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At least 199 records · Page 11Linked to original sources
PubMed1999

Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle.

Poly(ethylenimine) (PEI) samples of various molecular weights and pHs were used to transfect endothelial cells to achieve levels of gene expression for comparison. PEIs with nominal molecular weights of 600, 1200, 1800, 10,000, and 70,000 Da were examined at pHs of 5. 0, 6.0, 7.0, and 8.0, and the results were recorded in terms of transfection efficiencies at 24, 48, 68, 92, and 120 h post-transfection. Trials were performed on the human endothelial cell-derived cell line EA.hy 926. We found that, for the polymers tested, transfection efficiency increased as the molecular weight of PEI increased. Representative values of PEIs at pH 6 and molecular weight 70,000 produced average transfection efficiencies of 25.6 +/- 7.9% (n = 8) at the greatest average expression levels, while PEI of molecular weight 10,000 yielded efficiencies of only 11.4 +/- 1.7% (n = 6). Transfection efficiencies for molecular weight 1,800 PEI were essentially zero, and PEIs of lower molecular weights produced no transfection at all. In contrast, the pH of the PEI solutions had no discernible effect on transfection. Optimal expression of the green fluorescent protein reporter occurred between 2 and 3 days post-transfection. The amount of reporter expression also was noted, as determined by the brightness of fluorescing cells under UV. The data obtained demonstrate that the molecular weight of the PEI carrier has an effect on transfection efficiency while the pH of the PEI solutions prior to DNA complexation has no such effect.

Chromatography, Gel↗
PubMed2000

Inhibition of hepatitis C virus (HCV)-RNA-dependent translation and replication of a chimeric HCV poliovirus using synthetic stabilized ribozymes.

Ribozymes are catalytic RNA molecules that can be designed to cleave specific RNA sequences. To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection, we designed and synthesized hammerhead ribozymes targeting 15 conserved sites in the 5' untranslated region (UTR) of HCV RNA. This region forms an internal ribosome entry site that allows for efficient translation of the HCV polyprotein. The 15 synthetic ribozymes contained modified nucleotides and linkages that stabilize the molecules against nuclease degradation. All 15 ribozymes were tested for their ability to reduce expression in an HCV 5' UTR/luciferase reporter system and for their ability to inhibit replication of an HCV-poliovirus (HCV-PV) chimera. Treatment with several ribozymes resulted in significant down-regulation of HCV 5' UTR/luciferase reporter expression (range 40% to 80% inhibition, P <.05). Moreover, several ribozymes showed significant inhibition (>90%, P <.001) of chimeric HCV-PV replication. We further show that the inhibitory activity of ribozymes targeting site 195 of HCV RNA exhibits a sequence-specific dose response, requires an active catalytic ribozyme core, and is dependent on the presence of the HCV 5' UTR. Treatment with synthetic stabilized anti-HCV ribozymes has the potential to aid patients who are infected with HCV by reducing the viral burden through specific targeting and cleavage of the viral genome.

Antiviral Agents↗
PubMed1991

Targeting of transgene expression to monocyte/macrophages by the gp91-phox promoter and consequent histiocytic malignancies.

A component of a heterodimeric cytochrome b, designated gp91-phox, is required for the microbicidal activity of phagocytic cells and is expressed exclusively in differentiated myelomonocytic cells (granulocytes; monocyte/macrophages). In an attempt to identify cis-elements responsible for this restricted pattern of expression, we produced transgenic mice carrying reporter genes linked to the human gp91-phox promoter. Immunohistochemical and RNA analyses indicate that 450 base pairs of the proximal gp91-phox promoter is sufficient to target reporter expression to a subset of monocyte/macrophages. Mice expressing simian virus 40 large tumor antigen under control of the gp91-phox promoter develop monocyte/macrophage-derived malignancies with complete penetrance at 6-12 mo of age and provide an animal model of true histiocytic lymphoma. As these transgenes are inactive in most phagocytic cells that express the endogenous gp91-phox-encoding gene, we infer that additional genomic regulatory elements are necessary for appropriate targeting to the full complement of phagocytes in vivo.

