Reflux changes in adenoidal hyperplasia: a controlled prospective study to investigate its aetiology.

OBJECTIVES: To compare pepsin, carbonic anhydrase III (CAIII), cyclooxygenase-2 (COX-2) and mucin 5AC (MUC5AC) expression in children with adenoid hypertrophy and normal controls. DESIGN: A non-randomised, controlled prospective study. SETTING: Two paediatric hospitals in Adelaide, South Australia. PARTICIPANTS: Children aged 2-10 years, 21 undergoing adenoidectomy and 12 controls undergoing routine dental surgery. MAIN OUTCOME MEASURES: We measured expression of pepsin, CAIII, COX-2 and MUC5AC levels by real-time RT-PCR, immunohistochemistry, and Western blot to determine any difference between children with hyperplastic adenoids and controls. RESULTS: Pepsin was not detected in any study or control adenoid by immunohistochemistry or Western blot. Real-time RT-PCR analysis showed a statistically significant difference between groups with respect to COX-2 (P = 0.027) and MUC5AC (P = 0.02) but no difference in CAIII expression (P = 0.414). A significant correlation was also found between COX-2 and MUC5AC expression (Kendall Tau = 0.4, P = 0.005). CONCLUSION: Our results suggest that the biochemical changes seen in adenoid hypertrophy are different to those seen in reflux-affected tissues. The decreased COX-2 and MUC5AC expression may be due to squamous metaplasia and other inflammatory changes associated with adenoid hypertrophy. Our findings infer there is little evidence of reflux being a major contributory factor in the pathophysiology of adenoidal hypertrophy.

Identification of dioxin-responsive genes in Hep G2 cells using differential mRNA display RT-PCR.

Differential mRNA display RT-PCR (DD RT-PCR) offers a tool to identify genes which are regulated or responsive to certain receptors or chemicals such as dioxin (TCDD). Treatment of Hep G2 cells with TCDD followed by DD analysis of a gel with series of different primers revealed a significantly different pattern from the control for a number of mRNAs. The differentially displayed mRNAs were isolated and reamplified. A GenBank search of four mRNAs revealed two known and two unknown sequences. Northern blot analysis revealed that two known sequences, fibrinogen gamma chain and plastin mRNAs were down regulated by TCDD in a time-dependent manner, whereas two unknown mRNAs were induced by TCDD treatment. The function of these genes in TCDD toxicity is not known; however, the application of DD RT-PCR in the studies of TCDD-induced responses could be very useful in the discovery of other unknown genes important for TCDD toxicity.

Localization of a pioglitazone response element in the adipocyte fatty acid-binding protein gene.

The thiazolidinediones are a class of antidiabetic compounds that increase the sensitivity of target tissues to insulin. An earlier study has shown that these compounds enhance the insulin-stimulated differentiation of 3T3-L1 cells and up-regulate expression of differentiation-dependent genes. We have observed that the mRNA encoding the adipocyte fatty acid-binding protein (aFABP) increases shortly after incubation of cells with pioglitazone, a thiazolidinedone analogue. The drug was found to enhance, in a time- and dose-dependent fashion, the expression of a chimeric gene that was constructed by fusing the aFABP promoter upstream of the chloramphenicol acetyltransferase (CAT) gene. To localize the sequence within the promoter that is responsive to pioglitazone, a series of chimeric genes containing sections of the aFABP promoter fused to the CAT gene were analyzed after transfection of 3T3-L1 cells. A section of DNA located at -5.2 kilobases and known to encompass a tissue-specific and differentiation-dependent enhancer element was found to confer responsiveness to the drug. Analysis of sequences in this region of the aFABP promoter by DNA gel retardation assays revealed the presence of a protein in nuclear extracts from drug-treated cells that bound to a specific sequence (ARE-6). The presence of the protein could be demonstrated in differentiated adipocytes, but the protein was present at only low levels in preadipocytes. Treatment of preadipocytes with pioglitazone resulted in the precocious appearance of this protein in nuclear extracts. Multiple copies of the ARE-6 sequence inserted upstream of a heterologous promoter linked to the CAT gene conferred pioglitazone responsiveness. The experiments reported in this study establish that the insulin-sensitizing agent pioglitazone up-regulates expression of the aFABP gene through an element located within a region of DNA responsible for tissue-specific and differentiation-dependent expression of the gene.

