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R Stein

Publications and source records attributed to R Stein.

At least 109 records · Page 6Linked to original sources
PubMed1997

P110delta, a novel phosphoinositide 3-kinase in leukocytes.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that have been implicated in signal transduction through tyrosine kinase- and heterotrimeric G-protein-linked receptors. We report herein the cloning and characterization of p110delta, a novel class I PI3K. Like p110alpha and p110beta, other class I PI3Ks, p110delta displays a broad phosphoinositide lipid substrate specificity and interacts with SH2/SH3 domain-containing p85 adaptor proteins and with GTP-bound Ras. In contrast to the widely distributed p110alpha and beta, p110delta is exclusively found in leukocytes. In these cells, p110alpha and delta both associate with the p85alpha and beta adaptor subunits and are similarly recruited to activated signaling complexes after treatment with the cytokines interleukin 3 and 4 and stem cell factor. Thus, these class I PI3Ks appear not to be distinguishable at the level of p85 adaptor selection or recruitment to activated receptor complexes. However, distinct biochemical and structural features of p110delta suggest divergent functional/regulatory capacities for this PI3K. Unlike p110alpha, p110delta does not phosphorylate p85 but instead harbors an intrinsic autophosphorylation capacity. In addition, the p110delta catalytic domain contains unique potential protein-protein interaction modules such as a Pro-rich region and a basic-region leucine-zipper (bZIP)-like domain. Possible selective functions of p110delta in white blood cells are discussed.

Amino Acid Sequence↗
PubMed1997

Characterization of detergent-insoluble complexes containing the cellular prion protein and its scrapie isoform.

Cells infected with prions contain both prion protein isoforms cellular prion protein (PrPC) and scrapie prion protein (PrPSc). PrPSc is formed posttranslationally through the pathological refolding of PrPC. In scrapie-infected ScN2a cells, the metabolism of both PrP isoforms involves cholesterol-dependent pathways. We show here that both PrPC and PrPSc are attached to Triton X-100-insoluble, low-density complexes or "rafts." These complexes are sensitive to saponin and thus probably contain cholesterol. This finding suggests that the transformation PrPC --> PrPSc occurs within rafts. It also reveals the existence of rafts in late compartments of the endocytic pathway, where most PrPSc resides. When Triton X-100 lysates of cells were incubated at 37 degrees C prior to density analysis, PrPC was still found in buoyant complexes, although it now failed to sediment at high speed. This property was shared by another glycophosphatidyl inositol protein, Thy-1, and also by the raft resident GM1. In one ScN2a clone and in the brain of a Syrian hamster with scrapie, Triton X-100 extraction at 37 degrees C permitted resolution of PrPC and PrPSc into two distinct peaks of different densities. This suggests that there are two populations of PrP-containing rafts and may permit isolation of PrPC-specific rafts from those containing PrPSc. Our findings reinforce the contention that rafts are involved in various aspects of PrP metabolism and in the "life cycle" of prions.

Animals↗
PubMed1997

Modulation of control mechanisms of dopamine-induced apoptosis--a future approach to the treatment of Parkinson's disease?

The cause for the progressive and selective degeneration of the dopaminergic (DA) nigrostriatal neurons in Parkinson's disease (PD) is still unknown. We suggest a novel approach, that links this neuronal degenerative process to inappropriate triggering of apoptosis, an active, controlled program of cellular self destruction, by excess oxidative stress mediated by DA metabolism. In support of this concept, we found that DA, the endogenous neurotransmitter, is capable of initiating apoptosis in cultured, postmitotic chick sympathetic neurons, an observation further extended to other cellular systems (PC-12 cells, cerebellar granular cells, thymocytes, splenocytes). In comparing the relative apoptosis-triggering potency of other mononamine neurotransmitters, DA was found to be the most active, whereas norepinephrine and serotonin had a moderate and a mild effects, respectively. This grading can be correlated with the relative involvement of the relevant neuronal systems (i.e., substantia nigra, locus ceruleus and raphe nuclei) in PD. We therefore hypothesize that neuronal degeneration in PD may be caused, at least in part, by a failure, either inherited or acquired, in cellular control systems of apoptosis, that may normally restrain the lethal potential of these endogenous neuro-transmitters and their potentially-toxic oxidation products. We therefore point at apoptosis-control systems as a critical scene of events, where the fate of nigrostriatal neurons is ultimately determined, and whose modulation may yield attenuation of the neuronal degenerative process. In support of this concept, we found that vector-driven stable expression of the proto-oncogene bcl-2, an inhibitor of apoptosis, can exert powerful cellular protection against DA toxicity in rat pheochromocytoma PC-12 cells. Furthermore, cell extracts from bcl-2-expressing cells were found to markedly inhibit in vitro oxidation of DA and production of DA-melanin. We also found that expression of bcl-2 can inhibit the decrease in intracellular reduced thiol (-SH) groups which we observed following exposure to DA. Research of the bcl-2 system and associated control mechanisms of apoptosis, possibly acting in association with intra-cellular anti-oxidant pathways, may therefore lead to novel therapeutic approaches for neuroprotection in PD.