Animals↗
PubMed1996

Silencing of the gene for the beta subunit of human chorionic gonadotropin by the embryonic transcription factor Oct-3/4.

The transcription factor Oct-3/4 may be important in maintaining embryonic cells in an undifferentiated state. It is probably down-regulated at about the time that human chorionic gonadotropin (hCG) is first expressed in embryonic trophectoderm. Here we report that Oct-3/4 strongly inhibits the hCGbeta subunit (hCGbeta) promoter in JAr choriocarcinoma cells. Oct-3/4 reduced chloramphenicol acetyltransferase (CAT) reporter expression from the -305hCGbeta promoter by about 90% in transient co-transfection assays, but had no effect on expression from the -249hCGbeta promoter. The -305/-249 hCGbeta fragment specifically bound synthetic Oct-3/4 protein as measured in electrophoretic mobility shift assays, and the Oct-3/4-binding site was localized around -270 by methylation interference footprinting. Site-directed mutagenesis of this binding site abolished Oct-3/4 repression. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGbeta messenger RNA and hCG protein to less than 10% of controls. We suggest that silencing of Oct-3/4 in trophectoderm is a prerequisite for hCG up-regulation in early human embryos at the time of maternal recognition of pregnancy.

Base Sequence↗
PubMed1995

Expression and activity of the newt Msx-1 gene in relation to limb regeneration.

The Msx-1 homeobox gene is expressed in various contexts during vertebrate development, including the progress zone of the avian and mouse limb bud. Expression of mouse Msx-1 in a cultured myogenic cell line conferred a transformed phenotype and inhibited fusion into myotubes. It has been proposed that Msx-1 expression is required to maintain certain cells in a proliferating and undifferentiated state and may be associated with the ability to regenerate limbs. Urodele amphibians such as the newt regenerate their limbs by formation of a growth zone or blastema, and we have isolated and sequenced newt Msx-1 (NvMsx-1) from a limb blastemal cDNA library. NvMsx-1 expression was detectable in RNA preparations from both limb and tail and their regeneration blastemas, although cultured cells established from limb blastemal mesenchyme gave negative results. When either COS cells or cultured newt blastemal cells were cotransfected with an expression vector for NvMsx-1 and reporter plasmids containing multiple homeobox protein binding sites, NvMsx-1 repressed reporter expression. If NvMsx-1 was expressed together with a marker enzyme in cultured newt blastemal cells, no significant difference in DNA synthesis was observed relative to control transfectants. When myogenic mononucleate cells were transfected with NvMsx-1 and subsequently exposed to low serum to promote fusion, the fraction of Msx-1 positive cells in myotubes was comparable to a control transfected population analysed in the same culture. These results indicate that although Msx-1 expression could be important for limb regeneration, it does not exert a cell-autonomous effect on proliferation or myogenic differentiation of cultured blastemal cells.

Amino Acid Sequence↗
PubMed1996

Characterization and functional analysis of the human inducible nitric oxide synthase gene promoter.

BACKGROUND: Nitric oxide has a wide variety of homeostatic and pathological effects. Control of the production of nitric oxide by the inducible form of the enzyme resides in the 5' promoter region of the gene. Although control of the murine isoform has been investigated, little is known about the functional aspects of the human analog. MATERIALS AND METHODS: A 3.9-kb 5' nontranslated region of the human gene was cloned, sequenced, and several reporter constructs prepared. The promoter-reporter constructs were transfected into human or murine monocytoid cells and reporter expression quantified following cytokine activation of the cells. The production of nitric oxide was also monitored. RESULTS: Although a murine promoter-reporter functioned efficiently in both human and mouse cells, the human constructs functioned only in human cells. The activity of the mouse construct increased progressively with the addition of activating cytokines, but the human promoter-reporter did not. Although interleukin 1 beta drove expression of the human inducible nitric oxide synthase reporter, actual expression of nitric oxide required both interleukin 1 beta and interferon-gamma. CONCLUSIONS: The data indicate that despite the significant homology between the human and mouse inducible nitric oxide synthase promoter sequence, control of the two genes is quite different. In addition to being more efficient in promoter activity, the murine promoter responds increasingly to cytokines that are not effective for the human analog. It is also apparent that human inducible nitric oxide synthase is controlled at both the level of transcription and post-translationally.

