High-level expression of a cloned HLA heavy chain gene introduced into mouse cells on a bovine papillomavirus vector.

A gene encoding the heavy chain of an HLA human histocompatibility antigen was isolated from a library of human DNA by recombination and selection in vivo. After insertion into a bovine papillomavirus (BPV) DNA expression vector, the gene was introduced into cultured mouse cells. Cells transformed with the HLA-BPV plasmids did not appear to contain extrachromosomal viral DNA, whereas BPV recombinants usually replicated as plasmids in transformed cell lines. Large amounts of HLA RNA were produced by the transformed cells, and the rate of synthesis of human heavy chain was several-fold higher than in the JY cell line, a well-characterized human lymphoblastoid cell line which expresses high levels of surface HLA antigen. Substantial amounts of human heavy chain accumulated in the transformed cells, and HLA antigen was present at the cell surface. These observations establish the feasibility of using BPV vectors to study the structure and function of HLA antigens and the expression of cloned HLA genes.

Ultrahigh resolution drug design I: details of interactions in human aldose reductase-inhibitor complex at 0.66 A.

The first subatomic resolution structure of a 36 kDa protein [aldose reductase (AR)] is presented. AR was cocrystallized at pH 5.0 with its cofactor NADP+ and inhibitor IDD 594, a therapeutic candidate for the treatment of diabetic complications. X-ray diffraction data were collected up to 0.62 A resolution and treated up to 0.66 A resolution. Anisotropic refinement followed by a blocked matrix inversion produced low standard deviations (<0.005 A). The model was very well ordered overall (CA atoms' mean B factor is 5.5 A2). The model and the electron-density maps revealed fine features, such as H-atoms, bond densities, and significant deviations from standard stereochemistry. Other features, such as networks of hydrogen bonds (H bonds), a large number of multiple conformations, and solvent structure were also better defined. Most of the atoms in the active site region were extremely well ordered (mean B approximately 3 A2), leading to the identification of the protonation states of the residues involved in catalysis. The electrostatic interactions of the inhibitor's charged carboxylate head with the catalytic residues and the charged coenzyme NADP+ explained the inhibitor's noncompetitive character. Furthermore, a short contact involving the IDD 594 bromine atom explained the selectivity profile of the inhibitor, important feature to avoid toxic effects. The presented structure and the details revealed are instrumental for better understanding of the inhibition mechanism of AR by IDD 594, and hence, for the rational drug design of future inhibitors. This work demonstrates the capabilities of subatomic resolution experiments and stimulates further developments of methods allowing the use of the full potential of these experiments.

Thyroid hormone and the d-type cyclins interact in regulating enterocyte gene transcription.

Thyroid hormone (T3) is an important regulator of gut mucosal development and differentiation, inducing intestinal alkaline phosphatase (IAP) and repressing lactase gene transcription. In contrast, cyclin D1 (CD1) appears to be a growth promoter in the gut, functioning to maintain the undifferentiated state. The present studies were designed to examine the effects of CD1 on T3 action within intestinal epithelia. Caco-2 cells were maintained in hypothyroid medium and transiently transfected with either rat lactase (3.0 kb) or human IAP (2.4 kb) luciferase (Luc) reporter plasmids. Cotransfections were carried out using two T3 receptor (TR) isoforms, TR"-1 and TR$-1, as well as plasmids expressing CD1, CD3, CA, or CB1. Cells were then treated +/- 10 nmol/L T3 for 24 hours and luciferase activity was determined. With T3 treatment, IAP-Luc activity was induced (TR"-1 = eightfold, TR$-1 = ninefold), but these effects were dramatically inhibited (> 50%) by CD1 and CD3. In contrast, CA and CB1 did not alter T3-mediated IAP gene activation. The ability of CD1 and CD3 to inhibit T3 action was also tested in the context of the lactase gene, which is negatively regulated by T3. As expected, lactase reporter gene activity was repressed by T3 treatment in the case of both receptor isoforms, TR"-1 = 30% and TR$-1 = 40%. In contrast to its effects on the IAP gene, CD1 did not inhibit T3-mediated changes in lactase reporter gene activity. The D-type cyclins (CD1 and CD3), but not CA or CB1, specifically inhibit T3-mediated activation of the IAP gene. In contrast, the D-type cyclins do not inhibit T3-mediated repression of the lactase gene. These studies have identified a novel molecular interaction that exists between the pathways of growth and differentiation within intestinal epithelia.

