[Clinical and endoscopic characteristics of adult celiac disease].

Objective: The study aimed to analyze the clinical and endoscopic characteristics of adult celiac disease (CD) to provide a scientific basis for more effective CD diagnosis and treatment. Methods: In this cross-sectional study, the clinical and endoscopic data of 96 adult CD patients treated in the Department of Gastroenterology of the People's Hospital of Xinjiang Uygur Autonomous Region from March 2016 to December 2021 were retrospectively collected and analyzed. Results: A total of 96 CD patients were diagnosed, including 33 men and 63 women. The average age was 47±14 years (range, 18-81 years). The disease occurred mainly in the age group of 31-60 years. The median course of the disease was 2.0 (0.2-40.0) years. There were 41 (42.7%) classical and 55 (57.3%) non-classical CD patients. All patients with classical CD showed chronic diarrhea, often accompanied by abdominal pain (46.3%, 19/41), abdominal distension (17.1%, 7/41), anemia (65.9%, 27/41), and chronic fatigue (48.8%, 20/41). The main manifestations of non-classical CD were chronic abdominal pain (58.2%, 32/55), abdominal distension (32.7%, 18/55), anemia (40.0%, 22/55), and osteopenia/osteoporosis (38.2%, 21/55). Compared with non-classical CD, anemia developed more frequently in classical CD, and the difference was statistically significant (P = 0.012). The incidence of complications in CD patients was 36.5% (35/96), and the main complications were thyroid disease (19.8%, 19/96), connective tissue disease (6.2%, 6/96), and kidney disease (6.2%, 6/96). There was no significant difference between classical and non-classical CD (P>0.05). The frequency of endoscopic manifestations in CD patients was 84.4% (81/96). Duodenal bulb endoscopy showed nodular changes (72.9%, 70/96), grooved changes (10.4%, 10/96), and focal villous atrophy (9.4%, 9/96). The main manifestations of descending endoscopy were the decrease, flattening, or disappearance of duodenal folds (43.8%, 42/96), scallop-like changes (38.5%, 37/96), and nodular changes (34.4%, 33/96). Conclusions: Adult CD patients are mostly female. CD occurred mainly in the age group of 31-60 years. The clinical manifestations were mainly those of non-classical CD. Some patients often had other autoimmune diseases. Patients with characteristic endoscopic manifestations should be warned about the possibility of developing CD. Clinicians should strengthen the understanding of CD and reduce the related rates of missed diagnosis.

Lesion-aware convolutional neural network for chest radiograph classification.

AIM: To investigate the performance of a deep-learning approach termed lesion-aware convolutional neural network (LACNN) to identify 14 different thoracic diseases on chest X-rays (CXRs). MATERIALS AND METHODS: In total, 10,738 CXRs of 3,526 patients were collected retrospectively. Of these, 1,937 CXRs of 598 patients were selected for training and optimising the lesion-detection network (LDN) of LACNN. The remaining 8,801 CXRs from 2,928 patients were used to train and test the classification network of LACNN. The discriminative performance of the deep-learning approach was compared with that obtained by the radiologists. In addition, its generalisation was validated on the independent public dataset, ChestX-ray14. The decision-making process of the model was visualised by occlusion testing, and the effect of the integration of CXRs and non-image data on model performance was also investigated. In a systematic evaluation, F1 score, sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) metrics were calculated. RESULTS: The model generated statistically significantly higher AUC performance compared with radiologists on atelectasis, mass, and nodule, with AUC values of 0.831 (95% confidence interval [CI]: 0.807-0.855), 0.959 (95% CI: 0.944-0.974), and 0.928 (95% CI: 0.906-0.950), respectively. For the other 11 pathologies, there were no statistically significant differences. The average time to complete each CXR classification in the testing dataset was substantially longer for the radiologists (∼35 seconds) than for the LACNN (∼0.197 seconds). In the ChestX-ray14 dataset, the present model also showed competitive performance in comparison with other state-of-the-art deep-learning approaches. Model performance was slightly improved when introducing non-image data. CONCLUSION: The proposed LACNN achieved radiologist-level performance in identifying thoracic diseases on CXRs, and could potentially expand patient access to CXR diagnostics.