Animals↗
PubMed2000

Green fluorescence protein as a transcriptional reporter for the long terminal repeats of the human immunodeficiency virus type 1.

Using the enhanced green fluorescence protein (EGFP), a transient reporter expression system was established to assess the transcriptional activity of the long terminal repeats (LTR) of primary isolates of the human immunodeficiency virus type 1 (HIV-1). Consistent with the conventional chloramphenicol acetyl transferase (CAT) reporter, EGFP expression, under the direction of HIV-1 LTR, was readily detected in the transient transfection and was elevated by co-transfection of HIV-1 tat-expression vector. Comparing to CAT, however, EGFP expression system has two advantages: (i) Using a fluorescence activated cell sorter (FACS), it was possible to simultaneously measure transfection efficiency and fluorescence intensity of the transfected live cells without the necessity of co-transfection of a reference plasmid for comparing the transcriptional activity of two promoters; and (ii) EGFP expression was readily detected at a DNA concentration where CAT activity was not detectable possibly because the transfectants could be 'gated'. On the other hand, at a higher concentration of DNA, CAT signal became more prominent than that of EGFP, possibly because the enzymatic activity of CAT 'amplified' the signal. EGFP fluorescence detected by FACS was a direct measurement of the expressed chromophore. It is concluded that the system is rapid, reproducible, convenient and useful for quantitative analysis of transcription.

Chloramphenicol O-Acetyltransferase↗
PubMed1995

Toddlers' emotion regulation behaviors: the roles of social context and family expressiveness.

Fifty-five toddlers (mean age = 17.7 months) were observed in a modified strange situation that included their preschool-age older siblings (mean age = 50.6 months). The toddlers were observed while interacting with their older siblings and again with the siblings and a stranger. Differences in the toddlers' emotional lability, latency to distress, self-soothing behavior, and comfort-seeking behavior across the two situations were examined. The linkage between the family's reported expression of positive emotion, sadness, and anger and the toddlers' emotion regulation behaviors was evaluated. The results revealed that the toddlers were more emotionally regulated in the presence of the siblings and the stranger than when they were alone with their siblings. In addition, mother-reported positive expressiveness within the family was related to higher levels of self-soothing behavior in the siblings-alone condition, and mother-reported sadness within the family was inversely related to toddlers' self-soothing behavior in both conditions. The results are discussed in terms of the importance of social-context factors and family expressiveness for the development of emotion regulation.

Adaptation, Psychological↗
PubMed1998

Regulation of human renin gene promoter activity: a new negative regulatory region determines the responsiveness to TNF alpha.