Multiple unilateral fibroadenomas.

Fibroadenomas account for the majority of breast biopsies performed today. The natural history of fibroadenomas varies. They are usually found as a solitary, 1-2 cm lesion that is described as being firm, rubbery, nontender, well-circumscribed, and found in women less than 30 years of age. We present an uncommon case of an adolescent female who had a cluster of fibroadenomas in one of her breasts, her treatment, and a review of the literature.

Characterization of the mouse insulin receptor gene promoter.

The 5' flanking region of the mouse insulin proreceptor gene was isolated, and the 5' boundary of the minimal promoter was mapped. Genomic clones encompassing greater than 30 kilobases of the gene contain the promoter and exons 1 and 2 interrupted by an approximately 20-kilobase intron at the codon for amino acid 7 of the alpha subunit. The nucleotide sequence of a 1.3-kilobase fragment containing 766 base pairs of the 5' flanking region and the entire first exon was determined. Two major transcription start sites were mapped by S1 nuclease analysis to sites located 469 and 424 nucleotides upstream from the initiation codon for translation. The 5' terminus of an insulin proreceptor cDNA, isolated from a mouse 3T3-L1 adipocyte cDNA library, corresponds to the 3'-most major start site of transcription. The 5' deletion mutants of the 5' flanking region of the proreceptor gene, linked upstream of the bacterial chloramphenicol acetyltransferase reporter gene, were transfected into 3T3-L1 preadipocytes and assayed for promoter activity. The 5' boundary of the minimal promoter, which directs unexpectedly high levels of reporter gene expression, maps to a region 22 base pairs upstream from the 3'-most major transcription start site.

Substrate phosphorylation catalyzed by the insulin receptor tyrosine kinase. Kinetic correlation to autophosphorylation of specific sites in the beta subunit.

The kinetics of insulin-stimulated autophosphorylation of specific tyrosines in the beta subunit of the mouse insulin receptor and activation of receptor kinase-catalyzed phosphorylation of a model substrate were compared. The deduced amino acid sequence of the mouse proreceptor was determined to locate tyrosine-containing tryptic peptides. Receptor was first incubated with unlabeled ATP to occupy nonrelevant autophosphorylation sites, after which [32P]autophosphorylation at relevant sites and attendant activation of substrate phosphorylation were initiated with [gamma-32P]ATP and insulin. Activation of substrate phosphorylation underwent an initial lag of 10-20 s during which there was substantial 32P-autophosphorylation of tryptic phosphopeptides p2 and p3, but not p1. Following the lag, incorporation of 32P into p1 and the activation of substrate phosphorylation increased abruptly and exhibited identical kinetics. The addition of substrate to the receptor prior to ATP inhibits insulin-stimulated autophosphorylation, and consequently substrate phosphorylation. Insulin-stimulated autophosphorylation of the receptor in the presence of substrate inhibited primarily the incorporation of 32P into p1 and drastically inhibited substrate phosphorylation. From Edman radiosequencing of 32P-labeled p1, p2, and p3 and the amino acid sequence of the mouse receptor, the location of each phosphopeptide within the beta subunit was determined. Further characterization of these phosphopeptides revealed that p1 and p2 represent the triply and doubly phosphorylated forms, respectively, of the region within the tyrosine kinase domain containing tyrosines 1148, 1152, and 1153. The doubly phosphorylated forms contain phosphotyrosines either at positions 1148 and 1152/1153 or positions 1152 and 1153. These results indicate that insulin stimulates sequential autophosphorylation of tyrosines 1148, 1152 and 1153, and that the transition from the doubly to the triply phosphorylated forms is primarily responsible for the activation of substrate phosphorylation.

