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Results for “Targeted expression”

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At least 271 records · Page 15Linked to original sources
PubMed2001

Specificity of signaling by STAT1 depends on SH2 and C-terminal domains that regulate Ser727 phosphorylation, differentially affecting specific target gene expression.

Complete activation of signal transducer and activator of transcription 1 (STAT1) requires phosphorylation at both Y701 and a conserved PMS(727)P sequence. S727 phosphorylation of STAT1 in interferon-gamma (IFN-gamma)-treated mouse fibroblasts occurred without a need for p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1 and 2 or c-Jun kinases, and required both an intact SH2 domain and phosphorylation of Y701. In contrast, UV irradiation-induced STAT1 phosphorylation on S727 required p38MAPK, but no SH2 domain- phosphotyrosine interactions. Mutation of S727 differentially affected IFN-gamma target genes, at the level of both basal and induced expression. Particularly strong effects were noted for the GBP1 and TAP1 genes. The PMS(727)P motif of STAT3 was phosphorylated by stimuli and signaling pathways different from those for STAT1 S727. Transfer of the STAT3 C-terminus to STAT1 changed the stimulus and pathway specificity of STAT1 S727 phosphorylation to that of STAT3. Our data suggest that STAT C-termini contribute to the specificity of cellular responses by linking individual STATs to different serine kinase pathways and through an intrinsically different requirement for serine phosphorylation at different target gene promoters.

3T3 Cells↗
PubMed2000

Heat-induced gene expression as a novel targeted cancer gene therapy strategy.

One of the main advantages of gene therapy over traditional therapy is the potential to target the expression of therapeutic genes in desired cells or tissues. To achieve targeted gene expression, we experimented with a new approach based on the long-established phenomenon of the heat shock response. By using the green fluorescence protein as a reporter gene, it was demonstrated that expression of a heterologous gene with a heat shock protein 70 promoter could be elevated to 500-1000-fold over background by moderate hyperthermia (39 degrees C to 43 degrees C) in tissue cultured cells. The heat-induced green fluorescence protein expression was first detectable at 3 h after heating and reached a maximum at 18-24 h. The expression dropped back to baseline within 72 h. In addition, when cells were infected with adenovirus vectors containing the heat-inducible interleukin 12 or tumor necrosis factor alpha genes and then heated (42 degrees C, 30 min), expression was at least 13,600- or 6.8 x 10(5)-fold over background, respectively. Intralesion injection of the interleukin-12-carrying adenovirus vector in a mouse melanoma tumor model caused significant tumor growth delay only with hyperthermia treatment. Our results therefore support heat-induced gene expression as a feasible approach for targeted cancer gene therapy.

Adenoviridae↗
PubMed2001

Xenopus Dishevelled signaling regulates both neural and mesodermal convergent extension: parallel forces elongating the body axis.

During amphibian development, non-canonical Wnt signals regulate the polarity of intercalating dorsal mesoderm cells during convergent extension. Cells of the overlying posterior neural ectoderm engage in similar morphogenetic cell movements. Important differences have been discerned in the cell behaviors associated with neural and mesodermal cell intercalation, raising the possibility that different mechanisms may control intercalations in these two tissues. In this report, targeted expression of mutants of Xenopus Dishevelled (Xdsh) to neural or mesodermal tissues elicited different defects that were consistent with inhibition of either neural or mesodermal convergent extension. Expression of mutant Xdsh also inhibited elongation of neural tissues in vitro in Keller sandwich explants and in vivo in neural plate grafts. Targeted expression of other Wnt signaling antagonists also inhibited neural convergent extension in whole embryos. In situ hybridization indicated that these defects were not due to changes in cell fate. Examination of embryonic phenotypes after inhibition of convergent extension in different tissues reveals a primary role for mesodermal convergent extension in axial elongation, and a role for neural convergent extension as an equalizing force to produce a straight axis. This study demonstrates that non-canonical Wnt signaling is a common mechanism controlling convergent extension in two very different tissues in the Xenopus embryo and may reflect a general conservation of control mechanisms in vertebrate convergent extension.

