Regulation of insulin gene transcription.

Recent studies of the insulin gene promoter and the transcription factors that regulate it have expanded our understanding of both how the production of insulin is restricted to the pancreatic beta -cell, and how that production is regulated by physiologic signals such as glucose. A picture is emerging in which an elaborate set of transcription factors binds to specific sequences along the promoter and recruits additional transcriptional co-activators to build a functional transcriptional activation complex that is unique to beta -cells. Surprisingly, however, genetic experiments in mice have demonstrated an unexpected degree of redundancy in the factors that control insulin gene expression, and have revealed the presence of a network of transcription factors that coordinate the expression of factors forming the insulin gene activation complex.

Intramolecular control of transcriptional activity by the NK2-specific domain in NK-2 homeodomain proteins.

The developmentally important homeodomain transcription factors of the NK-2 class contain a highly conserved region, the NK2-specific domain (NK2-SD). The function of this domain, however, remains unknown. The primary structure of the NK2-SD suggests that it might function as an accessory DNA-binding domain or as a protein-protein interaction interface.

Transcriptional and translational regulation of beta-cell differentiation factor Nkx6.1.

In the mature pancreas, the homeodomain transcription factor Nkx6.1 is uniquely restricted to beta-cells. Nkx6.

Expression of neurogenin3 reveals an islet cell precursor population in the pancreas.

Differentiation of early gut endoderm cells into the endocrine cells forming the pancreatic islets of Langerhans depends on a cascade of gene activation events controlled by transcription factors including the basic helix-loop-helix (bHLH) proteins. To delineate this cascade, we began by establishing the position of neurogenin3, a bHLH factor found in the pancreas during fetal development. We detect neurogenin3 immunoreactivity transiently in scattered ductal cells in the fetal mouse pancreas, peaking at embryonic day 15.

Beta-cell differentiation factor Nkx6.1 contains distinct DNA binding interference and transcriptional repression domains.

beta-Cell differentiation factor Nkx6.1 is a homeodomain protein expressed in developing and mature beta-cells in the pancreatic islets of Langerhans. To understand how it contributes to beta-cell development and function, we characterized its DNA binding and transactivation properties.

LMX1B transactivation and expression in nail-patella syndrome.

Lmx1b, a member of the LIM homeodomain protein family, is essential for the specification of dorsal limb fates at the zeugopodal and autopodal level in vertebrates. We and others have shown that a skeletal dysplasia, nail-patella syndrome (NPS), results from mutations in LMX1B. While it is a unique mesenchymal determinant of dorsal limb patterning during vertebrate development, the mechanism by which LMX1B mutations generate the NPS phenotype has not been addressed at a transcriptional level or correlated with its spatial pattern of gene expression.

The homeodomain of PDX-1 mediates multiple protein-protein interactions in the formation of a transcriptional activation complex on the insulin promoter.

Activation of insulin gene transcription specifically in the pancreatic beta cells depends on multiple nuclear proteins that interact with each other and with sequences on the insulin gene promoter to build a transcriptional activation complex. The homeodomain protein PDX-1 exemplifies such interactions by binding to the A3/4 region of the rat insulin I promoter and activating insulin gene transcription by cooperating with the basic-helix-loop-helix (bHLH) protein E47/Pan1, which binds to the adjacent E2 site. The present study provides evidence that the homeodomain of PDX-1 acts as a protein-protein interaction domain to recruit multiple proteins, including E47/Pan1, BETA2/NeuroD1, and high-mobility group protein I(Y), to an activation complex on the E2A3/4 minienhancer.

A pilot study of the management of uncomplicated candidemia with a standardized protocol of amphotericin B.

We evaluated an amphotericin treatment strategy on the basis of duration of candidemia and clinical findings. Patients without neutropenia who had uncomplicated candidemia received 200 mg of amphotericin B over 5-7 days if they had had 1 day of positive cultures (PC group). The clinical cure rate was 93% (95% confidence interval [CI], 77%-99%; n=29 episodes) in the SC group, with no relapses (median follow-up, 272 days).

Paired-homeodomain transcription factor PAX4 acts as a transcriptional repressor in early pancreatic development.

The paired-homeodomain transcription factor PAX4 is expressed in the developing pancreas and along with PAX6 is required for normal development of the endocrine cells. In the absence of PAX4, the numbers of insulin-producing beta cells and somatostatin-producing delta cells are drastically reduced, while the numbers of glucagon-producing alpha cells are increased. To gain insight into PAX4 function, we cloned a full-length Pax4 cDNA from a beta-cell cDNA library and identified a bipartite consensus DNA binding sequence consisting of a homeodomain binding site separated from a paired domain binding site by 15 nucleotides.

Biotin regulation of pancreatic glucokinase and insulin in primary cultured rat islets and in biotin-deficient rats.

Biotin has been reported to affect glucose homeostasis; however, its role on pancreatic islets of Langerhans has not been assessed. In this report, we demonstrate that physiologic concentrations of biotin stimulate glucokinase activity in rat islets in culture. Using the branched DNA (bDNA) assay, a sensitive signal amplification technique, we detected relative increases in glucokinase mRNA levels of 41.

Effect of retinoic acid on glucokinase activity and gene expression and on insulin secretion in primary cultures of pancreatic islets.

Retinoic acid has manifold effects on pancreatic beta-cells. Previously we reported that retinoic acid increases glucokinase activity and messenger RNA (mRNA) levels in the insulinoma cell line RIN-m5F; however, we could not rule out the possibility that the effect of retinoic acid on RIN-m5F glucokinase was inherent to the cell line or related to its differentiating capacity. In this report, we demonstrate that physiologic concentrations of retinoic acid stimulate glucokinase activity in both fetal islets and differentiated adult islets in culture.

