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PubMed1999

Upstream element of the sea urchin arylsulfatase gene serves as an insulator.

Insulator DNAs functionally isolate neighboring genes by blocking interactions between distal cis-regulatory elements and promoters. Here we report that a DNA fragment located in the upstream region of sea urchin, H. pulcherrimus, arylsulfatase (HpArs) gene blocks the interaction of the Ars enhancer when positioned between the enhancer and the target promoter, in an orientation dependent manner. The Ars insulator works only 3' to 5' direction and has no significant stimulatory or inhibitory effects on its own promoter. In transgenic Drosophila, the Ars insulator blocks the interaction between even-skipped stripe enhancer and its target promoter. The insulation mechanism operates also unidirectionally in Drosophila. We also show that the efficiency of transformation of HeLa cells is enhanced when the integrated gene is flanked by the Ars insulator, suggesting the sea urchin insulator overcomes the position-dependent transgene expression in mammalian cells. These results demonstrate that the mechanism of action of the insulator has been conserved throughout evolution.

Animals↗

PubMed1990

Early nicotinamide treatment in the NOD mouse: effects on diabetes and insulitis suppression and autoantibody levels.

Nicotinamide, a vitamin B group substance, has previously been shown to prevent diabetes and suppress insulitis in the NOD mouse. In order to further understand its mode of action, we have administered the vitamin orally to female NOD mice from weaning and have examined its effect cross-sectionally (days 40, 106 and 250) on the severity of insulitis, changes in T cell subpopulations, levels of islet cell antibodies (ICA) and insulin autoantibodies (IAA) and diabetes development. At day 40, the incidence and severity of insulitis were much lower in the nicotinamide group (n = 22) than in the control mice (n = 21; 23.8% versus 57.1%, 2.9 +/- 1.4% versus 9.6 +/- 2.6%, respectively). At day 106, the incidence of insulitis increased to 82% in the nicotinamide group (n = 11) and remained similar at day 250 (n = 14). At these two age groups all control mice developed insulitis. The insulitis score increased to about 20% at day 106 in the nicotinamide mice and remained the same at day 250. In the control animals, this score increased from 9.6 +/- 2.6% at day 40 to about 33% and 60% at days 106 and 250, respectively. Diabetes was not detected in the 14 animals maintained on nicotinamide while 4/14 control mice developed the disease with severe insulitis. Most of the immune cells infiltrating the islets were T cells, with higher numbers of L3T4 than Lyt2 cells. At days 40 and 106, the L3T4:Lyt2 cell ratios remained unchanged in both the nicotinamide and control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Changes in insulation of wetsuits during repetitive exposure to pressure.

This investigation was intended to a) establish a relationship between pressure and thickness (and hence insulation) of wetsuits during acute changes in pressure from 0.5 to 5 atmospheres absolute (ATA); b) determine the effect of repetitive compression-decompression (C-D) procedures on the insulation properties of wetsuits; and c) assess the possible recovery of insulation after cessation of repetitive C-D. Various wetsuits with different thicknesses and types of linings were selected. In all wetsuits, insulation and thickness were both reduced as similar, curvilinear functions of acutely increasing pressure in the range of 0.5-5.0 ATA. Effects of repetitive C-D (3 ATA) were studied in 5-mm-thick suit samples with various linings. The insulation of suits (0.100-0.120 degrees C.W-1.m-2) rapidly decreased after the first 5000 C-D. In suits without linings (skin suits), the insulation was reduced to 0.080 degrees C.W-1.m-2 at 5000 C-D and revealed no further reduction during the continuation of C-D cycles up to 30,000. Suits lined with single- or double-sided jersey showed a continuous decrease in the insulation and thickness as the number of cycles increased up to 12,000 and remained at a lowered value (0.060 degrees C.W-1.m-2) until the termination of C-D (30,000 times). A nearly complete recovery of thickness was seen 10 days after cessation of C-D treatment in the skin suit when C-D cycles were less than 3500; however, no recovery was observed in suits with linings (either single- or double-sided) in 25 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Atmospheric Pressure↗

PubMed1981

Toxicology of urea formaldehyde and polyurethane foam insulation.

