Biomedical subjects
K Docherty
Publications and source records attributed to K Docherty.
Insulin-releasing pituitary cells as a model for somatic cell gene therapy in diabetes mellitus.
Insulin delivery by somatic cell gene therapy was evaluated using murine pituitary AtT20MtIns-1.4 cells. These cells have been stably transfected to release human insulin by the introduction of a recombinant plasmid bearing a human preproinsulin cDNA under the control of a zinc-sensitive metallothionein promoter. 6 x 10(7) AtT20MtIns-1.4 cells were implanted subcutaneously into streptozotocin-diabetic mice immunosuppressed with cyclosporin A. Release of human insulin was assessed using a specific plasma human C-peptide assay. On days 1 and 2 after implantation human C-peptide concentrations were about 0.02 pmol/ml. Consumption of zinc sulphate solution (500 mg/l) as drinking fluid for days 3-5 increased plasma human C-peptide concentrations to 0.11 +/- 0.01 pmol/ml (mean +/- S.E.M.), n = 11, P < 0.01, and concentrations declined when zinc was discontinued. The extent of hyperglycaemia was slightly lower (P < 0.05) than in a group implanted with non-transfected AtT20 cells. The study was terminated after 9 days, and tumour-like aggregations of implanted cells were identified at autopsy. These comprised a large necrotic core with insulin-containing cells at the periphery. The study provides support for the view that somatic cell gene therapy offers a potential approach to insulin delivery in diabetes mellitus.
Negative regulation of transcription in eukaryotes.
Explore the source record for details and available documents.
The helix-loop-helix transcription factor USF (upstream stimulating factor) binds to a regulatory sequence of the human insulin gene enhancer.
Two important sequence elements, designated insulin enhancer binding site 1 (IEB1) or NIR and IEB2 or FAR, are involved in regulating expression of the rat insulin I gene. These elements bind a helix-loop-helix transcription factor, insulin enhancer factor 1 (IEF1). The IEB1 site is highly conserved among insulin genes but the IEB2 site is not conserved. To investigate the factors binding at the equivalent IEB1 and IEB2 sites in the human insulin gene enhancer, electrophoretic mobility shift assays were performed using a variety of cell extracts and probes specific for the homologous IEB1 and IEB2 sites. The results indicate that a factor with similar tissue distribution and binding characteristics to those of IEF1 binds to the IEB1 site in the human insulin gene, but that a separate factor, identified as the adenovirus major late transcription factor [MLTF, or upstream stimulating factor (USF)] binds to the IEB2 site.
Human insulin gene enhancer-binding proteins in pancreatic alpha and beta cell lines.
Electrophoretic mobility shift assays were performed using oligonucleotides corresponding to known protein binding sites within the human insulin gene enhancer and nuclear extracts from mouse pancreatic alpha and beta cell lines. The results demonstrate that a previously described factor, IUF-1, binds to three sites at -82 (the CT1 box), -215 (the CT2 box), and -319 (the CT3 box) in the human insulin gene enhancer. IUF-1 was present only in beta but not in alpha cells, while all other DNA-binding proteins were present in both cell lines. IUF-1 may therefore be an important determinant of insulin gene beta cell-specific expression.
Cell-specific gene expression in the islets of Langerhans: E boxes and TAAT boxes.
Explore the source record for details and available documents.
The human insulin gene-linked polymorphic region adopts a G-quartet structure in chromatin assembled in vitro.
The insulin gene-linked polymorphic region (ILPR), located 363 bp upstream of the human insulin gene, is composed of tandem repeats of the consensus sequence ACAGGGGT(G/C)(T/C)GGGG. It has previously been shown that an insulin gene fragment containing the ILPR adopts an altered DNA structure in vitro. Furthermore, oligonucleotides containing the consensus repeat sequence exhibit multiple quadriplex DNA structures. The present study was undertaken to determine whether such altered DNA structures existed within the ILPR when the insulin gene was assembled into chromatin in vitro. Chromatin assembly was achieved using histones and an extract from unfertilized eggs from Xenopus laevis. The presence of altered DNA conformations within the 5' region of the human insulin gene was investigated using the structural probe nuclease P1. Nuclease P1 recognized multiple distinct sites in the 5' flanking region of the human insulin gene in naked DNA. Most of these sites disappeared when the recombinant plasmid DNA was treated with histones and unfertilized egg extract. In the assembled DNA, the ILPR appeared as the major site of nuclease P1 hypersensitivity. Fine-mapping of the multiple reactive sites within the ILPR showed a pattern characteristic of G-quartet foldback structures similar to those that have been observed for telomeric DNA.
Insulin delivery by somatic cell gene therapy.