[High-frequency jet ventilation with a proximal injector in a case of surgery of the tracheal carina].

A 44-year-old man underwent surgery requiring medial sternotomy of resection of a tracheal carcina tumor. High frequency jet ventilation was used during tumor resection and reconstruction of the carina in order to shorten the time of surgery and to provide a nearly immobile and unobstructed surgical field. The only noteworthy complication was a tendency to respiratory acidosis that resolved without sequelae in the operating room once the trachea was closed.

A novel glucose-responsive element in the human insulin gene functions uniquely in primary cultured islets.

Insulin gene transcription is limited to the beta cells within the mammalian pancreas and, like insulin secretion, is regulated by glucose. Our previous studies in primary cultured beta cells suggested the presence of a strong glucose-responsive enhancer element between base pairs -341 and -260 of the human insulin promoter, the same region in which a transcriptional repressor had been identified in beta-cell tumor lines. In an attempt to map these promoter activities and resolve these conflicting data, we designed minienhancer constructs spanning this region, and tested them in primary cultured and immortalized cells.

Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic beta cells.

The endocrine pancreas is organized into clusters of cells called islets of Langerhans comprising four well-defined cell types: alpha beta, delta and PP cells. While recent genetic studies indicate that islet development depends on the function of an integrated network of transcription factors, the specific roles of these factors in early cell-type specification and differentiation remain elusive. Nkx2.

Exchange of perfluorodecalin for gas or oil: a model for avoiding slippage.

Background: The introduction of liquid perfluorocarbons as an intraoperative tool has greatly facilitated retinal re-apposition in giant retinal tears (GRT) and relieving retinotomies (RR). Slippage of the retina can occur during the exchange of heavy liquids for oil or gas, especially if the fill of perfluorocarbons is subtotal.

Methods: We have used a model eye chamber to study the surface interactions of perfluorodecalin (PFD) with silicone oil and air to evaluate possible mechanisms of slippage.

Identification of a human LMX1 (LMX1.1)-related gene, LMX1.2: tissue-specific expression and linkage mapping on chromosome 9.

LMX1 is a LIM-homeodomain (LIM-HD)-containing protein expressed selectively in insulin-producing beta-cell lines, and it it has been shown to activate insulin gene transcription. The human LMX1 gene was mapped by fluorescence in situ hybridization to chromosome region 1q22-q23, yet Church et al. (1994, Nat.

The endocrine secretion of human insulin and growth hormone by exocrine glands of the gastrointestinal tract.

The exocrine pancreas, liver, and submandibular glands of the rat were used to express and secrete two exogenous, human protein hormones (growth hormone and insulin) into blood at physiological concentrations. Transfection, expression, and secretion were achieved by the in vivo retrograde injection of plasmid DNA into the secretory ducts of these glands. Pancreatic acinar cells secreted physiological concentrations of growth hormone into the circulation, and its secretion was enhanced by cholinergic stimulation.

Genetic analysis reveals that PAX6 is required for normal transcription of pancreatic hormone genes and islet development.

We present genetic and biochemical evidence that PAX6 is a key regulator of pancreatic islet hormone gene transcription and is required for normal islet development. In embryos homozygous for a mutant allele of the Pax6 gene, Small eye (Sey(Neu)), the numbers of all four types of endocrine cells in the pancreas are decreased significantly, and islet morphology is abnormal. In the remaining islet cells, hormone production, particularly glucagon production, is markedly reduced because of decreased gene transcription.

Transcriptional synergy between LIM-homeodomain proteins and basic helix-loop-helix proteins: the LIM2 domain determines specificity.

LIM-homeodomain proteins direct cellular differentiation by activating transcription of cell-type-specific genes, but this activation requires cooperation with other nuclear factors. The LIM-homeodomain protein Lmx1 cooperates with the basic helix-loop-helix (bHLH) protein E47/Pan-1 to activate the insulin promoter in transfected fibroblasts. In this study, we show that two proteins originally called Lmx1 are the closely related products of two distinct vertebrate genes, Lmx1.

The beta cell transcription factors and development of the pancreas.

The pancreatic beta cell is the major source of circulating insulin in adult mammals. In the multistep process of insulin synthesis it is initiation of transcription that restricts insulin synthesis to the beta cell since all subsequent steps can be performed by other cell types. Many of the transcription factors that bind to the insulin promoter and activate insulin gene transcription have been isolated.

Regulation of insulin preRNA splicing by glucose.

Glucose tightly regulates the synthesis and secretion of insulin by beta cells in the pancreatic islets of Langerhans. To investigate whether glucose regulates insulin synthesis at the level of insulin RNA splicing, we developed a method to detect and quantify a small amount of RNA by using the branched DNA (bDNA) signal-amplification technique. This assay is both sensitive and highly specific: mouse insulin II mRNA can be detected from a single beta cell (betaTC3 cells or mouse islets), whereas 1 million non-insulin-producing alpha cells (alphaTC1.

Isolation, characterization, and chromosomal mapping of the human Nkx6.1 gene (NKX6A), a new pancreatic islet homeobox gene.

Nkx6.1 (gene symbol NKX6A), a new member of the NK homeobox gene family, was recently identified in rodent pancreatic islet beta-cell lines. The pattern of expression suggested that this gene product might be important for control of islet development and/or regulation of insulin biosynthesis.

Function of the human insulin promoter in primary cultured islet cells.

Pancreatic islet beta cells regulate the rate of insulin gene transcription in response to a number of nutrients, the most potent of which is glucose. To test for its regulation by glucose, the promoter sequence was isolated from the human insulin gene. When linked to chloramphenicol acetyltransferase and transfected into primary islet cultures, the human insulin promoter is activated by glucose.