Two types of foam insulation are in wide use. Urea formaldehyde foam is a relatively inexpensive, easily installed, and efficient insulation. Toxicity from this insulation is related to release of free formaldehyde into the home. Mild to incapacitating symptoms have been reported in occupants of urea formaldehyde-insulated homes. Airborne formaldehyde levels frequently have exceeded standards set for occupational exposure. The long-term consequences of such exposure are unknown. Because of publicity over the toxicity of urea formaldehyde foam, many physicians and patients have confused urea formaldehyde and polyurethane foam. Unlike urea formaldehyde, polyurethane foam is fully cured before construction. Toxicity occurs only during manufacture and curing. To date, there have been no reports to our knowledge of toxicity in occupants of polyurethane-insulated homes. However, toxicity caused by pyrolysis products may occur during combustion in homes insulated with either type of insulation. This report details 48 patients in whom complete medical data were obtained out of the first 100 patients contacting the Rocky Mountain Poison Center.

Construction Materials↗

PubMed1994

Effect of infection by lactic dehydrogenase virus on expression of intercellular adhesion molecule-1 on vascular endothelial cells of pancreatic islets in streptozotocin-induced insulitis of CD-1 mice.

Streptozotocin (SZ)-induced insulitis, which is an animal model for insulin-dependent diabetes mellitus, was suppressed by lactic dehydrogenase virus (LDV) infection. There were no differences in degenerative and necrotic changes of islet cells between SZ-treated mice and SZ-treated mice with LDV infection during the pre-insulitis phase. The degree of insulitis was more severe and the plasma glucose levels were higher in SZ-treated mice than in SZ-treated mice with LDV infection. Severe degenerative and necrotic changes of cells with mononuclear cell infiltration into the islets were seen in the SZ-treated mice. Infiltration of these cells into islets was less in SZ-treated mice with LDV infection. During the pre-insulitis phase, there was slight expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of vascular endothelial cells in and/or around islets in both groups. ICAM-1 expression on vascular endothelial cells increased in parallel to the degree of insulitis. The degree of this expression in SZ-treated mice was higher than in SZ-treated mice with LDV infection. These results suggest that expression of ICAM-1 on vascular endothelial cells in SZ-treated mice may be important for the development of insulitis. Also, decreased expression of ICAM-1 in the islets may be responsible for the inhibition of the development of insulitis seen in SZ-treated mice with LDV infection.

Animals↗

PubMed2006

Sea urchin arylsulfatase insulator exerts its anti-silencing effect without interacting with the nuclear matrix.

Chromatin insulators have been shown to stabilize transgene expression. Although insulators have been suggested to regulate the subcellular localization of chromosomes, it is still unclear whether this property is important for their anti-silencing activity. To investigate the underlying mechanisms governing the anti-silencing function of insulators, we studied the association of sea urchin arylsulfatase insulator (ArsI) with the nuclear matrix, which is a key component of the subnuclear localization of the genome. ArsI did not potentiate the nuclear matrix association with the transgene, even though it showed strong anti-silencing activity. This observation was in clear contrast to the results of the experiment using a human interferon-beta scaffold attachment region, in which the anti-silencing effect coincided with the enhanced matrix association. Chromatin immunoprecipitation analyses suggested that the absence of the matrix binding by ArsI was due to a lack of its binding to CCCTC-binding factor (CTCF), a protein known to be associated with matrix binding by chicken beta-globin insulator. Furthermore, ArsI maintained the nucleosome occupancy within the transgene at a constant level during long-term culture, although ArsI itself was not a nucleosome-excluding sequence. Taken together, these results suggest that this insulator exerts its anti-silencing activity by counteracting silencing-associated factors to maintain local chromatin environment, rather than by remodeling the subnuclear localization of the transgene locus.

Animals↗

PubMed2003

Insulation from viral transcriptional regulatory elements enables improvement to hepatoma-specific gene expression from adenovirus vectors.