BACKGROUND: The renin-angiotensin system has been known to regulate blood pressure and body fluid homeostasis. Several lines of evidence have shown that renin gene expression and release are up-regulated by beta-adrenergic stimulation, sodium depletion, and angiotensin converting enzyme inhibition, but down-regulated by cytokines. To further characterize the human renin gene (hREN) promoter structure, its regulation, and to identify an appropriate cell system for study, we examined five cell lines and investigated drug effects on the hREN promoter expression. METHODS: Using the hREN-luciferase reporter gene constructs in the DNA transfection assays, approximately 5 kb of the hREN 5' flanking region was assessed for promoter activity in five different cell lines. Regulation of the hREN promoter activity was investigated using Y-1 adrenal cells that were transfected with the hREN-luciferase DNA and were treated with forskolin, calcium ionophore A23187, phorbol ester, angiotensin II (Ang II), or cytokines. RESULTS: Transient transfection analysis showed that the 5 kb hREN 5' flanking DNA alone was able to confer significant promoter activity in Y-1 adrenal cells. In transfected Y-1 cells, luciferase reporter expression was induced by forskolin, suppressed by the calcium ionophore A23187, and phorbol ester in a dose-dependent manner, but was unaffected by angiotensin II (Ang II). However, when Y-1 reporter cells were transfected with human angiotensin II receptor type 1 (AT1) cDNA, hREN promoter activity was dose-dependently down-regulated by Ang II, which was blockable by losartan, an AT1-selective antagonist. Further studies also showed that hREN promoter activity in Y-1 cells was selectively down-regulated by TNF alpha. Deletion of the hREN promoter sequences between position -3916 and -2822 not only enhanced hREN promoter activity by approximately tenfold, but also caused a failure of down-regulation by TNF alpha. In contrast, neither interleukin (IL)-1 alpha, IL-1 beta, IL-2, nor IL-6 exerted any significant effect. CONCLUSIONS: Together the results suggest that TNF alpha is a negative regulator of the hREN expression in the adrenal cells, and that the TNF alpha responsiveness may be controlled by elements located between the positions -3916 and -2822 of the hREN promoter. Moreover, the Y-1 cell line may provide a valuable model system for studying renin gene regulation.

Adrenal Glands↗
PubMed1989

Control of granulocyte-macrophage colony-stimulating factor production in normal endothelial cells by positive and negative regulatory elements.

Granulocyte-macrophage (GM)-CSF is an acidic glycoprotein involved in the hemopoietic response to inflammation and in the functional activation of mature blood cells. The protein is produced in response to a number of inflammatory mediators by mesenchymal cells present in the hemopoietic microenvironment and at peripheral sites of inflammation. To understand the molecular basis for the regulation of GM-CSF gene expression, nuclear run-on assays and a functional analysis of potential regulatory sequences were performed in normal human endothelial cells. These studies suggest that GM-CSF is regulated by both transcriptional and post-transcriptional mechanisms. By using hybrid constructs containing a reporter gene and varying lengths of the regions flanking the human GM-CSF gene, a 14-bp sequence was identified in the region upstream of the GM-CSF cap site which increased reporter expression in response to a number of inflammatory mediators. In addition, despite a failure to detect GM-CSF-specific RNA or protein in unstimulated cells, basal transcription from the GM-CSF promoter was readily detectable. To account for this apparent discrepancy, sequences present in the 3' untranslated region of the GM-CSF gene were found to substantially reduce the level of reporter gene expression in a number of cell types. Homologous sequences are found in other genes which share a similar pattern of expression, and may provide the molecular basis for the coordinate regulation of multiple inflammatory response genes.

Base Sequence↗
PubMed1994

Use of genetic recombination as a reporter of gene expression.

An understanding of the patterns of gene expression in response to specific environmental signals can yield insight into a variety of complex biological systems such as microbial-host interactions, developmental cycles, cellular differentiation, ontogeny, etc. To extend the utility of the reporter gene fusion approach to such studies, we have constructed a gene expression reporter cassette that permits the generation of transcriptional fusions to tnpR encoding resolvase, a site-specific recombinase of the transposable element gamma delta. Induction of the transcriptional fusions results in production of resolvase, which in turn, catalyzes excision of a linked tetracycline-resistance reporter gene flanked by direct repeats of res, the DNA sequences at which resolvase functions. The loss of tetracycline resistance in descendant bacteria serves as a permanent and heritable marker of prior gene expression. This gene fusion approach will allow us to assay the induction of gene expression in as few as one cell. Additionally, gene expression can be assayed at a later time and/or different place from the inducing environment facilitating the study of gene expression in complex environments such as animal tissues.