Animals↗
PubMed1997

Advantage of residualizing radiolabels for an internalizing antibody against the B-cell lymphoma antigen, CD22.

LL2 is an anti-CD22 pan-B-cell monoclonal antibody which, when radiolabeled, has a high sensitivity for detecting B-cell, non-Hodgkin's lymphoma (NHL), as well as an antitumor efficacy in therapeutic applications. The aim of this study was to determine whether intracellularly retained radiolabels have an advantage in the diagnosis and therapy of lymphoma with LL2. In vitro studies showed that iodinated LL2 is intracellularly catabolized, with a rapid release of the radioiodine from the cell. In contrast, residualizing radiolabels, such as radioactive metals, are retained intracellularly for substantially longer. In vivo studies were performed using LL2-labeled with radioiodine by a non-residualizing (chloramine-T) or a residualizing method (dilactitol-tyramine, DLT), or with a radioactive metal (111In). The biodistribution of a mixture of 125I (non-residualizing chloramine-T compared to residualizing DLT), 111In-labeled LL2 murine IgG2a or its fragments [F(ab')2, Fab'], as well as its humanized, CDR-grafted form, was studied in nude mice bearing the RL human B-cell NHL cell line. Radiation doses were calculated from the biodistribution data according to the Medical International Radiation Dose scheme to assess the potential advantage for therapeutic applications. At all assay times, tumor uptake was higher with the residualizing labels (i.e., 111In and DLT-125I) than with the non-residualizing iodine label. For example, tumor/blood ratios of 111In-labeled IgG were 3.2-, 3.5- and 2.8-fold higher than for non-residualizing iodinated IgG on days 3, 7 and 14, respectively. Similar results were obtained for DLT-labeled IgG and fragments with residualized radiolabels. Tumor/organ ratios also were higher with residualizing labels. No significant differences in tumor, blood and organ uptake were observed between murine and humanized LL2. The conventionally iodinated anti-CD20 antibody, 1F5, had tumor uptake values comparable to those of iodinated LL2, the uptake of both antibodies being strongly dependent on tumor size. These data suggest that, with internalizing antibodies such as LL2, labeling with intracellularly retained isotopes has an advantage over released ones, which justifies further clinical trials with residualizing 111In-labeled LL2 for diagnosis, and residualizing 131I and 90Y labels for therapy.

Animals↗
PubMed1997

The use of Epstein-Barr virus-based shuttle vectors in rat PC12 cells.

1. The rat pheochromocytoma PC12 cell line has been a commonly used model for studies of neuronal development, function, and death. Thus the ability to transfect PC12 cells in an efficient manner and to manipulate their gene expression would enhance the usefulness of these cells. 2. We demonstrate that EBV-based vectors provide a useful expression system for gene manipulation in rat PC12 cells. 3. The EBV-based vectors replicate episomally in PC12 cells for at least 2 months, as evidence by their recovery from the transfected cells and by the digestion of the episomal plasmid with the isoschizomer MboI and DpnI restriction enzymes. 3. PC12 cells are efficiently transfected by EBV-based vectors both transiently and stably. 4. Transfection of PC12 cells with an EBV-based vector containing tau cDNA in the antisense orientation resulted in a decrease in the level of tau protein in the transfected cells. 5. The results demonstrate that EBV-based vectors can be a useful expression system for gene manipulation in PC12 cells.

Animals↗
PubMed1997

Dopamine-induced apoptosis is inhibited in PC12 cells expressing Bcl-2.

