRNA. These results demonstrate that the GFAT gene undergoes a late transcriptional response to EGF and that the provision of high glucose concentrations to the cells blocks this EGF activation. This effect of glucose does not appear to result from its metabolism through GFAT to glucosamine.

Case report: prolonged match chewing: an unusual case of tooth wear.

A case is reported of the diagnosis and clinical management of a patient with an obsessional neurosis resulting in extensive tooth and denture acrylic wear partially caused by prolonged match chewing.

Sequence of the functional human keratin K14 promoter.

The K14 keratin is an intermediate filament produced in squamous epithelia. This tissue-specific expression is directed by the promoter (pK14) of the K14 gene which has been used extensively to direct the expression of transgenes to the skin. Human K14 was cloned and the upstream sequence is presented. In transient transfections, pK14 directs expression of a luciferase reporter in keratinocytes much more potently than in breast cancer cells.