Adaptor Proteins, Signal Transducing↗
PubMed2026

PGC1α expression using targeted redox-responsive nanogels protects against prostate cancer in vivo.

Prostate cancer is among the most frequently diagnosed cancers in men in the UK and US. Increasing evidence implicates metabolic dysregulation as a critical driver of disease progression. Central to this process is peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) that promotes oxidative metabolism and mitochondrial biogenesis while inhibiting metastatic programs. This work investigated the therapeutic potential of PGC1α overexpression via mRNA delivery. Here, we report a prostate-specific, targeted disulphide-crosslinked nanogel system for intracellular delivery of mRNA encoding the N-terminal isoform of PGC1α (NT-PGC1α). Functionalization of the NGs with a peptide targeting prostate-specific membrane antigen (PSMA) enabled selective delivery of NT-PGC1α mRNA in PCa cells and 3D spheroid models. We confirmed sustained PGC1α expression, and increased mitochondrial protein content, indicative of enhanced mitochondrial biogenesis. These nanogels, which were prepared in situ using a nanopolymerization technique, exhibited high mRNA loading capacity, low cytotoxicity, and redox-responsive cargo release, enabling controlled cytosolic delivery following intracellular glutathione-mediated degradation. In vivo, systemic administration of the PSMA-targeted NT-PGC1α mRNA-loaded nanogels resulted in tumor-preferential accumulation and significant suppression of xenograft growth (by 73.4% relative to untreated control), with minimal systemic toxicity. This study presents the first example of a prostate-targeted, disulfide-crosslinked nanogel system for mRNA-mediated metabolic reprogramming in prostate cancer, and highlights its promise as a platform for future RNA-based targeted and precision stimuli-responsive cancer therapies.

Male↗
PubMed2001

Targeting the expression of anti-apoptotic proteins by antisense oligonucleotides.

Antisense oligonucleotide (ASO) biotechnology has been widely used to inhibit the expression of proteins involved in human disease. ASOs are designed to bind messenger RNA transcripts via Watson-Crick base-pairing and inhibit synthesis of targeted proteins. These proteins include protein kinases, growth factors, glutamate receptors, anti-apoptotic proteins, and proteins involved in genetic disorders. Non-mRNA targets such as the RNA component of the telomerase enzyme are also being explored. Pre-clinical and clinical trials using ASO biotechnology have progressed with standard ASOs such as phosphorothioates, but newer ASO analogs are rapidly being developed with the idea of enhancing specificity and biological activity. A current major research thrust is the design and testing of antisense oligonucleotides as anti-cancer drugs. The primary focus of this review is an analysis of recent uses of ASO biotechnology to inhibit anti-apoptotic gene expression in tumor cells.

Clinical Trials as Topic↗
PubMed1996

Protection from natural killer cell-mediated lysis by HLA-G expression on target cells.

The outermost layer of the human placenta is devoid of classical class I human leukocyte antigens (HLA-A, HLA-B, and HLA-C) and class II proteins (HLA-DR, HLA-DQ, and HLA-DP). Although this prevents recognition by maternal T lymphocytes, the lack of class I molecules leaves these cells susceptible to attack by natural killer (NK) cells. However, trophoblast cells directly in contact with the maternal tissues express the class I molecule HLA-G, which may be involved in protecting the trophoblast from recognition by NK cells. Here evidence is provided that expression of HLA-G is sufficient to protect otherwise susceptible target cells from lysis by activated NK1 and NK2 cell lines and clones that are specific for distinct groups of HLA-C alleles. The receptors on NK cells that recognize HLA-G are also identified.

CD56 Antigen↗
PubMed1993

Expression and targeting of a 47 kDa integral peroxisomal membrane protein of Candida boidinii in wild type and a peroxisome-deficient mutant of Hansenula polymorpha.

