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At least 541 records · Page 30Linked to original sources
PubMed2003

GLUT9 is differentially expressed and targeted in the preimplantation embryo.

During preimplantation development in the mouse, it is crucial that glucose metabolism not be compromised. Any decrease in glucose uptake at this stage in development can compromise the developing embryo. We have cloned another member of the glucose transporter family, GLUT9, which is expressed embryonically. Three different isoforms were identified. We have shown that two of the mouse GLUT9 isoforms transport glucose at a rate significantly greater than controls. Expression analysis of the preimplantation blastocyst identifies only the presence of the shorter GLUT9 isoform, RT-PCR and Western immunoblot confirmed this finding. A differential pattern of expression was seen with GLUT9 present at the plasma membrane in one- and two-cell zygotes and in an intracellular compartment in trophectoderm cells at a blastocyst stage. Although blocking GLUT9 expression during preimplantation development had no effect on glucose transport or apoptosis, transfer of these embryos into pseudopregnant mice resulted in increased pregnancy loss, suggesting that GLUT9 is critical for early preimplantation development.

Amino Acid Sequence↗
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↗
PubMed2003

Inhibition of prostate cancer growth by vitamin D: Regulation of target gene expression.

Prostate cancer (PCa) cells express vitamin D receptors (VDR) and 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits the growth of epithelial cells derived from normal, benign prostate hyperplasia, and PCa as well as established PCa cell lines. The growth inhibitory effects of 1,25(OH)(2)D(3) in cell cultures are modulated tissue by the presence and activities of the enzymes 25-hydroxyvitamin D(3) 24-hydroxylase which initiates the inactivation of 1,25(OH)(2)D(3) and 25-hydroxyvitamin D(3) 1alpha-hydroxylase which catalyses its synthesis. In LNCaP human PCa cells 1,25(OH)(2)D(3) exerts antiproliferative activity predominantly by cell cycle arrest through the induction of IGF binding protein-3 (IGFBP-3) expression which in turn increases the levels of the cell cycle inhibitor p21 leading to growth arrest. cDNA microarray analyses of primary prostatic epithelial and PCa cells reveal that 1,25(OH)(2)D(3) regulates many target genes expanding the possible mechanisms of its anticancer activity and raising new potential therapeutic targets. Some of these target genes are involved in growth regulation, protection from oxidative stress, and cell-cell and cell-matrix interactions. A small clinical trial has shown that 1,25(OH)(2)D(3) can slow the rate of prostate specific antigen (PSA) rise in PCa patients demonstrating proof of concept that 1,25(OH)(2)D(3) exhibits therapeutic activity in men with PCa. Further investigation of the role of calcitriol and its analogs for the therapy or chemoprevention of PCa is currently being pursued.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2004

Overexpression of the small mitochondrial Hsp22 extends Drosophila life span and increases resistance to oxidative stress.

Heat shock proteins (Hsp) are involved in protein folding, transport and stress resistance. Studies reporting an increased mRNA level of hsp genes in aged Drosophila suggest that expression of Hsp might be beneficial in preventing damages induced by aging. Because oxidative damage is often observed in aged organisms and mitochondria are sensitive to reactive oxygen species, we tested the hypothesis that increased levels of a small Hsp localized in mitochondria, Hsp22 of Drosophila melanogaster, could protect mitochondrial proteins and influence the aging process. We demonstrate that a ubiquitous or a targeted expression of Hsp22 within motorneurons increases the mean life span by more than 30%. Hsp22 shows beneficial effects on early-aging events since the premortality phase displays the same increase as the mean lifespan. Moreover, flies expressing Hsp22 in their motorneurons maintain their locomotor activity longer as assessed by a negative geotaxis assay. The motorneurons-targeted expression of Hsp22 also significantly increases flies' resistance to oxidative injuries induced by paraquat (up to 35%) and thermal stress (39% at 30 degrees C and 23% at 37 degrees C). These observations establish Hsp22 as a key player in cell-protection mechanisms against oxidative injuries and aging in Drosophila and corroborate the pivotal role of mitochondria in the process of aging.