Glucose-6-phosphate dehydrogenase: a "housekeeping" enzyme subject to tissue-specific regulation by hormones, nutrients, and oxidant stress.

The enzyme, glucose-6-phosphate dehydrogenase (G6PDH, EC1.1.1.49), has long been considered and studied as the archetypical X-linked "housekeeping" enzyme that is present in all cells, where it plays the key role in regulating carbon flow through the pentose phosphate pathway. Specifically, the enzyme catalyzes the first reaction in the pathway leading to the production of pentose phosphates and reducing power in the form of NADPH for reductive biosynthesis and maintenance of the redox state of the cell. It was in this latter function that the crucial importance of the enzyme was first appreciated with the description of the human deficiency syndrome. While the gene can be considered to be a constitutively expressed "housekeeping" gene in many tissues, there are several other tissues (liver, adipose, lung, and proliferating cells) wherein modulation of cellular G6PDH activity represents an important component of the integrated response to external stimuli (hormones, growth factors, nutrients, and oxidant stress). In this regard, adaptive regulation of G6PDH has been found to be exerted at transcriptional and posttranscriptional levels. However, the regulation observed is tissue-specific, which elicits the central question of this review, "How can the G6PDH gene be constitutively expressed in some tissues while displaying adaptive regulation in others when there exists a single transcription unit for the gene?" Future studies utilizing cloned genomic fragments of the human and other mammalian G6PDH genes should provide answers to this question.

Isolation and sequence of a rat glucose-6-phosphate dehydrogenase promoter.

A 935 bp fragment of the rat glucose-6-phosphate dehydrogenase (G6PDH) gene containing promoter activity was isolated using the polymerase chain reaction (PCR). This fragment was sequenced and primer extension analysis showed a transcription initiation site in agreement with the human and mouse genes. Computer analysis of the sequence showed a 60% and 78% similarity to the human and mouse G6PDH sequences, respectively. A TATA box element, TTAAAT, was found and shown to be 100% similar to the human and mouse TATA box elements. Based on sequence comparison, some putative transcriptional regulatory elements were also found.

Adipocyte fatty acid-binding protein: regulation of gene expression in vivo and in vitro by an insulin-sensitizing agent.

Pioglitazone, a thiazolidinedione, is a novel antidiabetic compound that can lower blood glucose in diabetic rodents by increasing insulin sensitivity in target tissues. We have previously demonstrated that pioglitazone can enhance the insulin- or insulin-like growth factor-1-regulated differentiation of 3T3-L1 cells, a cell line that undergoes morphological and biochemical differentiation to mature adipocytes [Mol. Pharmacol. 41:393-398 (1992)]. In this study, we have examined the effect of pioglitazone on the expression of the adipocyte fatty acid-binding protein (aFABP) in ob/ob mice and 3T3-L1 cells. Administration of the drug to mice was observed to cause a dose-dependent increase in aFABP mRNA expression in epididymal fat, which was correlated with a decrease in blood glucose and insulin levels. Treatment of 3T3-L1 cells with pioglitazone enhanced aFABP expression in a time-dependent fashion. To explore a possible direct effect of pioglitazone on aFABP expression, a chimeric gene was constructed containing the aFABP promoter fused upstream of the bacterial reporter gene for chloramphenicol acetyltransferase. After transfection into 3T3-L1 cells and selection of stable transformants, regulation of the chimeric gene was studied. Pioglitazone, in combination with insulin or insulin-like growth factor-1, was observed to elicit a dose-dependent increase in expression, indicating a role for pioglitazone in regulating transcription of the aFABP gene. Several thiazolidinedione analogs were tested for their ability to induce the expression of the chimeric gene, and it was found that activity in this assay paralleled the structure-activity relationships observed for enhancement of 3T3-L1 cell differentiation. These observations on control of aFABP gene expression by pioglitazone suggest possible mechanisms by which cellular sensitivity to insulin may be regulated.