1. Degeneration of nigrostriatal dopaminergic neurons is the major pathogenic substrate of Parkinson's disease (PD). It is assumed that the lethal trigger is the accumulation of oxidative reactive species generated during metabolism of the natural neurotransmitter dopamine. 2. We have recently shown that dopamine is capable of inducing programmed cell death (PCD) or apoptosis in cultured postmitotic chick sympathetic neurons and rat PC12 pheochromocytoma cells. 3. The bcl-2 gene encodes a protein which blocks physiological PCD in many mammalian cells. In an attempt to elucidate further the mechanism of dopamine toxicity, we examined the potential protective effect of bcl-2 in PC12 cells which were transfected with the protooncogene. 4. In our experiments, Bcl-2 producing cells showed a marked resistance to dopamine toxicity. The percentage of nuclear condensation and DNA fragmentation visualized by the end-labeling method following dopamine treatment was significantly lower in bcl-2 expressing cells. Bcl-2 did not protect PC12 cells against toxicity induced by exposure to dopamine-melanin. Extracts of PC12 cells containing Bcl-2 inhibited dopamine autooxidation and formation of dopamine-melanin. Furthermore, the presence of Bcl-2 protected cells from thiol imbalance and prevented thiol loss following exposure to dopamine. 5. The protective effects of Bcl-2 against dopamine toxicity may be explained, in part, by its action as an antioxidant and by its interference in the production of toxic agents. The possible protection by Bcl-2 against neuronal degeneration caused by dopamine may play a role in the pathogenesis of PD and may provide a new direction for the development of neuroprotective therapies.

Animals↗
PubMed1997

The proto-oncogene Bcl-2 inhibits cellular toxicity of dopamine: possible implications for Parkinson's disease.

It is currently believed that excessive oxidant stress induced by metabolism of dopamine (DA), plays a major role in the pathogenesis of the selective nigrostriatal neuronal loss in Parkinson's disease. We recently showed that the neurotransmitter DA, in physiological concentrations, is capable of initiating apoptosis in cultured, post-mitotic sympathetic neurons. Bcl-2 is a proto-oncogene that blocks apoptosis. We now report that Bcl-2 is a powerful inhibitor of DA toxicity in PC-12 pheochromocytoma cells. We induced stable expression of Bcl-2 in PC-12 cells by transfection with recombinant pCMV5 expression vector, containing mouse bcl-2 (coding-sequence) cDNA. Cells expressing Bcl-2 manifested marked resistance to otherwise lethal (300 uM) in vitroconcentrations of DA. This protective effect was reflected in the trypan-blue test of cell survival, 3 H-thymidine incorporation and inhibition of the characteristic apoptotic morphologic alterations in scanning electron microscopic studies. Bcl-2 and associated control systems of apoptosis may have an important physiological role in restraining the apop-tosis-triggering potential of DA in nigrostriatal neurons. This novel field of research may yield insights into the pathogenesis of Parkinson's disease and lead to development of novel therapeutic approaches.

Journal Article↗
PubMed1997

Skeletal muscle fibre type transformation following spinal cord injury.

Following spinal cord injury (SCI), upper motor neuron paralysed muscles lose the normal type I (slow) and II (fast) fibre mosaic pattern and become predominantly composed of type II (fast glycolytic) fibres). The majority of the research demonstrating this fibre type shift was based on pH sensitive myofibrillar ATPase staining techniques on muscle from longstanding paraplegics and quadriplegics. The purpose of this study was to describe muscle fibre type changes over a wide time spectrum post SCI using immunofluorescent techniques which may be more sensitive to change. A total of 19 vastus lateralis muscle biopsy specimens were obtained from 12 SCI subjects representing time points of 0.5-219 months post SCI. Fast and slow myosin heavy chain isoform distribution was determined on single muscle fibres for each of the biopsy specimens. Early post SCI (< 1 month) myosin heavy chain (MCH) isoform composition remained relatively stable. A transitional period was seen between 1 and 20 months post SCI wherein there was a progressive drop in the proportion of slow MHC isoform fibres and a rise in the proportion that co-expressed both the fast and slow MHC isoform. By approximately 70 months post SCI a new steady state had been reached characterized by almost exclusively fast MHC isoform expression. This research has demonstrated that post SCI muscle type II transformation occurs in stages and commences earlier than previously appreciated. Interventions aimed at preventing or minimizing the transformation would need to be instituted within weeks post SCI.

Adenosine Triphosphatases↗
PubMed1997

Erythroid response to treatment with G-CSF plus erythropoietin for the anaemia of patients with myelodysplastic syndromes: proposal for a predictive model.