Myoglobin A79G polymorphism association with exercise-induced skeletal muscle damage.

We assessed the role of A79G, a polymorphism of the myoglobin gene (MB), in susceptibility to exercise-induced skeletal muscle damage. Between January 2012 and December 2014, a total of 166 cases with exercise-induced skeletal muscle damage and 166 controls were recruited into our study. Genotyping of MB A79G was carried out using polymerase chain reaction coupled with restriction fragment length polymorphism. Using unconditional logistic regression analysis, we found that the GG genotype of MB A79G was associated with higher risk of exercise-induced muscle damage compared with the wild-type genotype, and the OR (95%CI) was 2.91 (1.20-7.59). Compared with the AA genotype, the AG+GG genotype was associated with a significantly increased risk of exercise-induced muscle damage for those with blood lactic acid ≥1.80 mM (OR = 2.05; 95%CI = 1.09-3.88). In conclusion, we found that the A79G polymorphism of the MB gene plays an important role in influencing the development of exercise-induced skeletal muscle damage.

Prevalence of Toxoplasma infection in first-episode schizophrenia and comparison between Toxoplasma-seropositive and Toxoplasma-seronegative schizophrenia.

OBJECTIVE: To compare the prevalence of Toxoplasma infection between the first-episode schizophrenia and the controls and to compare the clinical features between the Toxoplasma-seronegative and Toxoplasma-seropositive patients with schizophrenia. METHOD: The rate of serum reactivity to Toxoplasma in 600 schizophrenia, 600 affective disorders, and 400 controls was investigated. The clinical symptoms of the schizophrenia patients were scored and compared. RESULTS: The rate of IgG antibody, not IgM in the schizophrenia patients, was higher than the control groups, and the odds ratio of schizophrenia associated with IgG antibody was 2.22-5.12. The affective disorders did not differ in the rate of IgG or IgM antibody from the normal or the physical disease control. The seropositive schizophrenia patients had higher scores on the positive subscale and three components of Positive and Negative Symptoms Scale than the seronegative patients. CONCLUSION: This study lent further weight to the hypothesis that exposure to Toxoplasma may be a risk factor for schizophrenia.

Exogenous nitric oxide induces apoptosis in Toxoplasma gondii tachyzoites via a calcium signal transduction pathway.

The mechanism by which nitric oxide (NO)-dependent cytotoxicity acts against Toxoplasma gondii tachyzoites is poorly understood. An NO donor, sodium nitroprusside (SNP), was used to induce death in T. gondii tachyzoites in vitro as a model for investigating (i) whether NO is capable of inducing apoptosis-like death in tachyzoites and (ii) whether a calcium signal transduction pathway is involved. Exposure to 2 mM SNP resulted in a pattern of tachyzoite death that shares many features with metazoan apoptosis and it may involve a calcium signal transduction pathway. Motility and cell survival in these parasites showed a gradual decline with increasing levels of SNP. Features common to metazoan apoptosis are observed after exposure to 2 mM SNP. Ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetra-acetic acid (EGTA), Verapamil and bis-(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid/acetoxymethyl ester (BAPTA/AM) partially increased the cell survival concomitant with decreased [Ca2+]i in cells exposed to SNP. An NO scavenger (N-acetylcysteine), the analogue of SNP (devoid of NO), inhibited the rate of apoptosis after SNP treatment compared with SNP treatment without scavenger, but alone did not induce apoptosis. Taken together, the results indicate that SNP is capable of inducing apoptosis in T. gondii tachyzoites via a calcium signal transduction pathway.

Mn(2+) ions reduce the enzymatic and pharmacological activities of bothropstoxin-I, a myotoxic Lys49 phospholipase A(2) homologue from Bothrops jararacussu snake venom.