The feasibility of somatic cell gene therapy as a method of insulin delivery has been studied in mice. Murine pituitary AtT20 cells were transfected with a human preproinsulin DNA in a plasmid containing a metallothionein promoter and a gene conferring resistance to the antibiotic G418. The AtT20MtIns-1.4 clone of cells was selected because of its higher insulin-releasing activity compared with other clones. After culturing for 24 h in Dulbecco's medium containing 10 mM glucose, the AtT20MtIns-1.4 cells released human insulin at about 5 ng/10(6) cells per 24 h. Insulin release was not significantly altered by raised concentrations of glucose, potassium or calcium, but insulin release was increased by 20 mM arginine, 5 mM isomethylbutylxanthine and 90 microM zinc. AtT20MtIns-1.4 cells (2 x 10(6)) were implanted intraperitoneally into non-diabetic athymic nude (nu/nu) mice, and the mice were made diabetic by injection of streptozotocin after 7 days. Release of human insulin in vivo was assessed using a specific plasma human C-peptide assay. Human C-peptide concentrations were maintained at about 0.1 pmol/ml throughout the 29 days of the study. The development of streptozotocin-induced hyperglycaemia was delayed in recipients of the cells releasing human insulin, compared with a control group receiving an implant of non-transfected cells. At autopsy the implanted AtT20MtIns-1.4 cells in each recipient had formed a tumour-like aggregation, with an outer region of insulin-containing cells.(ABSTRACT TRUNCATED AT 250 WORDS)
How is the developmental timing and tissue-specificity of insulin gene expression controlled?
Explore the source record for details and available documents.
Sequence requirements for processing of proinsulin in transfected mouse pituitary AtT20 cells.
To investigate the sequence requirements for proteolytic processing of prohormones at pairs of basic amino acids, normal and mutant proinsulins were expressed in the mouse pituitary corticotrophic cell line AtT20. The extent of processing was determined by h.p.l.c. analysis of insulin-like immunoreactivity secreted into the media of transfected cells. In this model system, normal proinsulin was efficiently processed to insulin. The mutant des-38-62-proinsulin, in which all but six amino acids of the C-peptide were deleted, was also processed to insulin but less efficiently than the wild-type. The mutant Lys64-Arg65 to Thr64-Arg65 was partially processed to insulin, while the mutant Arg31-Arg32 to Arg31-Gly32 was not processed at either site. These results indicate: (i) that a six-amino-acid spacer between the two pairs of basic amino acids in proinsulin is sufficient to permit processing at both sites; (ii) that the endoproteinase responsible for cleavage at the Lys64-Arg65 site will also recognize Thr64-Arg65; (iii) that the endoproteinase responsible for cleavage at the Arg31-Arg32 site will not recognize Arg31-Gly32; and (iv) that the change Arg31-Arg32 to Arg31-Gly32 affects processing at the Lys64-Arg65 site.
A member of the eukaryotic subtilisin family (PC3) has the enzymic properties of the type 1 proinsulin-converting endopeptidase.
PC3, a mammalian homologue of the yeast subtilisin-like proteinase Kex2, was expressed in Xenopus oocytes and its activity was characterized. PC3 cleaved human proinsulin at one of the two dibasic sites (KTRR32 but not LQKR65). The specificity, inhibitor profile, pH optimum (5.5) and Ca(2+)-dependence (K0.5 = 2.5-3 mM) paralleled those of the insulin-granule type 1 endopeptidase activity, suggesting a role for PC3 in the conversion of prohormones.
A consensus repeat sequence from the human insulin gene linked polymorphic region adopts multiple quadriplex DNA structures in vitro.
A hypervariable region consisting of repeats of a 14 base pair (bp) consensus sequence ACAGGGGT(G/C)(T/C)GGGG is located 363 bp upstream of the human insulin gene. Different repeat numbers of this oligonucleotide give rise to a polymorphism, and so this region is commonly known as the insulin gene linked polymorphic region (ILPR). Here we present evidence, based on the mobility in non-denaturing polyacrylamide gels of two dissimilarly sized oligonucleotides containing the ILPR consensus sequence, that this sequence can adopt a number of quadriplex DNA structures in vitro.
Cyclic AMP produces desensitization of prostacyclin and adenosine A2 receptors in hybrid cell lines but does not affect Gs function.
Prostacyclin and adenosine A2 receptors stimulate adenylate cyclase activity in the related somatic hybrid cell lines NG108-15 and NCB20. The role of cAMP in the desensitization of these receptors has been examined. Pretreatment for 17 h with forskolin or 8-bromo-cAMP had the same effect in both cell lines. There was no change in the response to sodium fluoride or forskolin, suggesting that the function of Gs and adenylate cyclase were unaffected by increased levels of cAMP. Receptor responses were affected however; the maximum response to N-ethylcarboxamidoadenosine (an A2 receptor agonist) was reduced by 30-40%, there was a small but consistent shift to the right of the dose-response curve for iloprost (a stable analogue of prostacyclin) and [3H]iloprost binding studies revealed a loss of prostacyclin receptors. However, the loss of receptor responsiveness was much smaller than that which occurs following pretreatment with prostacyclin or adenosine A2 receptor agonists (Keen et al. (1989) Biochem. Pharmacol. 38, 3827-3833; Kelly et al. (1990) Br. J. Pharmacol. 99, 309-316) suggesting that cAMP may not play a major role in agonist mediated desensitization.