A 47 kDa integral peroxisomal membrane protein (PMP47) of Candida boidinii was expressed in wild type (WT) and a temperature-sensitive (Ts6) peroxisome-deficient (per) mutant of Hansenula polymorpha. The subcellular location of PMP47 appeared to be dependent on the level of expression. At low expression levels PMP47 was sorted to the peroxisomal membrane; however, in Ts6 cells grown at restrictive temperatures (which lack intact peroxisomes) PMP47 was solely located in small cytosolic aggregates, together with homologous H. polymorpha PMP's. At enhanced expression levels, however, part of the protein also became incorporated into mitochondria, both in transformed WT and Ts6 cells.

Blotting, Western↗
PubMed2016

Matrix stiffness promotes cartilage endplate chondrocyte calcification in disc degeneration via miR-20a targeting ANKH expression.

The mechanical environment is crucial for intervertebral disc degeneration (IDD). However, the mechanisms underlying the regulation of cartilage endplate (CEP) calcification by altered matrix stiffness remain unclear. In this study, we found that matrix stiffness of CEP was positively correlated with the degree of IDD, and stiff matrix, which mimicked the severe degeneration of CEP, promoted inorganic phosphate-induced calcification in CEP chondrocytes. Co-expression analysis of the miRNA and mRNA profiles showed that increasing stiffness resulted in up-regulation of miR-20a and down-regulation of decreased ankylosis protein homolog (ANKH) during inorganic phosphate-induced calcification in CEP chondrocytes. Through a dual luciferase reporter assay, we confirmed that miR-20a directly targets 3'-untranslated regions of ANKH. The inhibition of miR-20a attenuated the calcium deposition and calcification-related gene expression, whereas the overexpression of miR-20a enhanced calcification in CEP chondrocytes on stiff matrix. The rescue of ANKH expression restored the decreased pyrophosphate efflux and inhibited calcification. In clinical samples, the levels of ANKH expression were inversely associated with the degeneration degree of CEP. Thus, our findings demonstrate that the miR-20a/ANKH axis mediates the stiff matrix- promoted CEP calcification, suggesting that miR-20a and ANKH are potential targets in restraining the progression of IDD.

3' Untranslated Regions↗
PubMed2000

Suppression of gene expression by targeted disruption of messenger RNA: available options and current strategies.

At least three different approaches may be used for gene targeting including: A) gene knockout by homologous recombination; B) employment of synthetic oligonucleotides capable of hybridizing with DNA or RNA, and C) use of polyamides and other natural DNA-bonding molecules called lexitropsins. Targeting mRNA is attractive because mRNA is more accessible than the corresponding gene. Three basic strategies have emerged for this purpose, the most familiar being to introduce antisense nucleic acids into a cell in the hopes that they will form Watson-Crick base pairs with the targeted gene's mRNA. Duplexed mRNA cannot be translated, and almost certainly initiates processes which lead to its destruction. The antisense nucleic acid can take the form of RNA expressed from a vector which has been transfected into the cell, or take the form of a DNA or RNA oligonucleotide which can be introduced into cells through a variety of means. DNA and RNA oligonucleotides can be modified for stability as well as engineered to contain inherent cleaving activity. It has also been proven that because RNA and DNA are very similar chemical compounds, DNA molecules with enzymatic activity could also be developed. This assumption proved correct and led to the development of a "general-purpose" RNA-cleaving DNA enzyme. The attraction of DNAzymes over ribozymes is that they are very inexpensive to make and that because they are composed of DNA and not RNA, they are inherently more stable than ribozymes. Although mRNA targeting is impeccable in theory, many additional considerations must be taken into account in applying these strategies in living cells including mRNA site selection, drug delivery and intracellular localization of the antisense agent. Nevertheless, the ongoing revolution in cell and molecular biology, combined with advances in the emerging disciplines of genomics and informatics, has made the concept of nontoxic, cancer-specific therapies more viable then ever and continues to drive interest in this field.

Animals↗
PubMed1997

Cross-clade human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte responses in HIV-infected Zambians.