Bothropstoxin-I (BthTX-I), a myotoxic Lys49 phospholipase A(2) (PLA(2)) homologue isolated from Bothrops jararacussu snake venom, causes a range of biological effects, including myonecrosis, mouse paw edema, irreversible neuromuscular blockade and lysis of cell cultures. Among eight divalent cations assayed, Mn(2+) was the most effective in reducing mouse paw edema induced by BthTX-I (25 microg). Preincubating BthTX-I with Mn(2+) (1.0mM) reduced mouse paw edema by 70% and myotoxicity by 60% in mice injected i.m. with 50 microg toxin. Mn(2+) (50 microl of a 1mM solution) administered within 1min at the site of toxin injection was still but less effective in antagonising BthTX-I-induced myotoxicity. Mn(2+) (1.0mM) completely prevented BthTX-I (1.4 microM)-induced neuromuscular blockade in the mouse phrenic-nerve diaphragm preparation. Mn(2+) (0.25mM) protected about 85% of NB41A3 cells from lysis when coincubated with BthTX-I (1.0 microM) for 25h. Preincubation with 0.25mM Mn(2+) increased the sensitivity of the cells to subsequent lysis by BthTX-I in the absence of Mn(2+). BthTX-I (1 microM) caused extensive fatty acid release (from 0.8% of the total radiolabeled lipid in control cells to 56% with toxin) when incubated with the NB41A3 cell line for 25h. PLA(2) activity observed in cell cultures after addition of BthTX-I was considerably reduced by 0.25mM Mn(2+). Mn(2+) ions constitute a promising agent to assess the action mechanism and the effects of enzymatic inhibition on the pharmacological activity of Lys49 PLA(2) homologues.

[Dynamic changes in amino acid and glucose in culture medium with adult Schistosoma japonicum].

OBJECTIVE: To observe the dynamic changes in the contents of amino acid(AA), glucose(Gluc) and triglyceride(TG) in the culture medium containing adult Schistosoma japonicum. METHODS: The contents of AA, Gluc and TG in the culture medium during the incubation period for d0 to 6 d were detected by amino acid automatic analyzer and automatic biochemical analyzer. RESULTS: The contents of Arg, Thr, Met, and Lys and Gluc were reduced, Asp and Ala increased apparently. CONCLUASION: Increasing the levels of Arg, Thr, Met Lys and Gluc, reducing the levels of Asp and Ala, and changing the culture medium in time might be in favor of the in vitro cultivation of S. japonicum.

[The effect of nitric oxide donor on the DNA content in Toxoplasma gondii tachyzoite].

OBJECTIVE: To determine the role of sodium nitroprusside (SNP) in regulating DNA synthesis of Toxoplasma gondii tachyzoites. METHODS: Hypodiploid peak of tachyzoite DNA induced by SNP was assessed according to DNA fragmentation. The effect of SNP on appearance of hypodiploid peak and the effect of Ca2+ on the growth of tachyzoites were evaluated. The intracellular Ca2+ chelator (BAPTA/AM), antagonist of Ca2+ channel (verapamil) and the extracellular Ca2+ chelator (EGTA) were used. The change of DNA content was measured by flow cytometry. RESULTS: SNP inhibited DNA synthesis of tachyzoites in a dose- and time-dependent pattern. The antiproliferative effect of SNP on tachyzoites was inhibited by verapamil, EGTA and BAPTA/AM. The inhibition of the growth of tachyzoites by SNP was associated with increased subploid peak through a Ca(2+)-dependent mechanism. CONCLUSION: SNP induced a hypodiploid peak in tachyzoites by altering the Ca2+ concentration in the plasma of tachyzoite, resulting in damages of the parasite.

[Effect of acryl thiourea on liver pathologic changes in mice infected with Schistosoma japonicum].