The human insulin gene linked polymorphic region exhibits an altered DNA structure.
Regulation of transcription of the human insulin gene appears to involve a series of DNA sequences in the 5' region. Hypersensitivity to DNA structural probes has previously been demonstrated in regulatory regions of cloned genomic DNA fragments, and been correlated with gene activity. To investigate the structure of the DNA in the human insulin gene, bromoacetaldehyde and S1 nuclease were reacted with a supercoiled plasmid containing a 5kb genomic insulin fragment. Both probes revealed the human insulin gene linked polymorphic region (ILPR), a region (-363) upstream of the transcriptional start site which contains multiple repeats of a 14-15mer oligonucleotide with the consensus sequence ACAGGGGT(G/C)(T/C)GGGG, as the major hypersensitive site. Fine mapping and electron microscopic analysis both show a very different behaviour of the two DNA strands in the region of the ILPR and suggest the G-rich strand may be adopting a highly structured conformation with the complementary strand remaining largely single stranded.
1992 R.D. Lawrence Lecture. The regulation of insulin gene expression.
Explore the source record for details and available documents.
RNA-protein interactions in the 5' untranslated region of preproinsulin mRNA.
A comparison between species of the 5' untranslated region of preproinsulin mRNA revealed conserved sequences associated with a potential stem-loop structure. The present study was undertaken to determine whether specific protein interactions exist with mRNA sequences involved in the formation or stabilization of this structure in the 5' untranslated region. 32P-Labelled RNA probes corresponding to sequences from this region were synthesized by an in-vitro transcription reaction and used in electrophoretic mobility shift and u.v.-crosslinking studies with cytoplasmic protein extracts from a number of cell lines. Specific protein-RNA interactions were mapped to a sequence located between nucleotides -21 and -50 upstream of the AUG start codon. A number of proteins of molecular mass 25 kDa, 40kDa, 46kDa, 58kDa, 69kDa, 97kDa, 110kDa and 160kDa were specifically crosslinked to this sequence. The observed specific protein-RNA interactions in the 5' untranslated region may affect the activity of preproinsulin mRNA.
Analysis of DNA structure in the human insulin gene-linked polymorphic region in vivo.
An altered DNA structure exists within the hypervariable region located 360 bp upstream of the human insulin gene. The aim of the present study was to determine whether this structure exists in the insulin gene in vivo, and whether its presence is related to the expression of the insulin gene. However, since there were no clonal human beta-cell lines available for such studies, the human insulin gene was transfected into a rat insulinoma-derived beta-cell line and several human insulin-expressing clones were selected. One such cell line was treated in vivo with the DNA structural probe bromoacetaldehyde and the chromosomal DNA was extracted. Following digestion with TaqI and subsequent digestion with S1-nuclease to cleave at the bromoacetaldehyde-reactive sites, the DNA was subjected to agarose gel electrophoresis, and insulin gene fragments were detected by Southern blot analysis. Bromoacetaldehyde generated subfragments of 2500, 1700 and 800 bp in the human insulin gene isolated from the rat beta-cell line, while the human insulin gene in the non-expressing HeLa cell line was unreactive to bromoacetaldehyde. These results suggest that an altered structure might exist in the insulin gene-linked polymorphic region of the human insulin gene in vivo, and that this structure may play a role in the expression of the insulin gene.
Site-directed mutagenesis and expression of PC2 in microinjected Xenopus oocytes.
The biosynthesis and post-translational maturation of PC2, a neuroendocrine-specific Kex2-like endoprotease, following expression in Xenopus oocytes is described. The initial translation product was a 75-kDa membrane-associated protein which was released from the oocytes as a glycosylated 71-kDa protein. During extended chase periods, the extracellular 71-kDa protein was converted to a mature 68-kDa product. A deletion mutant lacking a putative COOH-terminal amphipathic helix was still membrane-associated, suggesting that this domain was not essential for attachment of PC2 to membranes. Two putative proregion cleavage site mutants were also constructed. Conversion of the 75-kDa peptide to the 71-kDa peptide involved cleavage at the sequence Lys-Arg-Arg-Arg (amino acids 78-81), since mutation of this sequence to Lys-Val-Arg-Leu resulted in the secretion of the 75-kDa peptide. Extracellular conversion of the 71-kDa peptide to the 68-kDa peptide involved cleavage at the sequence Arg-Lys-Lys-Arg (amino acids 106-109), since deletion of this tetrabasic sequence resulted in secretion of the 71-kDa peptide without further conversion to the 68-kDa form. Finally, a mutation which changed a catalytically important Asp to Asn did not affect processing of proPC2. These results may be relevant to our understanding of mechanisms in the intracellular sorting and maturation of proPC2 in neuroendocrine cells.