Platelet-derived growth factor induces interleukin-1 receptor gene expression in Balb/c 3T3 fibroblasts.

Our previous studies have demonstrated that platelet-derived growth factor (PDGF) modulated cellular responses to interleukin-1 (IL-1). In this communication, we show that PDGF regulates expression of IL-1 receptor (IL-1 R) gene. Treatment of quiescent cultures of Balb/c 3T3 fibroblasts with PDGF produced 20-30-fold stimulation of IL-1 R mRNA with a concomitant increase in cell surface 125I-binding. IL-1 R mRNA accumulation occurred after an initial lag period and with a time course preceding the increase in 125I-IL-1 binding to cells. Induction of IL-1 R mRNA was blocked by inhibitors of protein synthesis, suggesting that a product of a gene expressed immediately after PDGF addition is required for IL-1 R gene expression. These latter data provide evidence for an ordered sequence of expression of PDGF-inducible "immediate early" gene(s) and IL-1 R gene. These results suggest that in connective tissues, PDGF may be an important determinant in initiating and maintaining cellular responses to IL-1. Such responses may have important consequences in the actions of IL-1 under normal and pathological conditions such as arthritis and atherosclerosis.

Crystallization of purified recombinant human interleukin-1 beta.

The gene for human interleukin-1 beta was cloned from SK-hep-1 hepatoma cellular RNA and expressed at high levels in Escherichia coli both as the naturally processed form (rIL-1 beta) and as a variant with an additional sequence of three amino acids on the N-terminus (rIL-1 beta +). Expressed protein was purified to homogeneity by a sequence of steps, which included low pH incubation, adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 fast-performance liquid chromatography (FPLC) column, and anion exchange chromatography on QAE Sepharose. The final step provided a biologically active protein that migrates on two-dimensional (2-D) gels as a single spot with a pI of 6.7 +/- 0.2 and a molecular mass of 17,500 daltons. Concentrated solutions of rIL-1 beta have produced crystals by ammonium sulfate precipitation. The crystals are tetragonal, show the symmetry of space group P4(1) or its enantiomer, have lattice constants of a = 58.46 (1) and c = 77.02 (3) A, and scatter to at least 2 A resolution. A structure determination based on these crystals is under way.

The effect of temperature-sensitive RNA mutants on the transcription products from cloned ribosomal protein genes of yeast.

The levels of four ribosomal protein (rp) mRNAs in different mutant strains were determined by hydridization of radiolabeled cloned genes to RNA fractionated on CH3HgOH gels and transferred to DBM paper. Two ribosomal protein genes (rp 51 and rp 52) controlled by the locus RNA2 have dramatically decreased mRNA levels after a shift-up to the nonpermissive temperature in a strain carrying the rna2 mutation (ts368). Two ribosomal protein genes not controlled by the RNA2 locus and several control nonribosomal protein genes are relatively unaffected by the temperature shift in this strain. Other genes in the vicinity of one of the rna2-sensitive ribosomal protein genes (th rp 51 gene) are insensitive to the rna2 gene product, suggesting that all ribosomal protein genes do not occur in clusters and that the RNA2 gene product does not affect a large region of chromatin. In ts368 at the nonpermissive temperature, the concentration of higher molecular weight transcripts complementary to the rp 51 and the rp 52 plasmids is increased. Analysis of the rp 51 plasmid transcripts reveals that the temperature-induced higher molecular weight transcripts differ from the mature rp 51 mRNA by the presence of an intron. This observation and the kinetics with which the concentration of the various rp 51 transcripts change after a temperature shift suggest that the effect of rna2 may be at the level of processing of rp mRNA.