OBJECTIVE: To observe the effect of acryl thiourea, an inhibitor of phenol oxidase, on pathological changes in the liver of mice infected with Schistosoma japonicum. METHODS: From day 22 to day 42 postinfection with cercariae, the mice of the acryl thiourea group were each injected i.p. with acryl thiourea at a dose of 300 mg/kg every other day. The mice were killed on the 42nd day postinfection to observe the pathological changes in the liver. RESULTS: Compared to the infected control group, the liver tissue of the acryl thiourea group showed scattered foci of inflammatory cell infiltration, the mean diameter and area of the foci were significantly reduced (P < 0.01), and there were no eggs in the center of the foci except for some granules. CONCLUSION: After i.p. injections of acryl thiourea, no typical egg granuloma was found in the liver of infected mice. This was possibly due to the inhibition of schistosome phenol oxidase activity and so the female adult schistosomes could not produce normal eggs.

Sequential repression and activation of the CCAAT enhancer-binding protein-alpha (C/EBPalpha ) gene during adipogenesis.

CCAAT enhancer-binding protein-alpha (C/EBPalpha) functions as a pleiotropic transcriptional activator of adipocyte genes during adipogenesis. Nuclear factor C/EBP undifferentiated protein (CUP), an isoform of activator protein-2alpha (AP-2alpha), binds to repressive elements in the C/EBPalpha gene promoter, silencing the gene until late in the differentiation program. The CUP regulatory element overlaps a Sp (GT-box) element in the promoter to which Sp3 (or Sp1) can bind. Binding by Sp3 or Sp1 and CUP/AP2-alpha is mutually exclusive. Sp3 is a strong transcriptional activator of the C/EBPalpha gene promoter in 3T3-L1 preadipocytes and Schneider cells, this activation being repressed by CUP/AP-2alpha. Sp3 is expressed throughout differentiation, whereas CUP/AP-2alpha, which is expressed only by preadipocytes, is down-regulated during differentiation coincident with transcription of the C/EBPalpha gene. Thus, CUP/AP-2alpha delays access of Sp3 to the Sp regulatory element, preventing premature expression of C/EBPalpha and thereby interference by C/EBPalpha (which is antimitotic) with mitotic clonal expansion, an essential early event in the differentiation program.

Role of the CCAAT enhancer binding proteins (C/EBPs) in adipocyte differentiation.

Members of the C/EBP family of transcription factors play essential roles in the adipocyte differentiation program. Treatment of growth-arrested 3T3-L1 preadipocytes with appropriate hormonal agents causes the cells to synchronously reenter the cell cycle and to undergo mitotic clonal expansion. Expression of C/EBPbeta and delta occur early in clonal expansion, later followed by C/EBPalpha (which is anti-mitotic) as the cells exit the cell cycle begin to express adipocyte genes. C/EBPalpha serves as transcriptional activator of many adipocyte genes whose expression produce the adipocyte phenotype. Recent work in this laboratory has focussed on the roles of C/EBPbeta and delta in the differentiation program, in particular the mechanisms by which they activate transcription of the C/EBPalpha gene. Several regulatory elements, both repressive and activating, in proximal promoter of the gene have been identified. The cognate transacting factors that interact with these elements have been characterized and their functions elucidated. These factors have been incorporated into a model for a cascade that leads to transcriptional activation of the C/EBPalpha gene and the terminal steps in the differentiation program.

Pyridinylimidazole compound SB 203580 inhibits the activity but not the activation of p38 mitogen-activated protein kinase.

p38 MAPK is a Ser/Thr protein kinase activated by various inflammatory cytokines and a variety of stress stimuli. It is involved in many physiological processes, including the production of inflammatory cytokines. We have previously reported the design and synthesis of a series of pyridinylimidazole compounds that are selective inhibitors of p38 MAPK. These compounds, exemplified by SB 203580, are exceptionally effective in cell-based assays, including the inhibition of inflammatory cytokine production. SB 203580 is widely used as a tool to dissect the role of p38 MAPK in various physiological processes. It has previously been established that SB 203580 acts primarily to block the catalytic activity of p38 MAPK. However, it has been suggested that in cells, the compounds could also inhibit p38 MAPK activation by virtue of their ability to bind to the inactive enzyme. We undertook careful studies to definitively demonstrate that treatment with SB 203580 had no effect on Thr(180) and Tyr(182) phosphorylation, and hence activation of p38 in vivo. SB 203580, however, potently inhibited the activity of p38 MAPK as demonstrated by the inhibition of the activation of MAPKAP K2, a specific physiological substrate of p38 MAPK. This was observed regardless of stimuli or cell type. Identical results were obtained when the p38 MAPK cascade was partially reconstituted in vitro. Thus, our data clearly indicate that SB 203580 specifically inhibits the activity of p38 MAPK but not its activation by upstream MAPKK.