We previously reported that the HS-4 insulator, derived from the chicken beta-globin locus, was able to shield a downstream inducible promoter from viral enhancers or silencers present in the genome of adenovirus vectors. In this study, we constructed two recombinant adenoviruses (Ad) that express an alkaline phosphatase (AP) reporter gene driven by an alpha-fetoprotein (AFP) enhancer/promoter with and without HS-4 insulator (Ad.HS4.AFP-AP and Ad.AFP-AP). The insulated vector, Ad.HS4.AFP-AP, conferred significantly higher AP expression than Ad.AFP-AP in all AFP-producing hepatocellular carcinoma cell lines (HepG2, Hep3B, and HuH7) examined. AP expression from Ad.HS4.AFP-AP was specific to hepatoma cells and barely detectable in AFP-negative tumor cell lines and normal human cells, including human hepatocytes. Intravenous infusion of viral vectors into mice with liver metastasis derived from Hep3B hepatoma cells resulted in AP expression exclusively localized to tumor cells. The number of tumor cells with detectable AP expression was significantly higher in mice infused with Ad.HS4.AFP-AP than in mice that received the non-insulated vector. This study demonstrates that the HS-4 insulator in the context of an Ad vector can increase the activity of the AFP promoter, while maintaining its tumor-specificity in vitro and in vivo. Considering that the anti-tumor activity of oncolytic vectors often depends on the level of pro-apoptotic or suicide gene expression, insulators might be a useful tool to improve the efficacy and specificity of these vectors.

Adenoviridae↗

PubMed2006

RNA interference machinery influences the nuclear organization of a chromatin insulator.

RNA interference (RNAi) is a conserved silencing mechanism that can act through alteration of chromatin structure. Chromatin insulators promote higher-order nuclear organization, thereby establishing DNA domains subject to distinct transcriptional controls. We present evidence for a functional relationship between RNAi and the gypsy insulator of D. melanogaster. Insulator activity is decreased when Argonaute genes required for RNAi are mutated, and insulator function is improved when the levels of the Rm62 helicase, involved in double-stranded RNA (dsRNA)-mediated silencing and heterochromatin formation, are reduced. Rm62 interacts physically with the DNA-binding insulator protein CP190 in an RNA-dependent manner. Finally, reduction of Rm62 levels results in marked nuclear reorganization of a compromised insulator. These results suggest that the RNAi machinery acts as a modulator of nuclear architecture capable of effecting global changes in gene expression.

Animals↗

PubMed2003

Visualization of chromatin domains created by the gypsy insulator of Drosophila.

Insulators might regulate gene expression by establishing and maintaining the organization of the chromatin fiber within the nucleus. Biochemical fractionation and in situ high salt extraction of lysed cells show that two known protein components of the gypsy insulator are present in the nuclear matrix. Using FISH with DNA probes located between two endogenous Su(Hw) binding sites, we show that the intervening DNA is arranged in a loop, with the two insulators located at the base. Mutations in insulator proteins, subjecting the cells to a brief heat shock, or destruction of the nuclear matrix lead to disruption of the loop. Insertion of an additional gypsy insulator in the center of the loop results in the formation of paired loops through the attachment of the inserted sequences to the nuclear matrix. These results suggest that the gypsy insulator might establish higher-order domains of chromatin structure and regulate nuclear organization by tethering the DNA to the nuclear matrix and creating chromatin loops.

Animals↗

PubMed2006

Identification of genomic sites that bind the Drosophila suppressor of Hairy-wing insulator protein.

Eukaryotic genomes are divided into independent transcriptional domains by DNA elements known as insulators. The gypsy insulator, a 350-bp element isolated from the Drosophila gypsy retrovirus, contains twelve degenerate binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Su(Hw) associates with over 500 non-gypsy genomic sites, the functions of which are largely unknown. Using a bioinformatics approach, we identified 37 putative Su(Hw) insulators (pSIs) that represent regions containing clustered matches to the gypsy insulator Su(Hw) consensus binding sequence. The majority of these pSIs contain fewer than four Su(Hw) binding sites, with only seven showing in vivo Su(Hw) association, as demonstrated by chromatin immunoprecipitation. To understand the properties of the pSIs, these elements were tested for enhancer-blocking capabilities using a transgene assay system. In a complementary set of experiments, effects of the pSIs on transcriptional regulation of genes at the natural genomic location were determined. Our data suggest that pSIs have complex genomic functions and, in some cases, establish insulators. These studies provide the first direct evidence that the Su(Hw) protein contributes to the regulation of gene expression in the Drosophila genome through the establishment of endogenous insulators.

Animals↗

PubMed2005

The Mcp element from the bithorax complex contains an insulator that is capable of pairwise interactions and can facilitate enhancer-promoter communication.