GATA family transcription factors activate lactase gene promoter in intestinal Caco-2 cells.

The GATA family of transcription factors regulate tissue-specific patterns of gene expression during development. We have characterized the interaction between GATA proteins and the lactase gene promoter. Nuclear protein bound to the lactase gene GATA region cis element (-97 to -73) was analyzed by electrophoretic mobility shift assays (EMSA) and supershift assays with GATA antibodies. Lactase promoter activities were assayed in Caco-2 cells transfected with wild-type and mutated luciferase promoter-reporter constructs and GATA-4/5/6 expression constructs. EMSA with the GATA region probe yields a specific DNA-protein complex that requires the GATA factor binding site WGATAR. The complex is recognized by GATA-4- and GATA-6-specific antibodies. GATA-4/5/6 expression constructs are able to activate transcription driven by the wild-type promoter, but not by a promoter in which the GATA binding site is mutated, in Caco-2 and nonintestinal QT6 cells. GATA factor binding to the lactase cis element correlates with functional promoter activation. We conclude that each of the GATA family zinc finger proteins expressed in the intestine, GATA-4, -5, and -6, can interact with the lactase promoter GATA element and can function to activate the promoter in Caco-2 cells.

Evolving asymmetric hypertrophic pyloric stenosis associated with histologic evidence of eosinophilic gastroenteritis.

The most frequently occurring and important cause of gastric outlet obstruction in the neonate and young infant is infantile hypertrophic pyloric stenosis (IHPS). A reported association of IHPS and eosinophilic gastroenteritis [1] raises interesting questions about the possible etiologic relationship between the two entities. It is plausible that the observed sonographic pyloric muscular wall thickness in IHPS may in part be dependent on the degree and duration of an allergic gastroenteropathy. A recent report suggests that endoscopy may be a more reliable diagnostic method than sonography in the patient with evolving IHPS [2]. Our recent experience with a patient with evolving IHPS supports the findings described in these prior reports.

The homeodomain protein Cdx2 regulates lactase gene promoter activity during enterocyte differentiation.

BACKGROUND & AIMS: Lactase is the intestinal disaccharidase responsible for digestion of lactose, the predominant carbohydrate in milk. Transcription of the lactase gene is activated during enterocyte differentiation. We have characterized the interaction between the lactase promoter and Cdx2, a homeodomain protein involved in regulating intestinal development and differentiation. METHODS: Nuclear protein bound to the lactase gene cis element, CE-LPH1, was analyzed by electrophoretic mobility shift assays and supershifts with Cdx2 antibody. Lactase promoter activities were assayed in cells transfected with luciferase reporter constructs and a Cdx2 expression construct. RESULTS: Electrophoretic mobility shift assay with CE-LPH1 yields a specific DNA/protein complex that requires the caudal-related protein binding site, TTTAC. The complex is recognized by Cdx2 antibody and is more abundant in differentiated enterocytes. A Cdx2 expression construct is able to activate transcription driven by the wild-type, but not a mutated, promoter and results in increased endogenous lactase messenger RNA. CONCLUSIONS: The homeodomain protein Cdx2 interacts with the lactase promoter and is capable of activating transcription of the endogenous gene. In contrast to a previous report, Cdx2 interaction with the lactase promoter correlates with enterocyte differentiation. These conclusions are consistent with the role of Cdx2 in regulating intestinal cell differentiation.

Differentiation-induced gene expression in 3T3-L1 preadipocytes. Characterization of a differentially expressed gene encoding stearoyl-CoA desaturase.