Isolation and structural characterization of a cytotoxic L-amino acid oxidase from Agkistrodon contortrix laticinctus snake venom: preliminary crystallographic data.

We have purified a cytotoxic L-amino acid oxidase (LAO) from Agkistrodon contortrix laticinctus snake venom by means of Superdex-200 gel filtration, followed by phenyl-Sepharose CL-4B chromatography. The purified enzyme (ACL LAO) is a dimer on gel filtration, with a M(r) of 60,000 for the monomer as estimated by SDS-PAGE. LAO activity was tested against 15 amino acids, but only 9 were oxidized by the enzyme, suggesting that it presents some degree of specificity. ACL LAO has apoptosis-inducing activity in an HL-60 cell culture assay. After 24 h treatment with 25 micrograms/ml of ACL LAO, the typical DNA fragmentation pattern of apoptotic cells was observed on agarose gel electrophoresis. NMR analysis showed the presence of a flavin mononucleotide prosthetic group. To solve its 3-D structure, crystals of the purified protein were grown in 0.1 M Tris-HCl, pH 8.5, and 2 M (NH(4))(2)SO(4). Diffraction data collected to 3.5 A showed that the protein crystallized in the tetragonal system, with unit cell a = b = 103.22 A, c = 183.45 A. This is the first report of preliminary crystallization data for a snake venom L-amino acid oxidase.

Repressive effect of Sp1 on the C/EBPalpha gene promoter: role in adipocyte differentiation.

Expression of C/EBPalpha is required for differentiation of 3T3-L1 preadipocytes into adipocytes. Previous investigations indicated that transcription of the C/EBPalpha gene is sequentially activated during differentiation, initially by C/EBPbeta and C/EBPdelta and later by C/EBPalpha (autoactivation). These events are mediated by a C/EBP regulatory element in the promoter of the C/EBPalpha gene. This article presents evidence that members of the Sp family, notably Sp1, act repressively on the C/EBPalpha promoter prior to the induction of differentiation. Sp1 was shown to bind to a GC box at the 5' end of the C/EBP regulatory element in the C/EBPalpha promoter and, in so doing, to competitively prevent binding to and transactivation of the promoter by the C/EBPs. One of the differentiation inducers methylisobutylxanthine (a cAMP phosphodiesterase inhibitor) or Forskolin, both of which increase the cellular cAMP level, causes down-regulation of Sp1. This decrease in Sp1 level early in the differentiation program appears to facilitate access of C/EBPbeta and/or C/EBPdelta to the C/EBP regulatory element and, thereby, derepression of the C/EBPalpha gene.

An ergonomic design and performance evaluation of pipettes.

This paper describes the results of an investigation of the differences in performance, postures, strains on hand-arm-shoulder musculature, and subjective ratings of three pipettes (models A, B, and C). Both models A and B were pipettes available on the market. Model C was developed for this study of an ergonomically designed pipette. The gripping posture of the three models was distinct both in the anatomical and in the functional sense. Working with models A and B required a four-finger grasp with a thumb operated plunger. Model C required a finger-palmar power grip and the plunger was operated by the fingers. Performance evaluation of the different pipettes in different tasks indicated that using the proposed model C resulted in a 2-3% lower fault rate, a 10% shorter completion time, and the highest subjective ratings among the three. Postural analysis results indicated that when using model C, the shoulder was the least abducted, the wrist was the least extended, and the wrist was the least radially extended. Model C appeared to provide the greatest opportunity for delicate adjustments of posture in response to the activity of the skin receptors and reduced the strains on the upper body musculature, justifying the ergonomic input into the design.