Previous studies have shown that approximately 40% of patients with myelodysplastic syndrome (MDS) and anaemia respond to treatment with human recombinant granulocyte-CSF (G-CSF) plus erythropoietin (epo). The present study was designed to investigate pre-treatment variables for their ability to predict erythroid responses to this treatment. 98 patients with MDS (30 RA, 31 RARS, 32 RAEB, five RAEB-t) were treated with a combination of G-CSF (0.3-3.0 microg/kg/d, s.c.) and epo (60-300 U/kg/d, s.c.) for at least 10 weeks. Minimum criteria for erythroid response was a 100% reduction of red blood cell (RBC) transfusion need or an increase in haemoglobin level of > or = 1.5 g/dl. 35 patients (36%) showed responses to treatment. Medium duration of response was 11-24 months. In multivariate analysis, serum erythropoietin levels and initial RBC-transfusion need retained high statistical significance (P < 0.01). Using pre-treatment serum epo levels as a ternary variable (< 100, 100-500 or > 500 U/l) and RBC transfusion need as a binary variable (< 2 or > or = 2 units per month), the analysis provided a predictive score for erythroid response. This score divided patients into three groups: one group with a high probability of erythroid responses (74%), one intermediate group (23%) and one group with poor responses to treatment (7%). This predictive scoring system could be used in decisions regarding use of these cytokines for treating the anaemia of MDS, both for defining patients who should not be given the treatment and for selecting patients for inclusion in prospective trials.

Aged↗
PubMed1997

Urinary diversion in children and young adults using the Mainz Pouch I technique.

OBJECTIVES: To determine the late complications and consequences for renal function, vitamin and acid-base metabolism after application of the Mainz Pouch I (MZP-I) technique in children and young adults. PATIENTS AND METHODS: To November 1994, the MZP-I procedure was carried out in 463 patients at our institution, 91 of whom were children and adolescents (< or = 20 years old) using bladder augmentation in 21 and a continent cutaneous stoma in 70. A minimum follow-up of 1 year was possible in 87 patients or 163 renal units (RUs) with a mean of 5.5 years (range 1-10.5). RESULTS: At the last examination, 23 of 55 (42%) preoperatively dilated RUs had improved. 131 of the 163 RUs (80%) were stable and nine RUs (5.5%) showed a slight clinical asymptomatic increase in the upper tract dilatation. Through an extraperitoneal flank incision, 11% of the RUs which developed stenosis at the ureterocolic anastomosis were successfully reimplanted (16% in patients with neurogenic disorders, 17% with pre-operative irradiation and 5% in the remaining patients). Two of 32 patients with an intussuscepted and invaginated ileal nipple required re-operation due to incontinence, but none of the patients with an appendiceal stoma were incontinent. Open revision of a stomal stenosis was performed in three and endoscopic treatment in nine patients. In 54 patients, the levels of vitamins A, B1, B2, B6, E, folic and bile acid were within normal ranges. There was no significant decrease in vitamin B12 levels after operation. In none of the patients with normal pre-operative creatinine values had the levels increased and none developed severe acidosis or bowel neoplasm. CONCLUSION: The MZP-I is recommended as a technique for bladder augmentation or continent urinary diversion in children and young adults, with an acceptable complication rate which offers long-term protection of the upper urinary tract.

Acid-Base Equilibrium↗
PubMed1997

Hepatocyte nuclear factor 3beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene.

The mammalian homeobox gene pdx-1 is expressed in pluripotent precursor cells in the dorsal and ventral pancreatic bud and duodenal endoderm, which will produce the pancreas and the rostral duodenum. In the adult, pdr-1 is expressed principally within insulin-secreting pancreatic islet beta cells and cells of the duodenal epithelium. Our objective in this study was to localize sequences within the mouse pdx-1 gene mediating selective expression within the islet. Studies of transgenic mice in which a genomic fragment of the mouse pdx-1 gene from kb -4.5 to +8.2 was used to drive a beta-galactosidase reporter showed that the control sequences sufficient for appropriate developmental and adult specific expression were contained within this region. Three nuclease-hypersensitive sites, located between bp -2560 and -1880 (site 1), bp -1330 and -800 (site 2), and bp -260 and +180 (site 3), were identified within the 5'-flanking region of the endogenous pdx-1 gene. Pancreatic beta-cell-specific expression was shown to be controlled by sequences within site 1 from an analysis of the expression pattern of various pdr-1-herpes simplex virus thymidine kinase promoter expression constructs in transfected beta-cell and non-beta-cell lines. Furthermore, we also established that this region was important in vivo by demonstrating that expression from a site 1-driven beta-galactosidase reporter construct was directed to islet beta-cells in transgenic mice. The activity of the site 1-driven constructs was reduced substantially in beta-cell lines by mutating a hepatocyte nuclear factor 3 (HNF3)-like site located between nucleotides -2007 and -1996. Gel shift analysis indicated that HNF3beta present in islet beta cells binds to this element. Immunohistochemical studies revealed that HNF3beta was present within the nuclei of almost all islet beta cells and subsets of pancreatic acinar cells. Together, these results suggest that HNF3beta, a key regulator of endodermal cell lineage development, plays an essential role in the cell-type-specific transcription of the pdx-1 gene in the pancreas.