Aging↗
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↗
PubMed2002

Human umbilical cord blood NK T cells kill tumors by multiple cytotoxic mechanisms.

Natural killer (NK) T cells are restricted by CD1d and play an important role in the rejection of malignant tumors, but how kill these tumors is unclear. To investigate this, we cultured Valpha24+CD4+ NK T cells in human umbilical cord blood, which was enriched by immunomagnetic beads. In short-term (4 h) cytotoxicity assays, the NK T cells could kill only those targets expressing CD1d. In longer cytotoxicity assays (20 h), however, the NK T cells were able to kill all the tumors, regardless of CD1d expression. When each of the perforin, Fas-FasL, and TNF-alpha cytotoxic mechanisms were blocked, it was apparent that perforin killing dominated in both the short- and long-term assays. In the short-term assay, perforin killing required that the targets expressed CD1d, but killing was more efficient if Fas was present because then the Fas-FasL mechanism was also used. Thus, cells that lacked Fas and CD1d and were not killed in the 4-h assay, were instead lysed in 20-h assay through a combination of perforin and TNF-alpha killing. NK T cells can kill tumor targets by perforin, Fas-FasL, and TNF-alpha mechanisms. TNF-alpha killing requires longer contact between effectors and targets, suggesting that TNF-alpha acts by enhancing perforin killing.

Antigens, CD1↗
PubMed2001

Cloning and Expression of Targeting CuZn-SOD to Central Nervous System.

Oxygen-derived free radicals are thought to be involved in the pathogenesis of a wide range of neurological disorders. Targeted delivery of CuZn-SOD to neurons in central nervous system may have therapeutic value in such diseases. The gene encoding human CuZn-SOD was fused to tetanus toxin fragment C geneto construct a fusion gene, then it was cloned into prokaryotic expression vector pET-22b( ) and baculovirus vector pFastBacHTb, and was expressed in E.coli and Tn-5B1-4 cells, respectively. The recombinant fusion protein has a subunit molecular mass of 68 kD and is recognized by both anti-CuZn-SOD and anti-tetanustoxin antibody. By pyrogallol autooxidation assay, it is shown that the CuZn-SOD moiety retains substantial enzymatic activity, where the TTC moiety might deliver the fusion protein to neurons in central nervous system. So, CuZn-SOD/TTC may be a useful agent for the targeted delivery of CuZn-SOD to neurons.

Journal Article↗
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↗
PubMed2001

[The construction and evaluation of antisense RNA expression vector targeting at HCV 5'NCR].

Antisense RNA is an important field of antisense technique. In order to explore a novel approach for the treatment of hepatitis C virus (HCV) infection and prove the availability of transgenic cellular model HepG2. 9706, an antisense RNA was designed targeting at the highly conserved 5'NCR and translation initiation site of HCV RNA at nt positions 13-397. It was inserted into the downstream of SV40 promotor sequence of pGL3 control vector in which luciferase gene is deleted. The antisense RNA expression vector (pHCV-asR) was constructed and identified by PCR, endonucleases reaction and DNA sequencing. Its expression in transfected HepG2 cells was tested by RT-PCR. To evaluate the inhibitory activities of pHCV-asR, HepG2. 9706 cells were transfected using this construct via Lipofectin method. Luciferase activity in cell lysates was measured for quantitative determining antiviral effects within the cells. The results showed that the inserted sequence of the pHCV-asR is the same as the designed sequence and can express in HepG2 cells. It was also found that pHCV-asR in HepG2. 9706 has a dose-dependent inhibitory effects on luciferase expression controlled by HCV 5'NCR with a inhibitory rate of 57%.

5' Untranslated Regions↗
PubMed2002

Induction of apoptosis in HeLa cells with siRNA expression vector targeted against bcl-2.