LYS49 phospholipase A2 myotoxins lyse cell cultures by two distinct mechanisms.

Three Lys49 phospholipase A2 (PLA2) myotoxins from Agkistrodon contortrix laticinctus (ACLMT), Bothrops jararacussu (bothropstoxin-I) and Bothrops asper (myotoxin II) snake venoms are enzymatically inactive on artificial substrates, yet addition of these toxins to cell cultures causes the release of fatty acids derived from the hydrolysis of membrane phospholipids. Bothropstoxin-I treated with p-bromophenacyl bromide is no longer enzymatically active on cell cultures, suggesting the toxin, not tissue PLA2, may hydrolyze the phospholipids. The NB41A3 cell line is sensitive to lysis by ACLMT by two separate mechanisms. The first mechanism is predominant at lower concentrations of ACLMT (0.1-0.5 microM) and over long incubation periods (24 h) with toxin. This mechanism is antagonized by methylprednisolone (MePDN). The second is predominant at higher concentrations of toxin (1-5 microM) incubated over a short period (1 h) and is not antagonized by MePDN. There is no correlation between enzymatic activity and toxicity at the higher concentrations (5 microM; 1 h) when the enzymatic activity of ACLMT is compared with a noncytolytic PLA2 from Naja naja atra venom (1 microM). However, over a 24 h period, triglyceride formation relative to fatty acids remaining free is about 10-fold greater for ACLMT (ratio about 40:1) than for the PLA2 from Naja naja atra venom (ratio about 4:1), suggesting the two enzymes act on substrates associated with different cellular compartments under this condition. Therefore, two mechanisms of Lys49 PLA2-induced myonecrosis exist and these are dependent on toxin concentration. The MePDN-sensitive mechanism associated with triglyceride accumulation correlates with myotoxicity.

Derepression of the C/EBPalpha gene during adipogenesis: identification of AP-2alpha as a repressor.

During adipogenesis, CCAAT/enhancer binding protein alpha (C/EBPalpha) serves as a pleiotropic transcriptional activator of adipocyte genes. Previously, we identified dual repressive elements in the C/EBPalpha gene and a putative transacting factor (C/EBPalpha undifferentiated protein, or CUP) expressed by preadipocytes, but not adipocytes, that bind to these elements. In the present investigation, CUP was purified 17,000-fold from nuclear extracts of 3T3-L1 preadipocytes. Amino acid sequence and mass spectral analysis of tryptic peptides derived from purifed CUP (molecular mass approximately 50 kDa) revealed that the repressor is (or contains) an isoform of the transcription factor, AP-2alpha. Electrophoretic mobility shift and Western blot analysis on purified CUP and preadipocyte nuclear extracts confirmed the identity of CUP as AP-2alpha. Both AP-2alpha protein and CUP binding activity are expressed by preadipocytes and then decrease concomitantly during differentiation of 3T3-L1 preadipocytes into adipocytes. Consistent with a repressive role of AP-2alpha/CUP, an AP-2alpha1 expression vector, cotransfected with a C/EBPalpha promoter-reporter construct into 3T3-L1 adipocytes, inhibited reporter gene transcription. Taken together with previous results, these findings suggest that in preadipocytes the C/EBPalpha gene is repressed by AP-2alpha/CUP, which, upon induction of differentiation, is down-regulated, allowing expression of the gene.

Melittin and phospholipase A2 from bee (Apis mellifera) venom cause necrosis of murine skeletal muscle in vivo.