We have examined cross-clade HIV-specific cytotoxic T-lymphocyte (CTL) activity in peripheral blood of eight Zambian individuals infected with non-B-clade human immunodeficiency virus type 1 (HIV-1). Heteroduplex mobility assay and partial sequence analysis of env and gag genes strongly suggests that all the HIV-infected subjects were infected with clade C HIV-1. Six of eight C-clade HIV-infected individuals elicited CTL activity specific for recombinant vaccinia virus-infected autologous targets expressing HIV gag-pol-env derived from B-clade HIV-1 (IIIB). Recognition of individual recombinant HIV-1 B-clade vaccinia virus-infected targets expressing gag, pol, or env was variable among the patients tested, indicating that cross-clade CTL activity is not limited to a single HIV protein. These data demonstrate that HIV clade C-infected individuals can mount vigorous HIV clade B-reactive CTL responses.

Cross Reactions↗
PubMed1999

Genetic dissection of behavior: modulation of locomotion by light in the Drosophila melanogaster larva requires genetically distinct visual system functions.

The Drosophila larva modulates its pattern of locomotion when exposed to light. Modulation of locomotion can be measured as a reduction in the distance traveled and by a sharp change of direction when the light is turned on. When the light is turned off this change of direction, albeit significantly smaller than when the light is turned on, is still significantly larger than in the absence of light transition. Mutations that disrupt adult phototransduction disrupt a subset of these responses. In larvae carrying these mutations the magnitude of change of direction when the light is turned on is reduced to levels indistinguishable from that recorded when the light is turned off, but it is still significantly higher than in the absence of any light transition. Similar results were obtained when these responses were measured in strains where the larval photoreceptor neurons were ablated by mutations in the glass (gl) gene or by the targeted expression of the cell death gene head involution defective (hid). A mutation in the homeobox gene sine oculis (so) that ablates the larval visual system, or the targeted expression of the reaper (rpr) cell death gene, abolishes all responses to light detected as a change of direction. We propose the existence of an extraocular light perception that does not use the same phototransduction cascade as the adult photoreceptors. Our results indicate that this novel visual function depends on the blue-absorbing rhodopsin Rh1 and is specified by the so gene.

Animals↗
PubMed1996

Emotion-specific processing deficit in focal brain-damaged patients.

Focal brain-damaged patients (left hemisphere damage, right hemisphere damage) and hospitalized general medical patients were asked to sort test photographs into target expressions of four facial emotions, happy, sad, fear and anger. In a second task, patients were asked to match neutral photographs with these target emotion expressions in a forced-choice format. Patients were also asked to rate their mood state on a two-dimensional affect grid. Right hemisphere-damaged patients were significantly inaccurate in matching the test-target expressions of facial emotions in comparison to left hemisphere-damaged or general medical patients. Analysis of error scores indicated a bias toward negative emotions by left hemisphere-damaged patients. Congruent to their mood state, left hemisphere-damaged patients also attributed "sadness" on neutral state of expression significantly more often than in right hemisphere-damaged patients.

Adult↗
PubMed2000

Stage-specific expression and targeting of cyst wall protein-green fluorescent protein chimeras in Giardia.

In preparation for being shed into the environment as infectious cysts, trophozoites of Giardia spp. synthesize and deposit large amounts of extracellular matrix into a resistant extracellular cyst wall. Functional aspects of this developmentally regulated process were investigated by expressing a series of chimeric cyst wall protein 1 (CWP1)-green fluorescent protein (GFP) reporter proteins. It was demonstrated that a short 110 bp 5' flanking region of the CWP1 gene harbors all necessary cis-DNA elements for strictly encystation-specific expression of a reporter during in vitro encystation, whereas sequences in the 3' flanking region are involved in modulation of steady-state levels of its mRNA during encystation. Encysting Giardia expressing CWP1-GFP chimeras showed formation and maturation of labeled dense granule-like vesicles and subsequent incorporation of GFP-tagged protein into the cyst wall, dependent on which domains of CWP1 were included. The N-terminal domain of CWP1 was required for targeting GFP to regulated compartments of the secretory apparatus, whereas a central domain containing leucine-rich repeats mediated association of the chimera with the extracellular cyst wall. We show that analysis of protein transport using GFP-tagged molecules is feasible in an anaerobic organism and provides a useful tool for investigating the organization of primitive eukaryotic vesicular transport.