Alkaline Phosphatase↗
PubMed2000

Analysis of dipeptidyl peptidase IV gene regulation in transgenic mice: DNA elements sufficient for promoter activity in the kidney, but not the intestine, reside on the proximal portion of the gene 5'-flanking region.

The dipeptidyl peptidase IV (DPPIV) gene encodes a brush border membrane exopeptidase that is expressed in a tissue-restricted fashion. To examine the regulation of DPPIV transcription in various tissues in vivo, we examined the expression of DPPIV 5'-flanking region (promoter)-human growth hormone reporter constructs in transgenic mice. These mice exhibited cell-type specific reporter expression in kidney. Surprisingly, however, only very low to non-detectable levels of reporter were found in small intestine. These results indicate that DNA elements sufficient for DPPIV expression in kidney, but not intestine, reside in the 5'-flanking region of the gene.

5' Untranslated Regions↗
PubMed1991

Infantile hereditary neuropathy with hypomyelination: report of two siblings with different expressivity.

We report the cases of two siblings both affected by inherited sensory-motor neuropathy of a demyelinative nature but with markedly different severity and pathological findings. The clinical, neurophysiological and morphological features in these two cases were consistent with the diagnosis of Hereditary Motor Sensory Neuropathy type 3 (HMSN 3), according to the classification of Dyck, with different expressivity. These results raise the still unsettled question of the phenotypic variants in inherited neuropathies. In fact the most severely affected of our cases had clinical and neurophysiological findings identical to those reported in cases of Congenital Hypomyelination Neuropathy (CHN), but the morphological picture in the sural nerve was inconsistent with this diagnosis. The criteria for the diagnosis and the reported cases of CHN have been reviewed.

Biopsy, Needle↗
PubMed2000

Glucocorticoid-inducible retrovector for regulated transgene expression in genetically engineered bone marrow stromal cells.

Transplantable bone marrow stromal cells can be utilized for cell therapy of mesenchymal disorders. They can also be genetically engineered to express synthetic transgenes and subsequently serve as a platform for systemic delivery of therapeutic proteins in vivo. Inducible production of therapeutic proteins would markedly enhance the usefulness of stromal cells for cell therapy applications. We determined whether synthetic corticosteroid hormones can be used to tightly control transgene expression via the glucocorticoid response pathway in primary bone marrow stromal cells. This regulatory mechanism does not require the presence of potentially immunogenic prokaryotic or chimeric "Trans-activators." Further, synthetic corticosteroids are pharmaceutical agents that can be readily used in vivo. We designed a self-inactivating retroviral vector in which expression of the green fluorescent protein (GFP) reporter is controlled by a minimal synthetic promoter composed of five tandem glucocorticoid response elements upstream of a TATAA box. Vesicular stomatitis virus G-pseudotyped retroparticles were synthesized and utilized to transduce cultured cell lines and primary rat bone marrow stromal cells. We have shown that primary rat bone marrow stromal cells could be efficiently engineered with our GRE-containing retrovector, basal reporter expression was low in the absence of exogenous synthetic corticosteroids, and GFP expression was dexamethasone inducible and reversible. To summarize, this strategy allows dexamethasone-induced, "on-demand" transgene expression from transplantable genetically engineered bone marrow stromal cells.

Animals↗
PubMed1999

The integrin alpha10 subunit: expression pattern, partial gene structure, and chromosomal localization.

Herein we report the cloning of cDNAs and incompletely processed hnRNAs from endothelia and heart that encode the alpha10 subunit forming part of the novel collagen type II-binding integrin alpha10beta1 of chondrocytes. Analysis of hnRNA clones and reported expressed sequence tags revealed the positions of 17 putative intron-exon splice junctions shared with those of the p150,95 (ITGAX) gene. Human alpha10 transcripts of 5.4 and 1.8 kb were not restricted to chondrocytes but, instead, were widely expressed in a panel of 24 tissue types, where the highest expression was found in muscle and heart. The human alpha10 subunit gene (ITGA10) was localized to band q21 of chromosome 1.