Melittin and phospholipase A2 (PLA2) from bee (Apis mellifera) venom were rested for their ability to induce necrosis of skeletal muscle cells after intramuscular injection into mice. Light and electron microscopic examination of tissue indicated that both melittin (4 micrograms/g) and bee venom PLA2 (4 micrograms/g) caused necrosis of skeletal muscle cells within 30 min after i.m. injection. Early changes in the cells consisted of delta lesions, indicating a ruptured plasma membrane, and hypercontraction of myofibrils. By 24 hr the affected cells appeared as an amorphous mass of disorganized and disrupted myofibrils contained in an intact basal lamina. To ensure that the myotoxic activity of the melittin preparation was not due to contaminating. PLA2 activity, the preparation was treated with p-bromophenacyl bromide (p-BPB), a known inhibitor of PLA2 activity. The p-BPB-treated melittin was determined to have no detectable PLA2 activity using a sensitive muscle cell culture assay, and it still induced myonecrosis, although to a lesser extent and of a slower onset. Additionally, p-BPB treatment of purified bee venom PLA2 completely inhibited its myotoxic activity. These results indicate that both melittin and bee venom PLA2 are capable of inducing necrosis of skeletal muscle cells upon i.m. injection, and that the catalytic and myotoxic activities of bee venom PLA2 are inihibited by p-BPB. Also, melittin and contaminating PLA2 in the melittin fraction may be acting synergistically to induce a stronger and more rapid myotoxic effect than occurs with either alone.

A subpopulation of estrogen receptors are modified by O-linked N-acetylglucosamine.

Estrogen receptors (ER) are ligand-inducible transcription factors regulated by Ser(Thr)-O-phosphorylation. Many transcription factors and eukaryotic RNA polymerase II itself are also dynamically modified by Ser(Thr)-O-linked N-acetylglucosamine moieties (O-GlcNAc). Here we report that subpopulations of murine, bovine, and human estrogen receptors are modified by O-GlcNAc. O-GlcNAc moieties were detected on insect cell-expressed, mouse ER (mER) by probing with bovine milk galactosyltransferase, followed by structural analysis. Wheat germ agglutinin-Sepharose affinity chromatography also readily detected terminal GlcNAc residues on subpopulations of ER purified from calf uterus, from human breast cancer cells (MCF-7), or from mER produced by in vitro translation. These data suggest that greater than 10% of these populations of estrogen receptors bear O-GlcNAc. Site mapping of insect cell expressed mER localized one major site of O-GlcNAc addition to Thr-575, within a PEST region of the carboxyl-terminal F domain. Based upon their relative resistance to both hexosaminidase and to in vitro galactosylation, O-GlcNAc moieties appear to be largely buried on native mER. This dynamic saccharide modification, like phosphorylation, may play a role in modulating the dimerization, stability, or transactivation functions of estrogen receptors.

Repression of transcription mediated by dual elements in the CCAAT/enhancer binding protein alpha gene.

During adipocyte differentiation, the expression of C/EBPalpha is activated, which in turn serves to transcriptionally activate numerous adipocyte genes. A previous search for cis elements that regulate transcription of the C/EBPalpha gene led to the identification of a potential repressive element within the proximal 5' flanking region of the gene. Nuclear extracts from 3T3-L1 preadipocytes, but not adipocytes, were found to contain a factor, CUP (C/EBPalpha undifferentiated protein), that binds to this site (the CUP-1 site). In the present investigation, we show that C/EBPalpha promoter-luciferase constructs containing both the proximal 5' flanking and the entire 5' untranslated regions of the gene exhibit an expression pattern during adipocyte differentiation comparable to that of the endogenous C/EBPalpha gene. Mutation of the CUP-1 site in these constructs had little effect on reporter gene expression; however, when this mutation was combined with deletion of the 5' untranslated region, reporter gene expression by preadipocytes was dramatically up-regulated. Consistent with this finding, a second CUP binding site (the CUP-2 site) was identified in the 5' untranslated region. Although mutation of either CUP element in constructs containing both the 5' flanking and 5' untranslated region had little effect on reporter gene transcription, mutation of both CUP elements markedly activated transcription. Thus, it appears that dual CUP regulatory elements repress transcription of the C/EBPalpha gene prior to induction of the adipocyte differentiation program.