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific gene silencing in animal and plant cells. In mammalian cells, 21- or 22-nucleotide (nt) RNAs with 2-nt 3' overhangs small inhibitory RNAs (siRNAs) exhibit an RNAi effect. Very recently, we and others have developed a vector-based siRNA expression system that can induce RNAi in mammalian cells. In this report, to apply this system to oncogene therapy, we tried to suppress the expression of the bcl-2 gene, which is known as a key molecule in the regulation of apoptosis or programmed cell death, by using the siRNA expression system. Western blotting analysis revealed that this siRNA expression vector against bcl-2 suppressed the expression of the bcl-2 protein. Furthermore, HeLa cells which were transiently transfected with the siRNA expression vector against bcl-2 and were subsequently treated with doxorubicin efficiently underwent apoptosis, concomitant with the repression of the bcl-2 gene. These results demonstrate that the siRNA expression vector against bcl-2 has a potential as therapeutic agent for a variety of cancers by down-regulating bcl-2 gene expression.

Apoptosis↗
PubMed1997

S100A1 and S100B expression and target proteins in type I diabetes.

Calcium receptor proteins are an essential link between hormones that alter intracellular calcium levels and the generation of cellular responses. However, there is no information available regarding the role of calcium receptor proteins, in particular the S100 family, in insulin action and/or diabetes. This study examines the effects of streptozotocin-induced type I diabetes on the expression of the individual S100A1 and S100B isoforms as well as their binding proteins. Diabetes did not increase (or initiate) S100B expression in any non-S100B-expressing tissue (skeletal muscle, heart, kidney, liver, spleen, and pancreas). In all S100B-expressing tissues examined (brain, white fat, and testes), S100B protein levels increased approximately 2-fold while steady state S100B messenger RNA (mRNA) levels decreased. S100A1-expressing tissues exhibited increased (kidney and lung), decreased (skeletal muscle), and unchanged (brain and heart) S100A1 protein levels. While noncoordinate changes in S100A1 protein and steady state mRNA levels were observed in heart, other S100A1-expressing tissues (brain, slow twitch skeletal muscle, and kidney) exhibited coordinate changes in S100A1 protein and steady state mRNA levels. Altogether, these results suggest that the effects of diabetes on S100 expression are isoform as well as tissue-specific. Gel overlay analysis of the S100-binding protein profile revealed both increases and decreases in binding proteins in all tissues examined. In summary, changes in the expression of S100A1, S100B, and S100-binding proteins occur in type I diabetes and represent important molecular events in the effects of insulin/insulin insufficiency on cell function.

Animals↗
PubMed2003

Rare expression of target antigens for immunotherapy on disseminated tumor cells in breast cancer patients without overt metastases.

Occult disseminated tumor cells are the major cause of relapse in patients with primary operable breast cancer but detection and characterization of these few cells is difficult. Applying immunohistochemistry, an immunomagnetic enrichment technique (IET) and immunocytochemistry (IC), we studied 58 breast cancer patients without overt metastases for the frequency of cytokeratin-positive (CK+) bone marrow (BM) cells coexpressing the epithelial adhesion molecule 17-1A (EpCAM) and c-erbB-2 and analyzed the primary tumor for these antigens as a strategy for additional immunotherapy. The primary tumors were analyzed for the target antigens by a pathologist. Dissemination of CK+ cells was studied in 4-6 x 10(6) BM cells by IC alone. For characterization of CK+ cells, 10-15 x 10(6) BM cells were incubated with microbeads coupled to antibodies detecting the target antigens, labelled cells were separated on selection columns and the positively (BM cells carrying the target antigen) and negatively (BM cells without target antigen) selected fractions were stained for CK+ cells. The effectiveness of these methods was confirmed in cell culture models. 17-1A was detected in all primary tumors and c-erbB-2 overexpression (2+, 3+) was found in 25/58 tissue samples. In total, analyzing 15-20 x 10(6) BM cells in each patient, the detection rate for CK+ cells in the BM was 69% (40/58 patients). Interestingly, analysis of the positive and negative enrichment fractions showed that the 17-1A antigen was coexpressed on CK+ cells in only 6 patients and c-erbB-2/CK+ cells were found in only one patient. Although 17-1A and c-erbB-2 were frequently detected in the primary tumor, these antigens were rarely expressed on CK+ BM cells. Whether the applied IET is not able to detect low amounts of these target antigens has to be clarified. Nevertheless, applying cell-cycle independent protocols in clinical trials requires careful elucidation of those patients who might benefit from these therapies.

Antigens, Neoplasm↗