Chromatin insulators, or boundary elements, appear to control eukaryotic gene expression by regulating interactions between enhancers and promoters. Boundaries have been identified in the 3' cis-regulatory region of Abd-B, which is subdivided into a series of separate iab domains. Boundary elements such as Mcp, Fab-7, and Fab-8 and adjacent silencers flank the iab domains and restrict the activity of the iab enhancers. We have identified an insulator in the 755-bp Mcp fragment that is linked to the previously characterized Polycomb response element (PRE) and silences the adjacent genes. This insulator blocks the enhancers of the yellow and white genes and protects them from PRE-mediated repression. The interaction between the Mcp elements, each containing the insulator and PRE, allows the eye enhancer to activate the white promoter over the repressed yellow domain. The same level of white activation was observed when the Mcp element combined with the insulator alone was interposed between the eye enhancer and the promoter, suggesting that the insulator is responsible for the interaction between the Mcp elements.

ATP-Binding Cassette Transporters↗

PubMed2006

Study of long-distance functional interactions between Su(Hw) insulators that can regulate enhancer-promoter communication in Drosophila melanogaster.

The Su(Hw) insulator found in the gypsy retrotransposon is the most potent enhancer blocker in Drosophila melanogaster. However, two such insulators in tandem do not prevent enhancer-promoter communication, apparently because of their pairing interaction that results in mutual neutralization. Furthering our studies of the role of insulators in the control of gene expression, here we present a functional analysis of a large set of transgenic constructs with various arrangements of regulatory elements, including two or three insulators. We demonstrate that their interplay can have quite different outcomes depending on the order of and distance between elements. Thus, insulators can interact with each other over considerable distances, across interposed enhancers or promoters and coding sequences, whereby enhancer blocking may be attenuated, cancelled, or restored. Some inferences concerning the possible modes of insulator action are made from collating the new data and the relevant literature, with tentative schemes illustrating the regulatory situations in particular model constructs.

Animals↗

PubMed2004

Insulator dynamics and the setting of chromatin domains.

The early discovery of cis-regulatory elements able to promote transcription of genes over large distances led to the postulate that elements, termed insulators, should also exist that would limit the action of enhancers, LCRs and silencers to defined domains. Such insulators were indeed found during the past fifteen years in a wide range of organisms, from yeast to humans. Recent advances point to an important role of transcription factors in insulator activity and demonstrate that the operational observation of an insulator effect relies on a delicate balance between the "efficiency" of the insulator and that of the element to be counteracted. In addition, genuine insulator elements now appear less common than initially envisaged, and they are only found at loci displaying a high density of coding or regulatory information. Where this is not the case, chromatin domains of opposing properties are thought to confront each other at "fuzzy" boundaries. In this article, we propose models for both fixed and fuzzy boundaries that incorporate probabilistic and dynamic parameters.

Animals↗

PubMed2006

Transplantation of bone marrow genetically engineered to express proinsulin II protects against autoimmune insulitis in NOD mice.

BACKGROUND: Type 1 diabetes (T1D) is a T-cell-dependent autoimmune disease resulting from destructive inflammation (insulitis) of the insulin-producing pancreatic beta-cells. Transgenic expression of proinsulin II by a MHC class II promoter or transfer of bone marrow from these transgenic mice protects NOD mice from insulitis and diabetes. We assessed the feasibility of gene therapy in the NOD mouse as an approach to treat T1D by ex vivo genetic manipulation of normal hematopoietic stem cells (HSCs) with proinsulin II followed by transfer to recipient mice. METHODS: HSCs were isolated from 6-8-week-old NOD female mice and transduced in vitro with retrovirus encoding enhanced green fluorescent protein (EGFP) and either proinsulin II or control autoantigen. Additional control groups included mice transferred with non-manipulated bone marrow and mice which did not receive bone marrow transfer. EGFP-sorted or non-sorted HSCs were transferred into pre-conditioned 3-4-week-old female NOD mice and insulitis was assessed 8 weeks post-transfer. RESULTS: Chimerism was established in all major lymphoid tissues, ranging from 5-15% in non-sorted bone marrow transplants to 20-45% in EGFP-sorted bone marrow transplants. The incidence and degree of insulitis was significantly reduced in mice receiving proinsulin II bone marrow compared to controls. However, the incidence of sialitis in mice receiving proinsulin II bone marrow and control mice was not altered, indicating protection from insulitis was antigen specific. CONCLUSIONS: We show for the first time that ex vivo genetic manipulation of HSCs to express proinsulin II followed by transplantation to NOD mice can establish molecular chimerism and protect from destructive insulitis in an antigen-specific manner.