Amino Acid Sequence↗
PubMed1997

CREB-binding protein/p300 are transcriptional coactivators of p65.

CBP (CREB-binding protein) and p300 are versatile coactivators that link transcriptional activators to the basal transcriptional apparatus. In the present study, we identify CBP and p300 as coactivators of the nuclear factor-kappaB (NF-kappaB) component p65 (RelA). Consistent with their role as coactivators, both CBP and p300 potentiated p65-activated transcription of E-selectin and VCAM-1-CAT reporter constructs. The N- and C-terminal domains of both CBP/p300 functionally interact with a region of p65 containing the transcriptional activation domain as demonstrated by mammalian two-hybrid assays. Direct physical interactions of CBP/p300 with p65 were demonstrated by glutathione S-transferase fusion protein binding, and coimmunoprecipitation/Western blot studies. The adenovirus E1A 12S protein, which complexes with CBP and p300, inhibited p65-dependent gene expression. Reporter gene expression could be rescued from E1A inhibition by overexpression of CBP or p300. CBP and p300 act as coactivators of p65-driven gene activation and may play an important role in the cytokine-induced expression of various immune and inflammatory genes.

Adenovirus E1A Proteins↗
PubMed1990

Functional expression of dihydropyridine-insensitive calcium channels during PC12 cell differentiation by nerve growth factor (NGF), oncogenic ras, or src tyrosine kinase.

1. Recombinant retroviruses were used to introduce a temperature-sensitive v-src gene and oncogenic c-Ha-ras into PC12 cells, and stable cell lines expressing these genes were established. 2. As previously reported, expression of v-src (Alema et al., 1985) or c-Ha-ras (Noda et al., 1985) in PC12 cells results in neurite outgrowth resembling that induced by NGF. We report here that v-src but not oncogenic c-Ha-ras induces a stable morphologic neuronal differentiation similar to treatment with NGF. Oncogenic c-Ha-ras-induced neurite outgrowth is not stable with long-term culture, rather the cells revert to an undifferentiated morphology with altered cell cycle kinetics. 3. The stable neuronal phenotype induced by v-src and NGF is characterized by the functional expression of dihydropyridine-insensitive calcium currents.

Animals↗
PubMed1994

Cloning and characterization of cDNAs encoding oat PF1: a protein that binds to the PE1 region in the oat phytochrome A3 gene promoter.

In monocotyledons, the expression of the oat phytochrome A gene (PHYA) is down-regulated by phytochrome itself. This autoregulatory repression is the most rapid light-induced effect on gene expression reported in plants to date. A functional analysis of the oat PHYA3 gene minimal promoter in a rice transient expression assay has identified two promoter elements, PE1 and PE3, that interact synergistically in positive regulation. We have isolated an oat cDNA clone (pO2) that encodes a DNA-binding protein that binds to the PE1 region of the oat PHYA3 gene promoter. The in vitro binding properties of the pO2-encoded protein, towards DNA probes containing either the PE1 sequence or linker-substitution mutations in PE1, correlate with the activity of these DNA elements in the rice transient expression assay. These mutations are known to abolish expression of a reporter gene in vivo. Binding of these linker-substitution mutants to the pO2-encoded protein in vitro was lower by one to two orders of magnitude than the binding of the native PE1 region. We suggest, therefore, that the pO2 clone may encode the putative nuclear factor, oat PF1, that is involved in positive regulation of PHYA3 by binding to PE1 in vivo. pO2 encodes a 170-amino-acid-long protein that contains three repeats of the 'AT-hook' DNA-binding motif found in high mobility group I-Y (HMGI-Y) proteins. Oat PF1 is highly similar to rice PF1 and to the protein encoded by soybean cDNA SB16. They all have a strong similarity in their N-terminus to the pea H1 histone, and the presence of several AT-hook DNA-binding motifs in their C-terminal halves.

Avena↗