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Results for “Targeted expression”

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At least 253 records · Page 14Linked to original sources
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↗
PubMed1993

Alteration of Caenorhabditis elegans gene expression by targeted transformation.

We have produced strains carrying a synthetic fusion of parts of two vitellogenin genes, vit-2 and vit-6, integrated into the Caenorhabditis elegans genome. In most of the 63 transformant strains, the plasmid sequences are integrated at random locations in the genome. However, in two strains the transgene integrated by homologous recombination into the endogenous vit-2 gene. In both cases the reciprocal exchange between the chromosome and the injected circular plasmid containing a promoter deletion led to switching of the plasmid-borne promoter and the endogenous promoter, with a reduction in vit-2 expression. Thus in nematodes, transforming DNA can integrate by homologous recombination to result in partial inactivation of the chromosomal locus. The simplicity of the event and its reasonably high frequency suggest that gene targeting by homologous recombination should be considered as a method for directed inactivation of C. elegans genes.

Animals↗
PubMed1996

Evidence from normal expression and targeted misexpression that bone morphogenetic protein (Bmp-4) plays a role in mouse embryonic lung morphogenesis.

Epithelial-mesenchymal interactions are critical for the branching and differentiation of the lung, but the mechanisms involved are still unclear. To investigate this problem in mouse embryonic lung, we have studied the temporal and spatial expression of genes implicated in the morphogenesis of other organs. At 11.5 days p.c., hepatocyte nuclear factor-3beta (Hnf-3beta) is expressed uniformly throughout the epithelium, while Wnt-2 expression is confined to the distal mesenchyme. Sonic hedgehog (Shh) transcripts are found throughout the epithelium, with high levels in the distal tips of the terminal buds, while bone morphogenetic protein-4 (Bmp-4) transcripts are localized at high levels in the distal tips of the epithelium, with lower levels in the adjacent mesenchyme. Epithelial expression is also seen for Bmp-7, but transcripts are less dramatically upregulated at the distal tips. The Type I Bone morphogenetic protein receptor gene (Bmpr/Tfr-11/Brk-1) is expressed at low levels in the epithelium and in the distal mesenchyme. To investigate the role of Bmp-4 in lung development, we have misexpressed the gene throughout the distal epithelium of transgenic lungs using a surfactant protein C enhancer/promoter. From 15.5 days p.c., transgenic lungs are smaller than normal, with grossly distended terminal buds and, at birth, contain large air-filled sacs which do not support normal lung function. Labeling with BrdU reveals an inhibition of epithelia] proliferation in 15.5 days p.c. transgenic lungs. A small but significant stimulation of proliferation of mesenchymal cells is also observed, but this is accompanied by an increase in cell death. In situ hybridization with riboprobes for the proximal airway marker, CC10, and the distal airway marker, SP-C, shows normal differentiation of bronchiolar Clara cells but a reduction in the number of differentiated Type II cells in transgenic lungs. A model is proposed for the role of BMP4 and other signalling molecules in embryonic lung morphogenesis.

Animals↗
PubMed2001

A humanized monoclonal antibody constructed from intronless expression vectors targets human hepatocellular carcinoma cells.

An anti-human hepatocellular carcinoma (HCC) monoclonal antibody, hHP-1, was genetically humanized from a murine monoclonal antibody. In this study, a concept of positional template approach was applied to design the amino acid sequence of hHP-1's variable region, and synthetic DNA fragments for protein expression were produced through overlapping PCR from single strand oligonucleotides. Synthetic DNA fragments and human antibody constant region cDNA were used to construct two CMV promotor-based expression vectors for the antibody light and heavy chains, in which the variable region was connected directly to the constant region without an intron sequence. Completely assembled humanized antibody was successfully expressed in mammalian cells as IgG1 kappa molecules and purified using protein A affinity column. The immunogenicity of the hHP1 was estimated by the amino acid sequence and determined through a HAMA (human anti-murine antibody) serum reaction assay. Results indicated that the immunogenicity of hHP-1 was significantly reduced. In vitro binding activity assay showed that the hHP-1 had retained its binding function to a human HCC SMMC-7721 cell-line, without cross binding to other human normal tissues. Immunofluorescence staining showed that hHP-1 had a strong binding activity to SMMC cells. A competitive binding assay showed that the relative binding activity of hHP-1 was approximately 25% binding activity of the original murine antibody. Our results indicate that a humanized antibody could be produced using intronless vectors and expressed as a complete IgG1 kappa antibody. Hence we believe that hHP-1 could be a potential candidate for HCC treatment.

Amino Acid Sequence↗
PubMed2001

Peripheral blood stem cell allograft rejection mediated by CD4(+) T lymphocytes recognizing a single mismatch at HLA-DP beta 1*0901.

Little is known about the molecular characteristics of alloantigens recognized by alloreactive T cells mediating hematologic stem cell graft rejection. In particular, it has never been shown that such alloantigens can be encoded by HLA-DP beta alleles. Indeed, matching for HLA-DP antigens is generally not considered to be of functional importance for the outcome of allogeneic bone marrow or peripheral blood stem cell transplantation. In this study, a case of peripheral blood stem cell allograft rejection was investigated in which the patient and donor differed for a single mismatch at HLA-DP in the rejection direction. Patient-derived T lymphocytes circulating at the time of rejection showed direct ex vivo cytotoxic activity against donor-derived B-lymphoblastoid cells as well as other HLA-DP beta 1*0901--expressing targets. The presence of HLA-DP beta 1*0901--specific effectors in vivo was further confirmed by in vitro stimulation experiments. CD4(+) T-cell lines and clones with specific cytotoxic activity against HLA-DP beta 1*0901--expressing targets including donor B-lymphoblastoid cells were generated both by nonspecific and by donor-specific in vitro stimulation. Taken together, these data demonstrate that HLA-DP can be the target antigen of cytotoxic CD4(+) T lymphocytes involved in peripheral blood stem cell allograft rejection. (Blood. 2001;98:1122-1126)

Adult↗