Animals↗

PubMed1997

Stem cell gene therapy, position effects and chromatin insulators.

Low efficiency of gene transfer is the main obstacle for a clinically effective gene therapy at the level of the pluripotent hematopoietic stem cell. Another important aspect of stem cell gene therapy, the actual expression of the transduced genes, has only been investigated adequately in very few studies, mainly for globin genes. Transcriptional silencing and position effects due to negative effects of surrounding chromatin on the expression of randomly integrated vector sequences may seriously jeopardize the success of current gene therapy strategies, even if transduction efficiency can be significantly improved. We propose the incorporation of chromatin insulators in the design of gene therapy vectors to overcome the problem of position effects. Chromatin insulators are protein-binding DNA elements that lack intrinsic promoter/enhancer activity but shelter genes from transcriptional influence of surrounding chromatin. The best characterized insulators are from Drosophila. We hypothesize that the important cellular function of chromatin organization is evolutionarily conserved and that human homologs to Drosophila insulator binding proteins such as the suppressor of Hairy-wing exist and can be cloned. Using these putative proteins, it should be possible to identify corresponding minimal binding sites with insulator activity. The design and incorporation of effective chromatin insulator sequences in the next generation of gene therapy vectors should lead to improved and more predictable expression of therapeutic transgenes and constitute an important step toward clinically effective gene therapy.

Animals↗

PubMed2001

An insulator element from the sea urchin Hemicentrotus pulcherrimus suppresses variation in transgene expression in cultured tobacco cells.

Specialized DNA sequences known as insulators protect genes from both the positive and negative influences of nearby chromatin. Many insulators have been identified in various species; however, few function in multiple species. We have shown that an insulator from the Ars (arylsulfatase) gene of the sea urchin Hemicentrotus pulcherrimus functions in plant cells. Normally, expression of an introduced chimeric GUS gene is inactivated in approximately 30% of transformed tobacco BY2 clones. Transgenes containing the Ars insulator, however, were expressed in all transformed tobacco BY2 cells. The insulator did not affect the copy number, the chromosomal position of transgene integration or maximum expression levels. These results suggest that the insulator functions to suppress the variation normally associated with transgene expression in tobacco BY2 cells.

Animals↗

PubMed2005

Effects of muscle potential depression and muscle stimulation caused by different insulation coating configurations on cardiac pacemakers.

Insulation coating was added to the external pacemaker surface to prevent unnecessary electric current leakage to the periphery because the pulse generator body is used as an anode in unipolar pacing. However, a model without insulation coating has recently been used, so we studied the effects on muscle potential inhibition and muscle stimulation of pacemakers in unipolar pacing with different parts of the pacemaker body coated with insulation. Case comparisons were made for the following models: insulated except for the center of one side (33, group C), insulated except for the peripheral zone (10, group E), and noncoated models (11, group N). The muscle detection threshold voltage, muscle detection threshold pulse duration, muscle potential sensing threshold (MP), and lead resistance were measured. A comparison was made of the amount of energy (En) needed to reach the muscle stimulation threshold. For MP values, there was no significant statistical difference between group C and E, whereas a significant difference was present between group C and N and between group E and N. For En values, there was a significant difference between group C and E and between group C and N, but there was no significant difference between group E and N. The muscle potential sensing threshold dose not have a change in group E and much muscle stimulation energy is needed. The muscle potential sensing threshold was low in group N, requiring much muscle stimulation energy. Based on these results, it is usually not necessary to coat the pacemaker with insulation for unipolar pacing.

Aged↗

PubMed1988

Delay in onset of insulitis in NOD mice following a single injection of CD 4 and CD 8 antibodies.

Non-obese diabetic (NOD) mice injected with 500 micrograms of both CD 4 and CD 8 antibodies at 5 weeks of age did not develop insulitis until 18 weeks of age, 12 weeks later than the onset of insulitis in parallel uninjected controls. Injection with both antibodies at 2 weeks or 4 weeks of age protected from insulitis for 10 and 14 weeks respectively. Insulitis was not delayed in onset in animals injected at any age with one antibody only, or who were injected at birth. Injection after the onset of insulitis achieved partial but incomplete clearing of islet infiltrates. Salivary gland infiltrates (sialitis) were also delayed in animals injected with both CD 4 and CD 8 antibodies though the degree of protection was less pronounced than that seen for insulitis.

Age Factors↗