3' Untranslated Regions↗
PubMed1997

Ectopic expression of target genes may represent an inherent limitation of RT-PCR assays used for micrometastasis detection: studies on the epithelial glycoprotein gene EGP-2.

Our objective was to develop and study the feasibility of a quantitative, nested reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of micrometastatic, epithelial tumor cells using the epithelial glycoprotein EGP-2 gene as a target. Several carcinoma cell lines and peripheral blood samples of 10 healthy volunteers were screened for levels of EGP-2 mRNA. The assay included EGP-2 competitor molecules, carrying an internal deletion, that had been titrated by limiting dilution. Seven carcinoma cell lines showed a wide spectrum of EGP-2 mRNA expression levels, with the highest values (20-100 molecules/cell) seen in 3 breast-cancer cell lines. Unexpectedly, a consistent low level of EGP-2 mRNA expression (0.0004 molecules/cell) was observed in peripheral blood mononuclear cells, probably representing ectopic non-functional expression. Because of this background level, spiking experiments with T47D breast-carcinoma cells added to blood mononuclear cells exhibited a detection limit that was not better than approximately one tumor cell in 2 x 10(4) normal cells. Together with the considerable variation of EGP-2 transcript levels that is observed in different carcinoma cell lines, the extent of expression in normal blood cells would prevent a reliable estimation of low numbers of carcinoma cells in clinical samples. A similar situation might well apply for other target genes. This emphasizes the need for a critical evaluation of the different steps involved in the methods used for RT-PCR detection of micrometastatic tumor cells.

Adolescent↗
PubMed1993

Enzyme release assay of human NK cell activity using beta-galactosidase-expressing K562 target cell line.

In the present report, we established a K562 cell line useful for an enzyme release assay of human natural killer (NK) activity. Human myelogenous leukemia cell line, K562, was transfected with a plasmid carrying Escherichia coli beta-galactosidase (beta-gal) gene. A colony that permanently expresses the enzyme activity was isolated, and designated K562/Zneo. Incubation of K562/Zneo cells (1 x 10(4)) with nonadherent human peripheral blood lymphocytes (PBL) resulted in the release of beta-gal activity depending on the incubation time and the number of effector cells. Released beta-gal activity was assayed sensitively by using 4-methylumbelliferyl-beta-D-galactoside, a fluorescent substrate. The cytolytic activity of PBL was augmented significantly when the cells were preincubated with interleukin-2 for 20 h. This enzyme release assay showed a comparable sensitivity to that of 51Cr release assay. Thus, K562/Zneo cell line is thought to be useful for the nonradioactive assay of human NK and lymphokine-activated killer activities.

Cytotoxicity, Immunologic↗
PubMed1990

Selective induction of toxicity to human cells expressing human immunodeficiency virus type 1 Tat by a conditionally cytotoxic adenovirus vector.

The human immunodeficiency viruses (HIVs) primarily infect CD4+ T lymphocytes, leading eventually to the development of a systemic immune dysfunction termed acquired immunodeficiency syndrome (AIDS). An attractive strategy to combat HIV-mediated pathogenesis would be to eliminate the initial pool of infected cells and thus prevent disease progression. We have engineered a replication-defective, conditionally cytotoxic adenovirus vector, Ad-tk, whose action is dependent on the targeted expression of the herpes simplex virus type 1 thymidine kinase gene (tk), cloned downstream of the HIV-1 long terminal repeat, in human cells expressing the HIV-1 transcriptional activator Tat. Infection of Tat-expressing human HeLa or Jurkat cells with Ad-tk resulted in high-level tk expression, which was not deleterious to the viability of these cells. However, in the presence of the antiherpetic nucleoside analog ganciclovir, Ad-tk infection resulted in a massive reduction in the viability of these Tat-expressing cell lines. As adenoviruses are natural passengers of the human lymphoid system, our results suggest adenovirus vector-based strategies for the targeted expression, under the control of cis-responsive HIV regulatory elements, of cytotoxic agents in HIV-infected cells for the therapy of HIV-mediated pathogenesis.

Adenoviruses, Human↗