Previous studies have shown that differentiation of 3T3-L1 preadipocytes leads to the activation of transcription of an unidentified gene which encodes a 4.9-kilobase (kb) mRNA. Several cDNAs that include the complete sequence of this mRNA were obtained and used to isolate and characterize the gene. Analysis of the nucleotide and amino acid sequences of both cDNA and genomic clones revealed that the gene encodes the mouse stearoyl-CoA desaturase (SCD), an enzyme known to be expressed upon differentiation of 3T3-L1 preadipocytes. The predicted amino acid sequence (355 residues) of the mouse 3T3-L1 adipocyte SCD exhibits 92% identity to that of the rat liver SCD. There is also a high degree of nucleotide sequence identity between the mouse and rat mRNAs in their unusually long approximately 3.5-kb 3'-untranslated regions. Mice fed a diet containing unsaturated triacylglycerides express SCD mRNA only in adipose tissue, whereas mice starved and refed a fat-free diet, express SCD mRNA in both liver and adipose tissue. The mouse gene for the desaturase spans approximately 15 kb and contains 6 exons and 5 introns with all intron-exon junctions conforming to the GT/AG splicing rule. As determined by S1 nuclease mapping and primer extension analysis, the transcriptional initiation site maps 152 nucleotides upstream from the initiation methionine codon. A canonical promoter "TATA" box is located 30 base pairs upstream of the Cap site. A typical "CCAAT" box sequence is not present in the adjacent 5'-flanking region; however, there is a GC-rich sequence (at nucleotide -215) similar to the binding site for the nuclear transcription factor Sp1. Upstream from the transcriptional initiation site are elements with homology (approximately 75%) to the putative fat-specific transcriptional element FSE2 and core consensus sequences for cAMP and glucocorticoid regulatory elements. A chimeric construct, containing 363 base pairs of 5'-flanking sequence and 30 nucleotides of 5'-untranslated sequence of the mouse SCD gene ligated to the bacterial chloramphenicol acetyltransferase gene, was transfected into 3T3-L1 cells. When cells were induced to differentiate into adipocytes, expression of the SCD chloramphenicol acetyltransferase gene increased approximately 63-fold, suggesting that the SCD promoter region contains elements that mediate the response to adipogenic agents which induce differentiation.

Expression of the differentiation-induced gene for fatty acid-binding protein is activated by glucocorticoid and cAMP.

We have isolated and characterized a fragment of the gene encoding adipose fatty acid-binding protein (gene 422) from a 3T3-L1 adipocyte genomic library. The 5'-flanking sequence of the 422 gene contains potential regulatory regions for adipose-specific expression. At position -120 there is a fat-specific element that occurs in several genes expressed as preadipocytes differentiate, and at position -393 there is a glucocorticoid regulatory element core sequence. Chimeric constructs were prepared by ligating 858 base pairs or 248 base pairs of 5'-flanking sequence and 22 nucleotides of 5'-untranslated sequence of the 422 gene to the bacterial gene encoding chloramphenicol acetyltransferase (CAT); these constructs (delta 858.CAT and delta 248.CAT) were transfected into 3T3-L1 preadipocytes. When differentiation was initiated by the adipogenic agents methylisobutylxanthine (a cAMP phosphodiesterase inhibitor), dexamethasone, and insulin, expression of both constructs increased, reaching maximal levels within 24 hr. Both constructs were maximally induced 48 hr before appreciable accumulation of the endogenous 422 mRNA. Expression of delta 858.CAT, but not of delta 248.CAT, was induced by dexamethasone, which correlates with deletion of the potential glucocorticoid regulatory element. Expression of both constructs was induced by 8-bromoadenosine 3',5'-cyclic monophosphate, thus implicating the first 248 base pairs of 5'-flanking sequence of the 422 gene in the response to cAMP. Indirect effects by the adipogenic factors on CAT protein or mRNA synthesis and turnover were ruled out, since replacing the 5'-flanking region of the 422 gene constructs with viral promoters abolished the effects of dexamethasone and 8-bromoadenosine 3',5'-cyclic monophosphate on CAT expression. We conclude that the first 858 base pairs of 5'-flanking sequence of the 422 gene contains elements that mediate activation by dexamethasone and cAMP.