Animals↗
PubMed1997

Functional characterization of the transactivation properties of the PDX-1 homeodomain protein.

Pancreas formation is prevented in mice carrying a null mutation in the PDX-1 homeoprotein, demonstrating a key role for this factor in development. PDX-1 can also bind to and activate transcription from cis-acting regulatory sequences in the insulin and somatostatin genes, which are expressed in pancreatic islet beta and delta cells, respectively. In this study, we compared the functional properties of PDX-1 with those of the closely related Xenopus homeoprotein XIHbox8. Analysis of chimeras between PDX-1, XIHbox8, and the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that their transactivation domain was contained within the N-terminal region (amino acids 1 to 79). Detailed mutagenesis of this region indicated that transactivation is mediated by three highly conserved sequences, spanning amino acids 13 to 22 (subdomain A), 32 to 38 (subdomain B), and 60 to 73 (subdomain C). These sequences were also required by PDX-1 to synergistically activate insulin enhancer-mediated transcription with another key insulin gene activator, the E2A-encoded basic helix-loop-helix E2-5 and E47 proteins. These results indicated that N-terminal sequences conserved between the mammalian PDX-1 and Xenopus XIHbox8 proteins are important in transcriptional activation. Stable expression of the PDX-1 deltaABC mutant in the insulin- and PDX-1-expressing betaTC3 cell line resulted in a threefold reduction in the rate of endogenous insulin gene transcription. Strikingly, the level of the endogenous PDX-1 protein was reduced to very low levels in these cells. These results suggest that PDX-1 is not absolutely essential for insulin gene expression in betaTC3 cells. We discuss the possible significance of these findings for insulin gene transcription in islet beta cells.

Amino Acid Sequence↗
PubMed1997

Changes in tumor vascular permeability in response to experimental radioimmunotherapy: a comparative study of 11 xenografts.

Single and fractionated doses of radioimmunotherapy (RAIT) and standard chemotherapy (0.6 mg 5-FU/day and 0.36 leucovorin/day on days 1-5) result in decreases in vascular permeability (VP) in the GW-39 human colonic xenograft. The effect of a single dose of RAIT (MN-14 anticarcinoembryonic antigen, Mu-9 anticolon-specific antigen, PAM-4 anti-MUC-1, RS-7 and RS-11 antiepithelial glycoprotein labeled with 131I) has also been evaluated in 10 other tumors. Fourteen days after a fixed 1,500-cGy dose of RAIT, 3 colonic tumors (LS174T, HT-29 and MOSER) all exhibited decreases in VP (58, 75 and 70%, respectively). Two colonic (LoVo and GS-7) and 1 breast tumor (MDA-468) did not exhibit any change in VP, and 1 lung (CALU-3), 1 cervical (ME-180), 1 pancreatic (CaPan-1) and 1 breast cancer line (ZR-75) exhibited increases in tumor VP (214, 289, 170 and 139%, respectively). The differences in VP response to RAIT do not appear to be related to the type of tumor, the size of tumor or the antigen being targeted by RAIT. The differences in tumor VP response to RAIT are discussed in terms of the ability to achieve significant tumor accretion of a second dose of radioantibody on a multiple-dosing regimen. We have begun to investigate the mechanism(s) which regulate the varying responses of tumor VP to RAIT by assessing the role that nitric oxide plays. Administration of arginine, a substrate for nitric oxide synthase, results in increases in both baseline and RAIT-modified VP in GW-39 and ME-180 tumors.

Animals↗
PubMed1997

Treatment of patients with bladder exstrophy or incontinent epispadias. A long-term follow-up.

