We investigated the role of galectin-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptotic death in human breast carcinoma BT549 cells. We observed that parental galectin-3 null BT549 cells (BT549(par)) as well as control vector transfected (BT549(neo)) cells were resistant to TRAIL, while galectin-3 cDNA-transfected BT549 cells (BT549(gal-3)) were sensitive ...

This unit presents two methods of calcium phosphate-based eukaryotic cell transfection that can be used for both transient and stable transfections. In these protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. A HEPES-buffered solution is used to form a calcium ...

Differentiated, human submucosal-gland carcinoma, Calu-3 cell monolayers were used as in vitro model for the airway epithelium. Internalised phage were selected from a recombinant pComb8 phage library by repetitive cycles of bio-panning on Calu-3 monolayers, protease K degradation, cell-lysis and amplification. After four selection rounds, sequence analysis of 15 enriched ...

Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 is an autosomal dominant spinocerebellar degeneration characterized by a wide range of clinical manifestations. Unstable CAG trinucleotide repeat expansion in the MJD gene has been identified as the pathologic mutation of MJD. In this study, human SK-N-SH neuroblastoma cells stably transfected with full-length MJD ...

The goal of this study is to investigate whether TLR2 mediates hBD2 induction through NF-kappaB in response to bacterial components in the human airway epithelia. We showed that hTLR2 is expressed in the airway epithelial cells by immunohistochemical staining and RT-PCR. The biology of hTLR2 in this context was studied ...

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), a selenium-dependent glutathione peroxidase, can interact with lipophilic substrates, including the phospholipid hydroperoxides, fatty-acid hydroperoxides, and cholesteryl ester hydroperoxides, and reduce them to hydroxide compounds. We studied the functional role of endogenous PHGPx in regulation of 12(S)-lipoxygenase and cyclooxygenase 1 activities in human epidermoid carcinoma ...

Human gastric cancer SNU 484 cells express mutant p16, which migrates slower than the wild-type p16. We constructed an expression vector containing human p16 cDNA to evaluate the cytotoxic effects of exogenous p16 expression on SNU 484 cell proliferation and to explore the potential use of p16 in cancer gene ...

Platelet-derived endothelial cell growth factor (PD-ECGF) was isolated as an endothelial mitogen from platelets and demonstrated to have angiogenic activity and thymidine phosphorylase (TP) activity. It was reported that the overexpression of PD-ECGF occurred with the rapid tumor growth in vivo. In this study, we transfected PD-ECGF into the head ...

Transfection of cells by electroporation is a widely used and efficient method. Recently, it has been shown that single neurons in brain slice cultures can be transfected using micropipettes loaded with plasmid DNA expression constructs. However, the transfection efficiencies were very low. Routine employment of single-cell electroporation (SCE) for transfection ...

Human connexins 26 and 30 were expressed either through the bicistronic pIRES-EGFP expression vector or as EYFP-tagged chimeras. When transiently transfected in communication-incompetent HeLa cells, hCx26-pIRES transfectants were permeable to dyes up to 622 Da, but were significantly less permeable to 759 Da molecules. Under the same conditions, permeability of ...

5-Lipoxygenase activating protein (FLAP) functions as a facilitator of 5-lipoxygenase (5-LOX) activity. However, on the basis of the induction of apoptosis by the FLAP inhibitor MK886 in cells lacking 5-LOX, it is possible that this fatty acid-binding protein has other activities. This study was designed to examine potential roles of ...

The gef gene, found in Escherichia coli DNA, encodes a small (50 amino acids) protein which is related to cell-killing functions. We used the MS-36 melanoma cell line as an experimental model to examine the usefulness of the gef gene as a new strategy for cancer therapy. We transfected MS-36 ...

Here we report the generation of stable, selectable Drosophila S2 cell lines using the UAS-GAL4 system. Cloning of the hygromycin resistance gene into the pUAST vector and cotransfection with other pUAST constructs in S2 cells results in coexpression of up to four different proteins under hygromycin selection. Protein expression is ...

Stable cell transfection is used for the expression of exogenous genes or cDNAs in eukaryotic cells. Selection of these transfectants requires a dominant selectable marker. A variety of such markers has been identified and is currently in use. However, many of these are not suitable for all cell types or ...

Progesterone is an important regulator of growth and differentiation in breast tissues. In this study, the effect of progesterone on cell differentiation was evaluated in the estrogen receptor-negative and progesterone receptor (PR)-negative MDA-MB-231 cell line which was transfected with PR-complementary DNA. Morphological changes were analyzed at the ultrastructural level by ...

Efficient screening for ligands of seven-transmembrane, G-protein-coupled receptors, whether transfected or endogenously expressed, often involves cell-based reporter assays. Here we describe the development of reporter gene assays in HeLa cells. The reporter construct includes a synthetic multifunctional promoter with several different response motifs (NF-kappaB, STAT, and AP-1) and hence efficiently ...