OBJECTIVE: To determine the late outcome concerning urinary continence, late complications, sexuality, and fertility in patients with the exstrophy-epispadias complex. METHODS: Until July 1994, 115 patients underwent surgical treatment at our institution. The mean follow-up period in 102 patients is 16.7 years. Urinary diversion was performed in 88 patients, a modified Young-Dees procedure in 8, a sling plasty in 3, and genital reconstruction alone in 3 patients. RESULTS: The present continence rates are 96% for rectal reservoirs, 97% for Mainz pouch 1, and 67% for the modified Young-Dees procedure. The upper tracts have remained stable, and no bowel neoplasms have developed. 16 of 17 women > or = 18 years of age are satisfied with the cosmetic result after genital reconstruction. All adults engage in sexual intercourse; 5 women have delivered 7 children by cesarean section. 30 of 32 male adults are satisfied with the cosmetic result of the reconstructed external genitalia. Penile deviation was present in 11, distressing in 2 patients. After genital reconstruction 9 developed epididymitis, necessitating 2 orchiectomies and 3 vasectomies. No patient with reconstruction of the external genitalia can ejaculate normally or has fathered children, whereas the ejaculation was normal in 3 who did not undergo genital reconstruction. Furthermore, 2 of the 5 have fathered 4 children. CONCLUSIONS: Rectal reservoirs represent our urinary diversion of choice. After failed reconstruction/insufficient anal sphincter, a Mainz pouch I is constructed. The cosmetic results achieved by genital reconstruction are satisfactory. In women, antefixation of the uterus should be performed before or together with an introitus plasty to prevent uterine prolapse. In men, however, surgery is performed at the expense of fertility.

Adult↗
PubMed1997

Differentiation of new insulin-producing cells is induced by injury in adult pancreatic islets.

The ability of the adult pancreas to generate new insulin (beta) cells has been controversial because of difficulties in unequivocally identifying the precursor population. We recently determined that beta cells were generated during development from precursors that expressed the homeodomain-containing transcription factor pancreas duodenum homeobox gene-1 (PDX-1). To investigate whether PDX-1+ stem cells are present in adult pancreas, we examined two animal models of diabetes. One model was produced by injecting adult mice with streptozotocin (SZ), a toxin that produces hyperglycemia due to rapid and massive beta cell death. After SZ-mediated elimination of existing IN+/PDX-1+ cells, a population of somatostatin (SOM)+/PDX-1+ cells, a cell type thought to represent an embryonic islet precursor cell, appeared in islets. The appearance of SOM+/PDX-1+ cells was followed in time by the differentiation to SOM+/IN+/PDX-1+ cells. SOM+/PDX-1+ cells also appeared in islets of nonobese diabetic mice, a strain of mice in which beta cell destruction is immune-mediated. Our findings establish the existence of PDX-1+ beta cell precursors in the adult pancreas and indicate that their differentiation is induced by islet injury.

Animals↗
PubMed1997

Analysis of the role of E2A-encoded proteins in insulin gene transcription.

Pancreatic beta-cell type-specific transcription of the insulin gene is mediated, in part, by factors in the basic helix-loop-helix (bHLH) family that act on a site within the insulin enhancer, termed the E1-box. Expression from this element is regulated by a heteromeric protein complex containing ubiquitous (i.e. the E2A- and HEB-encoded proteins) and islet-enriched members of the bHLH family. Recent studies indicate that the E2A- and HEB-encoded proteins contain a transactivation domain, termed AD2, that functions more efficiently in transfected beta-cell lines. In the present report, we extend this observation by demonstrating that expression of full-length E2A proteins (E47, E12, and E2/5) activates insulin E element-directed transcription in a beta-cell line-selective manner. Stimulation required functional interactions with other key insulin gene transcription factors, including its islet bHLH partner as well as those that act on the RIPE3b1 and RIPE3a2 elements of the insulin gene enhancer. The conserved AD2 domain in the E2A proteins was essential in this process. The effect of the E2A- and HEB-encoded proteins on insulin gene expression was also analyzed in mice lacking a functional E2A or HEB gene. There was no apparent difference in insulin production between wild type, heterozygote, and homozygous mutant E2A or HEB mice. These results suggest that neither the E2A- or HEB-encoded proteins are essential for insulin transcription and that one factor can substitute for the other to impart normal insulin E1 activator function in mutant animals.

Animals↗