Muc4/Sialomucin complex (SMC) acts as an intramembrane ligand for the receptor tyrosine kinase ErbB2, inducing a limited phosphorylation of the receptor. Because Muc4/SMC is found at the apical surfaces of polarized epithelial cells and ErbB2 is often basolateral, the question arises as to whether these components become associated in polarized ...

Duchenne muscular dystrophy (DMD), caused by the absence of dystrophin, is associated with decreased muscle cell proliferation. An increased p21 mRNA level in DMD patients may be involved in the process. In this context we are interested to improve the proliferation of primary human skeletal muscle cells (SkMC) by a ...

Aldehyde dehydrogenase 3A1 (ALDH3A1) is one of the most abundant proteins found in corneal epithelial cells of mammalian species, with several postulated protective roles that include detoxification of peroxidic aldehydes, scavenging of free radicals, and direct absorption of ultraviolet (UV) radiation. In the present study, the protective role of ALDH3A1 ...

AIM: To investigate the expression level of ZNRD1 gene in gastric cancer cells SGC7901 and gastric cancer MDR (multidrug resistant) cells SGC7901/VCR, and to observe the drug sensitizing and proliferation effect of ZNRD1 antisense nucleic acid transduction on SGC7901/VCR cells. METHODS: Amplification of sequences encoding ZNRD1 from SGC7901/VCR cDNA by ...

Electroporation--the use of high-voltage electric shocks to introduce DNA into cells--can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques. In this unit, the describes the electroporation of mammalian ...

Chitosan has the potential for DNA complexation and is useful as a non-viral vector for gene delivery. Highly purified low molecular weight chitosan (LMWC) was prepared. Lactobionic acid (LA) bearing galactose group was coupled with LMWC for liver-specificity. A series of galactosylated-LMWC (gal-LMWC) samples covering a range of galactose group ...

BACKGROUND/AIM: It has been reported previously in cases of adenosquamous carcinoma of the lung in Okinawa, a subtropical island 2000 km south of mainland Japan, that the squamous cell carcinoma components were positive for human papillomavirus (HPV) by non-isotopic in situ hybridisation (NISH). The adenocarcinoma cells adjacent to the squamous ...

After loading cells in culture with acetylcholine (ACh), it was possible to identify cells that express a calcium-dependent release mechanism and cells that do not release. Mediatophore transfection restored the release capability of non-releasing cells. The transfection of choline acetyltransferase and the vesicular ACh transporter (VAChT) in cells that have ...

Oncogenic human papillomavirus (HPV) plays a possible aetiological role in a subset of head and neck cancers, particularly in tonsillar carcinomas. For establishing a model to study mechanisms involved in HPV-associated tonsillar carcinogenesis, normal human tonsillar epithelial (HTE) cells were transfected with full-length HPV-16 DNA. The transfections produced four immortalised ...

The INI1 gene, which encodes a functionally uncharacterized protein component of the hSWI/SNF chromatin remodeling complex, is often mutated or deleted in malignant rhabdoid tumor (MRT). Two isoforms of INI1, that differ by the variable inclusion of nine amino acids, potentially are produced by differential RNA splicing. To determine the ...

Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82-kDa surface glycoprotein (gp82) that has been implicated in host cell invasion. gp82-mediated interaction of metacyclic forms with target cells induces in both cells activation of the signal transduction pathways, leading to intracellular Ca(2+) mobilization, which is required for parasite internalization. ...

A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms ...

It was established that remarkable changes in the N-glycosylation are induced in immortalized cancer cells. Whether changes were induced in human stromal cells immortalized by transfection with the human telomerase catalytic subunit (hTert) cDNA was examined by lectin blot analysis. Morphological appearance and growth rate of the gene-transfected stromal cells ...

To characterize the properties of presenilin (PS) 1- and PS2-associated gamma-secretases, we established stable transfectants overexpressing amyloid precursor protein and wild-type (wt) or a number of mutant (mt) PS1 or PS2. Quantification of the intracellular amyloid beta-protein (Abeta) levels in mtPS1 and mtPS2 cell lines revealed the presence of two ...

The reference bioassay for lactogens is the Nb2 cell proliferation assay, whose extreme sensitivity allows the detection of very low amounts of lactogenic activity in biologic fluids. The use of rat Nb2 cells raises the problem of species specificity when analyzing lactogens of other origin, including human lactogenic hormones for ...

To determine whether cellular components of tissue-engineered cardiovascular structures are derived from cells harvested and seeded onto an acellular scaffold, or from cells originating from surrounding tissue (e.g., proximal and distal anastomosis), cellular retroviral transfection with green fluorescent protein (GFP) was used. Ovine endothelial cells (ECs) were transfected with a ...

We searched by a cDNA subtraction screen for differentially expressed transcripts in MCF-7 mammary carcinoma cells grown on tenascin-C versus fibronectin. On tenascin-C, cells had irregular shapes with many processes, whereas on fibronectin they were flat with a cobble stone-like appearance. We found elevated levels of 14-3-3 tau transcripts and ...

Calcium phosphate transfection is a widely used method to produce recombinant proteins in mammalian cells. However, the mechanisms involved in plasmid DNA transfer to the nucleus of the transfected cells remain poorly understood and result in great variation of transfection efficiency. The role of the cell cycle in transfection by ...

Immunotoxins consist of a target-cell-specific binding moiety, chemically or recombinantly linked to a cytotoxic component. A number of different immunotoxins (IT) have increasingly been evaluated for immunotherapy. Since these foreign proteins are highly immunogenic in human, we have developed recombinant IT using the human ribonuclease angiogenin. Due to their potential ...

Human papillomavirus type 16 (HPV-16) is associated with development of anogenital squamous cell cancers (SCCs) and their precursor, intraepithelial neoplasia (IN). Few approaches to the treatment of IN to prevent SCC are targeted specifically to HPV. We have designed an HPV-specific therapy using the herpes simplex virus type 1 thymidine ...

A highly efficient and specific small interfering (siRNA) (PsiR4) for the serine/threonine kinase Pim-1 has been generated that silences the expression of a Pim1-green fluorescent protein (GFP) fusion gene at low nanomolar concentrations (approximately 5 nM). Only one of four siRNAs tested against Pim-1 had high potency, whereas the three ...

Transfection of mammalian cells with preformed small interfering RNAs (siRNAs) permits a transient and often specific reduction of gene expression. It is possible to rapidly examine the uptake of siRNAs by transfection with fluorescently labeled siRNAs. We examined the apparent uptake of such siRNAs by several leukemic cell lines after ...

To evaluate the specific inhibition of antisense u-PAR on the u-PAR expressions in highly invasive cell subclones and to determine its blocking function in the invasion by those cells, a cDNA fragment of u-PAR obtained by RT-PCR was inserted into a plasmid vector named pcDNA3 in antisense orientation. Then the ...

Previously we reported the development of a novel expression system with Tat/TAR-oriP vectors and HKB11 cell line, which supports high level protein expression (Cho et al. Cytotechnology 2001, 37, 23-30). In the present study, we further demonstrated that HKB11 cells are suitable for high throughput expression (microgram scale) of genomic ...

Hexokinase is the first enzyme involved in the glycolysis process that produces glucose phosphorylate. Our previous study reported on our cloning of mouse Hk1-s (mHk1-s) cDNA, which were expressed only in testis cells, and noted that this cDNA has a spermatogenic cell-specific region (SSR) that replaces the porin binding domain ...

Poly-L-lysine with 40% of the epsilon -amino groups substituted with lactosyl residues facilitated the internalization of lactosylated poly-L-lysine/cDNA complexes into cystic fibrosis (CF) and non-CF airway epithelial cells. It was previously shown that lactosylated poly-L-lysine enhanced the transfer of cDNA into the cell nucleus, resulting in transfection. The cell entry ...

BACKGROUND: We have studied gene transfer efficiency of glycosylated polylysines and glycosylated polyethylenimines as vectors in immortalized differentiated airway gland serous cells and primary cultures of human airway surface epithelial cells. METHODS AND RESULTS: In both cell types, lactosylated PEI was more efficient for gene transfer than unsubstituted PEI and ...

To study the role of the metastasis associated protein S100A4, an osteosarcoma cell line (OHS) with a high level of this protein was transfected with a vector containing a ribozyme that degrades S100A4 mRNA and, as controls, OHS cells were transfected with the vector alone. We have followed up our ...

To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly invasive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer invasion assay and colony formation assay ...

The choroid plexus plays a wide range of roles in brain development, maturation, aging process, endocrine regulation, and pathogenesis of certain neurodegenerative diseases. To facilitate in vitro study, we have used a gene transfection technique to immortalize murine choroidal epithelial cells. A viral plasmid (pSV3neo) was inserted into the host ...

Expression of polo-like kinase-1 (PLK1), which has several functions in mitotic progression, is elevated in a broad range of human tumors. To investigate the role of PLK1 in neoplastic proliferation, we used the technique of RNA interference. Cells from several different cancer cell lines (MCF-7 breast cancer cells, HeLa S3 ...

To investigate the significance of sialylation and sulfation of lactosylceramide in transformed cells, we established ganglioside GM3- and lactosylsulfatide (SM3)-reconstituted cells by transfecting cDNAs of GM3 synthase and cerebroside sulfotransferase into the J5 subclone of 3LL Lewis lung carcinoma cells. The J5 clone was selected for the transfection of these ...

Biodegradable microparticles represent a promising carrier system for the efficient delivery of therapeutic macromolecules to phagocytic professional antigen-presenting cells (APC). Solid lipid microparticles (SLM) consisting of a tripalmitin matrix were prepared using a novel micromixer-based solvent extraction process. A positive surface charge was introduced by the incorporation of cationic lipids ...

PURPOSE: Brain-derived neurotrophic factor (BDNF) has been reported to protect retinal neurons against ischemia and light-induced damage. In the current study, the BDNF gene was transfected into iris pigment epithelial (IPE) cells of Long-Evans rats, and the neuroprotective ability of the IPE cells transfected with the BDNF gene against N-methyl-D-aspartate ...